野生金荞麦类黄酮次生代谢分子调控研究
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摘要
金荞麦(Fagopyrum cymosum(Trev．)Meisn)是蓼科(Polygonaceae)荞麦属(Fagopyrum Mill．)多年生草本植物，是一种重要的资源植物，为国家二级保护植物。原产于我国西南，分布于陕西、江苏、浙江、江西、河南、湖北、湖南、广西、广东、四川、重庆及云南等省区。野生金荞麦中蕴涵着丰富的优异基因，金荞麦的块根活性提取物(主要为类黄酮次生代谢产物)是多种重要的抗癌药物和癌预防药物的主要成分之一。目前，由于人工栽培的金荞麦产量和品质的限制，对野生金荞麦资源造成了极大的破坏，这就使野生金荞麦生物活性物的工厂化生产显得十分迫切。国内外已经展开了资源收集、多样性、生理特性、育种、药效学以及生态环境等方面的研究，基于活性成分合成的功能基因的研究，国内外还未见报道。
在本研究中，通过获得高黄酮含量愈伤系，建立野生金荞麦的RACE cDNA文库，从中快速分离克隆与药效成分积累相关的类黄酮次生代谢产物的关键酶基因(DFR，IFR)和转录调控因子P基因，并转化烟草超量表达进行功能的初步验证，为下一步转化野生金荞麦调控其类黄酮次生代谢，同时为实现其次生代谢产物的工厂化生产打下基础。
1、金荞麦高黄酮含量愈伤系的获得
以消毒金荞麦种子萌发的无菌苗的茎与叶片作为外植体，分别在MS添加2.0mg／L 6-BA和0.1mg／L NAA的培养基和MS添加1.0mg／L 6-BA和0.5mg／L NAA的固体培养基上经系统诱导并连续继代3次，获得了大量的稳定愈伤组织。采用目视法初步筛选出生长较快、不易褐化、颜色较深或稳定并且继代稳定的4个愈伤系。其中红色和褐色愈伤系主要来源于茎的诱导，而浅黄色和白色系则来源于叶的诱导用野生金荞麦无菌苗的茎和叶片作外植体。用分光光度法直接筛选出高类黄酮含量的红色系，其类黄酮含苗为6.3804mg／g，是含量最低的白色系的3.5倍。获得的来源于茎的高类黄酮含量的红色愈伤系为进一步的细胞系悬浮培养和药用次生代谢产物的规模化生产奠定了基础，同时，也为研究金荞麦类黄酮次生代谢的分子调控提供了优良的材料。
2、金荞麦愈伤系cDNA文库的构建
利用改良的CTAB结合LiCl沉淀方法从资源植物金荞麦愈伤组织中提取出完整性好、纯度高的总RNA。用SMART cDNA文库构建试剂盒成功构建了金荞麦红色愈伤系cDNA文库。原始文库滴度达到1.5×10~6 pfu／ml，扩增文库滴度约为7.0×10~9 pfu／ml，重组率达98％，插入片段在0.5 kb以上，平均约1.1 kb。通过PCR检测，从总文库中检测到了低丰度表达的P基因的特异片段，说明所构建的文库覆盖度高、代表性强。综合分析所构建的cDNA文库基本指标，表明已获得高质量的金荞麦愈伤系cDNA文库，可以用于进一步的基因克隆，这是第一次正式报道金荞麦cDNA文库的构建。
3、FcDFR基因的克隆与功能初步分析
(1)采用RACE结合cDNA文库筛选的方法，根据获得的DFR同源探针序列，我们从金荞麦cDNA文库中克隆到了DFR蛋白基因，命名为FcDFR(登录号：EF522145)。
(2) FcDFR基因有一个长85bp的内含子，有1个78bp的5′-UTR区、1个201bp的3′-UTR区及一个1026个核苷酸的开放阅读框。通过Southern杂交分析，推测在金荞麦基因组中DFR基因是1—2个基因的小家族，FcDFR基因是单拷贝基因。
(3)运用生物信息学手段对FcDFR编码蛋白FcDFR的结构特点与性质进行了分析，FcDFR富含疏水氨基酸，在N端存在一个NADP结合位点“VTGASGFVGSWLVMRLLEHGY”，还存在一个底物结合特异性决定的氨基酸基序“TVNVEEKQKPVYDETCWSDVDFCRRV”，推测它可能以DHQ或DHK为底物催化生成无色翠雀素和无色天竺葵素。它没有信号肽，可能定位于质膜，具有酶的典型三级结构特征。
(4)构建金荞麦FcDFR基因植物表达载体pC2301-FcDFR，用农杆菌介导的叶盘法将FcDFR基因导入烟草超量表达。通过报告基因GUS活性组织化学染色筛选、PCR检测以及RT-PCR检测。证实FcDFR基因已经整合进烟草基因组并获表达的转基因烟草95株。
(5)对95个转基因烟草叶片总黄酮进行测定，在所有检测的转基因烟草中，不同株系的总黄酮含量有一定差异，大部分叶片总黄酮含量都有不同程度的提高，最高可达对照的1.8倍，其中有78株显著增加，表明在这些株系中FcDFR基因得到了有效表达，促进烟草总黄酮的有效积累，这表明FcDFR基因具有生物活性，其超量表达对增加叶片中类黄酮的含量作用明显。17个株系总黄酮和对照烟草总黄酮含量没有显著差异。
4、FcMYBP1基因的克隆与功能初步分析
(1)用RACE方法筛选文库采用RACE结合cDNA文库筛选的方法，根据玉米P基因及Myb转录调控因子R2R3家族的R2、R3保守区域获得的同源探针，我们从金荞麦cDNA文库中克隆到了一个长1159个核苜酸R2R3类MYB蛋白基因，根据同源性比对分析，分离得到的基因可能是金荞麦类黄酮代谢途径(花色素和原花色素支路)的调控基因，命名为FcMYBP1(登录号：EF522147)。与FcMYBP1蛋白同源性最高的是AAY51377(矮牵牛PH4，61％)，AAX51291(葡萄MYB5b，55％)，AF336278(棉花BNLGHi233，66％)，AAS68190(葡萄的MYB5a，61％)和CAJ90831(葡萄的MYBPA1，76％)。
(2) FcMYBP1基因有一个长81bp的内含子，有1个111bp的5′-UTR区、1个250bp的3′-UTR区及一个798个核苷酸的开放阅读框，编码一个长265个氨基酸的蛋白质。通过Southern杂交分析，推测在金荞麦基因组中FcMYBP1基因可能有1—2个拷贝。
