与紫云英转脂蛋白AsE246相互作用靶蛋白的筛选及其基因表达特征

Screening of target protein interacting with lipid transfer protein AsE246 of Astragalus sinicus and expression characteristics of corresponding encoding gene

摘要:

摘要: 【目的】AsE246是我们首次报道的紫云英根瘤特异表达的非特异性转脂蛋白（nsLTP1: non specific lipid transfer protein 1）编码基因。本实验旨在筛选和鉴定与AsE246相互作用的宿主植物靶蛋白，并分析靶基因在共生和胁迫条件下的表达特征。【方法】利用酵母双杂交技术、小范围杂交技术及实时荧光定量PCR，筛选与AsE246的相互作用蛋白，并定量分析靶基因在结瘤与固氮过程中的时空表达特性。【结果】获取一个阳性克隆，其cDNA序列经Blast分析表明：候选靶蛋白是一个DnaJ-like蛋白，该蛋白相应基因命名为AsDJL1。AsE246与AsDJL1在酵母体内确实相互作用。AsDJL1在固氮根瘤中特异性增强表达，在NaCl胁迫下表达水平显著提高，在(NH4)2SO4胁迫下表达水平显著下降。【结论】本实验是筛选与LTP相互作用蛋白的首次报道。获得了直接的实验证据表明互作基因AsDJL1与AsE246具有高度相似的表达特征和功能，为深入研究二者的相互作用及其在共生固氮和应答环境胁迫中的调控机制，提供了一定的工作基础和理论依据。

Abstract:

Abstract: [Objective] AsE246 was a novel nodule-specific expressed nodulin gene encoding non-specific lipid transfer protein 1 (nsLTP1) of A. sinicus. We screened and identified host plant target protein interacting with AsE246, and characterized the expression patterns of target gene under symbiotic and stress conditions. [Methods] Yeast two-hybrid system, small-scale yeast hybridization test and real-time PCR technique were used to capture the host target protein that interacts with the bait protein AsE246, and to quantitatively analyze the temporal and spatial expression characteristics of target gene during root nodule development and nitrogen fixation process. [Results] One positive clone was obtained, its cDNA insert sequence and Blast analysis showed that: the target protein was a DnaJ-like protein, thus the corresponding encoding gene was named as AsDJL1. AsDJL1 was specifically-enhancing expressed in nitrogen-fixing root nodules, and significantly increased under NaCl stress while significantly decreased under (NH4)2SO4 stress. [Conclusion] This work was the first report on the isolation of proteins interacting with LTP. The work obtained some direct and convincing evidences to show the interacting gene demonstrated high similarity as AsE246 in expression patterns and functions involved. The present progress provided a basic foundation and theoretical basis to undertake any further investigation into their interaction, and regulation mechanism associated with symbiotic nitrogen fixation or response to environmental stress.