【作者】 庄晓峰；

【导师】 李德葆；

【作者基本信息】 浙江大学 ， 植物病理与分子生物学， 2003， 博士

【摘要】 1 本研究对受稻瘟病菌诱导水稻叶组织特异表达cDNA文库进行了大规模测序，共获得有效EST序列15396条，其中14975条EST序列己被国际公共数据库——GenBank数据库接受。对13316条具有Poly(A)尾的序列进行了冗余度分析，共获得5633条独立基因，占全部序列的42.3％。其中低丰度表达基因(表达频率≤2)4503条，中丰度表达基因(2＜表达频率＜20)1074条，高丰度表达基因(表达频率≥20)56条。 5633条独立基因与GenBank水稻数据库进行BLASTn比对，首次建立了受稻瘟病菌诱导水稻叶组织特异表达数据库。通过对数据的生物信息学分析发现，未知基因3628条，占64.4％；有功能注释的基因642条，占11.40％；有染色体定位信息的1560条，占27.7％。 通过对所有已知功能基因的分析发现，数据库中与己知植物抗逆相关的蛋白有66类。涉及苯丙烷类代谢、氧代谢、氧化还原反应、病程相关蛋白以及植物抵御高温胁迫、低温胁迫、光胁迫、水胁迫、植物创伤等相关的蛋白。证实水稻防御稻瘟病菌侵染的抗病过程是涉及众多基因共表达的一个复杂的网络体系，并且与其它生物因子、非生物因子胁迫处理之间存在着密切的联系。 2 有意思的是：通过与稻瘟病菌全基因组序列数据库和水稻全基因组序列数据库的BLASTn比对，对5633条独立基因进行筛选，共检出来源于稻瘟病菌的基因16条。对这些基因的生物信息学分析结果表明，除3个未知基因外，Mgr003编码的环孢菌素(Cyclophilin)和Mgr011编码的P型ATP酶与稻瘟病菌侵染早期附着孢形成相关；Mgr007编码的半乳糖氧化酶和Mgr016编码的β-葡萄糖苷酶参与识别和降解水稻表皮细胞；C-4甲基固醇氧化酶(Mgr005)和δ-固醇C-甲基转移酶(Mgr009)参与稻瘟病菌致病性相关的膜蛋白——麦角固醇的合成；还有可能与稻瘟病菌侵染过程中信号传导相关的质膜钠离子应答蛋白(Mgr002)和丝氨酸／苏氨酸蛋白激酶(Mgr015)；与蛋白合成相关的核糖体L12蛋白(Mgr014)和延伸因子2(Mgr010)；与真核细胞细胞体内的蛋白质的运输和分泌相关的外被体蛋白(Mgr006)；参与脂肪酸生物合成的烯酞还原酶（Mgr008）等。从受稻瘟病菌诱导水稻叶组织中筛选到的这些基因可能在病原与寄主的相互作用中起重要作用。 3利用前期大规模测序的表达序列标签，制备完成容量为1106条独立基因的CDNA微阵列，对抗瘟性近等基因系H7R和H7S的表达谱分析发现：在水稻样本受到稻瘟病菌侵染8小时后，众多的基因表达发生变化，在980组有效数据中，有170个基因的表达发生显著改变，其中106个基因上调表达，64个基因下调表达。 为克隆与抗病相关的新基因，选取10个差异表达明显的未知基因进行了生物信息学分折，除氯原酸辅酶A－3－h甲基转移酶和肉桂醇脱氢酶是在木质化过程中参与木质素形成的关键基因外，其它基因编码的蛋白也可能参与抗病相关的生理生化反应。 从10个差异表达基因中选取具有BTB／POZ结构域的J001e09作为候选基因，结合分子生物学实验方法，通过电于拼接克隆了一个在稻瘟病菌诱导早期表达的新基因osBTB，并获得该基因的全长cDNA序列（974hp）和DNA序列（sl74bp）。Northerll杂交结果显示该基因在水稻受稻瘟病菌诱导早期表达，与水稻抵御病原菌侵入密切相关。 OSBTde因的RNA微阵列分析结果表明：＄基因受稻瘟病菌．非生物胁迫和植物激素等诱导表达；与9个抗病基因的RNA微阵列聚类分析结果发现，该基因的表达与WK的表达状况类似，结合前期的BLASTPLh对结果，推测OSBTds因具有类f叨皿PK的蛋白激酶活性，在植物信号传导中发挥重要作用。更多还原

