大豆组成型三重反应基因GmCTR1的克隆与功能分析

doi:10.3864/j.issn.0578-1752.2017.16.003

摘要/Abstract

摘要： 【目的】植物激素乙烯参与植物的生长发育以及生物与非生物胁迫过程。组成型三重反应基因CTR1作为乙烯受体下游一个负调控因子，结合乙烯受体共同参与乙烯的信号转导途径。本研究通过对大豆（Glycine max）的组成型三重反应基因CTR1进行克隆和诱导表达分析，以及在大豆发状根中过表达该基因后进行疫霉根腐病的抗性分析，探究CTR1在大豆与疫霉菌互作过程中的功能。【方法】利用同源克隆的方法，以拟南芥的AtCTR1序列为探针，在大豆基因组数据库中搜索同源性最高的基因即为大豆的GmCTR1（Glyma.13G151100），并从Williams82中将GmCTR1克隆出来。对GmCTR1进行多重序列比对、系统进化分析和疫霉菌的诱导表达分析。构建植物过表达载体pBinGFP2: GmCTR1。应用发根农杆菌介导的遗传转化方法获得大豆发状根，经GFP荧光筛选得到大豆过表达GmCTR1的阳性发状根和转入空质粒的阴性对照发状根。对过表达GmCTR1发状根和阴性对照发状根侵染疫霉菌后，检测病斑长度、疫霉生物量和抗性相关基因的表达。【结果】根据同源序列比对结果，将与拟南芥AtCTR1（AT5G03730）同源性最高（75.3%）的大豆基因Glyma.13G151100命名为GmCTR1，从Williams82中克隆得到GmCTR1的CDS序列。该基因的CDS序列全长2 511 bp，编码836个氨基酸的丝氨酸/苏氨酸蛋白激酶，蛋白质量为92.35 kD，等电点为6.51。多重序列比对结果显示，GmCTR1氨基酸具有CTR1同源蛋白的典型结构域和关键特征。构建系统发育树进行进化分析发现，GmCTR1与菜豆（Phaseolus vulgaris）、蒺藜苜蓿（Medicago truncatula）的CTR1亲缘关系十分相近，在同一个分支上，且与菜豆的亲缘关系最近。荧光定量PCR结果表明，在大豆疫霉菌P6497侵染后，GmCTR1受诱导逐渐上调表达，在侵染48 h后表达水平达到最高，随后表达量降低。利用酶切连接方法将GmCTR1的CDS序列构建到植物过表达载体pBinGFP2中，PCR和酶切验证获得了重组质粒pBinGFP2: GmCTR1。对发状根接种疫霉菌后发现，过表达GmCTR1减弱了对疫霉根腐病的抗性，疫霉菌侵染36 h后过表达发状根与对照相比病斑长度显著增长。过表达GmCTR1发状根中疫霉菌的生物量与对照相比增加，与大豆抗病反应相关基因的表达水平显著降低。【结论】GmCTR1在大豆与疫霉菌的互作过程中发挥一定的负调控作用。

关键词: 大豆, GmCTR1, 表达分析, 疫霉根腐病抗性, 大豆发状根

Abstract: 【Objective】The plant hormone ethylene involved in plant growth, development, biotic and abiotic stress processes. The CONSTITUTIVE TRIPLE RESPONSE1 gene acts as a key negative regulator of ethylene receptors and participates in ethylene signal transduction pathway by combining ethylene receptor. In order to conduct a preliminary study on the function of GmCTR1, GmCTR1 was cloned and its expression was analyzed, and then transformed into soybean hairy roots for analyzing resistance to Phytophthora root rot of soybean.【Method】Based on the sequence of AtCTR1 (AT5G03730), Glyma.13G151100 which with the highest homology was selected by BLAST, and it was named GmCTR1. The coding sequence of GmCTR1 was isolated from Williams82. Sequence alignment, phylogenetic analysis were performed. The expression level of GmCTR1 uponinfection of Phytophthora sojae wasanalyzed by qRT-PCR. The plant overexpression vector pBinGFP2:GmCTR1 was constructed. Then the biological function of GmCTR1 against P. sojae was explored through transforming soybean hairy roots in agrobacterium-mediated method. The transgenic overexpressing hairy roots and empty-vector hairy roots were obtained by GFP fluorescence screening. Then the lesion length, accumulation of P. sojae andthe relative expression level of resistance related genes were measured.【Result】 Based on the sequence of AtCTR1 (AT5G03730), the gene Glyma.13G151100 which with the highest homology was selected by BLAST, and it was named GmCTR1. The coding sequence of GmCTR1 was isolated from Williams82. The CDS of GmCTR1 is 2 511 bp in length. GmCTR1 is a serine/threonine protein kinase encoding 836 amino acids. The molecular weight is 92.35 kD, and the isoelectric point is 6.51. Multiple sequence alignment of GmCTR1 and other CTR1s showed that GmCTR1 contains the typical domain of CTR1 protein. Phylogenetic analysis indicated that the CTR1s from Phaseolus vulgaris, Medicago truncatula were highly similar to GmCTR1 which located in the same branch. qRT-PCR analysis showed that the expression level of GmCTR1 was up-regulated upon infection by P. sojae. There was the highest expression level at 48 h post infection, and then the expression level decreased slightly. The CDS of GmCTR1 was constructed into the overexpression vector pBinGFP2. The recombinant plasmid was confirmed by bacteria PCR and enzyme digested. After inoculation of P. sojae, the resistance was significantly decreased in overexpressed hairy roots. Compared with the empty-vector hairy roots, there are longer lesion length at 36 hpi. Expression analysis showed that there are more accumulation of P. sojae inoverexpressing hairy roots. Besides, the expression level of resistance related genes is significantly reduced in overexpressing hairy roots.【Conclusion】GmCTR1 may act as a negative regulator in the interactions between soybean and the P. sojae.

Key words: Glycine max, GmCTR1, expression and analysis, Phytophthora root rot resistance, soybean hairy root

张楠楠，薛冬，崔晓霞，赵晋铭，郭娜，王海棠，邢邯. 大豆组成型三重反应基因GmCTR1的克隆与功能分析[J]. 中国农业科学, 2017, 50(16): 3082-3091.

ZHANG NanNan, XUE Dong, CUI XiaoXia, ZHAO JinMing, GUO Na, WANG HaiTang, XING Han. Cloning and Functional Analysis of the CTR1 in Soybean[J]. Scientia Agricultura Sinica, 2017, 50(16): 3082-3091.

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