龙眼体胚发生过程中的CDC48和GPX基因克隆与表达
|
摘要
本研究以龙眼(Dimocarpus longan Lour.)“红核子”胚性愈伤组织(Embryogenic callus, EC)为材料,对以下8个方面进行研究:①龙眼胚性愈伤组织CDC48基因(cell division cycle 48)cDNA和DNA克隆;②CDC48基因的原核表达;③龙眼胚性愈伤组织CDC48基因的生物信息学分析;④龙眼胚性愈伤组织GPX (glutathione peroxidase)cDNA和DNA克隆;⑤GPX基因的原核表达;⑥龙眼胚性愈伤组织GPX的生物信息学分析;⑦以龙眼UBQ和EF-1a 2个基因共同作为内参基因,利用荧光定量PCR技术分析龙眼体胚发生过程各阶段培养物中龙眼CDC48和GPX基因转录水平表达变化;⑧分析龙眼体胚发生过程各阶段培养物及逆境胁迫处理下龙眼EC GPX活性变化。主要研究结果如下:
1.龙眼胚性愈伤组织CDC48基因cDNA和DNA全长的获得
以龙眼EC为材料,应用同源克隆结合RACE技术,获得了龙眼胚性愈伤组织CDC48的cDNA全长为为2620 bp,其中5'UTR为17 bp,3'UTR为187 bp,3’端还含有13个poly(A)尾。该序列与登录GenBank的其它植物CDC48基因有很高的同源性。序列分析发现拼接的cDNA含有一个2415 bp的开放阅读框,编码805个氨基酸,ATG为起始密码子、TAG为终止密码子。将此基因命名为DLCDC48,并在GenBank上登录,登录号为EU606206。从DNA水平克隆也得到了龙眼胚性愈伤组织的CDC48基因,并且经测序证实该基因无内含子,在GenBank上登录,登录号为:FJ590953。
2.龙眼胚性愈伤组织CDC48基因原核表达
根据龙眼EC CDC48全长cDNA的序列分析,在可能的ORF区域设计一对添加限制性内切酶的特异引物,进行ORF片段扩增并将其克隆到表达载体pET-28 a中。将构建好的表达载体在大肠杆菌表达宿主BL21(DE3)中进行融合表达。经过诱导,SDS-PAGE分析发现宿主菌中有一个分子量约为89 kD的新蛋白出现。该诱导表达的蛋白分子量与理论推导龙眼EC CDC48的分子量89.5 kD相近。
3.龙眼胚性愈伤组织CDC48基因的生物信息学分析
应用生物信息学软件对龙眼胚性愈伤组织CDC48的核苷酸序列和氨基酸序列进行了分析。结果表明: CDC48蛋白的分子量是89564.8 Da,理论等电点pI 4.92,是不具跨膜结构域的亲水性胞质蛋白,不具有信号肽,主要定位在细胞核上,有3个区域最有可能形成卷曲螺旋,由40.87%的a螺旋、15.28%的延伸链和43.85%的不规则卷曲组成,磷酸化位点有39个。保守结构域与功能域分析龙眼CDC48具有两个典型的ATPase模块,以及含有CDC48特有的N端。通过功能的预测与分析,推测与细胞的分裂有关系。通过其氨基酸序列的系统进化树分析,CDC48的进化在一定程度上反应了植物的进化。此外,还对CDC48酶分子三维立体结构等进行了预测和分析。
4.龙眼胚性愈伤组织GPX基因cDNA和DNA全长的获得
以龙眼EC为材料,应用同源克隆结合RACE技术,获得了龙眼胚性愈伤组织GPX的cDNA全长cDNA全长为947 bp, 5'UTR为195bp,3'UTR为245 bp,区域还含有典型的加尾信号AATAA和poly(A)尾。该序列与登录GenBank的其它植物GPX基因有很高的同源性。序列分析发现拼接的cDNA含有一个504 bp的开放阅读框,编码168个氨基酸,ATG为起始密码子、TAA为终止密码子。将此基因命名为DLGPX,并在GenBank上登录,登录号为EU364813。从DNA水平克隆也得到了龙眼胚性愈伤组织的GPX基因,1736 bp核苷酸序列,有ATG起始密码子和TAA终止密码子,并在GenBank上登陆,登陆号为:EU680970。通过DNAMAN 6.0软件分析,GPX基因由5个外显子和4个内含子组成,所有内含子的剪切位点均符合真核生物GT-AG规则。
5.龙眼胚性愈伤组织GPX基因原核表达
根据龙眼EC GPX全长cDNA的序列分析,在可能的ORF区域设计一对添加限制性内切酶的特异引物,进行ORF片段扩增并将其克隆到表达载体pET-28 a中。将构建好的表达载体在大肠杆菌表达宿主BL21(DE3)中进行融合表达。经过诱导,SDS-PAGE分析发现宿主菌中有一个分子量约为23 kD的新蛋白出现。本研究所用的酶切位点为SacI和XhoI,把载体上额外翻译的38个氨基酸一起表达。龙眼GPX编码基因产物的理论推导分子量18.54 kD,加上额外翻译的氨基酸理论推导分子量为4.08 KD(38个氨基酸理论推导分子量为39.45 KD),总共为22.62KD,与本研究结果相符。
6.龙眼胚性愈伤组织GPX基因生物信息学分析
运用生物信息学软件对龙眼胚性愈伤组织GPX基因的核苷酸序列和氨基酸序列进行了分析。结果表明:GPX蛋白的分子量是18541.16Da,理论等电点pI 7.18,是不具跨膜结构域的亲水性胞质蛋白,不具有信号肽,细胞主要定位在细胞质上,有1个区域最有可能形成卷曲螺旋,由27.98%的a螺旋,20.249%的延伸链和51.79%的不规则卷曲组成,磷酸化位点有13个。龙眼胚性愈伤组织GPX氨基酸序列含有PHGPX的两个特征序列,推测所克隆到的可能是GPXs家族中保护膜免受损伤的PHGPX的cDNA序列。此外,还对GPX酶分子三维立体结构等进行了预测和分析。
7.龙眼胚性愈伤组织CDC48和GPX基因转录水平表达分析
用龙眼胚性培养物中UBQ和EF-1a 2个基因共同作为内参基因,利用荧光定量PCR技术分析龙眼体胚发生过程各阶段培养物龙眼CDC48和GPX基因转录水平表达变化。分析结果显示:龙眼CDC48在体胚发生过程中都有不同程度的表达,其中胚性紧实球形结构阶段表达量最大,其次是胚性愈伤组织,最低的是球形胚;龙眼GPX在体胚发生过程中也均有不同程度的表达,其中胚性紧实球形结构阶段表达量最大,其次是子叶胚,最低的是胚性愈伤组织。
8.龙眼体胚发生过程中GPX酶活性变化
GPX活性在龙眼体胚发生过程中也均有不同程度的表达,其中胚性紧实球形结构阶段表达量最大,其次是子叶胚,最低的是胚性愈伤组织,这与龙眼体胚发生过程中基因相对定量表达结果是一致的;以龙眼胚性细胞系LC2为材料,研究了在NaCl、光和温度胁迫下龙眼胚性愈伤组织GPX酶活性的变化规律。分析结果显示:在一定逆境胁迫下,细胞内GPX可以起到应答外界刺激的作用,且活性呈规律表达,推测GPX活性变化与胚性细胞抗逆性正相关,是植物在逆境胁迫下防御ROS伤害的主要标志之一。
总之,本研究克隆了与体胚发生过程有密切相关的2个基因,进行了表达分析,并比较全面地预测和分析了它们的结构和功能,加深了对龙眼体胚发生机制的理解,为构建龙眼体胚发生的基因网络提供新的资料,为研究龙眼体胚发生过程中的细胞发育调控和活性氧的清除等方面的机理提供有价值的证据,为利用基因工程方法进行龙眼和其它植物胚胎发育调控等方面的遗传改良提供基因资源。
In this experiment, the embryogenic calli (EC) of longan (Dimocarpus longan Lour. cv. Honghezi) were used as the materials to study the following eight aspects:①cDNA and DNA cloning of longan EC CDC48 gene (cell division cycle 48 gene);②prokaryotic expression of CDC48 gene;③analysis of bioinformatics of longan EC CDC48 gene;④c DNA and DNA cloning of longan EC GPX (glutathione peroxidase gene);⑤p rokaryotic expression of GPX gene;⑥a nalysis of bioinformatics of longan EC GPX;⑦u sing both UBQ and EF-1a genes in longan as the reference genes, the transcriptional expression levels of CDC48 and GPX genes in the embryogenic cultures at different developmental stages during longan somatic embryogenesis were analyzed by fluorescence quantitative PCR;⑧analysis the changes of longan GPX activities at different stages during longan somatic embryogenesis and under stress treatment. The main results showed as follows:
1. Obtaining full length of cDNA and DNA of CDC48 gene from longan EC
Using longan EC as the material, the full length of cDNA (2620 bp ) of CDC48 from longan EC was obtained by homology cloning combined with RACE technology, which contained 17bp 5'UTR,187bp 3'UTR, and 3'-end involved 13 poly(A)tails. The results showed the sequence of CDC48 gene from longan EC was highly homologous with that of other plants reported in GenBank. Sequence analysis revealed that splicing of the cDNA contained a 2415 bp open reading frame, encoded 805 amino acids, and ATG was the initiation codon and TAG was the stop codon. The gene was named as DLCDC48 and registered in GenBank. The registered No. was EU606206. The DNA of CDC48 gene which had no intron by sequence analysis was also cloned from longan EC, which was also registered in GenBank, and the registered No. was FJ590953.
2. Prokaryotic expression of longan EC CDC48 gene
According to sequence analysis of full-length cDNA of longan EC CDC48 gene, a pair of specific primers added in restriction enzymes were designed in the possible ORF region to amplify ORF, and then it was inserted into the expression vector pET-28 a, and finally the well constructed expression vector was taken into the E. coli expression host BL21 (DE3) for fusion expression. After induction, there was a new protein emerging from the host bacteria and the molecular weight was about 89 kD by SDS-PAGE analysis. The protein molecular weight of the induced expression was near with that of the theoretical derivation from longan EC CDC48.
3. Analysis of bioinformatics of longan EC CDC48 gene
Longan EC CDC48 nucleotide sequence and corresponding amino acid sequences were analyzed by using bioinformatics softwares. The results showed that the molecular weight of protein CDC48 was 89564.8 Da; the theoretical isoelectric point ( pI ) was 4.92; it was a kind of hydrophilic cytoplasmic protein without transmembrane domain and signal peptide, and was mainly located in the nucleus; three regions were most likely to form coiled-coil containing with 40.87%α-spiral, 15.28% extending chain and 43.85% irregular curl; and the phosphorylation sites were 39. The longan CDC48 gene had two typical ATPase modules and specific N-terminal by analysis of conservative structure domain and function domain. Through prediction and analysis, it was speculated that the function of this gene was relevant with cell division. By analyzing phylogenetic trees of its amino acid sequence, it was concluded that the evolution of CDC48 reflected the evolution of plants to some degree. Furthermore, the three-dimensional structure of CDC48 enzyme molecules was predicted and analyzed, etc..
