【作者】 高佳；

【导师】 戴良英；

【作者基本信息】 湖南农业大学 ， 植物病理学， 2009， 硕士

【摘要】 促分裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)是一类存在于所有真核生物中的丝氨酸/苏氨酸(Ser/Thr)蛋白激酶,在植物信号传导中起着重要作用。BWMK1是第1个被克隆鉴定的水稻MAPK基因,受稻瘟菌侵染和创伤强烈诱导表达。此外,BWMK1还受低温、干旱、黑暗和茉莉酸的诱导,而苯并噻二唑(BTH)、光照和水杨酸负调控其表达。BWMK1与植物防御、胁迫和生长发育相关并可能在植物不同的信号传导途径中起作用。为了对BWMK1和MAPK级链进行更深入的研究,我们通过酵母双杂交体系,以BWMK1为诱饵,筛选水稻cDNA文库,获得了7个互作蛋白:BWIP1-BWIP7。BLAST同源蛋白功能分析结果表明,BWIP1基因编码5-methyltetrahydropteroyltriglutamate—homocysteine S-methyltransferase,BWIP3基因编码肌醇磷酸合成酶,BWIP4基因编码一个水稻磷酸盐转运蛋白OsPT8,BWIP5基因编码一个含WD-40重复蛋白,BWIP6基因编码乙酰谷氨酸激酶2,BWIP2(Y2H12-2)和BWIP7基因未检索到相关功能。本研究中我们分别构建了Y2H12-2和OsPT8基因的RNA干涉及超表达载体,并对其功能进行了鉴定。通过根癌农杆菌介导法对2个基因进行遗传转化,并经过GUS、PCR以及RT-PCR鉴定,获得了大量阳性株系。对阳性后代进行白叶枯病菌抗性功能鉴定发现,Y2H12-2和OsPT8 RNA干涉株系均能提高对白叶枯病的抗性,Y2H12-2和OsPT8超表达株系对白叶枯病表现为更感。更多还原

【Abstract】 Mitogen-activated protein kinases(MAPKs) are the serine/threonine protein kinases in eukaryotes,they play an important role in plant signal transduction pathways.The first reported rice MAPK gene BWMK1 was transcriptionally activated by blast fungus infection and wounding,it was also induced by cold,drought,dark,and jasmonic acid (JA) treatments,but was repressed by light and salicylic acid(SA)/benzothiadiazole (BTH) treatments.It maybe related to plant defense,stress and growth development and may pay a role in cross-talk between different signaling pathways.We performed yeast two-hybrid(Y2H) to identify the proteins that interact with BWMK1.Rice cDNA library was screened using the BWMK1 cDNA as the bait.Seven different BWMK1 interacting proteins(BWIP1-BWIP7) were identified.BWIP1 encodes 5-methyltetrahydropteroyltri-glutamate—homocysteine S-methyltransferase,BWIP3 encodes myo-inositol phosphate synthease(MIPS),BWIP4 encodes a putative phosphate transporter(OsPT8),BWIP5 encodes a WD-40 repeat containing protein,BWIP6 encodes N-acetyl glutamate kinase 2 (NAGK2),but BWIP2(Y2H12-2) and BWIP6 shows no hits to known functional groups using the BLAST algorithm search.Among the seven BWIP genes,Y2H12-2 and OsPT8 were selected for functional analysis.RNAi and over-expression constructs were generated for the two genes.We obtained a lot of transgenic lines by Agrobacterium-mediated transformation method for transforming rice.They were verified by GUS,PCR and RT-PCR.Disease resistance of these lines to bacterial blight pathogen Xanthomonas oryzae pv.oryzae(Xoo) was evaluated.The result suggested us that both the RNAi lines of Y2H12-2 and OsPT8 showed enhanced resistance to Xoo,in contrary,both the over-expression lines of Y2H12-2 and OsPT8 made the rice more susceptible to Xoo than the wide type.更多还原