(3)运用生物信息学手段对FcMYBP1编码蛋白FcMYBP1的结构特点与性质进行了分析。在GeneBank中对FcMYBP1蛋白质进行保守结构域搜索和同源性分析，FcMYBP1氨基酸序列在N端存在保守的R2R3结合结构域，在C-端同源性低。在FcMYBP1的R2R3区中发现了一个和bHLH(basic helix-loop-helix，bHLH，碱性螺旋-环-螺旋)转录因子相互作用的结构域；FcMYBP1、VvMYB5a、PH4和AtMYB5的C端有两段保守序列：C1(130—138AA)，C2(214-229AA)；系统进化分析表明FcMYBP1和葡萄的VvMYB5a、矮牵牛PH4和拟南芥AtMYB5聚为一支。推测FcMYBP1和bHLH共同作用在类黄酮甚至苯丙氨酸代谢途径中起调控作用。
(4)构建金荞麦FcMYBP1基因植物表达载体pC2301-FcMYBP1，用农杆菌介导的叶盘法将FcMYBP1基因导入烟草超量表达。通过抗性筛选、报告基因GUS活性组织化学染色筛选、PCR检测以及RT-PCR检测，证实FcMYBP1基因已经整合进烟草基因组并获表达的转基因烟草80株。
(5)在80个转基因单株中，随机选择32个株系用于叶片总黄酮含量的检测，不同转基因株系的叶片总黄酮含量相差较大，其中有20株显著增加，最高为对照的3.2倍，8株变化差异不显著，4株显著下降。即有75％的转基因烟草叶片总黄酮含量有明显改变，说明FcMYBP1基因参与类黄酮代谢途径的调控。
Fagopyrum cymosum (Trev.) Meisn, one of perennial herbs of the genus Polygonaceae in the Fagopyrum Mill family, origins from southwest of china, and ranges from Shanxi, Jiangsu, Zhejiang, Jiangxi, Henan, Hunan, Hubei, Guangxi, Guangdong, Sichuan, Chongqing and Yunnan ect.. It is an important resource plant with rich nutrition and great medicinal values, and contains a large number of excellent genes. As an important traditional Chinese Medicinal Materials, the active extract of earthnut (secondary metabolites) of Fagopyrum cymosum is one of main constituents of many efficient drugs, and has prominent functions in anti-cancer, controlling tumour cell affect and transfering to lung, and anti-inflammatony antibacteria ect. At present, cultivated yield of Fagopyrum cymosum is restricted and cultivated species are lesser than wild type in nutrition value, efficient constituents and quantity for medicine, which leads to requesting for Fagopyrum cymosum largely, so that wild resource is destroyed greatly. In view of the conditions, it is ranked into the second degree of national protected plants. Domestically and abroad resource collection, diversity, physiological characteristic, breeding, pharmacodynamics and ecology environment and other aspects are outspread, however, in the light of function genes research relating to active components of medicine not reported.
In order to further explore worth of Fagopyrum cymosum and its excellent function genes, and protect wild resource, in this paper, acquisition of high flavonoids-producing red callus lines and construction cDNA Library of F. cymosum were reported. Furthermore, the cloning,structure and function analysis of the key gene(DFR) and transcription regulational gene(MYBP) in the pathway of flavoloid metabolism were performed. This research not only provided a useful tool for conserving excellent genetic resource, cloning genes and regulating research in the pathway of flavonoids biosynthesis, but laid the groundwork for future producing medicinal second metabolic products on a large scale.