【Abstract】 Establishment of Oryza sativa Specific ESTs Database Induced by Magnaporthe grisea and Identification of Resistance-related GenesPh. D. Candidate: Zhuang Xiaofeng (Plant pathology and Molecular biology) Directed by Prof. Li Debao1 In this study, a cDNA library from rice leaves induced by Magnaporthe griseawas sequenced randomly in a large scale. 15 396 ESTs were obtained, of which 14 975 have been released in dbEST (http://www. ncbi. nlm. nih. gov/dbEST). 5633 unique genes (42. 3 %) were identified through redundant analysis within 13 316 ESTs containing Poly (A) tail, among which 4503 genes were low redundancy (single or twice ) , 1074 genes were medium redundancy (3-19), and 56 genes were high redundancy (more than 20).The database of specific ESTs expressed in rice leaves induced by M. grisea was constructed for the first time by searching against Oryza sativa database in NCBI. In this database, 642 (11.4 %) were function annotated, 3628 unique genes (64.4%) were unknown, and other 1560 genes (27. 7%) were located in different chromosomes.It was found that 66 proteins were associated with plant resistance. Proteins were related to phenylpropanoid biosynthesis, oxygen metabolism, redox reaction, or belonged to pathogenesis-related protein, and some were resistant to stresses such as high temperature, chilling, light and wounding, etc. Therefore, it was concluded that rice defense response to disease was an complicated gene network, and closely related to biotic stress and abiotic stress.2 Interestingly, while compared to rice blast fungus genomic database and ricegenomic database, 16 genes originating from M. grisea were found from 5633 unique genes and picked out for further analysis. Bioinformatics analysis suggested that these gene were imported to pathogenesis-related development. Some of these genes, such as Cyclophilin (MgrOOS) and P-tpye ATPase(MgrOll), were involved in appressori urn formation. Galactose oxidase (Mgr007) and beta-glucosidase (Mgr016) were associated with recognizing host and decomposing host cell wall. C-4 methyl sterol oxidase(Mgr005) and delta(24)-sterol Omethyltransferase(Mgr009) were involved in the synthesis of ergosterol which is essential for pathogenicity of M. grisea. Plasma membrane sodium response 2(Mgr002) and serine/threonine kinase (Mgr015) may be involved in signal transduction during the infection. Ribosomalprotein L12 (Mgr014) and elongation factor 2(Mgr010) were important to protein synthesis. Coatomer (Mgr006) was attached to the trafficking of proteins and secretion within eukaryotic cells. CipB protein (MgrOOS) was involved in fatty acid biosynthesis. All the above genes may be involved in host-pathogen interaction in rice tissue.3 A cDNA array containing 1106 unique genes from 3 cDNA libraries was preparedto analyze the expression patterns of rice near iso-genic lines H7R (resistant) and H7S (susceptible). The results showed that 170 genes changed remarkably in 980 valid hybridization data, in which 106 genes were up-regulated and 64 were down-regulated after the rice samples induced by M. grisea for 8 h.In order to clone the new genes related to plant resistance to pathogen, ten genes up-regulated in cDNA array were analyzed further by bioinformatics. Except for caffeoyl-CoA 3-Q-methyltransferase (CCoAOMT) and cinnamyl alcohol dehydrogenase CAD), which were essential to lignin formation during lignif ication, all others may be participated in rice defense to disease. One clone CjOOleOQ) containing POZ/BTB domain was screened out for electronic cloning. By cloning in silico, a full-length cDNA sequence (1974bp) named OsBTByas cloned. A DNA sequence about 5174 bp was found through BLASTn to rice genome sequence. Northern hybridization confirmed that OsBTB was expressed at the early stages in response to pathogen infection.RNA array containing 755 rice samples was prepared to detect the expressions of OsBTB and 9 resistance-related genes (PAL, P-1, 3-glucanasc, polyubiquitin, SAM, SOD, phospholipase C, MARK, Pib, glycine-更多还原