苦荞WD40转录因子的基因克隆及其对花青素合成的影响

发布时间：2018-09-14 11:08

【摘要】：苦荞(Fagopyrum tataricum)为蓼科荞麦属双子叶禾谷类作物。苦荞作为一种药食两用作物,其最重要的生物活性成分为黄酮,以芦丁为代表的黄酮醇是其主要储存形式。然而,在遭受冷和紫外等胁迫时,另外一类黄酮花青素的合成则对苦荞逆境生理的稳定具有重要作用。植物花青素的生物合成在转录水平主要受到R2R3-MYB、bHLH和WD40等转录因子蛋白单独或者形成三元复合物的调控,其中转录因子WD40更多的是提供平台促进转录因子MYB和bHLH的互作。本研究以“西荞2号”苦荞为材料,克隆花青素调控相关转录因子FtWD40的cDNA序列,并鉴定其转录激活活性；分析花青素和FtWD40基因表达在苦荞中的组织特异性及其相关性,也分析了在苦荞逆境胁迫条件下二者变化趋势间的相关性；进一步通过转基因技术鉴定FtWD40过表达对烟草花青素合成的影响。本研究可望明确苦荞转录因子FtWD40对在花青素合成途径中的主要效应基因,加深对苦荞逆境胁迫中花青素合成调控分子机制的认识。主要取得以下研究结果：1、采用同源克隆法和RACE技术,获得1条苦荞WD40基因,命名为FtWD40。 FtWD40 ORF框为1035 bp,编码344个氨基酸残基,相对分子质量65.9 KDa;聚类分析结果显示,FtWD40和参与花青素代谢调控的AtTTGl和VvWD等聚为一簇。对花期苦荞根、茎、叶和花中的花青素含量,以及FtWD40基因表达的测定表明,花青素含量和FtWD40的基因表达量都具有相同的组织特异,均为花根叶茎,二者相关性系数为r=0.875(P0.05)。在冷胁迫和UV-B胁迫下,芽期苦荞花青素含量与FtWD40基因表达量的相关系数分别为r=0.785(P0.05)和r=0.891(P0.05)。以上结果表明,FtWD40可能参与了苦荞花青素合成的调控。2、构建pBridge-FtWD40真核表达载体,转化酵母AH109,经SD/-Trp/-His双缺培养筛选获得阳性转化子。经酵母单杂交系统中的β-半乳糖苷酶活性滤纸分析,结果表明FtWD40蛋白具有转录激活活性。3、构建pECMBIA1301-FtWD40植物表达载体,采用农杆菌侵染法转化烟草愈伤组织,进而对筛选获得的阳性转基因烟草中花青素含量及花青素合成途径中的NtPAL、NtCHS、NtANS、NtDFR、NtFLS和NtF3'H等基因的表达量进行了测定分析。FtWD40转基因烟草T-1、T-2和T-3株系中的花青素含量分别提高为对照平均的3.45倍、2.81倍和3.24倍(P0.01)。同时,qRT-PCR结果显示：NtPAL、NtCHS和NtF3'H的表达量没有显著变化(P0.05),NtDFR和NtANS的表达量分别提高为对照组的1.95倍和1.56倍(P0.01),而FLS的表达量则降低为对照的0.79倍(P0.05)。以上结果表明,FtWD40具有增强转基因烟草花青素合成的作用。
[Abstract]:Tartary buckwheat (Fagopyrum tataricum) is a dicotyledonous cereal crop of the genus Polygonaceae. Tartary buckwheat (Tartary buckwheat) is one of the most important bioactive components of Tartary buckwheat as a dual-purpose crop. The flavonol represented by rutin is the main storage form of Tartary buckwheat. However, under cold and UV stress, the synthesis of another flavonoid anthocyanin plays an important role in the stability of buckwheat stress physiology. The biosynthesis of plant anthocyanins is mainly regulated by transcription factor proteins such as R2R3-MYBnbHLH and WD40 alone or forming ternary complexes at the transcriptional level. The transcription factor WD40 provides a platform to facilitate the interaction of transcription factor MYB and bHLH. In this study, the cDNA sequence of anthocyanin regulating transcription factor FtWD40 was cloned and its transcriptional activation activity was identified, and the tissue specificity and correlation of anthocyanin and FtWD40 gene expression in Tartary buckwheat were analyzed. We also analyzed the correlation between the two changes under the stress of Tartary buckwheat, and further identified the effect of FtWD40 overexpression on anthocyanin synthesis by transgenic technology. In this study, we hope to clarify the main effector genes of buckwheat transcription factor FtWD40 in anthocyanin synthesis pathway, and to further understand the molecular mechanism of anthocyanin synthesis regulation in buckwheat stress. The main results are as follows: 1. Using homologous cloning and RACE technique, we obtained a WD40 gene from Tartary Buckwheat, named FtWD40.. The FtWD40 ORF frame was 1035 bp, encoding 344 amino acid residues, and the relative molecular weight of 65.9 KDa; cluster analysis showed that FtWD40 and AtTTGl and VvWD involved in the regulation of anthocyanin metabolism were clustered into a cluster. The content of anthocyanin in root, stem, leaf and flower of Tartary buckwheat at flowering stage and the expression of FtWD40 gene showed that the anthocyanin content and the gene expression of FtWD40 had the same tissue specificity, and the correlation coefficient between them was 0.875 (P0.05). Under cold stress and UV-B stress, the correlation coefficients between anthocyanin content and FtWD40 gene expression of Tartary buckwheat at bud stage were r = 0.785 (P0.05) and r ~ (0.891) (P0.05), respectively. These results suggested that FtWD40 might be involved in the regulation of anthocyanin synthesis of Tartary buckwheat. The eukaryotic expression vector of pBridge-FtWD40 was constructed and the transformed yeast AH109, was screened by SD/-Trp/-His double deficiency culture to obtain positive transformants. The activity of 尾-galactosidase in yeast single hybrid system was analyzed by filter paper. The results showed that FtWD40 protein had transcriptional activation activity. 3. PECMBIA1301-FtWD40 plant expression vector was constructed and transformed into tobacco callus by Agrobacterium tumefaciens. Furthermore, the anthocyanin content in the positive transgenic tobacco and the expression of NtPAL,NtCHS,NtANS,NtDFR,NtFLS and NtF3'H in the anthocyanin synthesis pathway were determined and analyzed. The anthocyanin content in T-1T-2 and T-3 lines of FtWD40 transgenic tobacco was increased, respectively. It was 3.45 times and 3.24 times as much as that of control (P0.01). At the same time, the results of qRT-PCR showed that there was no significant change in the expression of NtCHS and NtF3'H (P0.05). The expression of NtDFR and NtANS increased by 1.95 times and 1.56 times of the control group (P0.01), while the expression of FLS decreased to 0.79 times of the control group (P0.05). These results suggest that FtWD40 can enhance anthocyanin synthesis in transgenic tobacco.
【学位授予单位】：四川农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q943.2;S517

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