1. Acquisition of high flavonoids-producing red callus lines
Explants of stems and leaves were from sterile seedling of F. cymosum. When the stems were cultured on MS medium supplemented with 2.0mg/L 6-BA and 0.1 mg/L NAA, the induction rate was 100% after 12 days. When the leaves were cultured on MS medium supplemented with 1.0mg/L 6-BA and 0.5 mg/L NAA, the induction rate was 100% after 13 days. After continuous thrice clutures on respective medium of stems and leaves, the callus could be distinguished by colour with naked eyes into four cell lines , a red (stem1 callus line), a brown (stem2 callus line), a bright yellow (leaf1 callus line) and a white (leaf2 callus line) , representing respectively the different colour of metabolite accumulations. A sensitive spectrophotometric method has been developed for detecting the flavonoids in cultured cells. It revealed that the red line contained 6.3804 mg/g flavonoids which was 3.5 times more than the white line .
2. Construction of F. cymosum callus cDNA library
By modified CTAB extraction method and the LiCl precipitation method, total RNA With higher purity and integrality were isolated from F. cymosum callus. cDNA library was constructed with SMART cDNA Library Construction Kit. The titer of the primary library was 1.5×10~6 pfu/mL , in which 98% clones were recombinant and the inserted cDNA fragments ranged from 0.5 kb to 2 kb, 66% of them more than 1 kb. The amplified library has a titer of 7.0×10~9 pfu/mL. The positive signals of P gene, belonging to Myb R2R3 family and expressed at low abundance, were detected by degenerate PCR in the amplified library. The data indicated that the cDNA Library had high quality. These meant that a high quality cDNA library of F. cymosum callus was obtained.
3.The cloning and function analysis of FcDFR
Dihydroflavonol-4-reductase (DFR; EC1.1.1.219) catalyzes a key step late in the biosynthesis of anthocyanins, condensed tannins (proanthocyanidins), and other flavonoids. According to the obtained homologous probe from orther plant's DFRs, one DFR cDNA clones(FcDFR) were isolated from the F. cymosum using the RACE(rapid amplification of cDNA ends) methods to scanning cDNA library (GenBank accession No. EF522145/ EF522146).
