【作者】 孙前文；

【导师】 周道绣；

【作者基本信息】 华中农业大学 ， 生物化学与分子生物学， 2009， 博士

【摘要】 组蛋白修饰酶,包括组蛋白去乙酰化酶(Histone deacetylases,HDACs),组蛋白甲基转移酶(Histone methyltransferases,HMTs),以及组蛋白去甲基化酶(Histonedemethylase,HDMs)等,在动植物发育过程中发挥着重要的作用。然而在单子叶植物模式物种水稻中,对这些修饰酶的功能研究还相对较少。本课题针对组蛋白去乙酰化酶基因OsSRT1和组蛋白去甲基化酶基因JMJ706在水稻发育中的功能进行了深入的研究。OsSRT1属于Sirtuin类组蛋白去乙酰化酶,Western杂交检测该基因超量表达植株和RNAi植株的组蛋白修饰情况,发现组蛋白H3K9位点乙酰化修饰程度在RNAi植株中升高,而在超表达植株中则降低,证明此基因是一个组蛋白H3K9位点的去乙酰化酶,并且相关结果显示,此基因与H3K9位点的二甲基化修饰程度密切相关。进而通过自制OsSRT1抗体进行染色质免疫沉淀,利用高通量测序以寻找OsSRT1基因的靶位点。组蛋白去甲基化酶基因包括两个亚家族,LSD1和JMJ家族基因,进化树分析发现植物JMJ家族成员和动物相比,既存在进化的保守性,又有一定的差异。水稻JMJD2亚家族成员JMJ706为定位在异染色质区域的核蛋白,其突变后引起水稻花器官发育异常并导致H3K9me2及me3甲基化修饰程度积累。JMJ706全长cDNA互补转化其突变体jmj6后,转基因植株花发育异常的表型得以恢复;amiRNA抑制野生型JMJ706基因后,可产生类似jmj6的表型。RT-PCR检测结果显示,jmj6中OsMADS8基因的表达升高,OsMADS47和DH1基因的表达降低。染色质免疫沉淀结果显示,jmj6中OsMADS47和DDH1基因的启动子和编码区域的组蛋白H3K9me2和H3K9me3修饰程度积累。全基因组芯片结果显示,突变体与野生型相比:①苗期表达存在差异的基因相对较少;②3期幼穗中145个基因上升表达,97个基因下降表达;③表达存在差异的基因附近富集转座子、反转录转座子、重复序列、以及smRNA等可导致异染色质化的结构元件。分析突变体中表达量明显升高的基因Os03g02470的DNA甲基化情况,发现CG及CHG类DNA甲基化修饰程度降低。同时对野生型和突变体分别构建了两个不同发育时期的Small RNA文库,待进一步高通量测序。通过amiRNA技术分别敲除jmj6中组蛋白甲基转移酶SUVH亚家族的4个基因,发现转基因T0代部分转化植株中这4个基因的表达量明显下降,且穗的突变表型减弱,暗示SUVH家族基因与JMJ706可能执行相反的功能。更多还原

【Abstract】 Histone-modifying enzymes,including HDACs(Histone deacetylases),HMTs (Histone methyltransferases),and HDMs(Histone demethylases),play important roles in gene expression and development in animals as well as in plants.However,a few investigations of these enzymes have been reported in rice,the model species of monocots.This study deep analyzed the function of Histone deacetylase gene OsSRT1 and the Histone demethylase gene JMJ706 in rice development.The OsSRT1 belongs to Sirtuin subfamily of HDACs.Western blot analysis of its RNAi lines showed an increase of H3K9ace and a decrease of H3K9me2,while overexpressioning plants leads to a decrease of H3K9ace.The results supported OsSRT1 having histone deacetylase activity at H3K9 site in vivo,and also related to methylation at this site.In order to analyze the function of OsSRT1 in-depth,we produced its antibody successfully.The purifed antibodies were successfully used to ChIP assays,and the immunoprecipitated DNA were purified for high throughput deep-sequencing.LSD1 and JMJ family proteins are two classes of histone demethylation enzymes. Phylogenetic analysis of jmjC gene family showed that plant jmjC genes have both conserved and specific features compared with human homologues.JMJ706,a jmjC-domain-containing protein in rice,belongs to JMJD2 group,and encodes a heterochromatin-associated protein.Loss-of-function mutations of the JMJ706 gene lead to increased di- and trimethylations of H3K9 and affect the spikelet development.The spikelet mutant phenotype can be rescued by retransformating JMJ706 cDNA. Transgenic plants expressing an amiRNA of the JMJ706 showed a phenotype similarity to jmj6 mutants,mRNA expression level of 16 MADS-box genes and DH1 were analyzed by RT-PCR,and found OsMADS8 were induced,OsMADS47 and DH1 were reduced. ChIP-PCR showed the modification levels of H3K9me2 and H3K9me3 on the promoter and 5’regions of DH1 and OsMADS47 were more abundant in the mutants.Microarray analysis were performed to study the gene regulation mechanism of JMJ706.Compared the transcripts of two development stage of jmj6 mutant and the wild-type plants,we found:①60 genes were up-regulation at seedling stage,while 30 genes were down-regulation;②145 genes were up-regulation,97 genes were down-regulation at 3rd stage of panicle developmental;③there are many elements such as transposable element,DNA repeats,and small RNAs are found at chromosomal contexts of changed genes.We test the DNA methylation patterns of the up-regulated gene Os03g02470,and found symmetric DNA methylations(CG and CHG) were changed.Also we constructed two small RNA cDNA libraries for high throughput deep-sequencing.In order to analyze the functional relationship between HMTs and HDMs,we retransformed the amiRNAs of SUVH gene family members into jmj6 mutants.We observed the phenotype weaker in the retransgenic plants than in the jmj6 mutant.The real-time PCR results showed the expressions of amiRNA targeted genes were reduced in transgenic plants.更多还原