【作者】 朱廷恒；

【导师】 郑重；

【作者基本信息】 浙江大学 ， 植物病理学， 2003， 博士

【摘要】 病原物的侵染或某些化学诱导剂的处理都会激活植物自身的抗病防卫反应。植物防卫反应中，病程相关蛋白(pathogenesis-related proteins, PR proteins)的大量表达是一个显著特征。研究表明，PR基因的表达水平和抗病性之间紧密相关，有些PR蛋白在体外就有抗菌活性并且转PR蛋白基因的植物能提高抗病性。因此，PR蛋白基因的表达常作为分子水平上抗病反应的标志。化学诱导剂苯并噻二唑(benzothiadiazola, BTH)可诱导水稻产生系统获得抗性(systemic aquired resistance, SAR)，提高对稻瘟病和白叶枯病等的抗病性。本文报道了水稻中受BTH诱导表达的两个病程相关蛋白基因OsPR-4a和OsGlu的克隆和鉴定。 从水稻受BTH诱导表达的差减杂交cDNA文库中，选择一个预测编码PR-4蛋白的差别表达克隆bnhn-n18，以水稻受BTH诱导表达的全长cDNA文库DNA为模板，以基因特异引物和载体通用引物，通过PCR克隆了OsPR-4a的cDNA的缺失端。根据已知序列和克隆的缺失端序列拚接出OsPR-4a的全长cDNA。OsPR-4a全长cDNA为739bp，预测编码一个有151个氨基酸组成的蛋白，推测的分子量和等电点分别为16.47kD和4.42。从等电点看，OsPR-4a为一酸性蛋白。同源性分析表明，推测的OsPR-4a蛋白与已知的其他植物PR-4类蛋白在氨基酸水平上有大于60％的一致性。其中与大麦的PR-4蛋白相似性最高，达83％。OsPR-4a具有一个主要的结构域Barwin domain，属于Barwin家族。预测OsPR-4a蛋白N端的前25个氨基酸可能为信号肽并负责蛋白的胞外分泌。它与Barwin家族蛋白序列的一个显著特征是有6个保守的半胱氨酸，并能形成3个二硫键。根据OsPR-4a基因cDNA的非编码区序列设计引物对，经PCR扩增了其基因组序列，发现有一个944 bp的内含子。Sourthern分析表明，OsPR—4a以单拷贝存在于水稻基因组。 以Northern blot分析比较了供试的水稻抗病和感病近等基因系在稻瘟菌接种后OsPR-4a表达的差异。在不亲和互作中，接种36 h检测到OsPR-4a的表达；而亲和性互作在供试时间内没有检测到表达。对水稻稻瘟病感病品种幼苗在0.3mmol／L BTH处理后24 h作Northern blot分析，检测到OsPR-4a表达，而水处理对照在 48 h内未检测到表达。以 RTPCR检测 OsPR－4a在水稻SAR 中的表达动态，结果表明：水稻感病品种幼苗，在稻瘟菌接种后 36 hOsPR－4a表达量最高，72 h略有降低：在 0．3 mmol／L BTH处理 3 d后接种稻瘟菌，接种 3 6 h后 OsPR－4a表达量升高，并持续到 72 h。以上结果表明，水稻OsPR4a基因能被SAR诱发剂BTH和稻瘟病菌的侵染诱导表达，而且在水稻受BTH诱导处理后，稻瘟病菌的挑战接种能进一步强化该基因的表达。 克隆的水稻OsPR－4a基因在大肠杆菌中得到了表达，纯化的表达蛋白在体外对水稻稻瘟菌（M中卯porthe gFisea）和纹枯菌（Rh拓octonia solani）的生长都有一定的抑制作用。在抑菌实验中，对抑菌圈边缘的菌丝形态进行显微观察后发现，与正常菌丝相比，稻瘟病菌和纹枯病菌的菌丝有畸形和不规则扭曲。 用一对根据OsPR．4a基因序列和水稻基因组序列数据库相应序列设计的引物扩增到 OsPR下a基因上游 2257 hp的启动子序列。用 PLACE软件分析顺式作用元件，发现 OsPR－4a基因起始密码子上游 1．5 kb的区域内存在几个可能与 PR蛋白基因表达调控相关的顺式作用元件，如 W盒、PALboXA、GT结合序列、Dof结合序列和 Mybstl元件等。将 OsPR一a基因启动子区域及其 5’部分缺失构件与圳Z报道基因相连，并克隆进pCAMBIA139lZ载体中，用于分析启动子活性以及顺式作用元件在调控OsPR一a基因表达中的功能。 本研究还从水稻受BTH诱导表达的差减杂交CDNA文库中，选择一个预测编码p－l，3－葡聚糖酶的差别表达克隆bihn－nlo，以水稻受BTH诱导表达的全长CDNA文库DNA为模板，以基因特异引物和载体通用引物，通过PCR克隆了 OsGlu的 5’缺失端。用已知序列和Genebank搜索的该基因EST序列拚接出仇oU的全长c＊＊A。OSOS的全长。＊＊A为2们4抑，预测的0＊F编码一个有470个氨基酸组成的蛋白，推测的分子量和等电点分别为 50．3 kD和5．77。同源性分析表明，推测的OSGlll蛋白与己知的其他植物pl，3－葡聚糖酶有相似性，属于糖基水解酶家族 17哈lycosyl hydrolaseS family 7人更多还原

