小麦根中盐胁迫响应基因FBPA的克隆及鉴定
摘要
盐分是影响植物生长和产量的主要环境因子之一。在分子水平上,盐胁迫可以使植物中一些基因的表达状况发生改变,促进或抑制某些蛋白质的合成,提高其耐盐性。克隆植物耐盐相关基因并利用转基因技术培育植物耐盐品种为抗盐育种的研究开辟了广阔的应用前景。但由于植物耐盐性是一个受多基因控制的复杂的数量性状,其耐盐机制十分复杂,因此,有必要利用基因组学的方法分离并深入研究一些盐胁迫应答基因,并对这些基因进行植物转基因研究,以期培育出耐盐的转基因植物,从而更好地指导农业生产。
小麦是世界上分布最广、种植面积最大的粮食作物。耐盐小麦品种山融3号(SR3)是小麦品种济南177(Triticum aestivum L.2n=42)与其近缘野生种长穗偃麦草(Thinopyrum ponticum 2n=70)经过不对称体细胞杂交技术获得的体细胞杂种渐渗系。遗传及生理生化的分析表明,该品种含有长穗偃麦草染色体小片段,耐盐指数及各项生理生化指标均明显优于亲本小麦。山融3号不同于其它耐盐相关研究所利用的单一遗传背景的材料,其耐盐性由主效基因和微效基因共同控制。本实验室以山融3号小麦和济南177小麦为材料,利用抑制性差减杂交技术(suppressive subtractivehybridization,SSH)技术构建了山融3号和济南177盐胁迫响应的SSH cDNA文库。本研究根据小麦SSH cDNA文库中的相关信息,克隆得到一个在小麦根中对盐胁迫条件产生应答的果糖1,6-二磷酸醛缩酶基因(TaFBPA),并对其进行了初步鉴定和研究。
果糖1,6-二磷酸醛缩酶是生物体内碳代谢及糖代谢途径中的关键酶之一,它催化果糖1,6-二磷酸分解为磷酸二羟基丙酮和3-磷酸甘油醛及其逆反应。本研究利用嵌套PCR技术从小麦中克隆得到果糖1,6-二磷酸醛缩酶基因的全长cDNA序列,将其命名为TaFBPA。生物信息学分析表明该基因属于果糖1,6-二磷酸醛缩酶基因家族,该基因编码一个由358个氨基酸组成的蛋白质,该蛋白与玉米、拟南芥、水稻中同源基因的蛋白的相似性分别为88.55%、82.12%、77.99%。RT-PCR及Real-Time PCR分析结果表明,较高浓度的短期盐胁迫条件下,该基因在小麦根中发生明显的上调表达。构建植物瞬时表达载体,将该基因与绿色荧光蛋白基因融合在一起并转化拟南芥原生质体,结果表明,融合蛋白定位于细胞质中。构建原核表达载体,将该基因连入pZT32a载体并转化大肠杆菌菌株DE3进行原核表达,经IPTG诱导后,可以检测到目的蛋白大量表达,为下一步纯化融合蛋白并测定体外醛缩酶酶活性奠定基础。构建植物过量表达载体,利用农杆菌介导的花浸染方法转化拟南芥,获得转基因拟南芥后代。种子萌发实验表明,在盐胁迫条件下,与对照株系的种子相比,转基因株系的种子萌发延迟,且萌发率低,这表明过量表达TaFBPA基因的转基因拟南芥株系的种子对盐胁迫比较敏感。需要进一步的研究以期揭示TaFBPA基因参与植物盐胁迫应答的作用机理。
Salinity is one of the major environmental factors limiting plant growth and productivity. The expression of some genes will be changed by salt stress in the molecular level, which will promote or repress the synthesis of some relevant proteins to improve the ability to salt tolerance of the plant. It opens vast vistas for the plant slat-tolerance breeding research to isolate some relevant salt-tolerance genes from the plants and to develop slat-tolerance plant breeding by utilizing the technology of gene manipulation. However, the salt stress tolerance is controlled by quantitative trait loci (QTLs), and the mechanism of salt stress tolerance of plant is very sophisticated. Therefore, it is necessary to isolate and make an intensive study of some salt stress responsive genes, which could be transformed into plants for further studying. Finally, it is hoped that the salt-tolerant transgenic plants could be cultivated and be widely used for agricultural operation.