FcDFR contains a 201bp 3'-untranslated region and a 78bp 5'-untranslated region and a 1026-bp coding region encoding 341 amino acids with an MW and theoretical pI value of 38.60 kD and 5.91, respectively. The comparison between cDNA and genomic DNA sequences revealed that FcDFR is composed of two exons and one intron. Southern blot analysis indicated that DFR belongs to a small gene family, and FcDFR was a single copy in F. cymosum genomes.
A NADP-binding site (VTGASGFVGSWLVMRLLEHGY) and a substrate specificity motif (TVNVEEKQKPVYDETCWSDVDFCRRV), were observed in the deduced amino acid sequence of FcDFR contained C-terminal region. So that, FcDFR was postulated to have an activity to convert DHQ or DHK to leucodelphinidin or leucopelargonidin. The bioinformation analysis indicatied that FcDFR did not have a signal peptide, was localized in the plasma membrane, and had a typical tertiary tructure of enzyme.
FcDFR PCR products and pC2301 vector containing digestion sites were digested with Xba I and Sma I, and then FcDFR target fragments and pC2301 vector were collected and performed the ligation. This generated an recombinant expression vector plasmid pC2301-FcDFR. Using the leaf disc transformation procedure mediated by Agrobacterium tumefaciens EHA105 (including pC2301-FcDFR), recombinant plasmid were transformed into tobacco plants. 95 transgenical tobacco plantlets were obtained, in which the transgenic lines were identified by GUS GUS histochemical staining and PCR and RT-PCR analysis .
In 95 lines of transgenic plants, it was variable of total flavonoids contents in different lines. And many of them were improved in varied degree. The content of total flavonoids in leaves of 78 transgenic tobacco plants increased significantly, the highest being up to 1.8 folds, which indicated that the synthesis of total flavonoids was promoted by overexpress of FcDFR . However, the content of total flavonoids in 17 transgeniclines decreased.
4. The cloning and function analysis of FcMYBP1
According to the obtained homologous probe from the R2R3 conserved region in maize P gene and orther MYB genes, Using RACE on a F. cymosum callus cDNA library, we identified one clone, named FcMYBPl, encoding a putative R2R3 MYB protein. FcMYBP1 appeared to be a full-length cDNA of 1159 bp encoding a protein of 265 amino acids and contaied a 250 bp 3'-untranslated region and a 111 bp 5'-untranslated region. The comparison between cDNA and genomic DNA sequences revealed that FcMYBP1 is composed of two exons and one 81bp intron. Southern blot analysis indicated that FcMYBP1 belongs to a single copy gene in F. cymosum genomes. The FcMYBP1 protein sequence showed highly homology to PH4(61%) , VvMYB5b(55 %), BNLGHi233(66% ) , VvMYB5a(61%), and VvMYBPA1 (76%).
Through structure and property analysis of FcMYBP1 with bioinformational methods, We found that the amino acid sequence of FcMYBP1 showed great homology to other MYBP in the N-terminus, but not in the C-terminus. The R2R3 repeat region of FcMYBP1 is highly conserved and contains the motif [D/E]Lx2[R/K]x3Lx6Lx3R for interaction with bHLH proteins. Although the C-terminal region shows little homology to other MYBs, FcMYBP1, PH4, AtMYB5 and VvMYB5a share two conserved motifs in their C-terminal domains, C1(130—138AA) and C2 (214-229AA) . Phylogenetic analysis revealed that FcMYBP1 and VvMYB5a from Vitis vinifera formed a closely related subgroup. The putative function of FcMYBP1 may be involved in the control of different branches of the phenylpropanoid pathway in F. cymosum.
FcMYBP1 PCR products and pC2301 vector containing digestion sites were digested with Xba I and Sma I, and then FcMYBP1 target fragments and pC2301 vector were collected and performed the ligation. This generated an recombinant expression vector plasmid pC2301- FcMYBP1. Using the leaf disc transformation procedure mediated by Agrobacterium tumefaciens EHA105 (including pC2301- FcMYBP1), recombinant plasmid were transformed into tobacco plants. 80 transgenical tobacco plantlets were obtained, in which the transgenic lines were identified by GUS GUS histochemical staining and PCR and RT-PCR analysis .