【Abstract】 In response to the pathogens attack or chemical inducers treatment, plant can activate physiological defense mechanism that render limitation to pathogens and subsequent infection, Resistance induction is accompanied by the activation of a set of self-defensive genes involved in formation of chemical barriers. Among them, PRs (pathogenesis-related proteins) genes are the crucial one, and serve as important marker genes for defense activation. Benzothiadiazole (BTH) is an inducer of SAR (systemic aquired resistance) in rice and enchances the resistance to blast disease. In this study, we cloned two BTH-induced rice PR genes OsPR-4a and OsGlu, respectively. Possible roles of OsPR-4a in rice SAR and defense against Magnaporthe grisea are discussed.A cDNA clone,bnhn-n 18,encoding a putative PR-4 protein was selected from the BTH induced rice SSH(suppressed substractive hybridization) cDNA library. With the BTH-induced rice full-length cDNA library DNA as a template, the cDNA end of OsPR-4a was cloned by PCR with the gene specific primer and a vector universal primer. The assembled full-length cDNA for OsPR-4a is 739 bp, encoding a 151 amino acid protein with a predicted molecular mass of 16.47 kD and p/ of 4.42.The deduced protein has a main domain Barwin, and belongs to the Barwin family. Similar to other members of the family, the deduced OsPR-4a has a conserved pattern of six cysteines involved in 3 disulfide bonds, which distinguishBarwin family from others. Using the primers corresponding to the cDNA untranslating region, the OsPR-4a genomic sequence was cloned by PCR. The OsPR-4a genomic sequence include an intron with 944bp in length. Sourthern blot analysis showed OsPR-4a integrate in rice genome with a single copy.Expression of OsPR-4a in interaction of rice isogenic lines H8S (susceptible) and HSR (resistant) with Magnaporthe grisea was analyzed through Northern blot. The expression ofOsPR-4a was detected 36 h after inoculation in incompatible but not in compatible interaction at any testing time. Northern blot also showed that expression of OsPR-4a was detected 24 h after treatment with 0.3 mmol/L BTH, but was not detected in the water treatment during 48 hrs after treatment in seedlings of susceptible cultivar. The dynamic expression of OsPR-4a in susceptible rice seedlings was analyzed by RT-PCR. The highest expression of OsPR-4a was detected at 36 h after inoculation; When the seedlings pretreated with 0.3 mmol/L BTH were challenged with M.grisea 3d after the treatment, the highest expression of OsPR-4a was detected by RT-PCR at 36 h and retained to 72 h after inoculation. These results demonstrate that expression of OsPR-4a can be induced by SAR inducer BTH or blast pathogen. After BTH treatment, expression of OsPR-4a can be intensified upon subsequent challenge infection by M. grisea.The cloned OsPR-4a was expressed in Escherichia coli and purified protein show antifungal activity against rice pathogens M. grisea and Rhizoctonia solani.A promoter sequence with 2257 bp upstream of the rice OsPR-4a was amplified using two primers designed based on sequence of OsPR-4a and rice genome sequences database. Several cw-acting elements, including W-box, PAL-boxA, GT-1 elements and MybStl elements, were recognized by PLACE program. The promoter region sequence and its 5’ deletion constructs were fused to the gus reporter gene in pCAMBIA1391z vector for analyzing the promoter activity and the functions of the cis-acting elements in regulation of OsPR-4a expression.A cDNA clone,bnhn-n10,encoding a putative -1, 3-glucanase was selected from the BTH induced rice SSH cDNA library. With the BTH-induced rice full-length cDNA library DNA as a template, the 5’ cDNA fragment of OsGlu was cloned by PCR with the gene specific primer and a vector universal primer. The assembled full-length cDNA for OsGlu is 2034 bp, encoding a 470 amino acid protein with a predicted molecular mass of 50.3 kD and p/ of 5.77. Deduced OsGlu protein is homologous to plant -1, 3-glucanase and belongs to Glycosyl更多还原