【摘要】：轉(zhuǎn)錄因子可以和啟動(dòng)子互作影響基因表達(dá),對(duì)基因有重要的調(diào)控作用,bHLH家族轉(zhuǎn)錄因子是第二大轉(zhuǎn)錄因子。本實(shí)驗(yàn)圍繞大豆中bHLH04G、bHLH06G兩個(gè)轉(zhuǎn)錄因子對(duì)于皂苷合成和固氮結(jié)瘤的影響展開(kāi)研究。β-香樹(shù)脂醇合成酶是大豆皂苷合成的第一個(gè)關(guān)鍵酶,本團(tuán)隊(duì)竇富強(qiáng)同學(xué)用酵母單雜交篩選到與其互作的兩個(gè)轉(zhuǎn)錄因子bHLH04G和bHLH06G。大豆是重要的油料作物,其代謝調(diào)控的研究對(duì)于改良大豆品質(zhì)性狀有著重要的意義。本實(shí)驗(yàn)對(duì)這兩個(gè)基因分別構(gòu)建原核表達(dá)載體進(jìn)行原核表達(dá),發(fā)現(xiàn)這兩個(gè)蛋白均主要形成包涵體,嘗試不同的誘導(dǎo)條件并更換載體后,蛋白仍主要形成包涵體。此外,構(gòu)建RNAi載體培養(yǎng)轉(zhuǎn)基因大豆發(fā)根來(lái)研究這兩個(gè)轉(zhuǎn)錄因子對(duì)于皂苷合成和固氮結(jié)瘤的影響。通過(guò)對(duì)轉(zhuǎn)基因發(fā)根結(jié)瘤數(shù)統(tǒng)計(jì)和檢測(cè)結(jié)瘤相關(guān)基因表達(dá)情況,發(fā)現(xiàn)將這兩個(gè)基因分別沉默掉以后,結(jié)瘤數(shù)均顯著減少,結(jié)瘤相關(guān)基因表達(dá)量均降低。同時(shí)檢測(cè)轉(zhuǎn)基因發(fā)根皂苷合成關(guān)鍵基因AMS表達(dá)情況并提取皂苷檢測(cè)皂苷含量和成分的變化,發(fā)現(xiàn)將這兩個(gè)基因沉默掉以后,AMS的表達(dá)量明顯降低,皂苷元B、C和E的相對(duì)含量也有所降低。因此推測(cè)這兩個(gè)轉(zhuǎn)錄因子對(duì)大豆皂苷合成和結(jié)瘤固氮具有正向調(diào)控作用。為進(jìn)一步明確這兩個(gè)轉(zhuǎn)錄因子對(duì)大豆皂苷合成和結(jié)瘤固氮的調(diào)控機(jī)理,構(gòu)建RNAi-AMS載體培養(yǎng)轉(zhuǎn)基因發(fā)根。通過(guò)對(duì)轉(zhuǎn)基因發(fā)根結(jié)瘤數(shù)統(tǒng)計(jì)并檢測(cè)結(jié)瘤相關(guān)基因表達(dá)情況,發(fā)現(xiàn)將AMS沉默掉以后,結(jié)瘤數(shù)減少,結(jié)瘤相關(guān)基因的表達(dá)量降低。因此推測(cè)AMS也對(duì)大豆結(jié)瘤固氮有正向調(diào)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.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：S565.1

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