具有RNase活性的盐穗木病程相关蛋白HcPR10非生物胁迫抗性机理的研究

Pathogenesis-related (PR) proteins 10 are produced by plants in response to various abiotic and biotic stresses. The ribonuclease (RNase) activity is crucial for biotic defense, but the mechanism of abiotic tolerance is currently unknown. The salt-induced gene of HcPR10 was identified from halophyte Halostachys caspica which is mainly distributed in the Provinces of Xinjiang and encoded protein possessed the RNase activity in vitro. We hypothesize that HcPR10 protein with RNase activity might degrade tRNA and release combined cytokinins (CKs) to involve in abiotic stress. In order to evaluate the function of HcPR10 gene in biotic and abiotic stresses defense, HcPR10 gene will be transferred into the model plant Arabidopsis thaliana by floral dip method. The phenotypes and physiological and biochemical parameters were observed and measured in the condition of biotic stress and in the presence of salt, drought, cold or heat temperature in the transgenic plants. In addition, the types and concentrations of endogenous CKs in the transgenic and wild-type seedlings would be analyzed using HPLC method and compared. In an effort to further obtain insights into the mechanism underlying HcPR10-mediated abiotic tolerance, We will exploit RNA-Seq technology to analyze the functional categories (GO) and metabolic pathways (KEGG) of differentially expressed genes under the exogenous application of various CKs inhibitors between transgenic and wild-type Arabidopsis plants and the primary CKs response genes were evaluated by qRT-PCR. Meanwhile, combined with phenotype and abiotic stress tolerance in over-expression of HcPR10 plants with CKs inhibitors or not, above researches help us to understand defense function of HcPR10 and its abiotic tolerance mechanism.

RNase活性是病程相关蛋白PR10发挥生物胁迫的关键，但其非生物胁迫抗性的机制目前还不清楚。HcPR10是从荒漠盐生植物盐穗木中克隆获得的一个具有RNase活性的盐响应病程相关蛋白基因，为探讨HcPR10可能通过核酸酶活性降解tRNA从而释放了与之结合的细胞分裂素进而参与了植物对非生物胁迫的抗性。本项目以HcPR10为靶基因，采用花序浸染法获得转基因拟南芥，测定并比较细胞分裂素的种类和含量，观察转基因植株在正常条件的生长并鉴定其在病原体接种以及盐、旱、冷、热等非生物胁迫处理下的表型、生理数据，确定该基因防御生物和非生物学胁迫抗性的功能。采用细胞分裂素抑制剂处理转基因和野生型拟南芥，通过对两者转录组数据中差异表达基因的功能聚类和代谢通路及细胞分裂素响应相关基因表达变化的分析，结合细胞分裂素抑制剂下的转基因植物非生物胁迫表型和抗性，确定HcPR10与细胞分裂素的关系及非生物胁迫抗性的机制。

病程相关蛋白10在植物的生长发育及防御中发挥重要作用，但其分子机制尚不明确。我们从盐生植物盐穗木中克隆获得一个盐诱导的PR-10基因，命名为HcPR10。该基因在盐穗木不同的发育阶段均有表达，其编码的蛋白则定位于细胞核和细胞质中。随着HcPR10蛋白在转基因植物中积累，转基因拟南芥较野生型植株表现出提前开花，抽薹分枝数和单株角果数量显著增加的生长表型。通过RNA-seq分析，转基因植株中与细胞分裂素和应激相关的基因表达显著上调，且转基因植物中细胞分裂素水平的增加与植物生长发育表型间具有显著的相关性。此外，HcPR10蛋白主要表达在与细胞分裂素长距离转运相关的维管组织。晶体结构显示，HcPR10的深腔处可结合一个反式玉米素，具有细胞分裂素结合的特性。以上结果表明，HcPR10可作为细胞分裂素储库，调节植物体内细胞分裂素含量，进而促进细胞分裂相关信号转导，影响植物的生长、发育。此外，HcPR10也可通过调节与细胞分裂素响应相关的盐、旱基因表达，从而提高了植物的耐盐性和抗旱性。同时，HcPR10通过调节植物遭受生物攻毒后的RNase活性、胼胝质积累及防御相关基因的表达进而参与了转基因植株对生物胁迫的抗性。我们通过对HcPR10结构和功能的研究极大地促进了病程相关蛋白家族10的理解，也为培育具有高质量农艺性状的作物提供了候选基因。