In 32 random selective lines of transgenic plants, it was greatly variable of total flavonoid contents in different lines. The content of total flavonoids in 20 transgenic tobacco leaves increased significantly, the highest being up to 3.2 folds, and 8 lines showed similar contents with controls, and 4 tobacco leaves decreased significantly. In summary, the flavoloid contents of 75 % transgenic plants were altered greatly, which indicated that FcMYBP1 was involved in the pathway of flavonoid metabolisms.
在本研究中，通过获得高黄酮含量愈伤系，建立野生金荞麦的RACE cDNA文库，从中快速分离克隆与药效成分积累相关的类黄酮次生代谢产物的关键酶基因(DFR，IFR)和转录调控因子P基因，并转化烟草超量表达进行功能的初步验证，为下一步转化野生金荞麦调控其类黄酮次生代谢，同时为实现其次生代谢产物的工厂化生产打下基础。
1、金荞麦高黄酮含量愈伤系的获得
以消毒金荞麦种子萌发的无菌苗的茎与叶片作为外植体，分别在MS添加2.0mg／L 6-BA和0.1mg／L NAA的培养基和MS添加1.0mg／L 6-BA和0.5mg／L NAA的固体培养基上经系统诱导并连续继代3次，获得了大量的稳定愈伤组织。采用目视法初步筛选出生长较快、不易褐化、颜色较深或稳定并且继代稳定的4个愈伤系。其中红色和褐色愈伤系主要来源于茎的诱导，而浅黄色和白色系则来源于叶的诱导用野生金荞麦无菌苗的茎和叶片作外植体。用分光光度法直接筛选出高类黄酮含量的红色系，其类黄酮含苗为6.3804mg／g，是含量最低的白色系的3.5倍。获得的来源于茎的高类黄酮含量的红色愈伤系为进一步的细胞系悬浮培养和药用次生代谢产物的规模化生产奠定了基础，同时，也为研究金荞麦类黄酮次生代谢的分子调控提供了优良的材料。
2、金荞麦愈伤系cDNA文库的构建
利用改良的CTAB结合LiCl沉淀方法从资源植物金荞麦愈伤组织中提取出完整性好、纯度高的总RNA。用SMART cDNA文库构建试剂盒成功构建了金荞麦红色愈伤系cDNA文库。原始文库滴度达到1.5×10~6 pfu／ml，扩增文库滴度约为7.0×10~9 pfu／ml，重组率达98％，插入片段在0.5 kb以上，平均约1.1 kb。通过PCR检测，从总文库中检测到了低丰度表达的P基因的特异片段，说明所构建的文库覆盖度高、代表性强。综合分析所构建的cDNA文库基本指标，表明已获得高质量的金荞麦愈伤系cDNA文库，可以用于进一步的基因克隆，这是第一次正式报道金荞麦cDNA文库的构建。
3、FcDFR基因的克隆与功能初步分析
(1)采用RACE结合cDNA文库筛选的方法，根据获得的DFR同源探针序列，我们从金荞麦cDNA文库中克隆到了DFR蛋白基因，命名为FcDFR(登录号：EF522145)。
(2) FcDFR基因有一个长85bp的内含子，有1个78bp的5′-UTR区、1个201bp的3′-UTR区及一个1026个核苷酸的开放阅读框。通过Southern杂交分析，推测在金荞麦基因组中DFR基因是1—2个基因的小家族，FcDFR基因是单拷贝基因。
(3)运用生物信息学手段对FcDFR编码蛋白FcDFR的结构特点与性质进行了分析，FcDFR富含疏水氨基酸，在N端存在一个NADP结合位点“VTGASGFVGSWLVMRLLEHGY”，还存在一个底物结合特异性决定的氨基酸基序“TVNVEEKQKPVYDETCWSDVDFCRRV”，推测它可能以DHQ或DHK为底物催化生成无色翠雀素和无色天竺葵素。它没有信号肽，可能定位于质膜，具有酶的典型三级结构特征。
(4)构建金荞麦FcDFR基因植物表达载体pC2301-FcDFR，用农杆菌介导的叶盘法将FcDFR基因导入烟草超量表达。通过报告基因GUS活性组织化学染色筛选、PCR检测以及RT-PCR检测。证实FcDFR基因已经整合进烟草基因组并获表达的转基因烟草95株。
(5)对95个转基因烟草叶片总黄酮进行测定，在所有检测的转基因烟草中，不同株系的总黄酮含量有一定差异，大部分叶片总黄酮含量都有不同程度的提高，最高可达对照的1.8倍，其中有78株显著增加，表明在这些株系中FcDFR基因得到了有效表达，促进烟草总黄酮的有效积累，这表明FcDFR基因具有生物活性，其超量表达对增加叶片中类黄酮的含量作用明显。17个株系总黄酮和对照烟草总黄酮含量没有显著差异。
4、FcMYBP1基因的克隆与功能初步分析
(1)用RACE方法筛选文库采用RACE结合cDNA文库筛选的方法，根据玉米P基因及Myb转录调控因子R2R3家族的R2、R3保守区域获得的同源探针，我们从金荞麦cDNA文库中克隆到了一个长1159个核苜酸R2R3类MYB蛋白基因，根据同源性比对分析，分离得到的基因可能是金荞麦类黄酮代谢途径(花色素和原花色素支路)的调控基因，命名为FcMYBP1(登录号：EF522147)。与FcMYBP1蛋白同源性最高的是AAY51377(矮牵牛PH4，61％)，AAX51291(葡萄MYB5b，55％)，AF336278(棉花BNLGHi233，66％)，AAS68190(葡萄的MYB5a，61％)和CAJ90831(葡萄的MYBPA1，76％)。
