大豆早期共生信号传导途径基因的克隆及功能验证

发布时间：2018-11-09 18:10

【摘要】：大豆是世界上最主要的经济作物和最重要的油料作物之一。豆科植物与根瘤菌形成的共生固氮系统是自然界最重要的固氮方式,共生固氮因其高效性和经济性而在农业生产中具有举足轻重的作用。目前国内外关于豆科植物与根瘤菌共生固氮的研究大多集中于模式植物,很多早期结瘤基因已被鉴定,但对于大豆与根瘤菌共生结瘤固氮的研究较少。本研究通过对大豆早期结瘤基因Sym RK、CCa MK、NSP1、NSP2、NIN进行功能鉴定,期望对大豆-根瘤菌共生结瘤信号传导过程进行探索。主要研究结果如下:1.为了鉴定基因Gm Sym RKα/β、Gm CCa MKα/β、Gm NSP1α/β、Gm NSP2α及Gm NINα在大豆-根瘤菌共生结瘤过程中的功能,我们利用毛根转化技术,验证了其过量表达和下调表达对大豆植株结瘤数的影响。结果表明:Gm Sym RKα/β、Gm CCa MKα/β、Gm NSP1α/β、Gm NSP2α、Gm NINα过量表达后,植株结瘤数显著多于对照组;Gm Sym RKα/β、Gm CCa MKα/β基因下调表达后,植株结瘤数显著少于对照组。以上结果表明Gm Sym RKα/β、Gm CCa MKα/β、Gm NSP1α/β、Gm NSP2α、Gm NINα在大豆结瘤过程中均具有重要作用。2.我们利用毛根转化技术,验证了在蒺藜苜蓿突变体mtdmi3、mtnsp1-1及野生型A17中基因互补和过量表达对植株结瘤数的影响。结果表明:在mtdmi3中表达Gm CCa MKβ能够使植株恢复结瘤能力;在mtnsp1-1中表达Gm NSP1α/β均能够使植株恢复结瘤能力;同时,在野生型蒺藜苜蓿A17中过量表达Gm CCa MKβ、Gm NSP1α/β均能显著增加植株结瘤数目。以上结果说明,Gm CCa MKβ在蒺藜苜蓿中能够互补DMI3的生物学功能;Gm NSP1α/β在蒺藜苜蓿中均能互补NSP1的生物学功能。3.利用q RT-PCR检测了各基因表达情况的相互影响,结果表明:过量表达基因Gm Sym RK使Gm CCa MK、Gm NSP1及Gm NSP2的表达受到不同程度的上调,抑制Gm Sym RK的表达使Gm CCa MK、Gm NSP1及Gm NSP2的表达受到不同程度的下调;过量表达基因Gm CCa MK使Gm NSP1、Gm NSP2的表达受到不同程度的上调,抑制Gm CCa MK的表达使Gm NSP1、Gm NSP2的表达受到不同程度的下调;过量表达Gm NSP1使Gm NSP2及Gm NIN的表达受到强烈上调;过量表达Gm NSP2α使Gm CCa MK、Gm NIN的表达受到不同程度的下调;过量表达Gm NINα使Gm NSP2的表达受到强烈下调。以上结果说明,在早期结瘤信号传导途径中Gm Sym RK位于Gm CCa MK上游,Gm NSP1、Gm NSP2及Gm NIN位于Gm CCa MK下游;Gm NSP2α对Gm CCa MK可能存在负反馈调节;Gm NINα对Gm NSP2也可能存在负反馈调节。故大豆-根瘤菌早期共生结瘤信号传导途径与豆科模式植物相似。
[Abstract]:Soybean is one of the most important cash crops and oil crops in the world. The symbiotic nitrogen fixation system between legume plants and rhizobia is the most important nitrogen fixation mode in nature. Because of its high efficiency and economy symbiotic nitrogen fixation plays an important role in agricultural production. At present, most of the studies on symbiotic nitrogen fixation between legume and rhizobia are focused on model plants, many early nodulation genes have been identified, but there is little research on symbiotic nodule fixation of soybean and rhizobia. The purpose of this study was to investigate the signal transduction process of soybean-rhizobia symbiotic nodulation by identifying the function of soybean early nodulation gene Sym RK,CCa MK,NSP1,NSP2,NIN. The main results are as follows: 1. In order to identify the function of genes Gm Sym RK 伪 / 尾, Gm CCa MK 伪 / 尾, Gm NSP1 伪 / 尾, Gm NSP2 伪 and Gm NIN 伪 in the process of soybean rhizobia symbiotic nodulation, we used hairy root transformation technique. The effects of overexpression and down-regulation on nodule number of soybean plants were verified. The results showed that: Gm Sym RK 伪 / 尾, Gm CCa MK 伪 / 尾, Gm NSP1 伪 / 尾, Gm NSP2 伪, Gm NIN 伪 overexpression significantly increased the number of nodulation. After down-regulation of Gm Sym RK 伪 / 尾, Gm CCa MK 伪 / 尾 gene, the nodule number of the plant was significantly lower than that of the control group. The results showed that Gm Sym RK 伪 / 尾, Gm CCa MK 伪 / 尾, Gm NSP1 伪 / 尾 and Gm NSP2 伪, Gm NIN 伪 played an important role in the process of soybean nodulation. 2. The effects of gene complementation and overexpression on nodule number of alfalfa mutant mtdmi3,mtnsp1-1 and wild type A17 were studied by hairy root transformation technique. The results showed that the expression of Gm CCa MK 尾 in mtdmi3 could restore the nodulation ability of plants, and the expression of Gm NSP1 伪 / 尾 in mtnsp1-1 could restore the nodulation ability of plants. At the same time, overexpression of Gm CCa MK 尾 and Gm NSP1 伪 / 尾 in alfalfa A17 of wild type Tribulus terrestris significantly increased the number of nodulation. The results showed that, Gm CCa MK 尾 could complement the biological function of DMI3 in alfalfa of Tribulus terrestris, and Gm NSP1 伪 / 尾 could complement the biological function of NSP1 in alfalfa of Tribulus terrestris. 3. Q RT-PCR was used to detect the interaction of gene expression. The results showed that overexpression of gene Gm Sym RK increased the expression of Gm CCa MK,Gm NSP1 and Gm NSP2 in varying degrees, and inhibiting the expression of Gm Sym RK resulted in Gm CCa MK,. The expression of Gm NSP1 and Gm NSP2 was down-regulated in varying degrees. Overexpression of the gene Gm CCa MK increased the expression of Gm NSP1,Gm NSP2 in varying degrees, inhibited the expression of Gm CCa MK, down-regulated the expression of Gm NSP1,Gm NSP2 in varying degrees, and overexpressed Gm NSP1 made the expression of Gm NSP2 and Gm NIN strongly up-regulated. Overexpression of Gm NSP2 伪 resulted in down-regulation of Gm CCa MK,Gm NIN expression in varying degrees, and overexpression of Gm NIN 伪 resulted in strong down-regulation of Gm NSP2 expression. These results suggest that Gm Sym RK is located upstream of Gm CCa MK, Gm NSP1,Gm NSP2 and Gm NIN are downstream of Gm CCa MK in early nodulation signal transduction pathway, Gm NSP2 伪 may have negative feedback regulation on Gm CCa MK. Gm NIN 伪 may also have negative feedback regulation on Gm NSP2. Soybean-rhizobia early symbiotic nodule signal transduction pathway is similar to legume model plants.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S565.1

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