大豆蛋白激酶基因GmPKS4的鉴定、表达分析及其耐盐碱作用机制的研究

Saline-alkalization of soil is a serious worldwide environmental stress that limits plant growth and productivity. In northeast China, saline-alkaline soil is the consequent effect of hydrolysis of two carbonates NaHCO3 and Na2CO3, which bring about inordinately high soil pH. There were only a few species of alkali-resistant halophytic plants can survive in this land. Soybean [Glycine max (L.) Merrill], one of the most important oil and grain crops in China, plays an important role in agriculture industrial structure and people’s lives. However, natural calamities, especially the saline-alkaline stress limit soybean growth and productivity dramatically. Therefore, an increase in saline-alkaline tolerance of soybean is desirable and has been a major goal of variety breeding. PKS (SOS2-like protein kinases) are key protein kinases within calcium signal transduction network in plants under stress conditions.PKS are activated by Ca2+-SCaBP to convert inactive SOS1/NHX1 to an active form to mediate sodium balance. Our preliminary study showed that the expression of GmPKS4 gene was upregulated in the expression profiles of genes in leaves and roots of seedlings (two-leaf stage) of the soybean under salt and saline–alkaline stress conditions. In this project, in order to further clarify the role of GmPKS4 involved in the saline-alkaline tolerance of soybean, the transcripts of GmPKS4 gene in roots and leaves during drought, salt, saline-alkaline stresses are to be further assayed first.Then, we analyzed the sub-cellular location of GmPKS4 by transiently expression GmPKS4-GFP fusion protein in onion epidermal cells.Third,the aim to confirm the interaction protein of GmPKS4 gene, the yeast 2-hybrid system would be carried out. Furthermore, overexpression and reverse genetic technology are to be carried out to further ascertain the important role of GmPKS4 involved in saline-alkaline tolerance of soybean. The results obtained in this study will contribute importantly to better understanding of the molecular mechanism of GmPKS4 in soybean under saline-alkaline stress, and present a new biotechnological approach for increasing saline-alkaline tolerance of soybean by genetic engineering.

土壤盐碱化是抑制植物生长和降低农作物产量的重要环境因子之一。大豆是我国重要的经济作物，在农业产业结构和人民生活中占有重要地位。大豆因受多种自然灾害而减产，其中盐碱胁迫对其产量造成极大影响。因此，提高大豆耐盐碱能力，培育耐盐碱大豆新品种（系）具有重要意义。丝氨酸/苏氨酸蛋白激酶（PKS）是一类重要的蛋白，能够与钙离子结合蛋白SCaBP互作，调控下游基因的表达，从而在植物逆境信号转导中发挥重要作用。本项目拟在通过表达谱鉴定大豆耐盐碱关键基因，即蛋白激酶基因GmPKS4基础上，深入分析GmPKS4在盐碱等逆境下的表达情况及其与大豆耐盐碱的关系；同时利用酵母双杂交等技术，研究GmPKS4与SCaBP蛋白的互作类型；进而通过超表达、突变体回补及RNAi等鉴定GmPKS4的功能，明确其对下游基因SOS1/NHX1的调控效果，从而解析该基因在大豆耐盐碱分子机制中的作用，为抗盐碱性大豆分子育种奠定基础。

SOS2类蛋白激酶（SOS2-like protein kinases，PKS）编码丝氨酸/苏氨酸蛋白激酶，它属于一个基因家族，拟南芥中有25个PKS成员蛋白，除SOS2（CIPK24）外，其余的被命名为PKS2-PKS24。PKS蛋白与植物逆境胁迫应答和抗逆性密切相关。在本项目中，我们克隆了大豆GmPKS4基因，全长1317 bp，编码439个氨基酸，属于典型的SOS2类蛋白激酶；GmPKS4基因在盐、盐碱、碱、干旱和ABA胁迫下根和叶中的表达情况表明，它对不同逆境胁迫均有响应，根对盐胁迫最敏感，而在叶片中对碱胁迫响应更强烈；通过与绿色荧光蛋白（GFP）融合而在烟草叶肉细胞中瞬时表达，确认GmPKS4蛋白定位在细胞质与细胞核；以GmPKS4作为诱饵蛋白，对我们构建的盐、盐碱、干旱胁迫下大豆根和叶的混合酵母cDNA文库进行筛库，共筛选获得与其互作的蛋白20个，部分蛋白的相互作用验证仍在进行。另外，我们增加了对GmPKS4基因启动子的研究工作，克隆了该基因启动子1530 bp，经PlantCARE 和PLACE数据库分析发现，该基因启动子区具有多个逆境胁迫应答元件；转pGmPKS4-GUS烟草在不同逆境胁迫下的GUS染色与GUS活性分析发现，GmPKS4基因启动子可驱动GUS在碱、盐碱和干旱胁迫下强表达。最后，我们将GmPKS4在拟南芥、大豆发状根和大豆中进行超表达，通过对转基因植物的研究发现，GmPKS4基因的过表达能够提高植物的耐盐碱胁迫能力。我们对大豆GmPKS4在植物耐盐碱胁迫方面的研究，一方面为PKS基因家族的深入研究奠定了基础，另一方面可为大豆盐碱地分子育种提供候选基因资源。