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

Abstract

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

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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