大豆磷脂酶C基因PLC3的鉴定、表达分析及其耐盐碱作用机制的研究

The saline-alkaline soils which contain high amounts of NaHCO3 and Na2CO3 are more than 40 million hectares of lands throughout China. A significant proportion of recently cultivated agricultural land being salt affected owing to bad fertilization and irrigation, both of which cause concentrate the salts in the root zone. The salination and alkalization of soil brings the huge influence to agricultural, economical and environmental development. Therefore, finding the mechanism of saline-alkaline stress and increasing the tolerance of plants to saline-alkaline stress is urgently needed. During the past years, phospholiapse C were found for the critical roles in several abiotic stresses. It hydrolyzes phosphatidylinositol 4,5-bisphosphate and generates two second messengers, inositol 1,4,5-triphosphate (IP3) and 1,2-diacylglycerol (DAG). In this project, we found that several genes, especially phospholipase C3, were induced by salt and saline-alkaline treatment using high-throughput Illumina sequencing in soybean. Base on these, we are to be analyzed the expression profiles of phospholipase C3 during drought, salt, saline-alkaline stresses and find out the role of DAG and IP3 involved in saline-alkaline stress. To further determine the cellular role of phospholipase C3 under saline-alkaline condition, we would use overexpression and RNA interference strategy to confirm the function in saline-alkaline stress. All of these will provide a new opportunity to synthesize molecular and physiological knowledge to improve the saline-alkaline tolerance of plants relevant to food production and environmental sustainability; on the other hand, this study will provide a starting point for genetic engineering to increase saline-alkaline tolerance in soybean.

中国现有盐碱地面积为0.4亿公顷，因不良施肥与灌溉等因素致使上述盐碱地面积还在逐年扩大，土壤盐碱化给当地的农业生产、经济以及生态环境发展带来了巨大影响。因此，提高农作物的耐盐碱能力，培育耐盐碱作物新品种（系）尤为重要。近年来磷脂酶作为一个新的逆境响应因子受到人们广泛重视，其中磷脂酶C（PLC）能够水解磷脂酰肌醇4,5二磷酸，生成两个信使分子-肌醇三磷酸（IP3）和二酰甘油（DAG），在植物逆境信号转导中起着重要的调控作用。本项目拟在通过表达谱鉴定大豆耐盐碱关键基因，即磷脂酶C基因PLC3基础上，研究该基因在盐碱胁迫下的表达情况，分析PLC3基因通过第二信使IP3和DAG对大豆耐盐碱的调控作用，阐明其与大豆耐盐碱的关系；在此基础上，通过超表达和RNAi等反向遗传学方法进一步研究该基因的功能，明确其在大豆耐盐碱分子机制中的作用，为通过基因工程手段培育耐盐碱大豆新品系和盐碱地的有效利用奠定基础。

磷脂酰肌醇特异性的磷脂酶C（PI-PLC）能够水解磷脂酰肌醇-4,5-二磷酸生成1,4,5-肌醇三磷酸与二酰甘油，这在植物发育和非生物逆境应答中起着重要作用。但是，目前仍缺少对大豆磷脂酶C基因家族进行系统的表达分析以及功能鉴定。在本项目中，我们通过检索、筛选大豆基因组后发现大豆中共有12个磷脂酶C基因。大豆磷脂酶C基因家族成员分别分布在2,11,14和18号染色体上，并编码蛋白大小从58.8kDa到70.06kDa。同时荧光定量PCR分析发现大豆磷脂酶C的根与叶片均可以被盐、干旱以及盐碱处理所诱导。而在根中的磷脂酶C受ABA诱导表达显著升高。同时，我们分析了不同组织中大豆磷脂酶C基因家族成员的不同表达情况，发现GmPLC7在多数组织中均有较高表达，而GmPLC12在豆荚中特异的表达。另外，我们分析了大豆磷脂酶C基因的亚细胞定位情况，结果证实GmPLC10定位在细胞质膜上。最后，我们将GmPLC10在拟南芥以及大豆中进行过表达，同时在大豆中进行了CRISPR敲除。通过对转基因植株的研究发现，GmPLC10的过表达能够提高植物的耐旱及耐盐碱能力。我们对大豆磷脂酶C基因家族的研究为该基因家族的深入研究奠定了一定的基础。