TaCAD12和TaPK-R1过量表达转基因小麦的分子特性与功能分析

发布时间：2018-10-16 09:44

【摘要】：小麦是我国的主要粮食作物。小麦纹枯病是由禾谷丝核菌(Rhizoctonia cerealis)引起的小麦重要土传病害。冻害是影响小麦生长最主要的自然灾害之一。生产上,抗纹枯病、兼抗纹枯病与冻害小麦品种较为匮乏,因此分离克隆抗纹枯、耐冻害相关基因,研究其功能,为抗纹枯病及冻害育种提供理论基础,具有重要意义。本研究通过构建表达载体p20-Rssp2897:6MYC-TaCAD12、pAHC25-MYC-TaPK-R1,用基因枪转化法获得转TaCAD12、TaPK-R1基因小麦。获得以下研究结果：转TaCAD12基因小麦的研究结果：(1)对转TaCAD12基因小麦To-T2代进行PCR以及qRT-PCR分析,获得5个转TaCAD12基因小麦株系。(2)用Western blot分析5个转TaCAD12基因小麦株系中的阳性植株,结果表明这5个转基小麦株系中c-MYC-TaCAD12融合蛋白均被表达。(3)用WK207、R0301分别对转TaCAD12基因小麦T1、T2代进行接种,于收获期进行纹枯病鉴定,结果表明,在转基因小麦中过量表达TaCAD12基因,能提高对不同来源及致病力禾谷丝核菌的抗性。转TaPK-R1基因小麦的研究结果：(1)对转TaPK-Rl基因小麦To-T4代进行PCR、 RT-PCR以及qRT-PCR分析,获得3个转TaPK-R1基因小麦株系。(2)利用Western blot分析3个转TaPK-R1基因小麦株系中的阳性植株,结果表明这3个转基因小麦株系中c-MYC-TaPK-R1融合蛋白均被表达。(3)对T4代转基因小麦及受体进行低温处理,统计存活率、测定脯氨酸和电解质渗漏率含量,结果表明,过量表达TaPK-R1基因提高了转基因小麦对寒冷环境的适应性。(4)用R0301对转TaPK-R1基因小麦T1、T2代进行接种及纹枯病鉴定,用WK207对转TaPK-R1基因小麦T3、T4代进行接种及纹枯病鉴定,抗纹枯病鉴定结果表明,在转基因小麦中过表达TaPK-R1基因,能在不同程度上提高小麦对不同菌株禾谷丝核菌的抗性。(5)经过分子检测与接种抗病鉴定、低温胁迫处理,创制、选育出兼抗纹枯病与冻害的转TaPK-R1基因小麦新种质3份。
[Abstract]:Wheat is the main food crop in China. Wheat sheath blight is an important soil-borne disease of wheat caused by Rhizoctonia graminearum (Rhizoctonia cerealis). Freezing injury is one of the most important natural disasters affecting wheat growth. In the production of wheat varieties resistant to sheath blight and freezing injury, it is of great significance to isolate and clone the genes related to resistance to grain blight and freeze injury, to study its function, and to provide a theoretical basis for breeding for resistance to sheath blight and freezing injury. In this study, the expression vector p20-Rssp2897: 6MYC-TaCAD12 (pAHC25-MYC-TaPK-R1) was constructed, and the transgenic wheat was obtained by gene gun transformation. The following results were obtained: (1) PCR and qRT-PCR analysis were carried out on the To-T2 generation of TaCAD12 transgenic wheat, and 5 TaCAD12 transgenic wheat lines were obtained. (2) Western blot was used to analyze the positive plants in 5 TaCAD12 transgenic wheat lines. The results showed that the c-MYC-TaCAD12 fusion protein was expressed in all of the five transgenic wheat lines. (3) WK207,R0301 was used to inoculate the T _ 1 / T _ 2 generation of the transgenic wheat with TaCAD12 gene and identify the sheath blight at the harvest stage. The results showed that the TaCAD12 gene was overexpressed in the transgenic wheat. It can improve the resistance to Rhizoctonia graminearum from different sources and pathogenicity. The results were as follows: (1) three TaPK-R1 transgenic wheat lines were obtained by PCR, RT-PCR and qRT-PCR analysis on the To-T4 generation of TaPK-Rl transgenic wheat. (2) Western blot was used to analyze the positive plants in three TaPK-R1 transgenic wheat lines. The results showed that the c-MYC-TaPK-R1 fusion protein was expressed in all three transgenic wheat lines. (3) the low temperature treatment of T4 generation transgenic wheat and its receptor were used to calculate the survival rate and the content of proline and electrolyte leakage. Overexpression of TaPK-R1 gene improved the adaptability of transgenic wheat to cold environment. (4) inoculation and identification of sheath blight in T _ 1 and T _ 2 generation of transgenic wheat with TaPK-R1 gene were carried out with R0301, and inoculation and identification of sheath blight in T _ 3 and T _ 4 generation of transgenic wheat with TaPK-R1 gene with WK207. The results of identification of resistance to sheath blight showed that overexpression of TaPK-R1 gene in transgenic wheat could improve the resistance of wheat to different strains of Rhizoctonia graminearum to varying degrees. (5) the resistance to Rhizoctonia graminearum was identified by molecular detection and inoculation. Three new wheat germplasms with TaPK-R1 gene were selected, which were resistant to sheath blight and freezing injury.
【学位授予单位】：广西大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S512.1

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