4. Obtaining the full length of cDNA and DNA of GPX gene from longan EC
Using longan EC as the material, the full length of longan EC GPX gene 947 bp was obtained by homology cloning combined with RACE technology, which contained 195bp 5'UTR, 245bp 3'UTR with a typical add-tail signal AATAA and a poly (A) tail. The results showed that the sequence of GPX gene from longan EC was highly homologous with that of other plants reported in GenBank. Sequence analysis revealed that splicing of the cDNA contained a 504 bp open reading frame, encoded 168 amino acids, and ATG was the initiation codon and TAG was the stop codon. The gene was named as DLGPX, and registered in GenBank. The registered No. was EU364813. The DNA of GPX gene was cloned from longan EC, which had 1736 bp nucleotide sequence with ATG initiation codon and TAA stop codon. The gene was registered in GenBank and the No. was EU680970. By the analyses of the DNAMAN 6.0 software, the GPX gene consisted of 5 exons and 4 introns, and the splice sites of all introns were accorded with eukaryotic GT-AG rule. 5. Prokaryotic expression of longan EC GPX gene According to sequence analysis of full-length cDNA of longan EC GPX gene, a pair of specific primers added in restriction enzymes were designed in the possible ORF region to amplify ORF, and then it was inserted into the expression vector pET-28 a, and finally the well Constructed expression vector was taken into the E. coli expression host BL21 (DE3) for fusion expression. After induction, there was a new protein emerging from the host bacteria and molecular weight about 23 kD by SDS-PAGE analysis. In this study, SacI and XhoI were used as the enzyme restriction sites, and 38 amino acids translated additionally in vector were also expressed. The theoretical molecular weight of gene product encoded by longan GPX gene was 18.54 kD, added with the theoretical derivation molecular weight of translating additionally amino acids 4.08 KD(38 amino acids theoretical molecular weight is 39.45 KD), and the total was 22.62KD, which was accorded with the results of this experiment. 6. Analysis of bioinformatics of longan EC GPX gene Longan EC GPX nucleotide sequence and amino acid sequences were analyzed by using bioinformatics softwares. The results showed that the molecular weight of protein GPX gene was 89564.8 Da; the theoretical isoelectric point (pI ) was 7.18; it was a kind of hydrophilic cytoplasmic protein without transmembrane domain and signal peptide, and was mainly located in the cytoplasm; a region was most likely to form coiled-coil containing with 27.98%α-spiral, 20.249% extending chain and 51.79% irregular curl, and the phosphorylation sites were13. The amino acid sequence of longan EC GPX gene contained two characteristics sequences of PHGPX. It was speculated that the cloned gene was possibly the cDNA sequence of PHGPX which was protective to membrane injury in GPXs family. Furthermore, the three- dimensional structure of GPX enzyme molecules was predicted and analyzed, etc..
7. Analysis of the transcriptional expression levels of longan CDC48 and GPX genes by fluorescence quantitative PCR
Using both UBQ and EF-1a genes in longan as the reference genes, the transcriptional expression levels of CDC48 and GPX genes at different stages during longan somatic embryogenesis were analyzed by fluorescence quantitative PCR. The results showed that longan CDC48 expressed to some extents in the process of somatic embryogenesis; the expression level was the highest at the stage of tight-spherical embryogenic structure, next was at the stage of embryogenic callus(EC), and the lowest was at the stage of globular embryo; longan GPX also expressed to some extents in the process of somatic embryogenesis; the expression level was the highest at the stage of tight-spherical embryogenic structure, next was at the stage of cotyledon embryo, and the lowest was at the stage of embryogenic callus (EC).
8. Changes of GPX enzymatic activity in the process of longan somatic embryogenesis
In the process of longan somatic embryogenesis, GPX activity also expressed to some extents. The expression level was the highest at the stage of the tight-spherical embryogeic structure, next was at the stage of cotyledon embryos, and the lowest was at the stage of EC. The results were consistent with the results of gene relative quantitative expression in the process of longan somatic embryogenesis. Using longan EC lines LC2 as materials, the changing rules of GPX enzyme activity of longan EC under NaCl, light and temperature stress treatments were also studied in this experiment. The study indicated that GPX in cells played a role of response to external stimuli in face of adversity stress, and the expression of activity was regular. It was speculated that the changes of GPX activity and the resistance of embryogenic cells were of positive correlation, which was one of the main indicators of plant defensing ROS injury in adversity stress.
In conclusion, in this experiment two closely related genes in the process of somatic embryogenesis had been cloned. The expressions of the two genes were analyzed; and the structure and function were systematically predicted and analyzed; and the mechanism of somatic embryogenesis of longan was further understood. By this study, some new information could be provided for building the gene network of longan somatic embryogenesis; valuable evidences could be provided for exploring the mechanism of cell developmental regulation and removal of reactive oxygen in the process of longan somatic embryogenesis; and the gene resources could also be provided for genetic improvement of embryonic developmental regulation in longan and other plants, etc..
1.龙眼胚性愈伤组织CDC48基因cDNA和DNA全长的获得
以龙眼EC为材料,应用同源克隆结合RACE技术,获得了龙眼胚性愈伤组织CDC48的cDNA全长为为2620 bp,其中5'UTR为17 bp,3'UTR为187 bp,3’端还含有13个poly(A)尾。该序列与登录GenBank的其它植物CDC48基因有很高的同源性。序列分析发现拼接的cDNA含有一个2415 bp的开放阅读框,编码805个氨基酸,ATG为起始密码子、TAG为终止密码子。将此基因命名为DLCDC48,并在GenBank上登录,登录号为EU606206。从DNA水平克隆也得到了龙眼胚性愈伤组织的CDC48基因,并且经测序证实该基因无内含子,在GenBank上登录,登录号为:FJ590953。
2.龙眼胚性愈伤组织CDC48基因原核表达
根据龙眼EC CDC48全长cDNA的序列分析,在可能的ORF区域设计一对添加限制性内切酶的特异引物,进行ORF片段扩增并将其克隆到表达载体pET-28 a中。将构建好的表达载体在大肠杆菌表达宿主BL21(DE3)中进行融合表达。经过诱导,SDS-PAGE分析发现宿主菌中有一个分子量约为89 kD的新蛋白出现。该诱导表达的蛋白分子量与理论推导龙眼EC CDC48的分子量89.5 kD相近。
3.龙眼胚性愈伤组织CDC48基因的生物信息学分析
应用生物信息学软件对龙眼胚性愈伤组织CDC48的核苷酸序列和氨基酸序列进行了分析。结果表明: CDC48蛋白的分子量是89564.8 Da,理论等电点pI 4.92,是不具跨膜结构域的亲水性胞质蛋白,不具有信号肽,主要定位在细胞核上,有3个区域最有可能形成卷曲螺旋,由40.87%的a螺旋、15.28%的延伸链和43.85%的不规则卷曲组成,磷酸化位点有39个。保守结构域与功能域分析龙眼CDC48具有两个典型的ATPase模块,以及含有CDC48特有的N端。通过功能的预测与分析,推测与细胞的分裂有关系。通过其氨基酸序列的系统进化树分析,CDC48的进化在一定程度上反应了植物的进化。此外,还对CDC48酶分子三维立体结构等进行了预测和分析。
4.龙眼胚性愈伤组织GPX基因cDNA和DNA全长的获得
以龙眼EC为材料,应用同源克隆结合RACE技术,获得了龙眼胚性愈伤组织GPX的cDNA全长cDNA全长为947 bp, 5'UTR为195bp,3'UTR为245 bp,区域还含有典型的加尾信号AATAA和poly(A)尾。该序列与登录GenBank的其它植物GPX基因有很高的同源性。序列分析发现拼接的cDNA含有一个504 bp的开放阅读框,编码168个氨基酸,ATG为起始密码子、TAA为终止密码子。将此基因命名为DLGPX,并在GenBank上登录,登录号为EU364813。从DNA水平克隆也得到了龙眼胚性愈伤组织的GPX基因,1736 bp核苷酸序列,有ATG起始密码子和TAA终止密码子,并在GenBank上登陆,登陆号为:EU680970。通过DNAMAN 6.0软件分析,GPX基因由5个外显子和4个内含子组成,所有内含子的剪切位点均符合真核生物GT-AG规则。
5.龙眼胚性愈伤组织GPX基因原核表达
根据龙眼EC GPX全长cDNA的序列分析,在可能的ORF区域设计一对添加限制性内切酶的特异引物,进行ORF片段扩增并将其克隆到表达载体pET-28 a中。将构建好的表达载体在大肠杆菌表达宿主BL21(DE3)中进行融合表达。经过诱导,SDS-PAGE分析发现宿主菌中有一个分子量约为23 kD的新蛋白出现。本研究所用的酶切位点为SacI和XhoI,把载体上额外翻译的38个氨基酸一起表达。龙眼GPX编码基因产物的理论推导分子量18.54 kD,加上额外翻译的氨基酸理论推导分子量为4.08 KD(38个氨基酸理论推导分子量为39.45 KD),总共为22.62KD,与本研究结果相符。
6.龙眼胚性愈伤组织GPX基因生物信息学分析
运用生物信息学软件对龙眼胚性愈伤组织GPX基因的核苷酸序列和氨基酸序列进行了分析。结果表明:GPX蛋白的分子量是18541.16Da,理论等电点pI 7.18,是不具跨膜结构域的亲水性胞质蛋白,不具有信号肽,细胞主要定位在细胞质上,有1个区域最有可能形成卷曲螺旋,由27.98%的a螺旋,20.249%的延伸链和51.79%的不规则卷曲组成,磷酸化位点有13个。龙眼胚性愈伤组织GPX氨基酸序列含有PHGPX的两个特征序列,推测所克隆到的可能是GPXs家族中保护膜免受损伤的PHGPX的cDNA序列。此外,还对GPX酶分子三维立体结构等进行了预测和分析。
7.龙眼胚性愈伤组织CDC48和GPX基因转录水平表达分析
用龙眼胚性培养物中UBQ和EF-1a 2个基因共同作为内参基因,利用荧光定量PCR技术分析龙眼体胚发生过程各阶段培养物龙眼CDC48和GPX基因转录水平表达变化。分析结果显示:龙眼CDC48在体胚发生过程中都有不同程度的表达,其中胚性紧实球形结构阶段表达量最大,其次是胚性愈伤组织,最低的是球形胚;龙眼GPX在体胚发生过程中也均有不同程度的表达,其中胚性紧实球形结构阶段表达量最大,其次是子叶胚,最低的是胚性愈伤组织。
8.龙眼体胚发生过程中GPX酶活性变化
GPX活性在龙眼体胚发生过程中也均有不同程度的表达,其中胚性紧实球形结构阶段表达量最大,其次是子叶胚,最低的是胚性愈伤组织,这与龙眼体胚发生过程中基因相对定量表达结果是一致的;以龙眼胚性细胞系LC2为材料,研究了在NaCl、光和温度胁迫下龙眼胚性愈伤组织GPX酶活性的变化规律。分析结果显示:在一定逆境胁迫下,细胞内GPX可以起到应答外界刺激的作用,且活性呈规律表达,推测GPX活性变化与胚性细胞抗逆性正相关,是植物在逆境胁迫下防御ROS伤害的主要标志之一。
总之,本研究克隆了与体胚发生过程有密切相关的2个基因,进行了表达分析,并比较全面地预测和分析了它们的结构和功能,加深了对龙眼体胚发生机制的理解,为构建龙眼体胚发生的基因网络提供新的资料,为研究龙眼体胚发生过程中的细胞发育调控和活性氧的清除等方面的机理提供有价值的证据,为利用基因工程方法进行龙眼和其它植物胚胎发育调控等方面的遗传改良提供基因资源。
In this experiment, the embryogenic calli (EC) of longan (Dimocarpus longan Lour. cv. Honghezi) were used as the materials to study the following eight aspects:①cDNA and DNA cloning of longan EC CDC48 gene (cell division cycle 48 gene);②prokaryotic expression of CDC48 gene;③analysis of bioinformatics of longan EC CDC48 gene;④c DNA and DNA cloning of longan EC GPX (glutathione peroxidase gene);⑤p rokaryotic expression of GPX gene;⑥a nalysis of bioinformatics of longan EC GPX;⑦u sing both UBQ and EF-1a genes in longan as the reference genes, the transcriptional expression levels of CDC48 and GPX genes in the embryogenic cultures at different developmental stages during longan somatic embryogenesis were analyzed by fluorescence quantitative PCR;⑧analysis the changes of longan GPX activities at different stages during longan somatic embryogenesis and under stress treatment. The main results showed as follows:
1. Obtaining full length of cDNA and DNA of CDC48 gene from longan EC
Using longan EC as the material, the full length of cDNA (2620 bp ) of CDC48 from longan EC was obtained by homology cloning combined with RACE technology, which contained 17bp 5'UTR,187bp 3'UTR, and 3'-end involved 13 poly(A)tails. The results showed the sequence of CDC48 gene from longan EC was highly homologous with that of other plants reported in GenBank. Sequence analysis revealed that splicing of the cDNA contained a 2415 bp open reading frame, encoded 805 amino acids, and ATG was the initiation codon and TAG was the stop codon. The gene was named as DLCDC48 and registered in GenBank. The registered No. was EU606206. The DNA of CDC48 gene which had no intron by sequence analysis was also cloned from longan EC, which was also registered in GenBank, and the registered No. was FJ590953.
2. Prokaryotic expression of longan EC CDC48 gene
According to sequence analysis of full-length cDNA of longan EC CDC48 gene, a pair of specific primers added in restriction enzymes were designed in the possible ORF region to amplify ORF, and then it was inserted into the expression vector pET-28 a, and finally the well constructed expression vector was taken into the E. coli expression host BL21 (DE3) for fusion expression. After induction, there was a new protein emerging from the host bacteria and the molecular weight was about 89 kD by SDS-PAGE analysis. The protein molecular weight of the induced expression was near with that of the theoretical derivation from longan EC CDC48.