Common wheat (Triticum asetivum L.) is one of the most important and widely planted crops in the world. A new somatic hybrid introgression line Shanrong No.3 (hereafter SR3) has been generated in our lab from hybridization of common wheat cv. Jinan 177 with wheatgrass (Thinopyrum ponticum), a salt and drought tolerant grass relative to wheat. Cytological and molecular analysis showed that some nuclear and non-nuclear DNAs and even functional genes of donor Th. ponticum were introgressed into this line. SR3 had a significantly higher yield than its parent JN177. The salt-tolerance of SR3 is controlled by the major and minor genes together. As one of the materials for salt-stress tolerance research, SR3 is different from the others with single genetic background. In our lab, the suppressive subtractive hybridization (SSH) technology was used to construct a salt-stress responsive SSH cDNA library of SR3 and JN177 wheat. According to the relevant information of the SSH cDNA library, a salt-stress responsive gene, fructose 1, 6-bisphosphate aldolase gene in wheat root (TaFBPA), was isolated and identified in this study.
The fructose 1, 6-bisphosphate aldolase (FBPA) is one of the important enzymes in the carbon metabolism and sugar metabolism pathway of the organisms, which catalyses an aldol cleavage of fructose 1, 6-bisphophate to dihydroxyacetone-phosphate and glyceroldehyde 3-phosphate and a reversible aldol condensation. The full-length cDNA sequence of fructose 1, 6-bisphosphate aldolase gene (TaFBPA) was acquired in this study by Nested-PCR technology. According to the bio-informatic sequence analysis, TaFBPA belongs to the family FBPA. TaFBPA encodes a protein of 358 amino acids that shares high identity with the orthologs from Zea mays (88.55%), Arabidopsis thaliana (82.12%), Oryza sativa (77.99%), respectively. The obviously elevated levels of TaFBPA expression have been detected by RT-PCR and Real-Time PCR analysis when the wheat seedling roots exposed to high salinity stress. Arabidopsis transformation of construct expressing the TaFBPA fused with green fluorescent protein (GFP) reveals that the fusion protein is targeted to cytoplasm. The recombined plasmid with insertion of TaFBPA to vector pET32a was transformed into E.coli DE3 to analyze its expression. A large amount of fusion protein was expressed by the induction of IPTG and detected by SDS-PAGE, which makes a foundation for the further protein purification and aldolase assay. The plant over-expression vector of TaFBPA was constructed and then transformed Columbia (Co10) Arabidopsis lines with Agrobacterium-mediated floral-dip method. The experiment of seed germination shows that the seeds of transgenic lines have a lower germination rate and a delayed germination time comparing with the seeds of control lines, which demonstrates that the seeds of transgenic lines with the over-expression of TaFBPA are more sensitive to the salt stress condition. Further studies are needed to deeply illustrate the mechanism of the TaFBPA gene involving in the salt- stress responses in the plant.