(2) FcMYBP1基因有一个长81bp的内含子，有1个111bp的5′-UTR区、1个250bp的3′-UTR区及一个798个核苷酸的开放阅读框，编码一个长265个氨基酸的蛋白质。通过Southern杂交分析，推测在金荞麦基因组中FcMYBP1基因可能有1—2个拷贝。
(3)运用生物信息学手段对FcMYBP1编码蛋白FcMYBP1的结构特点与性质进行了分析。在GeneBank中对FcMYBP1蛋白质进行保守结构域搜索和同源性分析，FcMYBP1氨基酸序列在N端存在保守的R2R3结合结构域，在C-端同源性低。在FcMYBP1的R2R3区中发现了一个和bHLH(basic helix-loop-helix，bHLH，碱性螺旋-环-螺旋)转录因子相互作用的结构域；FcMYBP1、VvMYB5a、PH4和AtMYB5的C端有两段保守序列：C1(130—138AA)，C2(214-229AA)；系统进化分析表明FcMYBP1和葡萄的VvMYB5a、矮牵牛PH4和拟南芥AtMYB5聚为一支。推测FcMYBP1和bHLH共同作用在类黄酮甚至苯丙氨酸代谢途径中起调控作用。
(4)构建金荞麦FcMYBP1基因植物表达载体pC2301-FcMYBP1，用农杆菌介导的叶盘法将FcMYBP1基因导入烟草超量表达。通过抗性筛选、报告基因GUS活性组织化学染色筛选、PCR检测以及RT-PCR检测，证实FcMYBP1基因已经整合进烟草基因组并获表达的转基因烟草80株。
(5)在80个转基因单株中，随机选择32个株系用于叶片总黄酮含量的检测，不同转基因株系的叶片总黄酮含量相差较大，其中有20株显著增加，最高为对照的3.2倍，8株变化差异不显著，4株显著下降。即有75％的转基因烟草叶片总黄酮含量有明显改变，说明FcMYBP1基因参与类黄酮代谢途径的调控。
Fagopyrum cymosum (Trev.) Meisn, one of perennial herbs of the genus Polygonaceae in the Fagopyrum Mill family, origins from southwest of china, and ranges from Shanxi, Jiangsu, Zhejiang, Jiangxi, Henan, Hunan, Hubei, Guangxi, Guangdong, Sichuan, Chongqing and Yunnan ect.. It is an important resource plant with rich nutrition and great medicinal values, and contains a large number of excellent genes. As an important traditional Chinese Medicinal Materials, the active extract of earthnut (secondary metabolites) of Fagopyrum cymosum is one of main constituents of many efficient drugs, and has prominent functions in anti-cancer, controlling tumour cell affect and transfering to lung, and anti-inflammatony antibacteria ect. At present, cultivated yield of Fagopyrum cymosum is restricted and cultivated species are lesser than wild type in nutrition value, efficient constituents and quantity for medicine, which leads to requesting for Fagopyrum cymosum largely, so that wild resource is destroyed greatly. In view of the conditions, it is ranked into the second degree of national protected plants. Domestically and abroad resource collection, diversity, physiological characteristic, breeding, pharmacodynamics and ecology environment and other aspects are outspread, however, in the light of function genes research relating to active components of medicine not reported.