3. Analysis of bioinformatics of longan EC CDC48 gene
Longan EC CDC48 nucleotide sequence and corresponding amino acid sequences were analyzed by using bioinformatics softwares. The results showed that the molecular weight of protein CDC48 was 89564.8 Da; the theoretical isoelectric point ( pI ) was 4.92; it was a kind of hydrophilic cytoplasmic protein without transmembrane domain and signal peptide, and was mainly located in the nucleus; three regions were most likely to form coiled-coil containing with 40.87%α-spiral, 15.28% extending chain and 43.85% irregular curl; and the phosphorylation sites were 39. The longan CDC48 gene had two typical ATPase modules and specific N-terminal by analysis of conservative structure domain and function domain. Through prediction and analysis, it was speculated that the function of this gene was relevant with cell division. By analyzing phylogenetic trees of its amino acid sequence, it was concluded that the evolution of CDC48 reflected the evolution of plants to some degree. Furthermore, the three-dimensional structure of CDC48 enzyme molecules was predicted and analyzed, etc..
4. Obtaining the full length of cDNA and DNA of GPX gene from longan EC
Using longan EC as the material, the full length of longan EC GPX gene 947 bp was obtained by homology cloning combined with RACE technology, which contained 195bp 5'UTR, 245bp 3'UTR with a typical add-tail signal AATAA and a poly (A) tail. The results showed that the sequence of GPX gene from longan EC was highly homologous with that of other plants reported in GenBank. Sequence analysis revealed that splicing of the cDNA contained a 504 bp open reading frame, encoded 168 amino acids, and ATG was the initiation codon and TAG was the stop codon. The gene was named as DLGPX, and registered in GenBank. The registered No. was EU364813. The DNA of GPX gene was cloned from longan EC, which had 1736 bp nucleotide sequence with ATG initiation codon and TAA stop codon. The gene was registered in GenBank and the No. was EU680970. By the analyses of the DNAMAN 6.0 software, the GPX gene consisted of 5 exons and 4 introns, and the splice sites of all introns were accorded with eukaryotic GT-AG rule. 5. Prokaryotic expression of longan EC GPX gene According to sequence analysis of full-length cDNA of longan EC GPX gene, a pair of specific primers added in restriction enzymes were designed in the possible ORF region to amplify ORF, and then it was inserted into the expression vector pET-28 a, and finally the well Constructed expression vector was taken into the E. coli expression host BL21 (DE3) for fusion expression. After induction, there was a new protein emerging from the host bacteria and molecular weight about 23 kD by SDS-PAGE analysis. In this study, SacI and XhoI were used as the enzyme restriction sites, and 38 amino acids translated additionally in vector were also expressed. The theoretical molecular weight of gene product encoded by longan GPX gene was 18.54 kD, added with the theoretical derivation molecular weight of translating additionally amino acids 4.08 KD(38 amino acids theoretical molecular weight is 39.45 KD), and the total was 22.62KD, which was accorded with the results of this experiment. 6. Analysis of bioinformatics of longan EC GPX gene Longan EC GPX nucleotide sequence and amino acid sequences were analyzed by using bioinformatics softwares. The results showed that the molecular weight of protein GPX gene was 89564.8 Da; the theoretical isoelectric point (pI ) was 7.18; it was a kind of hydrophilic cytoplasmic protein without transmembrane domain and signal peptide, and was mainly located in the cytoplasm; a region was most likely to form coiled-coil containing with 27.98%α-spiral, 20.249% extending chain and 51.79% irregular curl, and the phosphorylation sites were13. The amino acid sequence of longan EC GPX gene contained two characteristics sequences of PHGPX. It was speculated that the cloned gene was possibly the cDNA sequence of PHGPX which was protective to membrane injury in GPXs family. Furthermore, the three- dimensional structure of GPX enzyme molecules was predicted and analyzed, etc..
7. Analysis of the transcriptional expression levels of longan CDC48 and GPX genes by fluorescence quantitative PCR
Using both UBQ and EF-1a genes in longan as the reference genes, the transcriptional expression levels of CDC48 and GPX genes at different stages during longan somatic embryogenesis were analyzed by fluorescence quantitative PCR. The results showed that longan CDC48 expressed to some extents in the process of somatic embryogenesis; the expression level was the highest at the stage of tight-spherical embryogenic structure, next was at the stage of embryogenic callus(EC), and the lowest was at the stage of globular embryo; longan GPX also expressed to some extents in the process of somatic embryogenesis; the expression level was the highest at the stage of tight-spherical embryogenic structure, next was at the stage of cotyledon embryo, and the lowest was at the stage of embryogenic callus (EC).
8. Changes of GPX enzymatic activity in the process of longan somatic embryogenesis
In the process of longan somatic embryogenesis, GPX activity also expressed to some extents. The expression level was the highest at the stage of the tight-spherical embryogeic structure, next was at the stage of cotyledon embryos, and the lowest was at the stage of EC. The results were consistent with the results of gene relative quantitative expression in the process of longan somatic embryogenesis. Using longan EC lines LC2 as materials, the changing rules of GPX enzyme activity of longan EC under NaCl, light and temperature stress treatments were also studied in this experiment. The study indicated that GPX in cells played a role of response to external stimuli in face of adversity stress, and the expression of activity was regular. It was speculated that the changes of GPX activity and the resistance of embryogenic cells were of positive correlation, which was one of the main indicators of plant defensing ROS injury in adversity stress.
In conclusion, in this experiment two closely related genes in the process of somatic embryogenesis had been cloned. The expressions of the two genes were analyzed; and the structure and function were systematically predicted and analyzed; and the mechanism of somatic embryogenesis of longan was further understood. By this study, some new information could be provided for building the gene network of longan somatic embryogenesis; valuable evidences could be provided for exploring the mechanism of cell developmental regulation and removal of reactive oxygen in the process of longan somatic embryogenesis; and the gene resources could also be provided for genetic improvement of embryonic developmental regulation in longan and other plants, etc..
引文
[1]安娜.龙眼体胚发生过程中特异表达小分子多肽的分离、鉴定与基因克隆(D).福州:福建农林大学硕士学位论文, 2006.
[2]陈春玲.龙眼体胚发生机理初步研究[D].福州:福建农林大学硕士学位论文, 2001.
[3]陈菁瑛,陈景耀.龙眼茎尖离体培养及其脱毒效果[J].植物生理学通讯, 1996, 32 (2): 126-127.
[4]陈义挺,赖钟雄,郭志雄,等.枇杷主要种类的RAPD分析[J].江西农业大学学报,2003, 25 (2): 258-261.
[5]崔凯荣,王晓哲,陈雄等.小麦体细胞发生中DNA,RNA和蛋白质的合成动态[J].核农学报, 1997,11(4):209-214.
[6]崔凯荣,邢更生,刘新民,等.细胞信号转导与植物体细胞胚发生[J].生命科学, 2002, l4(3): 171-175.
[7]崔凯荣,邢更生,胤克功,等.体细胞胚胎发生的生化基础[J].生命科学, 2001, 13(1):28-33.
[8]巩万奎.阿拉伯半乳糖蛋白及其在植物细胞分化中的作用[J].生物技术报. 1999,115(4)
[9]郭志雄.龙眼胚胎乙醇脱氢酶的分离纯化、鉴定及其cDNA克隆[D].福州:福建农林大学博士学位论文, 2006.
[10]韩立敏.丹参APX和GPX基因克隆及其表达分析[D].西安:陕西师范大学硕士论文,2007.
[11]何金环,李存法,梁月丽,等.植物细胞活性氧及其胞内信号转导[J].河南农业科学,2005,8:18-20.
[12]侯少范,薛泰麟,谭见安.高等植物中的谷胱甘肽过氧化物酶及其功能[J].科学通报,1994,39(6):553 -556.
[13]黄爱缨,吴珍龄.水稻谷胱甘肽氧化物酶的测定法[J].西南农业大学学报,1999, 21(4): 324 -327.
[14]蒋明义.水分胁迫下植物体内HO-的产生与细胞的氧化损伤[J].植物学报,1999,41(3): 229-234.
[15]赖呈纯,赖钟雄,何园,方智振,等.龙眼胚性培养物高分辨率蛋白质双向电泳技术[J].福建农林大学学报:自然科学版. 2008,37 (1): 37-41
[16]赖钟雄,陈春玲,黄素华,等.龙眼胚性愈伤组织的长期继代与染色体数目变异[J].福建农业大学学报.2001,30(1): 29-32.
[17]赖钟雄,陈振光.龙眼胚性悬浮细胞原生质体培养及其体胚发生再生植株[J].热带作物学报. 2002, 23 (3): 45-52.
[18]赖钟雄,潘良镇,陈振光.龙眼胚性细胞系的建立与保持[J].福建农业大学学报.1997b,26(2): 160-167.
[19]赖钟雄,潘良镇,陈振光.龙眼胚性愈伤组织的高频率体细胞胚胎发生[J].福建农业大学学报,1997, 26(3): 271-276.
[20]赖钟雄,潘良镇,陈振光.龙眼体细胞胚胎的高频率萌发与植株再生[J].福建农业大学学报, 1998, 27(1): 31-36
[21]赖钟雄.龙眼生物技术研究[M].福建科学技术出版社, 2003.
[22]赖钟雄.龙眼原生质体培养高效再生体系的研究[D].福州:福建农业大学博士学位论文, 1997.
[23]雷明光,张舒,张冰,等.谷胱甘肽磷脂氢过氧化物酶研究进展[J].生物学通报, 2005,40 (5): 1-3.
[24]李冬梅.龙眼体胚成熟机理研究[D].福州:福建农林大学硕士学位论文, 2003.
[25]李惠华,赖钟雄,陈桂信,等.龙眼胞浆形抗坏血酸过氧化物酶基因3’末端序列的同源克隆[J].农业生物技术学报, 2006,14(1):141-142.
[26]李惠华.龙眼体胚发生过程中APX的生理学与分子生物学研究[D].福州:福建农林大学硕士学位论文, 2005.
[27]李文君,刘进元,赵南明.苦瓜谷胱甘肽磷脂氢过氧化物酶cDNA的克隆及其特征分析[J].生物化学与生物物理进展, 2001,28 (6): 908-911.
[28]林小苹,赖钟雄,黄浅,等.不同光质对龙眼胚性愈伤组织生长和细胞膜保护酶活性的影响[J].福建农林大学学报, 2008, 37(3): 235-256.
[29]林玉玲,赖钟雄.龙眼体胚发生过程中实时定量PCR内参基因的筛选[J].2009,(待发表)
[30]林玉玲,赖钟雄.龙眼胚胎发育过程中SOD活性的变化亚热带农业研究,2007, 3(4): 267-270.
[31]刘成明,胡又厘,傅嘉欣.生物技术在荔枝龙眼繁殖和品种改良中的应用研究进展[J].中国南方果树,2003,32(6):30-34.
[32]刘家忠,龚明.植物抗氧化系统研究进展[J].云南师范大学学报,1999, 19(6): 1-11.
[33]刘丽娜,刘祥林,陈志玲.细胞周期调控的一个重要元素--CDC48[J].生物学通报, 2006, 46 (5):26-29.
[34]刘丽娜.烟草细胞周期相关基因NtCDC48的克隆与功能研究[D].北京:首都师范大学硕士学位论文,2006.
[35]刘丽娜.烟草NtCDC48基因的克隆及功能分析[J].自然科学进展.2006.16(12).1561-1567.
[36]刘招龙.亚硒酸钠对高温胁迫下梨苗部分酶活性的影响[J].宁德师专学报, 2006, 18(3): 237-239.
[37]龙桂友.柑橘低温诱导相关基因的克隆与表达分析[D].长沙:湖南农业大学博士论文,2007.
[38]苗雨晨,白玲,苗琛,等.植物谷胱甘肽过氧化物酶研究进展[J].植物学通报, 2005, 22 (3): 350-356.
[39]邵巍,赖钟雄,赖呈纯,等.龙眼胚性培养物APX同工酶的分析方法建立及其在龙眼体胚发生过程中的变化[J].福建农林大学学报(自然科学版),2008, 37(2): 140-144.
[40]邵巍.龙眼胚性培养物胞质型apx基因克隆及其表达研究[D].福州:福建农林大学硕士学位论文, 2008.
