大豆bHLH04G和bHLH06G转录因子的原核表达研究及对皂苷合成和结瘤固氮的影响
发布时间：2018-11-15 23:22
【摘要】：转录因子可以和启动子互作影响基因表达,对基因有重要的调控作用,bHLH家族转录因子是第二大转录因子。本实验围绕大豆中bHLH04G、bHLH06G两个转录因子对于皂苷合成和固氮结瘤的影响展开研究。β-香树脂醇合成酶是大豆皂苷合成的第一个关键酶,本团队窦富强同学用酵母单杂交筛选到与其互作的两个转录因子bHLH04G和bHLH06G。大豆是重要的油料作物,其代谢调控的研究对于改良大豆品质性状有着重要的意义。本实验对这两个基因分别构建原核表达载体进行原核表达,发现这两个蛋白均主要形成包涵体,尝试不同的诱导条件并更换载体后,蛋白仍主要形成包涵体。此外,构建RNAi载体培养转基因大豆发根来研究这两个转录因子对于皂苷合成和固氮结瘤的影响。通过对转基因发根结瘤数统计和检测结瘤相关基因表达情况,发现将这两个基因分别沉默掉以后,结瘤数均显著减少,结瘤相关基因表达量均降低。同时检测转基因发根皂苷合成关键基因AMS表达情况并提取皂苷检测皂苷含量和成分的变化,发现将这两个基因沉默掉以后,AMS的表达量明显降低,皂苷元B、C和E的相对含量也有所降低。因此推测这两个转录因子对大豆皂苷合成和结瘤固氮具有正向调控作用。为进一步明确这两个转录因子对大豆皂苷合成和结瘤固氮的调控机理,构建RNAi-AMS载体培养转基因发根。通过对转基因发根结瘤数统计并检测结瘤相关基因表达情况,发现将AMS沉默掉以后,结瘤数减少,结瘤相关基因的表达量降低。因此推测AMS也对大豆结瘤固氮有正向调控作用。
[Abstract]:Transcription factors can interact with promoters to influence gene expression and play an important role in gene regulation. BHLH family transcription factors are the second largest transcription factors. In this study, the effects of two transcription factors of bHLH04G,bHLH06G in soybean on saponins synthesis and nitrogen-fixing nodulation were studied. 尾 -vanillin alcohol synthase was the first key enzyme in soybean saponins synthesis. Our team, Dou Fuqiang, selected two transcription factors, bHLH04G and bHLH06G., which interact with them by yeast single hybridization. Soybean is an important oil crop, and the study of its metabolic regulation is of great significance in improving soybean quality traits. In this study, the two prokaryotic expression vectors were constructed for prokaryotic expression. It was found that the two proteins mainly formed inclusion bodies. After different induction conditions were tried and the vectors were replaced, the protein was still mainly formed inclusion bodies. In addition, RNAi vector was constructed to culture transgenic soybean hairy roots to study the effects of these two transcription factors on saponins synthesis and nitrogen-fixing nodulation. By counting and detecting the number of nodule related genes, we found that after silencing the two genes, the number of nodulation and the expression of nodulation related genes were significantly decreased. At the same time, the expression of AMS, the key gene of saponin synthesis in transgenic hair root, was detected and the content and composition of saponins were detected. It was found that after silencing the two genes, the expression of AMS was significantly decreased, and the expression of saponins B was significantly decreased. The relative contents of C and E also decreased. These two transcription factors may play a positive role in soybean saponin synthesis and nodulation nitrogen fixation. In order to further clarify the regulatory mechanism of these two transcription factors on soybean saponins synthesis and nodulation and nitrogen fixation, RNAi-AMS vector was constructed to culture transgenic hairy roots. By counting the number of nodule and detecting the expression of nodulation related genes, it was found that after silencing AMS, the number of nodulation and the expression of nodulation related genes were decreased. Therefore, it is speculated that AMS also has positive regulation effect on soybean nodulation and nitrogen fixation.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S565.1
本文编号：2334703
[Abstract]:Transcription factors can interact with promoters to influence gene expression and play an important role in gene regulation. BHLH family transcription factors are the second largest transcription factors. In this study, the effects of two transcription factors of bHLH04G,bHLH06G in soybean on saponins synthesis and nitrogen-fixing nodulation were studied. 尾 -vanillin alcohol synthase was the first key enzyme in soybean saponins synthesis. Our team, Dou Fuqiang, selected two transcription factors, bHLH04G and bHLH06G., which interact with them by yeast single hybridization. Soybean is an important oil crop, and the study of its metabolic regulation is of great significance in improving soybean quality traits. In this study, the two prokaryotic expression vectors were constructed for prokaryotic expression. It was found that the two proteins mainly formed inclusion bodies. After different induction conditions were tried and the vectors were replaced, the protein was still mainly formed inclusion bodies. In addition, RNAi vector was constructed to culture transgenic soybean hairy roots to study the effects of these two transcription factors on saponins synthesis and nitrogen-fixing nodulation. By counting and detecting the number of nodule related genes, we found that after silencing the two genes, the number of nodulation and the expression of nodulation related genes were significantly decreased. At the same time, the expression of AMS, the key gene of saponin synthesis in transgenic hair root, was detected and the content and composition of saponins were detected. It was found that after silencing the two genes, the expression of AMS was significantly decreased, and the expression of saponins B was significantly decreased. The relative contents of C and E also decreased. These two transcription factors may play a positive role in soybean saponin synthesis and nodulation nitrogen fixation. In order to further clarify the regulatory mechanism of these two transcription factors on soybean saponins synthesis and nodulation and nitrogen fixation, RNAi-AMS vector was constructed to culture transgenic hairy roots. By counting the number of nodule and detecting the expression of nodulation related genes, it was found that after silencing AMS, the number of nodulation and the expression of nodulation related genes were decreased. Therefore, it is speculated that AMS also has positive regulation effect on soybean nodulation and nitrogen fixation.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S565.1
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