大豆早期结瘤基因克隆及功能验证

发布时间：2018-10-19 19:05

【摘要】：大豆(Glycinemax)是产自中国的一种粮食作物,从古到今,深受人们的喜欢。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关系,通过根瘤菌吸收氮素,而为大豆生长发育提供氮素。在本研究中,通过对在根瘤中表达含量较高的三个转录因子GmTGA4、GmbHLH93、GmWRI1进行功能验证,从而探索豆科根瘤菌共生固氮信号传导机制,从而为知道豆科类根瘤固氮提供理论依据。本论文研究结果如下:为了鉴定GmTGA4、GmbHLH93、GmWRI1在豆科根瘤菌共生固氮信号传导机制中的作用,我们利用大豆转基因发根技术,验证了在过表达和基因沉默情况下对大豆根瘤数目的影响,结果表明:在过表达基因GmTGA4、GmbHLH93、GmWRI1后,通过与对照组对比,发现根部根瘤数量明显增加;在基因沉默GmTGA4、GmbHLH93、GmWRI1后,通过与对照组对比,发现根部根瘤数量明显减少。通过以上实验结果说明,GmTGA4、GmbHLH93、GmWRI1在大豆根瘤结瘤过程中起到了很重要的作用。利用荧光定量PCR技术验证结瘤信号传导过程中关键基因GmSymRK、GmNSP1、GmNODULIN27对 GmTGA4、GmbHLH93、GmWI1 的影响。结果表明:过量表达GmTGA4、GmbHLH93、GmWRI1,分别导致GmSymRK表达量升高、降低、升高。GmNSP1表达量升高、降低、升高。GmNODULIN27表达量升高、降低、升高。基因沉默GmTGA4、GmbHLH93、GmWRI1,分别导致GmSymRK表达量降低、GmNSP1表达量降低、GmNODULIN27表达量升高。基因沉默GmTGA4、GmbHLH93、GmWRI1,分别导致GmSymRK表达量降低,GmNSP1表达量降低、升高、升高,GmNODULIN27表达量升高。以上实验结果说明:GmTGA4、GmbHLH93、GmWRI1可能位于 GmSymRK上游,位于 GmNSP1、GmNODULIN27 游。为研究基因在组织中表达,分别克隆了GmTGA4 GmbHLH93、Gm、GmWRI1基因的5'端上游调控区域,成功的构建了 GmTGA4、GmbHLH93、GmWRI1启动子连接GUS基因检测上述基因的表达部位,将融合基因载体转入农杆菌K599诱导转基因发根,收集根瘤和发根,经过GUS染液染色发现,GmTGA4、GmbHLH93、GmWRI1能驱动GUS报告基因的表达,并在根瘤中特异性表达,从而说明了以上3个转录因子在定位在根瘤中并发挥了作用。为研究GmTGA4、GmbHLH93、GmWRI1的互作基因,我们成功的构建了诱饵载体,利用酵母双杂交技术筛选大豆根瘤文库,并成功筛选到与GmTGA4互作的GmTTG1蛋白,说明了GmTGA 在根瘤内部可能起到了调控黄酮类化合物代谢的作用。另外实验室已经利用大豆皂苷基因成功筛选到GmbHLH93并进行了验证,说明了GmbHLH9 在根瘤内部可能起到了调控皂苷合成的作用。
[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.
【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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