[41]王凤华,赖钟雄.龙眼胚性愈伤组织维生素C过氧化物酶基因(apx)及NADH脱氢酶(nad2)基因部分序列的克隆[J].应用与环境生物学报. 2005, 11(1): 45-48.
[42]王凤华.龙眼体细胞胚胎发生过程中的基因差异表达[D].福州:福建农林大学博士学位论文, 2002.
[43]王亚馥,崔凯荣,高清祥,等.小麦体细胞胚发生的蛋白质组分和过氧化物酶同工酶的变化[J].兰州大学学报,1993, 29 (3): 189~193.
[44]王亚馥,崔凯荣,汪丽虹,等.小麦组织培养中体细胞胚胎发生的细胞胚胎学及淀粉消长动态的研究[J].实验生物学报, 1993. 26 (3): 259- 267.
[45]魏文雄,杨永青.龙眼子叶胚状体的诱导和试管苗培育[J].福建师范大学学报(自然科学版), 1981, (2): 102-106.
[46]邢更妹,李彬等.植物体细胞胚胎发生中抗氧化系统代谢动态和程序性细胞死亡[J].生命科学, 2000, l2(5): 215-218.
[47]邢更妹,李杉,崔凯荣,等.植物体细胞胚发生中抗氧化系统代谢动态和程序性细胞死亡[J]生命科学.
[48]邢更生,崔凯荣,山仑,等.植物体细胞胚发生的分子基础[J].遗传, 1999, 21(1): 30-34.
[49]闫春燕,徐承水,张士璀.动物磷脂氢谷胱甘肽过氧化物酶研究进展[J].科技信息, 2008, 22: 366-367
[50]杨晓东,刘进元.萝卜磷脂氢谷胱甘肽过氧化物酶基因结构及其调控序列分析[J].生物化学与生物物理进展, 2005, 32(7):649-656.
[51]杨永青,陈永锋.焦核龙眼果实遗传性状及胚培养的研究[J].园艺学报,1987,14(4):217- 222.
[52]杨永青,魏文雄.龙眼花粉植株的诱导[J].遗传学报, 1984, 11(4): 288-293.
[53]殷奎德,马连菊,刘世强.逆境条件下植物活性氧(AOS)的研究进展[J].沈阳农业大学学报, 2003,21(2): 29-33.
[54]曾黎辉.龙眼遗传转化研究初报[D].福州:福建农业大学硕士学位论文.1998.
[55]曾黎辉,陈振光,吕柳新.发根农杆菌转化龙眼研究初报[J].福建农业大学学报, 2000, 29(1):27- 30.
[56]翟中和.细胞生物学[M].北京:高等教育出版社, 1995.
[57]张景萍,吴珍龄.几种植物中谷胱甘肽过氧化物酶活性测定[J].广西农业科学, 2004, 35(3): 177-178.
[58]郑启发,胡桂兵,陈大成,等.荔枝龙眼细胞悬浮培养和转基因研究[J].果树学报,2005,22(2):125-128.
[59]周键,孙建波,彭明.高等植物细胞胚胎发生的研究进展[J].中国农学通报2008, 24 (2): 129-133
[60]周毅峰,吴永尧,唐巧玉.硒对大豆叶片谷胱甘肽过氧化物酶和过氧化氢酶歧化酶活性的影响[J].湖北民族学院学报, 2005, 23(2): 127-129.
[61] Acquaviva C, Pines J. The anaphase promoting complex/cyclosome:APC/[J]. Journal of Cell Science, 2006, 119: 2401-2404.
[62] Albert B . Molecular Biology of Cell, Garland Publishing, Inc. N. Y. Acta Horticulturae.2001,558:149～154.
[63] Arthur J R. The glutathione peroxidases[J]. Cell Mol Life Sci, 2000, 57: 1825- 1835.
[64] Asada K. Radical production and scavenging in the chloroplasts[M]. In: Photo.
[65] Asada K. The rolel of ascorbate peroxidase and monodehydro ascorbate reductase in H202 scavenging in plants[M]. In: Oxidative Stress and the Molecular Biology ofAntioxidant Defenses. Cold Spring Harbor Laboratory Press, 1997: 715-735.
[66] Asada K. The water-water cycle in chloroplasts: scavenging of active oxygens and dissipation of excess photons[J]. Annual Review of Plant Physiology, 1999, 50: 601-639.
[67] Asada, Kiso K., Yoshikawa, K. Univalent reduction of molecular oxygen by spinach chloroplasts on illimination[J]. J Biol Chem, 1974, 249: 2175- 2181.
[68] Avsian-Kretchmer O, Gueta-Dahan Y, Lev-Yadun S, et al. The salt-stress signal transduction pathway that activates the gpxl promoter is mediated by intracellular H202, different from the pathway induced by extracellular H202[J]. Plant Physiology, 2004, 135: 1685-1696.
[69] Babst, M., Katzmann, D. J., Synder, W. B., Wendland, B. and Emr, S. D.. Endosome-associated complex, ESCRT-II, recruits transport machinery for protein sorting at the multivesicular body[J]. Dev CeLL, 2002,3:283-289.
[70] Babst, M., Wendland, B., Estapa, E. J. and Emr, S. D. The Vps4pAAA ATPase regulates membrane association of a Vps protein complex required for normal endosome function[J]. EMBO J. 1998,17, 2982-2993.
[71] Berget S M, Moore C, Sharp P A. Spliced segments at the 5' terminus of adenovirus 2 late mRNA.Proc Natl Acad Sci USA, 1977,74:3171-3175.
[72] Beuron, F, F1ynn, T C, Ma, J, Kondo, H, Zhang, X. and Freemont, P.S. Motions and negative cooperativity between p97 domain revealed by cryo-electron microscopy and quantised elastic deformational model[J]. J. MoL. BioL. 2003, 327: 619-629.
[73] Braun S, Matuschewski K, Rape M, et al. Role of the ubiquitin-selective CDC48 (UFDI/NPL14) chaperone(segregase ) in ERAD of OLEl and other substrates [J]. EMBO,2002,21:625-621.
[74] Chu FF, Doroshow JH, Esworthy RS Expression, characterization and tissue distribution of a new cellular selenium-dependent glutathione peroxidase, GSHPx-GI[J]. Journal of Biological Chemistry, 1993, 268: 2571- 2576.
[75] Claudia Magioli, Rosa Maria Barroco,Carla Andrea Ben. et al Somatic embryo formation in Arabidopsis and eggplant is associated with expression of a glycine-rich protein gene (Atgrp-5) [J]. Plant science,2001.161:559-567.
[76] Criqui M C, Jamet E, Parmentier Y, et al. Isolation and characterization of a plant cDNA showing homology to animal glutathione peroxidases[J]. Plant Molecular Biology, 1992, 18: 623-627.
[77] Dai, R. M. and Li, C.-C. H.. Valosin-containing protein is a multi- ubiquitin chain-targeting factor required in ubiquitin-proteasome degradation[J].Nat. CeLL BioL. 2001, 3 740-3744.
[78] Dai, R.-M., Chen, E., Longo, D. L., Gorbea, C. M. and Li, C.-C. H. Involvement of valosin-containing protein, an ATPase co-purified with Ikappa Balpha and 26S Proteasome, in ubiquitin-proteasome-mediated degradation of Ikappa Balpha[J]. J. BioL. Chem. 1998, 273, 3562-3573.
[79] Dalal, S. and Hanson, P I.. Membrane traffic: what drives the AAA motor[J]. Cell. 2001, 104. 5-8.
[80] Dat J F, Foyer C H, Scott I M. Changes in salicylic acid and antioxidants during Induced the rmo tolerance in mustard seedlings[J]. Plant Physiology, 1998, 118: 1455-1461.
[81] De Jong A J, Hendrichks T, Meijer E A, et al. Transient reduction in secreted 32 kD chitinase prevents somatic embryogenesis in the carrot variant ts II [J]. Devel Cenet, 1995, 16: 332- 343.
[82] Delaunay A, Pflieger D, Barrault M, et al. A thiol peroxidase is an H2O2 receptor and redox-transducer in gene activation[J].Cell,2002, 111: 471- 481.
[83] Depege N, Drevet J, Boyer N. Molecular cloning and characterization of tomato cDNAs encoding glutathione peroxidase-proteins[J]. European Journal of Biochemistry. 1998, 253: 445-451.
[84] Dixon, D.P., Skipsey, M., Grundy, N.M. and Edwards, R. Stress-induced protein S-glutathionylation inarabidopsis[J]. Plant Physi., 2005, 138: 2233-2244.
[85] Doke N. The oxidative burst: roles in signal transduction and plant stress[A] Scandalios JCx Oxidative Stress and the Molecular Biology of Anrioxidant Defenses[C]. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1997: 785-813.
[86] Dong J Z, Perras M R, Abrams S R er al. Characterization of three heat-shock-protein genes and their developmental regulation during somatic embryogenesis in white spruce [picea glauca(moench) voss] [J]. Planta, 1996, 200: 85-91.
[87] Dong J Z, Perras M R, Abrams S R er al. Expression of abundant mRNAs during somatic embryogenesis of white spruce[picea glauca(moench) voss] [J]. Planta, 1996, 199: 459-466.
[88] Dong J Z et al. Induced gene expression following ABA uptake in embryogenic suspension cultures of picea glauca[J]. Plant Physiol. Plant Physiol. Biochem., 1996, 34: 579-587.
[89] Drotar A., Phelps P., Fall R. Evidence for glutathione peroxidase activities in cultured plant cells [J]. Plant Science, 1985(42): 35-40.
[90] Du H, Simpson R J, Moritz R L, et al .Isolation of the protein backbone of an arabinogalactan-protein from the styles of nicotiana alata and characterization of a corresponding cDNA[J].Plant Cell, 1994,6: 1643-1653.
[91] Du H, Simpson R J, Clarke A E et al. Molecular characterization of a stigma-specific gene encoding an arabinogalactan-protein(AGP) from nicotiana alata[J]. Plant Journal. 1996, 313-323.
[92] Elledge S J. Mitotic arrest: Mad2 Provents Sleepy from waking up the APC[J].Science,1998,279,999-1000.
[93] Eshdat Y, Holland D, Faltin Z, et al. Plant glutathione peroxidases[J]. Physiologic Plantarum, 1997, 100: 234-240.
[94] Flohe,L. GLutathione peroxidase[J].Bosic Life.Sci , 1988 ,49 : 663.
[95] Foyer C H,Noctor G.Redox homeostasis and antioxidant signaling:A metabolic interface between stress perception and physiological responses[J].Plant Cell, 2005, 17: 1866—1875.
[96] Foyer, C.H. and Noctor,G Redox sensing and signaling associated with reactive oxygen in chloroplast, peroxisomes and mitochondria[J]. Physiol. Plant., 2003, 119: 355-264.
[97] Frohlich K U, Fries H W, Rudiger M, et al. Yeast cell cycle protein CDC48p shows full-length homology to the mammalian protein VCP and is a member of protein family involved in secretion, peroxisome formation, and gene expression[J]. J Cell Biol,1991, 114: 443-453
[98] Fujlmura, T., and Komamlne, A. The serial observation of embryogenesis in a carrot cell suspension culture[J]. New Phyiol,1980, 86: 13-218.
[99] Gomez, L.D., Noctor,G,Knight, M. and Foyer, C.H. Regulation of calcium signaling and gene expression by glutathione[J].J.Exp Bot., 2004 (55): 1851一1859.
[100] Hatzpoulos P, Fong F, Sung Z R.Absicsic acid regulation of DC8,a carrot embryogenic gene[J]. Plant Physiol,1990, 94: 690- 695.
[101] Helleboid S et al. Inhibition of direct somatic embryogenesis by alpha-difluoromethylarginine in a cichorium hybrid: effects on polyamine content and protein patterns[J]. Planta, 1995, 196: 571-576.
[102] Henry Y, Vain P, de Buyser J. Genetic analysis of in vitro plant tissue culture responses and regeneration capacities[J].Euphytica,1994,79: 45- 48..
[103] Herbette P,Lenne C,Leblanc N,et al. Two GPX-like proteins from Lycopersicon esculentum and Helianthus annuus are antioxidant enzyme with phospholipid hydroperoxide glutathione peroxidase and thioredoxin peroxidase activities[J]. Eur J Bio Chem, 2002, 269: 2414-2420.
[104] Hernandez J A, Corpas F J. Salt-induced oxidative stress in chloroplasts of pea plants[J]. Plant Science, 1995, 105: 151-167.