In order to further explore worth of Fagopyrum cymosum and its excellent function genes, and protect wild resource, in this paper, acquisition of high flavonoids-producing red callus lines and construction cDNA Library of F. cymosum were reported. Furthermore, the cloning,structure and function analysis of the key gene(DFR) and transcription regulational gene(MYBP) in the pathway of flavoloid metabolism were performed. This research not only provided a useful tool for conserving excellent genetic resource, cloning genes and regulating research in the pathway of flavonoids biosynthesis, but laid the groundwork for future producing medicinal second metabolic products on a large scale.
1. Acquisition of high flavonoids-producing red callus lines
Explants of stems and leaves were from sterile seedling of F. cymosum. When the stems were cultured on MS medium supplemented with 2.0mg/L 6-BA and 0.1 mg/L NAA, the induction rate was 100% after 12 days. When the leaves were cultured on MS medium supplemented with 1.0mg/L 6-BA and 0.5 mg/L NAA, the induction rate was 100% after 13 days. After continuous thrice clutures on respective medium of stems and leaves, the callus could be distinguished by colour with naked eyes into four cell lines , a red (stem1 callus line), a brown (stem2 callus line), a bright yellow (leaf1 callus line) and a white (leaf2 callus line) , representing respectively the different colour of metabolite accumulations. A sensitive spectrophotometric method has been developed for detecting the flavonoids in cultured cells. It revealed that the red line contained 6.3804 mg/g flavonoids which was 3.5 times more than the white line .
2. Construction of F. cymosum callus cDNA library
By modified CTAB extraction method and the LiCl precipitation method, total RNA With higher purity and integrality were isolated from F. cymosum callus. cDNA library was constructed with SMART cDNA Library Construction Kit. The titer of the primary library was 1.5×10~6 pfu/mL , in which 98% clones were recombinant and the inserted cDNA fragments ranged from 0.5 kb to 2 kb, 66% of them more than 1 kb. The amplified library has a titer of 7.0×10~9 pfu/mL. The positive signals of P gene, belonging to Myb R2R3 family and expressed at low abundance, were detected by degenerate PCR in the amplified library. The data indicated that the cDNA Library had high quality. These meant that a high quality cDNA library of F. cymosum callus was obtained.
3.The cloning and function analysis of FcDFR
Dihydroflavonol-4-reductase (DFR; EC1.1.1.219) catalyzes a key step late in the biosynthesis of anthocyanins, condensed tannins (proanthocyanidins), and other flavonoids. According to the obtained homologous probe from orther plant's DFRs, one DFR cDNA clones(FcDFR) were isolated from the F. cymosum using the RACE(rapid amplification of cDNA ends) methods to scanning cDNA library (GenBank accession No. EF522145/ EF522146).
FcDFR contains a 201bp 3'-untranslated region and a 78bp 5'-untranslated region and a 1026-bp coding region encoding 341 amino acids with an MW and theoretical pI value of 38.60 kD and 5.91, respectively. The comparison between cDNA and genomic DNA sequences revealed that FcDFR is composed of two exons and one intron. Southern blot analysis indicated that DFR belongs to a small gene family, and FcDFR was a single copy in F. cymosum genomes.