小麦是世界上分布最广、种植面积最大的粮食作物。耐盐小麦品种山融3号(SR3)是小麦品种济南177(Triticum aestivum L.2n=42)与其近缘野生种长穗偃麦草(Thinopyrum ponticum 2n=70)经过不对称体细胞杂交技术获得的体细胞杂种渐渗系。遗传及生理生化的分析表明,该品种含有长穗偃麦草染色体小片段,耐盐指数及各项生理生化指标均明显优于亲本小麦。山融3号不同于其它耐盐相关研究所利用的单一遗传背景的材料,其耐盐性由主效基因和微效基因共同控制。本实验室以山融3号小麦和济南177小麦为材料,利用抑制性差减杂交技术(suppressive subtractivehybridization,SSH)技术构建了山融3号和济南177盐胁迫响应的SSH cDNA文库。本研究根据小麦SSH cDNA文库中的相关信息,克隆得到一个在小麦根中对盐胁迫条件产生应答的果糖1,6-二磷酸醛缩酶基因(TaFBPA),并对其进行了初步鉴定和研究。
果糖1,6-二磷酸醛缩酶是生物体内碳代谢及糖代谢途径中的关键酶之一,它催化果糖1,6-二磷酸分解为磷酸二羟基丙酮和3-磷酸甘油醛及其逆反应。本研究利用嵌套PCR技术从小麦中克隆得到果糖1,6-二磷酸醛缩酶基因的全长cDNA序列,将其命名为TaFBPA。生物信息学分析表明该基因属于果糖1,6-二磷酸醛缩酶基因家族,该基因编码一个由358个氨基酸组成的蛋白质,该蛋白与玉米、拟南芥、水稻中同源基因的蛋白的相似性分别为88.55%、82.12%、77.99%。RT-PCR及Real-Time PCR分析结果表明,较高浓度的短期盐胁迫条件下,该基因在小麦根中发生明显的上调表达。构建植物瞬时表达载体,将该基因与绿色荧光蛋白基因融合在一起并转化拟南芥原生质体,结果表明,融合蛋白定位于细胞质中。构建原核表达载体,将该基因连入pZT32a载体并转化大肠杆菌菌株DE3进行原核表达,经IPTG诱导后,可以检测到目的蛋白大量表达,为下一步纯化融合蛋白并测定体外醛缩酶酶活性奠定基础。构建植物过量表达载体,利用农杆菌介导的花浸染方法转化拟南芥,获得转基因拟南芥后代。种子萌发实验表明,在盐胁迫条件下,与对照株系的种子相比,转基因株系的种子萌发延迟,且萌发率低,这表明过量表达TaFBPA基因的转基因拟南芥株系的种子对盐胁迫比较敏感。需要进一步的研究以期揭示TaFBPA基因参与植物盐胁迫应答的作用机理。
Salinity is one of the major environmental factors limiting plant growth and productivity. The expression of some genes will be changed by salt stress in the molecular level, which will promote or repress the synthesis of some relevant proteins to improve the ability to salt tolerance of the plant. It opens vast vistas for the plant slat-tolerance breeding research to isolate some relevant salt-tolerance genes from the plants and to develop slat-tolerance plant breeding by utilizing the technology of gene manipulation. However, the salt stress tolerance is controlled by quantitative trait loci (QTLs), and the mechanism of salt stress tolerance of plant is very sophisticated. Therefore, it is necessary to isolate and make an intensive study of some salt stress responsive genes, which could be transformed into plants for further studying. Finally, it is hoped that the salt-tolerant transgenic plants could be cultivated and be widely used for agricultural operation.
Common wheat (Triticum asetivum L.) is one of the most important and widely planted crops in the world. A new somatic hybrid introgression line Shanrong No.3 (hereafter SR3) has been generated in our lab from hybridization of common wheat cv. Jinan 177 with wheatgrass (Thinopyrum ponticum), a salt and drought tolerant grass relative to wheat. Cytological and molecular analysis showed that some nuclear and non-nuclear DNAs and even functional genes of donor Th. ponticum were introgressed into this line. SR3 had a significantly higher yield than its parent JN177. The salt-tolerance of SR3 is controlled by the major and minor genes together. As one of the materials for salt-stress tolerance research, SR3 is different from the others with single genetic background. In our lab, the suppressive subtractive hybridization (SSH) technology was used to construct a salt-stress responsive SSH cDNA library of SR3 and JN177 wheat. According to the relevant information of the SSH cDNA library, a salt-stress responsive gene, fructose 1, 6-bisphosphate aldolase gene in wheat root (TaFBPA), was isolated and identified in this study.
