【摘要】：大豆(Glycinemax)是產(chǎn)自中國的一種糧食作物,從古到今,深受人們的喜歡。大豆最主要的特征之一就是其具有生物固氮能力,大豆根部的根瘤和根瘤菌具有共生的關系,通過根瘤菌吸收氮素,而為大豆生長發(fā)育提供氮素。在本研究中,通過對在根瘤中表達含量較高的三個轉(zhuǎn)錄因子GmTGA4、GmbHLH93、GmWRI1進行功能驗證,從而探索豆科根瘤菌共生固氮信號傳導機制,從而為知道豆科類根瘤固氮提供理論依據(jù)。本論文研究結(jié)果如下:為了鑒定GmTGA4、GmbHLH93、GmWRI1在豆科根瘤菌共生固氮信號傳導機制中的作用,我們利用大豆轉(zhuǎn)基因發(fā)根技術,驗證了在過表達和基因沉默情況下對大豆根瘤數(shù)目的影響,結(jié)果表明:在過表達基因GmTGA4、GmbHLH93、GmWRI1后,通過與對照組對比,發(fā)現(xiàn)根部根瘤數(shù)量明顯增加;在基因沉默GmTGA4、GmbHLH93、GmWRI1后,通過與對照組對比,發(fā)現(xiàn)根部根瘤數(shù)量明顯減少。通過以上實驗結(jié)果說明,GmTGA4、GmbHLH93、GmWRI1在大豆根瘤結(jié)瘤過程中起到了很重要的作用。利用熒光定量PCR技術驗證結(jié)瘤信號傳導過程中關鍵基因GmSymRK、GmNSP1、GmNODULIN27對 GmTGA4、GmbHLH93、GmWI1 的影響。結(jié)果表明:過量表達GmTGA4、GmbHLH93、GmWRI1,分別導致GmSymRK表達量升高、降低、升高。GmNSP1表達量升高、降低、升高。GmNODULIN27表達量升高、降低、升高�；虺聊珿mTGA4、GmbHLH93、GmWRI1,分別導致GmSymRK表達量降低、GmNSP1表達量降低、GmNODULIN27表達量升高�；虺聊珿mTGA4、GmbHLH93、GmWRI1,分別導致GmSymRK表達量降低,GmNSP1表達量降低、升高、升高,GmNODULIN27表達量升高。以上實驗結(jié)果說明:GmTGA4、GmbHLH93、GmWRI1可能位于 GmSymRK上游,位于 GmNSP1、GmNODULIN27 游。為研究基因在組織中表達,分別克隆了GmTGA4 GmbHLH93、Gm、GmWRI1基因的5'端上游調(diào)控區(qū)域,成功的構(gòu)建了 GmTGA4、GmbHLH93、GmWRI1啟動子連接GUS基因檢測上述基因的表達部位,將融合基因載體轉(zhuǎn)入農(nóng)桿菌K599誘導轉(zhuǎn)基因發(fā)根,收集根瘤和發(fā)根,經(jīng)過GUS染液染色發(fā)現(xiàn),GmTGA4、GmbHLH93、GmWRI1能驅(qū)動GUS報告基因的表達,并在根瘤中特異性表達,從而說明了以上3個轉(zhuǎn)錄因子在定位在根瘤中并發(fā)揮了作用。為研究GmTGA4、GmbHLH93、GmWRI1的互作基因,我們成功的構(gòu)建了誘餌載體,利用酵母雙雜交技術篩選大豆根瘤文庫,并成功篩選到與GmTGA4互作的GmTTG1蛋白,說明了GmTGA 在根瘤內(nèi)部可能起到了調(diào)控黃酮類化合物代謝的作用。另外實驗室已經(jīng)利用大豆皂苷基因成功篩選到GmbHLH93并進行了驗證,說明了GmbHLH9 在根瘤內(nèi)部可能起到了調(diào)控皂苷合成的作用。
[Abstract]:Soybean (Glycinemax) is a kind of food crop produced in China, from ancient to present, deeply liked by people. One of the most important characteristics of soybean is its biological nitrogen fixation ability. The root nodule and rhizobia of soybean have symbiotic relationship, through which nitrogen is absorbed by rhizobia to provide nitrogen for the growth and development of soybean. In this study, the functional verification of three transcription factors (GmTGA4,GmbHLH93,GmWRI1) with high expression in rhizobia was carried out in order to explore the mechanism of symbiotic nitrogen fixation signal transduction by legume rhizobia, and to provide a theoretical basis for the understanding of nitrogen-fixing in leguminous nodule. The results are as follows: in order to investigate the role of GmTGA4,GmbHLH93,GmWRI1 in the signal transduction mechanism of symbiotic nitrogen fixation of rhizobia, we used soybean transgenic rooting technique to verify the effect of over-expression and gene silencing on the number of soybean nodule. The results showed that the number of root nodules was significantly increased after overexpression of gene GmTGA4,GmbHLH93,GmWRI1 compared with the control group, and that after gene silencing GmTGA4,GmbHLH93,GmWRI1, the number of root nodules was significantly decreased compared with the control group. The results show that GmTGA4,GmbHLH93,GmWRI1 plays an important role in the process of soybean nodule formation. Fluorescence quantitative PCR technique was used to verify the effect of GmSymRK,GmNSP1,GmNODULIN27, a key gene in the signal transduction of nodulation, on GmTGA4,GmbHLH93,GmWI1. The results showed that overexpression of GmTGA4,GmbHLH93,GmWRI1, resulted in the increase, decrease, increase of GmSymRK expression, the increase, decrease, increase of GmNSP1 expression, and the increase, decrease and increase of GmNODULIN27 expression. Gene silencing of GmTGA4,GmbHLH93,GmWRI1, resulted in the decrease of GmSymRK expression, the decrease of GmNSP1 expression and the increase of GmNODULIN27 expression. Gene silencing of GmTGA4,GmbHLH93,GmWRI1, resulted in the decrease of GmSymRK expression, the decrease of GmNSP1 expression, and the increase of GmNODULIN27 expression. The above results suggest that GmTGA4,GmbHLH93,GmWRI1 may be located upstream of GmSymRK and located in GmNSP1,GmNODULIN27 swim. In order to study the gene expression in tissues, the upstream regulatory region of GmTGA4 GmbHLH93,Gm,GmWRI1 gene was cloned, and the GmTGA4,GmbHLH93,GmWRI1 promoter linked to GUS gene was successfully constructed to detect the expression site of the above gene. The fusion gene vector was transferred into Agrobacterium tumefaciens K599 to induce transgenic hair root, and the root nodule and hair root were collected. By GUS staining, it was found that GmTGA4,GmbHLH93,GmWRI1 could drive the expression of GUS reporter gene and express it specifically in the root nodule. Therefore, the above three transcription factors play an important role in the localization of root nodules. In order to study the interaction gene of GmTGA4,GmbHLH93,GmWRI1, we successfully constructed bait vector, screened soybean root nodule library by yeast two-hybrid technique, and successfully screened GmTTG1 protein interacting with GmTGA4. It is suggested that GmTGA may play a role in regulating the metabolism of flavonoids in nodule. In addition, GmbHLH93 was successfully screened and verified by soybean saponin gene in the laboratory, indicating that GmbHLH9 may play a role in regulating saponin synthesis in the root nodule.
【學位授予單位】：華中農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S565.1

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