[105] Hershko, A. and Ciechanover, A. The ubiquitin system[J]. Annu. Rev.Biochem. 1998,67: 425-479.
[106] Higashi K, Shiota H, Kamada H. Patterns of expression of the genes for glutamine synthetase isoforms during somatic and zygotic embryogenesis in carrot[J]. Plant Cell Physiol, 1998, 39: 418- 424.
[107] Hippeli S. Mechanisms of oxygen activation during plant stress: biochemical effects of air pollutants[J]. Journal of Plant Physiology,1996, 148: 249-257.
[108] Hochstrasser M.. Ubiquitin-dependent protein degradation[J]. Annu Rev,Genef , 1996, 30: 405-439.
[109] Holland D, Ben-Hayyim G Faltin Z, et al. Molecular characterization of salt-stress-associated protein in Citrus: protein and cDNA sequence homology to mammalian glutathione peroxidase[J]. Plant Molecular Biology, 1993, 21:923-927.
[110] Holland D, Ben-Hayyim G,Faltin Z,et al.Molecular characterization of salt-stress-associated protein in Citrus:Protein and cDNA equence homology to mammalian glutathione peroxidase[J]. Plant:460-469.
[111] Hsieh W L,Wolniak S M.Isolation and characterization of a funcational A-type cyclin from maize[J]. Plant Molecular Biloogy, 1998, 37: 121-129.
[112] Hutchison. C J.Glover D D .Cell Cycle Control[M].Oxford;IRLOxford Univerisity Press,1995.
[113] JSmirnoff N. Antioxidant systems and plant response to the environment. In: Environment And Plant Metabolism[M]. Oxford, UK. BIOS Scientific Publishers, 1995: 217-243.
[114] Jung B G, Lee K O, Lee S S, et al. A Chinese cabbage cDNA with high sequence identity to phospholipid hvdroperoxide glutathione peroxidases encodes a novel isoform of thioredoxin-dependent peroxidase[J]. Journal of Biological Chemistry, 2002, 277: 12572-12578.
[115] Kastan M B .Check point Controls and Cancer[M]//J Tooze.Cancer Surveys.Vol 29.New York:Cold Spring Harbor Laboratory Press,1997.
[116] Kiyosue T et al. Cloning of a carrot cDNA for a member of the family of ADP-ribosylation factors(ARFs) and characterization of the binding of nucleotides by its product after expression in e-coli[J]. Plant Cell Physiol.1995. 36: 849-856.
[117] Kiyosue T, Yamaguchi-Shinozaki K,Shinozaki K, et al. Isolation and characterization of a cDNA that encodes ECP31, an embryogenie- cell protein from carrot[J]. Plant MoL Biol. 1992,19:239- 249.
[118] Kondo H, Rabouille C, Newman R, et al. p47 is a cofactor forp97-mediated membrane fusion[J]. Nature, 1997,388: 75-78.
[119] Kragh K M,Hendriks T, De Jong A J, et al. Characterization of chitinases able to rescue somatic embryos of the temperature-sensitive carrot variant is tsII[J]. Plant, Mol, Biol, 1996, 31 :631-645.
[120] Kreuger M,van Holst G .J.Arsbinogalactan protein epitopes insomatic embryogenesis of Daucus carota [J].Planta, 1995, 197:135-141.
[121] Lai Z X, Chen C L, Chen Z G. progress in biotechnology research in longan[J].. Acta Horticulturae, 2000b ,558: 137~141.
[122] Lai Z X, Ch C L, Zeng L H , et al. Somatic embryogenesis in longan [Dimocarpus longan Lour.][J]. Forestry Sciences. 2000a, 67: 415-432.
[123] Lee, R. C.,R. L. Feinbaum, and V. Ambros. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14[J]. Ce11, 1993,75 (5): 843-54.
[124] Li W J, Feng H, Fan J H, et al. Molecular cloning and expression of a phospholipid hydroperoxide glutathione peroxidase homolog in Oryza sativa[J]. Biochimica et Biophysics Acta, 2000, 1493: 225-230.
[125] Li WJ, Feng H, Fan JH, et al. Molecular cloning and expression of a phospholipid hvdroperoxide glutathione peroxidasehomolog in Oryza sativa[J]. Biochimica et Biophysica Acta, 2000,1493: 225-230.
[126] Lowry, O. H, Rosebrough, N. Y., Farr, A. L.,et al. Protein measurement with the folin phenol reagent[J]. Boil. Chem.,1951, 193: 265-275.
[127] Luisa C. Sunflower seedlings subjected to increasing water deficit stress, oxidative stress and defence mehanisms[J]. Physiologic Plantarum, 1995, 93: 25-30.
[128] Maiorino M, Thomas JP, Girotti AW, Ursini F Reactivity of phospholipid hydroperoxide glu tathione peroxidase with membrane and lipoproteinlipid hydroperoxides[J]. Free Radical Research, 1991, 12: 131-135.
[129] Mau S L et al. Cloning of a carrot cDNA for a member of the family of ADP-ribosylation factors(ARFs) and characterization of the binding of nucleotides by its product after expression in e-coli[J]. Plant Journal, 1995, 8:269-281.
[130] McNew, J. A., Parlati, F, Fukuda, R., Johnston, R. J., Paz, K., Paumet,R, Sollner, T. H. and Rothman, J. E. Compartmental specificity of cellular membrane fusion encoded in SNARE proteins[J]. Nature, 2000, 407, 153-159.
[131] Mehdy, M.C. Active oxygen species in plant defense against pathogens[J]. Plant Physiol., 1994, 105: 467-472.
[132] Milla MAR, Maurer A, Huete AR, Gustafson JP. Glutathione peroxidase genes in Arabidposis areubiquitvous and regulated by abiotic stresses though diverse signaling pathways[J]. The Plant Journal, 2003, 36: 602-615.
[133] Mimuria S, Takisawa H. Xenopus CDC45-dependent Loading of Ploymerase onto Chromatin under the control of S-Phase CDK[J]. EMBO J, 1998, 17: 5699-5707.
[134] Mittler,R. Oxidative stress, antioxidants and stress tolerance. Trends in plant science2002, 7:405-410.
[135] Moir D, Stewart B C, Osmond B C, et al. Cold sensitive cell division cycle mutants of yeast; Isolation, properties, and pseudoversion studies[J]. Genetics, 1982, 100; 547-563.
[136] Mullineaux PM, Karpinski S,.Jimenez A, Cleary SP Robinson C, Creissen GPIdentication of cDNA encoding plastid-targeted glutathione peroxidase[J]. The Plant ,Journal, 1998, 13: 370-379.
[137] Noctor G,Foyer C H. Ascorbate and glutathione: keeping active oxygen under control[J]. Annual review of plant physiology[J]. Plant Molecular Biology, 1998, 49: 249-279.
[138] NOCTOR G, GOMEZL,LE’NE VANACKER He, et al . Interactions Between Biosynthesis , Compartmentation and Transport in the Control of Glutathione Homeostasis and Signaling [J ] . Journal of Experimental Botany ,2002 ,53(372) :1283 - 1304.
[139] Ogura, T. and Wilkinson, A. J. AAA+ superfamily ATPasescommon structure-diverse function[J]. Genes CeLLs. 2001,6:575-597.
[140] Pacific R E.Hydrophobicity as the signal for selective degraclation of hydroxyl radical-modified hemoglobin by the multicatalytic proteinase complex,proteasome[J].J.Biol.Chem.,1993,268: 15405-15411
[141] Patel, S. and Latterich, M. The AAA team: related ATPases with diverse functions[J]. Trends Cell Biol. 1998, 8: 65-71.
[142] Patel, S. K., Indig, F. E., Olivieri, N., Levine, N. D. and Latterich, M. Organelle membrane fusion: a novel function for the syntaxin homolog Ufelp in ER membrane fusion[J]. Cell 1998,92, 611-620.
[143] Philip G. Woodman. a protein coping with multiple identities[J]. Journal of Cell Science. 2003,116: 4283-4290.
[144] Pine J .Cyclins and Cyclin-dependent kinases:a biochemical view[J].Biochem J 2003, 308:679-711.
[145] Rape, M., Hoppe, T., Gorr, I, Kalocay, M., Richly, H. and Jentsch, S. Mobilization of processed, membrane-tethered SPT23 transcription factor by CDC48(LJFD1/NPL4), a ubiquitin-selective chaperone[J].Cell2001, 107,667-677.
[146] Rocher C,Lalanne JL,ChaudiereJ Purification properties of a recombinant sulfur analog of murine selenium-glutathione peroxidase[J].European,Journal of Biochemistry 1992,205:900-960.
[147] Rockel, B., Jakana, J., Chiu, W. and Baumeister, W. Electron cryo-microscopy of VAT, the Archaeal p97/CDC48 homologue from The rmopLasma acidophiLum[J]. J. MoL. BioL.2002,317, 673-681.
[148] Roeckel D P, GagneG , Dufaure J P, et al. Molecular characterization organ distri-bution and stress-mediated induction of two glutathione peroxidase-encoding mRNAs in sunflower (Helianthus annuus) [J]. PhysiologicPlantarum. 1998, 103: 385-394.
[149] Roeckel-Drevet P, GagneG, de Labrouhe TD, Dufaure JP, Nicolas P, Drevet JRMolecular characterization organ distribution and stress-mediated induction of two glutathione peroxidase-encoding mRNAs in sunflower (Helianthus annuus). 1998
[150] Sabeh F, Wright T, Norton SJ Purification and characterization of a glutathione peroxidase from the Aloe vera plant[J]. Enzyme and Protein, 1993, 47:92-98.
[151] Schmidt E D L,Guzzo F, Toonen M A J, et al. A leucine- rich repeat containing receptor-like kinase marks somatic plant cells competent to form embryos[J]. Development, 1997,124:2049- 2062.
[152] Shah K Cadella T W Jr,van Erp H et al. Subcellular localization and oligomerization of the Arabidopsis thaliana somatic embryogenesis receptor kinase 1 protein[J]. J Mol Biol. 2001,309:641-655.
[153] Smallwood E A et al. Immunochemical comparison of membrance-associated and secreted arabinagalactan-proteins in rice and carrot[J]. Planta, 1996, 193:452-459
[154] Smith, T. M,M. K. Lee, C. I. Szabo, N. Jerome, M. McEuen, M. Taylor, L. Hood, and M. C. King.. Complete genomic sequence and analysis of 117 kb of human DNA containing the gene BRCA1. Genome Res1996, 6 (11):1029-49.
[155] Stadman TC. Selenocvsteine. Annual Review of Biochemistry, 1996, 65: 83-100.
[156] Sterk, P., Boolj, H.,Schellekens,G.A_et al.Cell-specific expression of the carrot EP2 lipidtransfer protein gene[J].Plant Cell 3.1991:907-921
[157] Stirling, C., Rothblatt, J., Hosobuchi, M., Deshaies, R. and Schekman, R. Protein translocation mutants defective in the insertion of integral membrane proteins into the endoplasmic reticulum[J]. MoL. BioL. CeLL1992, 3, 129-142.
[158] Sugimoto M, Furui S, Suzuki Y Molecular clon ing and characterization of a cDNA encoding putative phospholipid hvdroperoxide glutathione peroxidase from Spinach[J].Bioscience Biotechnology and Biochemistry, 1997, 61:1379-1381.
[159] Sugimoto M, Sakamoto WPutative phospho lipid hvdroperoxide glutathione peroxidase gene from Arabidopsis thaliana induced by oxidative stresss[J]. Genes & Genetic Systems, 1997,72:311-306.
[160] Talia B T, Gozal B H, Holland D, et al. A stress-associated citrus protein is a distinct plant phospholipid hydroperoxide glutathione peroxidase[J]. FEBS Letters,1995, 366:151-155.
[161] Taylor, H. A homeobox gene regulatory element is composed of distinct conserved. the formation of free radicals in plants exposed to ozone[J]. Physiologic Plantarum. 1996.
[162] Tee, M. K., G. O. Aza-Blanc, P. A et al. A promoter within intron 35 of the human C4A gene initiates abundant adrenal-specific transcription of a 1 kb RNA:location of a cryptic CYP21 promoter element[J]? Human Molecular Genetics.1995, 11(4): 2109-2116.
[163] Tsai B., Ye Y.,Rapoport T A. Retro-translocation of proteins from the endoplasmic reticulum into thecytosol[J].Nat. Rev. Mol. Cell Biol. 2002,3(4): 246-255.