The fructose 1, 6-bisphosphate aldolase (FBPA) is one of the important enzymes in the carbon metabolism and sugar metabolism pathway of the organisms, which catalyses an aldol cleavage of fructose 1, 6-bisphophate to dihydroxyacetone-phosphate and glyceroldehyde 3-phosphate and a reversible aldol condensation. The full-length cDNA sequence of fructose 1, 6-bisphosphate aldolase gene (TaFBPA) was acquired in this study by Nested-PCR technology. According to the bio-informatic sequence analysis, TaFBPA belongs to the family FBPA. TaFBPA encodes a protein of 358 amino acids that shares high identity with the orthologs from Zea mays (88.55%), Arabidopsis thaliana (82.12%), Oryza sativa (77.99%), respectively. The obviously elevated levels of TaFBPA expression have been detected by RT-PCR and Real-Time PCR analysis when the wheat seedling roots exposed to high salinity stress. Arabidopsis transformation of construct expressing the TaFBPA fused with green fluorescent protein (GFP) reveals that the fusion protein is targeted to cytoplasm. The recombined plasmid with insertion of TaFBPA to vector pET32a was transformed into E.coli DE3 to analyze its expression. A large amount of fusion protein was expressed by the induction of IPTG and detected by SDS-PAGE, which makes a foundation for the further protein purification and aldolase assay. The plant over-expression vector of TaFBPA was constructed and then transformed Columbia (Co10) Arabidopsis lines with Agrobacterium-mediated floral-dip method. The experiment of seed germination shows that the seeds of transgenic lines have a lower germination rate and a delayed germination time comparing with the seeds of control lines, which demonstrates that the seeds of transgenic lines with the over-expression of TaFBPA are more sensitive to the salt stress condition. Further studies are needed to deeply illustrate the mechanism of the TaFBPA gene involving in the salt- stress responses in the plant.
引文
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79.李晓燕,宋占午,董至贤,植物的盐胁迫生理.北京师范大学学报(自然科学版),2004.140(13):p.106-111.
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82.刘俊君,彭学贤,大肠杆菌mtlD基因和gutD基因的克隆、全序列测序和高效表达.生物工程学报,1995.11(2):p.157-161.
83.苗济文,马云瑞,罗代雄等,土壤盐分对宁夏春小麦的影响.西北农业学报,1995.4(3):p.81-84.
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85.苏永全,吕迎春,盐分胁迫对植物的影响研究简述.甘肃农业科技,2007.5(3):p.23-27.
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88.王军,权太勇,夏光敏,盐胁迫下小麦体细胞杂种与亲本小麦幼苗的生长量和Na~+、K~+含量比较.热带亚热带植物学报,2004.12(4):p.355-358.
89.王忠,植物生理学.北京:中国农业出版社,2000:p.287-290..p.331-335.
90.许兴,李树华,惠红霞等,NaCl胁迫对小麦幼苗生长、叶绿素含量及Na~+、K~+吸收的影响.西北植物学报,2002.22(2):p.278-284.
91.杨少辉,季静,王罡宋等,盐胁迫对植物影响的研究进展.分子植物育种,2006.4(3):p.139-142.
92.杨晓慧,蒋卫杰等,植物对盐胁迫的反应及其抗盐机理研究进展.山东农业大学学报,2006.37(2):p.302-305.
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94.张宏飞等,高等植物Na~+吸收、转运及细胞内Na~+稳态平衡研究进展.植物学通报,2007.24(5):p.561-571.
95.张晓宁,曲志才,沈大棱等,受NaCl诱导的盐藻果糖1,6-二磷酸醛缩酶cDNA的克隆及其在烟草中的表达.中国科学,2002.32(5):p.392-398.
96.赵可夫,植物抗盐生理.北京:中国科学技术出版社,1993:p.9-10.