水稻木聚糖酶XIP抑制蛋白家族基因的功能和表达调控研究

Xylanase inhibitor is a protein inhibiting the activity of endo-b-1,4-xylanase that degrades the plant cell wall. Three types of xylanase inhibitors, XIP(xylanase inhibitor protein)-type, TAXI (Triticum aestivum xylanase inhibitor)-type and TLXI (thaumatin-like xylanase inhibitor)-type, have been identified in cereals. Recently, xylanase inhibitors, occurring as large polymorphic families, have been thought to be involved in plant defense..Tokunaga and Esaka revealed the existence of rice XIP-family genes in 2007. We have being working on xylanase inhibitors for several years. We have obtained transgenic rice plants with over-expression of xylanase inhibitor gene and gene knocked-down plants by using transgenic techniques. Our results showed that rice xylanase inhibitor genes participated in rice resistance to disease, and the enhanced resistance accompanied with the increased level of xylanase inhibitors gene transcripts in T1 plants. But it remains unclear how and why rice XIP-family genes enhance rice resistance to disease. The aim of this project is to study biological functions of rice XIP-family genes in plant development and their expression regulation. .In this study, we will explore the relationship between the expression levels of xylanase inhibitor genes and pathogenesis-related (PR) genes as well as the genes related to JA signaling pathway using over-expression plants infected with M. grisea. We will attempt to illustrate the mechanism that how rice XIP-family genes modulate pathogenesis-related (PR) gene expression, and mediate resistance by JA-dependent pathway..The project will investigate the transcript profiles of xylanase genes in different tissues and development, and inducible expression patterns under biotic and abiotic stress, and hormones..Microarray analysis using the Affymetrix oligonucletide rice chip array will be performed to determine more xylanase inhibitor-related genes and their regulation mechanism. We will characterize the interactions between the cis-acting element of rice xylanase inhibitors gene promoter and transcription factor. We will construct the related expression vectors of the 5' deletion of the rice XIP gene promoter, and use transient expression system to express reporter genes and testing report gene activity to analysis the promoter activity. .Finally we will propose a model to illustrate biological functions of rice XIP-family genes and their expression regulation as well as the mechanism on their roles in plant defense. Our model will provide a new insight of rice plant resistance to diseases.

谷物内的木聚糖酶抑制蛋白以多基因家族形式存在，在植物防御中起作用，但是在植物生长发育中的其他作用以及表达调控不很清楚 ，因此开展水稻木聚糖酶XIP抑制蛋白基因家族的功能和表达调控研究非常有意义。我们已经克隆和体外表达了水稻木聚糖酶XIP抑制蛋白家族不同基因，并获得了过量表达和消减表达的转基因水稻株系，研究表明XIP家族基因过量表达可提高转基因水稻的抗病性。在此基础上，本项目将研究转基因水稻中XIP基因表达水平与抗病相关基因及茉莉酸信号传导途径相关基因表达的关系，探明XIP基因调控抗病的机理。还要从整体层面分析整个家族及家族成员的组织特异和诱导表达谱；利用超量表达株系进行水稻全基因组芯片扫描，分析差异表达的抗病相关转录因子；拟通过瞬时表达技术检测LUC的荧光表达量研究其表达调控方式。最后提出水稻木聚糖酶XIP抑制蛋白基因家族表达调控和抗病防御中的作用机制模型，为水稻抗病防御提供新思路。

1997年，Debyser等首次发现小麦中有木聚糖酶抑制蛋白存在，研究围绕木聚糖酶抑制蛋白可能在植物防御中发挥作用，本研究利用转基因技术证实了水稻XIP木聚糖酶抑制蛋白在植物抗病抗虫方面的作用；并分析了水稻抑制蛋白基因家族的表达及其表达调控。.主要研究结论如下：.(1) 构建了木聚糖酶抑制蛋白基因promotor::GUS载体，家族各成员在水稻根茎叶中的表达模式不同，对外界胁迫的响应方式也不一样。OsXIP和riceXIP可以应答稻飞虱、损伤、低温、MeJA和NaCl的诱导，OsXIP的表达变化比riceXIP显著；RIXI的表达受稻飞虱、损伤、低温和MeJA诱导，不受NaCl诱导。.(2) 获得了RiceXIP、 RIXI 和OsXIP基因过表达和消减表达的转基因植株，RIXI基因过量表达对水稻生长发育没有显著影响，但水稻感染稻瘟病后，RIXI基因的过量表达可以提高水稻中部分防御相关基因的表达，同时增强水稻对稻瘟病的抗性；riceXIP过表达转基因水稻中，防御褐飞虱显著提高。.(3) 运用基因表达谱分析RIXI基因过量表达单拷贝纯合株系与野生型水稻的mRNA，发现了大量差异表达基因。对差异基因按功能做了分类，发现这些差异基因与生物学过程和分子功能相关，主要为转录因子家族和防御相关蛋白基因。通过Pathway显著性富集结果显示代谢通路、次级代谢的生物合成、植物与病原菌互作和激素信号转导通路中差异表达基因最多，而且植物与病原菌互作途径通路中基因表达差异最显著，我们详细分析了植物与病原菌互作、信号转导系统中的差异基因。.(4) 检测启动子活性研究水稻木聚糖酶XIP抑制蛋白基因家族表达的调控方式,抑制蛋白基因的启动子都包含多个与生物和非生物胁迫相关的顺式作用元件。5'启动子缺失实验表明riceXIP和OsXIP二者的启动子上都有增强子和抑制子结合位点，riceXIP全长启动子的GUS活性最低，而最短的启动子GUS活性最高，而OsXIP与之正好相反，全长启动子的GUS活性最高。利用双荧光素酶报告系统,瞬时表达检测转录因子OsWRKY46、OsWRKY6和抑制蛋白RIXI启动子的转录激活作用，OsWRKY46通过结合RIXI启动子W-box元件负调控靶基因的表达而OsWRKY6正调控。