[164] Volkand S, Feieralxnd J. Photo inactivation of catalase at low temperature and its relevance to photosynthetic and peroxide metabolism in leaves[J]. Plant Cell and the Environment,1989, 12: 701-712.
[165] Weissman, A. M. Ubiquitin and proteasomes: themes and variations on ubiquitylation[J]. Nat. Rev. Mol. Cell. Biol. 2001, 2, 169-178.
[166] Willekens, H., Chamnongpol, S., Davey, M., Schraudner, M.,Langebartels, C., Montagu, M.V , Inze D. and Camp W.VCatalase is a sink for H202 and is indispensable for stress defence in C-3 plants[J].EMBO J. 1997, 16: 4806-4816.
[167] Wurtele, E.S., Wang, H., Durgerian, S., et al. Characterization of a gene that is expressed early in somatic embryogenesis of Daucus carota[J].Plant Physiol, 1993, 102: 303-312.
[168] Xinrong Fu, Christine Ng, Daorong Feng, and Chun Liang. CDC48p is required for the cell cycle commitment point at Start via degradation of the Gl- CDK inhibitor Farlp[J].The Journal of Cell Biology. 2003, 163: 21-26.
[169] Ye, Y., Meyer, H. H. and Rapoport, T. A. The AAA ATPase Cdc48/p97 and its partners transport proteins from the ER into the cytosol[J]. Nature2001, 414, 652-656.
[170] Ye, Y., Meyer, H. H. and Rapoport, T. A. Function of the p97-Ufdl-Np14 complex in retrotranslocation from the ER to the cytosol: dual recognition of nonubiquitinated polypeptide segments and polyubiquitin chains[J].J.Cell Biol. 2003, 162, 71-84.
[171] Yu K F Pauls K P. Identification of a RAPD marker associated with somatic embryogenesis in alfalfa[J].Plant Mol. Biol1993, 22: 267-277.
[172] Zeng L H, Chen Z G .and LüL X et al. In vitro trmisformation mediated by Ri plasmid of Agrohacteriuu rhizogenes and trmisgeuic plant regeneration of longan. Proceediugs of the first International Symposium ou Litchi and Longan[J]. Acta Horticulturae. 2001, 558:149-154.
[173] Zhang, J., X. Sun, Y. Qian, and L. E. Maquat.. Intron function in the nonsense-mediated decay of beta-globin mRNA: indications that pre-mRNA splicing in the nucleus can influence mRNA translation in the cytoplasm[J]. Rna 1998, 4 (7): 801-815 .
[174] Zhang, X., Shaw, A., Bates, P A., Newman, R. H., Shaw, A., Bates, P A.,Newman, R. H., Gowen, B., Orlova, E., Gorman, M. A. et al., Structure of the AAA ATPase[J]. MoL. CeLL2000, 6, 1473-1484.
[175] Zhu J K ,Bressan R A, Hasegawa P M. Loss of arabinogalactin-protein from the plasma membrane of NaCI- adapted cells[J]. Planta,1993,190: 221-226.
[176] Zimmerman J L. Somatic embryoenesis: A model for early development in high plants[J].Plant Cell,1993,5:1411-1423.
[177] Zuckerkandl, E. Junk DNA and sectorial gene repression[J].Gene 1997, 205 (1-2):323-43.
[2]陈春玲.龙眼体胚发生机理初步研究[D].福州:福建农林大学硕士学位论文, 2001.
[3]陈菁瑛,陈景耀.龙眼茎尖离体培养及其脱毒效果[J].植物生理学通讯, 1996, 32 (2): 126-127.
[4]陈义挺,赖钟雄,郭志雄,等.枇杷主要种类的RAPD分析[J].江西农业大学学报,2003, 25 (2): 258-261.
[5]崔凯荣,王晓哲,陈雄等.小麦体细胞发生中DNA,RNA和蛋白质的合成动态[J].核农学报, 1997,11(4):209-214.
[6]崔凯荣,邢更生,刘新民,等.细胞信号转导与植物体细胞胚发生[J].生命科学, 2002, l4(3): 171-175.
[7]崔凯荣,邢更生,胤克功,等.体细胞胚胎发生的生化基础[J].生命科学, 2001, 13(1):28-33.
[8]巩万奎.阿拉伯半乳糖蛋白及其在植物细胞分化中的作用[J].生物技术报. 1999,115(4)
[9]郭志雄.龙眼胚胎乙醇脱氢酶的分离纯化、鉴定及其cDNA克隆[D].福州:福建农林大学博士学位论文, 2006.
[10]韩立敏.丹参APX和GPX基因克隆及其表达分析[D].西安:陕西师范大学硕士论文,2007.
[11]何金环,李存法,梁月丽,等.植物细胞活性氧及其胞内信号转导[J].河南农业科学,2005,8:18-20.
[12]侯少范,薛泰麟,谭见安.高等植物中的谷胱甘肽过氧化物酶及其功能[J].科学通报,1994,39(6):553 -556.
[13]黄爱缨,吴珍龄.水稻谷胱甘肽氧化物酶的测定法[J].西南农业大学学报,1999, 21(4): 324 -327.
[14]蒋明义.水分胁迫下植物体内HO-的产生与细胞的氧化损伤[J].植物学报,1999,41(3): 229-234.
[15]赖呈纯,赖钟雄,何园,方智振,等.龙眼胚性培养物高分辨率蛋白质双向电泳技术[J].福建农林大学学报:自然科学版. 2008,37 (1): 37-41
[16]赖钟雄,陈春玲,黄素华,等.龙眼胚性愈伤组织的长期继代与染色体数目变异[J].福建农业大学学报.2001,30(1): 29-32.
[17]赖钟雄,陈振光.龙眼胚性悬浮细胞原生质体培养及其体胚发生再生植株[J].热带作物学报. 2002, 23 (3): 45-52.
[18]赖钟雄,潘良镇,陈振光.龙眼胚性细胞系的建立与保持[J].福建农业大学学报.1997b,26(2): 160-167.
[19]赖钟雄,潘良镇,陈振光.龙眼胚性愈伤组织的高频率体细胞胚胎发生[J].福建农业大学学报,1997, 26(3): 271-276.
[20]赖钟雄,潘良镇,陈振光.龙眼体细胞胚胎的高频率萌发与植株再生[J].福建农业大学学报, 1998, 27(1): 31-36
[21]赖钟雄.龙眼生物技术研究[M].福建科学技术出版社, 2003.
[22]赖钟雄.龙眼原生质体培养高效再生体系的研究[D].福州:福建农业大学博士学位论文, 1997.
[23]雷明光,张舒,张冰,等.谷胱甘肽磷脂氢过氧化物酶研究进展[J].生物学通报, 2005,40 (5): 1-3.
[24]李冬梅.龙眼体胚成熟机理研究[D].福州:福建农林大学硕士学位论文, 2003.
[25]李惠华,赖钟雄,陈桂信,等.龙眼胞浆形抗坏血酸过氧化物酶基因3’末端序列的同源克隆[J].农业生物技术学报, 2006,14(1):141-142.
[26]李惠华.龙眼体胚发生过程中APX的生理学与分子生物学研究[D].福州:福建农林大学硕士学位论文, 2005.
[27]李文君,刘进元,赵南明.苦瓜谷胱甘肽磷脂氢过氧化物酶cDNA的克隆及其特征分析[J].生物化学与生物物理进展, 2001,28 (6): 908-911.
[28]林小苹,赖钟雄,黄浅,等.不同光质对龙眼胚性愈伤组织生长和细胞膜保护酶活性的影响[J].福建农林大学学报, 2008, 37(3): 235-256.
[29]林玉玲,赖钟雄.龙眼体胚发生过程中实时定量PCR内参基因的筛选[J].2009,(待发表)
[30]林玉玲,赖钟雄.龙眼胚胎发育过程中SOD活性的变化亚热带农业研究,2007, 3(4): 267-270.
[31]刘成明,胡又厘,傅嘉欣.生物技术在荔枝龙眼繁殖和品种改良中的应用研究进展[J].中国南方果树,2003,32(6):30-34.
[32]刘家忠,龚明.植物抗氧化系统研究进展[J].云南师范大学学报,1999, 19(6): 1-11.
[33]刘丽娜,刘祥林,陈志玲.细胞周期调控的一个重要元素--CDC48[J].生物学通报, 2006, 46 (5):26-29.
[34]刘丽娜.烟草细胞周期相关基因NtCDC48的克隆与功能研究[D].北京:首都师范大学硕士学位论文,2006.
[35]刘丽娜.烟草NtCDC48基因的克隆及功能分析[J].自然科学进展.2006.16(12).1561-1567.
[36]刘招龙.亚硒酸钠对高温胁迫下梨苗部分酶活性的影响[J].宁德师专学报, 2006, 18(3): 237-239.
[37]龙桂友.柑橘低温诱导相关基因的克隆与表达分析[D].长沙:湖南农业大学博士论文,2007.
[38]苗雨晨,白玲,苗琛,等.植物谷胱甘肽过氧化物酶研究进展[J].植物学通报, 2005, 22 (3): 350-356.
[39]邵巍,赖钟雄,赖呈纯,等.龙眼胚性培养物APX同工酶的分析方法建立及其在龙眼体胚发生过程中的变化[J].福建农林大学学报(自然科学版),2008, 37(2): 140-144.
[40]邵巍.龙眼胚性培养物胞质型apx基因克隆及其表达研究[D].福州:福建农林大学硕士学位论文, 2008.
[41]王凤华,赖钟雄.龙眼胚性愈伤组织维生素C过氧化物酶基因(apx)及NADH脱氢酶(nad2)基因部分序列的克隆[J].应用与环境生物学报. 2005, 11(1): 45-48.
[42]王凤华.龙眼体细胞胚胎发生过程中的基因差异表达[D].福州:福建农林大学博士学位论文, 2002.
[43]王亚馥,崔凯荣,高清祥,等.小麦体细胞胚发生的蛋白质组分和过氧化物酶同工酶的变化[J].兰州大学学报,1993, 29 (3): 189~193.
[44]王亚馥,崔凯荣,汪丽虹,等.小麦组织培养中体细胞胚胎发生的细胞胚胎学及淀粉消长动态的研究[J].实验生物学报, 1993. 26 (3): 259- 267.
[45]魏文雄,杨永青.龙眼子叶胚状体的诱导和试管苗培育[J].福建师范大学学报(自然科学版), 1981, (2): 102-106.
[46]邢更妹,李彬等.植物体细胞胚胎发生中抗氧化系统代谢动态和程序性细胞死亡[J].生命科学, 2000, l2(5): 215-218.
[47]邢更妹,李杉,崔凯荣,等.植物体细胞胚发生中抗氧化系统代谢动态和程序性细胞死亡[J]生命科学.
[48]邢更生,崔凯荣,山仑,等.植物体细胞胚发生的分子基础[J].遗传, 1999, 21(1): 30-34.
[49]闫春燕,徐承水,张士璀.动物磷脂氢谷胱甘肽过氧化物酶研究进展[J].科技信息, 2008, 22: 366-367
[50]杨晓东,刘进元.萝卜磷脂氢谷胱甘肽过氧化物酶基因结构及其调控序列分析[J].生物化学与生物物理进展, 2005, 32(7):649-656.
[51]杨永青,陈永锋.焦核龙眼果实遗传性状及胚培养的研究[J].园艺学报,1987,14(4):217- 222.
[52]杨永青,魏文雄.龙眼花粉植株的诱导[J].遗传学报, 1984, 11(4): 288-293.
[53]殷奎德,马连菊,刘世强.逆境条件下植物活性氧(AOS)的研究进展[J].沈阳农业大学学报, 2003,21(2): 29-33.
[54]曾黎辉.龙眼遗传转化研究初报[D].福州:福建农业大学硕士学位论文.1998.
[55]曾黎辉,陈振光,吕柳新.发根农杆菌转化龙眼研究初报[J].福建农业大学学报, 2000, 29(1):27- 30.
[56]翟中和.细胞生物学[M].北京:高等教育出版社, 1995.
[57]张景萍,吴珍龄.几种植物中谷胱甘肽过氧化物酶活性测定[J].广西农业科学, 2004, 35(3): 177-178.
[58]郑启发,胡桂兵,陈大成,等.荔枝龙眼细胞悬浮培养和转基因研究[J].果树学报,2005,22(2):125-128.
[59]周键,孙建波,彭明.高等植物细胞胚胎发生的研究进展[J].中国农学通报2008, 24 (2): 129-133
[60]周毅峰,吴永尧,唐巧玉.硒对大豆叶片谷胱甘肽过氧化物酶和过氧化氢酶歧化酶活性的影响[J].湖北民族学院学报, 2005, 23(2): 127-129.
