野生大豆类受体蛋白激酶CBRLK与SNARE转运蛋白BET11a互作调控耐盐碱性的分子机理研究

In previous studies, we have characterized a Glycine soja Ca2+/CAM-binding receptor like protein kinase, GsCBRLK, which positively regulates plant salt-alkaline tolerance. We identified GsBET11a as an interactor of GsCBRLK by using yeast two hybrid and bimolecular fluorescence complementation assays. To understand the molecular mechanism for GsBET11a-GsCBRLK interaction, in this project, we will identify the key domains and amino acids required for their interaction, as well as the interaction specificity of GsCBRLK with soybean and Arabidopsis BET1 subfamily proteins. We are also interested in investigating the co-expression characteristics of GsBET11a and GsCBRLK, including the expression patterns under salt-alkaline stress, protein co-localization in plant cells and the spatial-temporal expression specificity. In addition, to study the biological and physiological function of GsBET11a in response to salt-alkaline stress, we will analyze the growth performance and physiological indices of the GsBET11a transgenic Arabidopsis and soybean, as well as the T-DNA insertion Arabidopsis mutants. We try our best to precisely determine the role of GsBET11a in salt-alkaline responses, by using both the overexpression and gene silence technology. To reveal the regulatory mechanism between GsBET11a and GsCBRLK, we will conduct two different and independent experiments to confirm which one is the upstream regulator. We wonder whether GsBET11a functions as the upstream regulator and controls the plasma membrane localization of GsCBRLK; or GsCBRLK functions as the upstream regulator and phosphorylates GsBET11a. Collectively, this project aims to reveal the interactive mechanism and molecular basis of GsCBRLK and GsBET11a in response to salt-alkaline stress, and further identify new salt-alkaline resistant genes. Our results will be of great benefit to elucidate the signal transduction and regulatory network in salt-alkaline responses, and provide candidate genes and theoretical support for molecular breeding of legume crop with superior salt-alkaline tolerance.

本项目在确定野生大豆耐盐碱蛋白激酶CBRLK与BET11a相互作用的基础上，首先通过Y2H确定二者互作结构域、关键氨基酸及互作特异性，揭示BET11a-CBRLK蛋白互作机制；通过盐碱胁迫表达模式、蛋白共定位和时空表达特异性分析，研究BET11a与CBRLK基因共表达特性；利用超量表达拟南芥和大豆及拟南芥突变体，通过盐碱胁迫表型和生理指标分析，从超量表达和基因沉默2个角度，解析BET11a基因耐盐碱功能；为探索蛋白上下游调控关系，将分析BET11a对CBRLK质膜定位的调控作用，及CBRLK对BET11a磷酸化作用，揭示二者蛋白互作在植物盐碱胁迫应答中的生物学意义。本项目以研究“BET11a-CBRLK蛋白互作机制和耐盐碱分子机理”这一问题为主线，从中挖掘耐盐碱新基因，在理论上将为阐明植物盐碱胁迫应答调控网络提供重要参考，在应用上将为提高作物耐盐碱性，开发利用盐碱地提供理论依据和基因资源。

土壤盐碱化严重制约我国作物生产和农业发展。课题组前期从野生大豆中鉴定了一个耐盐碱功能显著的类受体激酶GsCBRLK，并筛选获得了其互作蛋白GsBET11a。本项目对GsCBRLK-GsBET11a蛋白互作机制进行了深入研究，发现GsCBRLK与野生大豆和拟南芥所有BET1亚家族成员均存在相互作用，GsBET11a蛋白C端跨膜结构域是与GsCBRLK互作的关键区域，且TMD的完整性决定其与GsCBRLK蛋白相互作用；CRCK1s激酶与BET1s蛋白互作具有特异性，GsCBRLK蛋白N端可变结构域与GsBET11a互作。对GsBET11a和GsCBRLK共表达特性研究发现，二者具有重叠的亚细胞定位和组织表达特性，且GsBET11a表达也受高盐和苏打盐碱胁迫诱导。功能分析结果证实GsBET11a在拟南芥和大豆中超量表达可提高转基因植株的耐盐性，而AtBET11和AtBET12基因突变显著降低了拟南芥的耐盐性，且AtBET11/12双基因突变导致胚胎发育异常。进一步研究发现GsBET11a过量表达影响GsCBRLK蛋白在细胞内的定位。研究结果揭示了GsBET11a-GsCBRLK蛋白互作机制，明确了GsBET11a基因耐盐碱功能，并初步阐明了GsBET11a-GsCBRLK介导的耐盐碱分子机理；补充完善了盐碱胁迫蛋白互作网络，为深入研究植物耐盐碱分子机制奠定了重要基础，并为豆科作物耐盐碱分子育种提供了重要基因资源。