A NADP-binding site (VTGASGFVGSWLVMRLLEHGY) and a substrate specificity motif (TVNVEEKQKPVYDETCWSDVDFCRRV), were observed in the deduced amino acid sequence of FcDFR contained C-terminal region. So that, FcDFR was postulated to have an activity to convert DHQ or DHK to leucodelphinidin or leucopelargonidin. The bioinformation analysis indicatied that FcDFR did not have a signal peptide, was localized in the plasma membrane, and had a typical tertiary tructure of enzyme.
FcDFR PCR products and pC2301 vector containing digestion sites were digested with Xba I and Sma I, and then FcDFR target fragments and pC2301 vector were collected and performed the ligation. This generated an recombinant expression vector plasmid pC2301-FcDFR. Using the leaf disc transformation procedure mediated by Agrobacterium tumefaciens EHA105 (including pC2301-FcDFR), recombinant plasmid were transformed into tobacco plants. 95 transgenical tobacco plantlets were obtained, in which the transgenic lines were identified by GUS GUS histochemical staining and PCR and RT-PCR analysis .
In 95 lines of transgenic plants, it was variable of total flavonoids contents in different lines. And many of them were improved in varied degree. The content of total flavonoids in leaves of 78 transgenic tobacco plants increased significantly, the highest being up to 1.8 folds, which indicated that the synthesis of total flavonoids was promoted by overexpress of FcDFR . However, the content of total flavonoids in 17 transgeniclines decreased.
4. The cloning and function analysis of FcMYBP1
According to the obtained homologous probe from the R2R3 conserved region in maize P gene and orther MYB genes, Using RACE on a F. cymosum callus cDNA library, we identified one clone, named FcMYBPl, encoding a putative R2R3 MYB protein. FcMYBP1 appeared to be a full-length cDNA of 1159 bp encoding a protein of 265 amino acids and contaied a 250 bp 3'-untranslated region and a 111 bp 5'-untranslated region. The comparison between cDNA and genomic DNA sequences revealed that FcMYBP1 is composed of two exons and one 81bp intron. Southern blot analysis indicated that FcMYBP1 belongs to a single copy gene in F. cymosum genomes. The FcMYBP1 protein sequence showed highly homology to PH4(61%) , VvMYB5b(55 %), BNLGHi233(66% ) , VvMYB5a(61%), and VvMYBPA1 (76%).
Through structure and property analysis of FcMYBP1 with bioinformational methods, We found that the amino acid sequence of FcMYBP1 showed great homology to other MYBP in the N-terminus, but not in the C-terminus. The R2R3 repeat region of FcMYBP1 is highly conserved and contains the motif [D/E]Lx2[R/K]x3Lx6Lx3R for interaction with bHLH proteins. Although the C-terminal region shows little homology to other MYBs, FcMYBP1, PH4, AtMYB5 and VvMYB5a share two conserved motifs in their C-terminal domains, C1(130—138AA) and C2 (214-229AA) . Phylogenetic analysis revealed that FcMYBP1 and VvMYB5a from Vitis vinifera formed a closely related subgroup. The putative function of FcMYBP1 may be involved in the control of different branches of the phenylpropanoid pathway in F. cymosum.
FcMYBP1 PCR products and pC2301 vector containing digestion sites were digested with Xba I and Sma I, and then FcMYBP1 target fragments and pC2301 vector were collected and performed the ligation. This generated an recombinant expression vector plasmid pC2301- FcMYBP1. Using the leaf disc transformation procedure mediated by Agrobacterium tumefaciens EHA105 (including pC2301- FcMYBP1), recombinant plasmid were transformed into tobacco plants. 80 transgenical tobacco plantlets were obtained, in which the transgenic lines were identified by GUS GUS histochemical staining and PCR and RT-PCR analysis .
In 32 random selective lines of transgenic plants, it was greatly variable of total flavonoid contents in different lines. The content of total flavonoids in 20 transgenic tobacco leaves increased significantly, the highest being up to 3.2 folds, and 8 lines showed similar contents with controls, and 4 tobacco leaves decreased significantly. In summary, the flavoloid contents of 75 % transgenic plants were altered greatly, which indicated that FcMYBP1 was involved in the pathway of flavonoid metabolisms.
引文
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