[61] Acquaviva C, Pines J. The anaphase promoting complex/cyclosome:APC/[J]. Journal of Cell Science, 2006, 119: 2401-2404.
[62] Albert B . Molecular Biology of Cell, Garland Publishing, Inc. N. Y. Acta Horticulturae.2001,558:149～154.
[63] Arthur J R. The glutathione peroxidases[J]. Cell Mol Life Sci, 2000, 57: 1825- 1835.
[64] Asada K. Radical production and scavenging in the chloroplasts[M]. In: Photo.
[65] Asada K. The rolel of ascorbate peroxidase and monodehydro ascorbate reductase in H202 scavenging in plants[M]. In: Oxidative Stress and the Molecular Biology ofAntioxidant Defenses. Cold Spring Harbor Laboratory Press, 1997: 715-735.
[66] Asada K. The water-water cycle in chloroplasts: scavenging of active oxygens and dissipation of excess photons[J]. Annual Review of Plant Physiology, 1999, 50: 601-639.
[67] Asada, Kiso K., Yoshikawa, K. Univalent reduction of molecular oxygen by spinach chloroplasts on illimination[J]. J Biol Chem, 1974, 249: 2175- 2181.
[68] Avsian-Kretchmer O, Gueta-Dahan Y, Lev-Yadun S, et al. The salt-stress signal transduction pathway that activates the gpxl promoter is mediated by intracellular H202, different from the pathway induced by extracellular H202[J]. Plant Physiology, 2004, 135: 1685-1696.
[69] Babst, M., Katzmann, D. J., Synder, W. B., Wendland, B. and Emr, S. D.. Endosome-associated complex, ESCRT-II, recruits transport machinery for protein sorting at the multivesicular body[J]. Dev CeLL, 2002,3:283-289.
[70] Babst, M., Wendland, B., Estapa, E. J. and Emr, S. D. The Vps4pAAA ATPase regulates membrane association of a Vps protein complex required for normal endosome function[J]. EMBO J. 1998,17, 2982-2993.
[71] Berget S M, Moore C, Sharp P A. Spliced segments at the 5' terminus of adenovirus 2 late mRNA.Proc Natl Acad Sci USA, 1977,74:3171-3175.
[72] Beuron, F, F1ynn, T C, Ma, J, Kondo, H, Zhang, X. and Freemont, P.S. Motions and negative cooperativity between p97 domain revealed by cryo-electron microscopy and quantised elastic deformational model[J]. J. MoL. BioL. 2003, 327: 619-629.
[73] Braun S, Matuschewski K, Rape M, et al. Role of the ubiquitin-selective CDC48 (UFDI/NPL14) chaperone(segregase ) in ERAD of OLEl and other substrates [J]. EMBO,2002,21:625-621.
[74] Chu FF, Doroshow JH, Esworthy RS Expression, characterization and tissue distribution of a new cellular selenium-dependent glutathione peroxidase, GSHPx-GI[J]. Journal of Biological Chemistry, 1993, 268: 2571- 2576.
[75] Claudia Magioli, Rosa Maria Barroco,Carla Andrea Ben. et al Somatic embryo formation in Arabidopsis and eggplant is associated with expression of a glycine-rich protein gene (Atgrp-5) [J]. Plant science,2001.161:559-567.
[76] Criqui M C, Jamet E, Parmentier Y, et al. Isolation and characterization of a plant cDNA showing homology to animal glutathione peroxidases[J]. Plant Molecular Biology, 1992, 18: 623-627.
[77] Dai, R. M. and Li, C.-C. H.. Valosin-containing protein is a multi- ubiquitin chain-targeting factor required in ubiquitin-proteasome degradation[J].Nat. CeLL BioL. 2001, 3 740-3744.
[78] Dai, R.-M., Chen, E., Longo, D. L., Gorbea, C. M. and Li, C.-C. H. Involvement of valosin-containing protein, an ATPase co-purified with Ikappa Balpha and 26S Proteasome, in ubiquitin-proteasome-mediated degradation of Ikappa Balpha[J]. J. BioL. Chem. 1998, 273, 3562-3573.
[79] Dalal, S. and Hanson, P I.. Membrane traffic: what drives the AAA motor[J]. Cell. 2001, 104. 5-8.
[80] Dat J F, Foyer C H, Scott I M. Changes in salicylic acid and antioxidants during Induced the rmo tolerance in mustard seedlings[J]. Plant Physiology, 1998, 118: 1455-1461.
[81] De Jong A J, Hendrichks T, Meijer E A, et al. Transient reduction in secreted 32 kD chitinase prevents somatic embryogenesis in the carrot variant ts II [J]. Devel Cenet, 1995, 16: 332- 343.
[82] Delaunay A, Pflieger D, Barrault M, et al. A thiol peroxidase is an H2O2 receptor and redox-transducer in gene activation[J].Cell,2002, 111: 471- 481.
[83] Depege N, Drevet J, Boyer N. Molecular cloning and characterization of tomato cDNAs encoding glutathione peroxidase-proteins[J]. European Journal of Biochemistry. 1998, 253: 445-451.
[84] Dixon, D.P., Skipsey, M., Grundy, N.M. and Edwards, R. Stress-induced protein S-glutathionylation inarabidopsis[J]. Plant Physi., 2005, 138: 2233-2244.
[85] Doke N. The oxidative burst: roles in signal transduction and plant stress[A] Scandalios JCx Oxidative Stress and the Molecular Biology of Anrioxidant Defenses[C]. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1997: 785-813.
[86] Dong J Z, Perras M R, Abrams S R er al. Characterization of three heat-shock-protein genes and their developmental regulation during somatic embryogenesis in white spruce [picea glauca(moench) voss] [J]. Planta, 1996, 200: 85-91.
[87] Dong J Z, Perras M R, Abrams S R er al. Expression of abundant mRNAs during somatic embryogenesis of white spruce[picea glauca(moench) voss] [J]. Planta, 1996, 199: 459-466.
[88] Dong J Z et al. Induced gene expression following ABA uptake in embryogenic suspension cultures of picea glauca[J]. Plant Physiol. Plant Physiol. Biochem., 1996, 34: 579-587.
[89] Drotar A., Phelps P., Fall R. Evidence for glutathione peroxidase activities in cultured plant cells [J]. Plant Science, 1985(42): 35-40.
[90] Du H, Simpson R J, Moritz R L, et al .Isolation of the protein backbone of an arabinogalactan-protein from the styles of nicotiana alata and characterization of a corresponding cDNA[J].Plant Cell, 1994,6: 1643-1653.
[91] Du H, Simpson R J, Clarke A E et al. Molecular characterization of a stigma-specific gene encoding an arabinogalactan-protein(AGP) from nicotiana alata[J]. Plant Journal. 1996, 313-323.
[92] Elledge S J. Mitotic arrest: Mad2 Provents Sleepy from waking up the APC[J].Science,1998,279,999-1000.
[93] Eshdat Y, Holland D, Faltin Z, et al. Plant glutathione peroxidases[J]. Physiologic Plantarum, 1997, 100: 234-240.
[94] Flohe,L. GLutathione peroxidase[J].Bosic Life.Sci , 1988 ,49 : 663.
[95] Foyer C H,Noctor G.Redox homeostasis and antioxidant signaling:A metabolic interface between stress perception and physiological responses[J].Plant Cell, 2005, 17: 1866—1875.
[96] Foyer, C.H. and Noctor,G Redox sensing and signaling associated with reactive oxygen in chloroplast, peroxisomes and mitochondria[J]. Physiol. Plant., 2003, 119: 355-264.
[97] Frohlich K U, Fries H W, Rudiger M, et al. Yeast cell cycle protein CDC48p shows full-length homology to the mammalian protein VCP and is a member of protein family involved in secretion, peroxisome formation, and gene expression[J]. J Cell Biol,1991, 114: 443-453
[98] Fujlmura, T., and Komamlne, A. The serial observation of embryogenesis in a carrot cell suspension culture[J]. New Phyiol,1980, 86: 13-218.
[99] Gomez, L.D., Noctor,G,Knight, M. and Foyer, C.H. Regulation of calcium signaling and gene expression by glutathione[J].J.Exp Bot., 2004 (55): 1851一1859.
[100] Hatzpoulos P, Fong F, Sung Z R.Absicsic acid regulation of DC8,a carrot embryogenic gene[J]. Plant Physiol,1990, 94: 690- 695.
[101] Helleboid S et al. Inhibition of direct somatic embryogenesis by alpha-difluoromethylarginine in a cichorium hybrid: effects on polyamine content and protein patterns[J]. Planta, 1995, 196: 571-576.
[102] Henry Y, Vain P, de Buyser J. Genetic analysis of in vitro plant tissue culture responses and regeneration capacities[J].Euphytica,1994,79: 45- 48..
[103] Herbette P,Lenne C,Leblanc N,et al. Two GPX-like proteins from Lycopersicon esculentum and Helianthus annuus are antioxidant enzyme with phospholipid hydroperoxide glutathione peroxidase and thioredoxin peroxidase activities[J]. Eur J Bio Chem, 2002, 269: 2414-2420.
[104] Hernandez J A, Corpas F J. Salt-induced oxidative stress in chloroplasts of pea plants[J]. Plant Science, 1995, 105: 151-167.
[105] Hershko, A. and Ciechanover, A. The ubiquitin system[J]. Annu. Rev.Biochem. 1998,67: 425-479.
[106] Higashi K, Shiota H, Kamada H. Patterns of expression of the genes for glutamine synthetase isoforms during somatic and zygotic embryogenesis in carrot[J]. Plant Cell Physiol, 1998, 39: 418- 424.
[107] Hippeli S. Mechanisms of oxygen activation during plant stress: biochemical effects of air pollutants[J]. Journal of Plant Physiology,1996, 148: 249-257.
[108] Hochstrasser M.. Ubiquitin-dependent protein degradation[J]. Annu Rev,Genef , 1996, 30: 405-439.
[109] Holland D, Ben-Hayyim G Faltin Z, et al. Molecular characterization of salt-stress-associated protein in Citrus: protein and cDNA sequence homology to mammalian glutathione peroxidase[J]. Plant Molecular Biology, 1993, 21:923-927.
[110] Holland D, Ben-Hayyim G,Faltin Z,et al.Molecular characterization of salt-stress-associated protein in Citrus:Protein and cDNA equence homology to mammalian glutathione peroxidase[J]. Plant:460-469.
[111] Hsieh W L,Wolniak S M.Isolation and characterization of a funcational A-type cyclin from maize[J]. Plant Molecular Biloogy, 1998, 37: 121-129.
[112] Hutchison. C J.Glover D D .Cell Cycle Control[M].Oxford;IRLOxford Univerisity Press,1995.
[113] JSmirnoff N. Antioxidant systems and plant response to the environment. In: Environment And Plant Metabolism[M]. Oxford, UK. BIOS Scientific Publishers, 1995: 217-243.
[114] Jung B G, Lee K O, Lee S S, et al. A Chinese cabbage cDNA with high sequence identity to phospholipid hvdroperoxide glutathione peroxidases encodes a novel isoform of thioredoxin-dependent peroxidase[J]. Journal of Biological Chemistry, 2002, 277: 12572-12578.
[115] Kastan M B .Check point Controls and Cancer[M]//J Tooze.Cancer Surveys.Vol 29.New York:Cold Spring Harbor Laboratory Press,1997.
[116] Kiyosue T et al. Cloning of a carrot cDNA for a member of the family of ADP-ribosylation factors(ARFs) and characterization of the binding of nucleotides by its product after expression in e-coli[J]. Plant Cell Physiol.1995. 36: 849-856.
[117] Kiyosue T, Yamaguchi-Shinozaki K,Shinozaki K, et al. Isolation and characterization of a cDNA that encodes ECP31, an embryogenie- cell protein from carrot[J]. Plant MoL Biol. 1992,19:239- 249.
[118] Kondo H, Rabouille C, Newman R, et al. p47 is a cofactor forp97-mediated membrane fusion[J]. Nature, 1997,388: 75-78.
[119] Kragh K M,Hendriks T, De Jong A J, et al. Characterization of chitinases able to rescue somatic embryos of the temperature-sensitive carrot variant is tsII[J]. Plant, Mol, Biol, 1996, 31 :631-645.
[120] Kreuger M,van Holst G .J.Arsbinogalactan protein epitopes insomatic embryogenesis of Daucus carota [J].Planta, 1995, 197:135-141.
[121] Lai Z X, Chen C L, Chen Z G. progress in biotechnology research in longan[J].. Acta Horticulturae, 2000b ,558: 137~141.
[122] Lai Z X, Ch C L, Zeng L H , et al. Somatic embryogenesis in longan [Dimocarpus longan Lour.][J]. Forestry Sciences. 2000a, 67: 415-432.
[123] Lee, R. C.,R. L. Feinbaum, and V. Ambros. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14[J]. Ce11, 1993,75 (5): 843-54.
[124] Li W J, Feng H, Fan J H, et al. Molecular cloning and expression of a phospholipid hydroperoxide glutathione peroxidase homolog in Oryza sativa[J]. Biochimica et Biophysics Acta, 2000, 1493: 225-230.
[125] Li WJ, Feng H, Fan JH, et al. Molecular cloning and expression of a phospholipid hvdroperoxide glutathione peroxidasehomolog in Oryza sativa[J]. Biochimica et Biophysica Acta, 2000,1493: 225-230.
[126] Lowry, O. H, Rosebrough, N. Y., Farr, A. L.,et al. Protein measurement with the folin phenol reagent[J]. Boil. Chem.,1951, 193: 265-275.
[127] Luisa C. Sunflower seedlings subjected to increasing water deficit stress, oxidative stress and defence mehanisms[J]. Physiologic Plantarum, 1995, 93: 25-30.
[128] Maiorino M, Thomas JP, Girotti AW, Ursini F Reactivity of phospholipid hydroperoxide glu tathione peroxidase with membrane and lipoproteinlipid hydroperoxides[J]. Free Radical Research, 1991, 12: 131-135.
[129] Mau S L et al. Cloning of a carrot cDNA for a member of the family of ADP-ribosylation factors(ARFs) and characterization of the binding of nucleotides by its product after expression in e-coli[J]. Plant Journal, 1995, 8:269-281.
[130] McNew, J. A., Parlati, F, Fukuda, R., Johnston, R. J., Paz, K., Paumet,R, Sollner, T. H. and Rothman, J. E. Compartmental specificity of cellular membrane fusion encoded in SNARE proteins[J]. Nature, 2000, 407, 153-159.
[131] Mehdy, M.C. Active oxygen species in plant defense against pathogens[J]. Plant Physiol., 1994, 105: 467-472.
[132] Milla MAR, Maurer A, Huete AR, Gustafson JP. Glutathione peroxidase genes in Arabidposis areubiquitvous and regulated by abiotic stresses though diverse signaling pathways[J]. The Plant Journal, 2003, 36: 602-615.
[133] Mimuria S, Takisawa H. Xenopus CDC45-dependent Loading of Ploymerase onto Chromatin under the control of S-Phase CDK[J]. EMBO J, 1998, 17: 5699-5707.
[134] Mittler,R. Oxidative stress, antioxidants and stress tolerance. Trends in plant science2002, 7:405-410.
[135] Moir D, Stewart B C, Osmond B C, et al. Cold sensitive cell division cycle mutants of yeast; Isolation, properties, and pseudoversion studies[J]. Genetics, 1982, 100; 547-563.
[136] Mullineaux PM, Karpinski S,.Jimenez A, Cleary SP Robinson C, Creissen GPIdentication of cDNA encoding plastid-targeted glutathione peroxidase[J]. The Plant ,Journal, 1998, 13: 370-379.
[137] Noctor G,Foyer C H. Ascorbate and glutathione: keeping active oxygen under control[J]. Annual review of plant physiology[J]. Plant Molecular Biology, 1998, 49: 249-279.
[138] NOCTOR G, GOMEZL,LE’NE VANACKER He, et al . Interactions Between Biosynthesis , Compartmentation and Transport in the Control of Glutathione Homeostasis and Signaling [J ] . Journal of Experimental Botany ,2002 ,53(372) :1283 - 1304.
[139] Ogura, T. and Wilkinson, A. J. AAA+ superfamily ATPasescommon structure-diverse function[J]. Genes CeLLs. 2001,6:575-597.
[140] Pacific R E.Hydrophobicity as the signal for selective degraclation of hydroxyl radical-modified hemoglobin by the multicatalytic proteinase complex,proteasome[J].J.Biol.Chem.,1993,268: 15405-15411
[141] Patel, S. and Latterich, M. The AAA team: related ATPases with diverse functions[J]. Trends Cell Biol. 1998, 8: 65-71.
[142] Patel, S. K., Indig, F. E., Olivieri, N., Levine, N. D. and Latterich, M. Organelle membrane fusion: a novel function for the syntaxin homolog Ufelp in ER membrane fusion[J]. Cell 1998,92, 611-620.
[143] Philip G. Woodman. a protein coping with multiple identities[J]. Journal of Cell Science. 2003,116: 4283-4290.
[144] Pine J .Cyclins and Cyclin-dependent kinases:a biochemical view[J].Biochem J 2003, 308:679-711.
[145] Rape, M., Hoppe, T., Gorr, I, Kalocay, M., Richly, H. and Jentsch, S. Mobilization of processed, membrane-tethered SPT23 transcription factor by CDC48(LJFD1/NPL4), a ubiquitin-selective chaperone[J].Cell2001, 107,667-677.
[146] Rocher C,Lalanne JL,ChaudiereJ Purification properties of a recombinant sulfur analog of murine selenium-glutathione peroxidase[J].European,Journal of Biochemistry 1992,205:900-960.
[147] Rockel, B., Jakana, J., Chiu, W. and Baumeister, W. Electron cryo-microscopy of VAT, the Archaeal p97/CDC48 homologue from The rmopLasma acidophiLum[J]. J. MoL. BioL.2002,317, 673-681.
[148] Roeckel D P, GagneG , Dufaure J P, et al. Molecular characterization organ distri-bution and stress-mediated induction of two glutathione peroxidase-encoding mRNAs in sunflower (Helianthus annuus) [J]. PhysiologicPlantarum. 1998, 103: 385-394.
[149] Roeckel-Drevet P, GagneG, de Labrouhe TD, Dufaure JP, Nicolas P, Drevet JRMolecular characterization organ distribution and stress-mediated induction of two glutathione peroxidase-encoding mRNAs in sunflower (Helianthus annuus). 1998
[150] Sabeh F, Wright T, Norton SJ Purification and characterization of a glutathione peroxidase from the Aloe vera plant[J]. Enzyme and Protein, 1993, 47:92-98.
[151] Schmidt E D L,Guzzo F, Toonen M A J, et al. A leucine- rich repeat containing receptor-like kinase marks somatic plant cells competent to form embryos[J]. Development, 1997,124:2049- 2062.
[152] Shah K Cadella T W Jr,van Erp H et al. Subcellular localization and oligomerization of the Arabidopsis thaliana somatic embryogenesis receptor kinase 1 protein[J]. J Mol Biol. 2001,309:641-655.
[153] Smallwood E A et al. Immunochemical comparison of membrance-associated and secreted arabinagalactan-proteins in rice and carrot[J]. Planta, 1996, 193:452-459
[154] Smith, T. M,M. K. Lee, C. I. Szabo, N. Jerome, M. McEuen, M. Taylor, L. Hood, and M. C. King.. Complete genomic sequence and analysis of 117 kb of human DNA containing the gene BRCA1. Genome Res1996, 6 (11):1029-49.
[155] Stadman TC. Selenocvsteine. Annual Review of Biochemistry, 1996, 65: 83-100.
[156] Sterk, P., Boolj, H.,Schellekens,G.A_et al.Cell-specific expression of the carrot EP2 lipidtransfer protein gene[J].Plant Cell 3.1991:907-921
[157] Stirling, C., Rothblatt, J., Hosobuchi, M., Deshaies, R. and Schekman, R. Protein translocation mutants defective in the insertion of integral membrane proteins into the endoplasmic reticulum[J]. MoL. BioL. CeLL1992, 3, 129-142.
[158] Sugimoto M, Furui S, Suzuki Y Molecular clon ing and characterization of a cDNA encoding putative phospholipid hvdroperoxide glutathione peroxidase from Spinach[J].Bioscience Biotechnology and Biochemistry, 1997, 61:1379-1381.
[159] Sugimoto M, Sakamoto WPutative phospho lipid hvdroperoxide glutathione peroxidase gene from Arabidopsis thaliana induced by oxidative stresss[J]. Genes & Genetic Systems, 1997,72:311-306.
[160] Talia B T, Gozal B H, Holland D, et al. A stress-associated citrus protein is a distinct plant phospholipid hydroperoxide glutathione peroxidase[J]. FEBS Letters,1995, 366:151-155.
[161] Taylor, H. A homeobox gene regulatory element is composed of distinct conserved. the formation of free radicals in plants exposed to ozone[J]. Physiologic Plantarum. 1996.
[162] Tee, M. K., G. O. Aza-Blanc, P. A et al. A promoter within intron 35 of the human C4A gene initiates abundant adrenal-specific transcription of a 1 kb RNA:location of a cryptic CYP21 promoter element[J]? Human Molecular Genetics.1995, 11(4): 2109-2116.
[163] Tsai B., Ye Y.,Rapoport T A. Retro-translocation of proteins from the endoplasmic reticulum into thecytosol[J].Nat. Rev. Mol. Cell Biol. 2002,3(4): 246-255.
[164] Volkand S, Feieralxnd J. Photo inactivation of catalase at low temperature and its relevance to photosynthetic and peroxide metabolism in leaves[J]. Plant Cell and the Environment,1989, 12: 701-712.
[165] Weissman, A. M. Ubiquitin and proteasomes: themes and variations on ubiquitylation[J]. Nat. Rev. Mol. Cell. Biol. 2001, 2, 169-178.
[166] Willekens, H., Chamnongpol, S., Davey, M., Schraudner, M.,Langebartels, C., Montagu, M.V , Inze D. and Camp W.VCatalase is a sink for H202 and is indispensable for stress defence in C-3 plants[J].EMBO J. 1997, 16: 4806-4816.
[167] Wurtele, E.S., Wang, H., Durgerian, S., et al. Characterization of a gene that is expressed early in somatic embryogenesis of Daucus carota[J].Plant Physiol, 1993, 102: 303-312.
[168] Xinrong Fu, Christine Ng, Daorong Feng, and Chun Liang. CDC48p is required for the cell cycle commitment point at Start via degradation of the Gl- CDK inhibitor Farlp[J].The Journal of Cell Biology. 2003, 163: 21-26.
[169] Ye, Y., Meyer, H. H. and Rapoport, T. A. The AAA ATPase Cdc48/p97 and its partners transport proteins from the ER into the cytosol[J]. Nature2001, 414, 652-656.
[170] Ye, Y., Meyer, H. H. and Rapoport, T. A. Function of the p97-Ufdl-Np14 complex in retrotranslocation from the ER to the cytosol: dual recognition of nonubiquitinated polypeptide segments and polyubiquitin chains[J].J.Cell Biol. 2003, 162, 71-84.
[171] Yu K F Pauls K P. Identification of a RAPD marker associated with somatic embryogenesis in alfalfa[J].Plant Mol. Biol1993, 22: 267-277.
[172] Zeng L H, Chen Z G .and LüL X et al. In vitro trmisformation mediated by Ri plasmid of Agrohacteriuu rhizogenes and trmisgeuic plant regeneration of longan. Proceediugs of the first International Symposium ou Litchi and Longan[J]. Acta Horticulturae. 2001, 558:149-154.
[173] Zhang, J., X. Sun, Y. Qian, and L. E. Maquat.. Intron function in the nonsense-mediated decay of beta-globin mRNA: indications that pre-mRNA splicing in the nucleus can influence mRNA translation in the cytoplasm[J]. Rna 1998, 4 (7): 801-815 .
[174] Zhang, X., Shaw, A., Bates, P A., Newman, R. H., Shaw, A., Bates, P A.,Newman, R. H., Gowen, B., Orlova, E., Gorman, M. A. et al., Structure of the AAA ATPase[J]. MoL. CeLL2000, 6, 1473-1484.
[175] Zhu J K ,Bressan R A, Hasegawa P M. Loss of arabinogalactin-protein from the plasma membrane of NaCI- adapted cells[J]. Planta,1993,190: 221-226.
[176] Zimmerman J L. Somatic embryoenesis: A model for early development in high plants[J].Plant Cell,1993,5:1411-1423.
[177] Zuckerkandl, E. Junk DNA and sectorial gene repression[J].Gene 1997, 205 (1-2):323-43.