mTERF蛋白pTAC15调控拟南芥叶绿体基因的转录终止
发布时间：2018-07-20 12:22
【摘要】：质体编码的聚合酶(PEP)是叶绿体中主要的聚合酶,负责绝大部分叶绿体基因的转录。PEP是由叶绿体编码的核心亚基以及细胞核编码的外周蛋白组成。pTAC15/mTERF8是PEP复合物(也称TAC复合物)成员之一。本文研究了pTAC15在质体基因表达过程中的作用。pTAC15含有8个保守的线粒体转录终止因子(mTERF)结构域,属于线粒体转录终止因子(mTERF)家族的成员。烟草体内瞬时表达结果表明,pTAC15:GFP融合蛋白定位于叶绿体中。进一步利用pTAC15:MYC转基因植株进行免疫实验分析表明,pTAC15是一个类囊体定位的叶绿体蛋白。结合BN-PAGE,2-D电泳和免疫杂交的实验,pTAC15:MYC出现在660kD的超分子复合物中,同PEP复合物核心亚基rpoB共迁移。酵母双杂交实验表明,pTAC15在PEP复合物中与另一个成员pTAC10相互作用。为了分析pTAC15在叶绿体基因表达过程中的作用,分离鉴定到ptac15的突变体。qRT-PCR分析表明,突变体中绝大部分叶绿体基因的表达水平与野生型相比没有明显差异,只有psbJ的转录水平上出现了上调。环式PCR的实验结果表明,野生型中叶绿体psbJ的转录终止位于终止密码子后第94和95的核苷酸位置,而ptac15突变体中psbJ的转录终止除了位于终止密码子后第94和95的核苷酸位置外,还出现了其他异常的转录终止位点。这些结果表明,pTAC15的缺失干扰了叶绿体基因组中psbJ的转录终止。凝胶阻滞(EMSA)实验以及体内的免疫共沉淀产物qPCR分析表明,pTAC15能够特异性结合质体基因psbJ的3’端序列。进一步的体外生化实验结果表明,pTAC15蛋白具有转录终止的作用。这些研究结果表明,拟南芥叶绿体蛋白pTAC15作为PEP复合物中的一员,能够特异性结合叶绿体基因psbJ的3'末端,参与该基因转录终止的作用。
[Abstract]:Plastid encoded polymerase (PEP) is the main polymerase in chloroplasts. PEP, which is responsible for the transcription of most chloroplast genes, is composed of the core subunit encoded by chloroplast and the peripheral protein encoded by nucleus. PTAC15 / mTERF8 is one of the members of PEP complex (also called TAC complex). The role of pTAC15 in plastid gene expression. PTAC15 contains eight conserved mitochondrial transcriptional termination factor (mTERF) domains and belongs to the mitochondrial transcriptional termination factor (mTERF) family. Transient expression in tobacco indicated that pTAC15: GFP fusion protein was located in chloroplast. The further immunoassay of pTAC15: MYC transgenic plants showed that pTAC15 was a thylakoid located chloroplast protein. In combination with BN-PAGEG 2-D electrophoresis and immuno-hybridization, pTAC15: MYC appeared in 660kD supramolecular complex and co-migrated with the core subunit rpoB of PEP complex. Yeast two-hybrid experiments showed that pTAC15 interacted with another member pTAC10 in PEP complex. In order to analyze the role of pTAC15 in chloroplast gene expression, the ptac15 mutant. QRT-PCR analysis showed that there was no significant difference in the expression level of most chloroplast genes between the mutant and wild type. Only psbJ transcriptional levels were up-regulated. The results of cyclic PCR showed that the transcriptional termination of chloroplast PSBJ in wild type was located at the 94th and 95th nucleotides after the termination of codon, while in the ptac15 mutant, the transcriptional termination of PSBJ was at the nucleotide position of 94th and 95th after the termination of codon. There are also other abnormal transcriptional termination sites. These results suggest that the deletion of pTAC15 interferes with the transcriptional termination of psbJ in chloroplast genome. Gel block assay (EMSA) and qPCR analysis of immunoprecipitation products showed that pTAC15 could specifically bind to the 3'terminal sequence of plastid gene psbJ. Further in vitro biochemical results showed that pTAC 15 protein had transcriptional termination. These results suggest that the chloroplast protein pTAC15, as a member of the PEP complex, can specifically bind to the 3'terminal of the chloroplast gene psbJ and play a role in the transcriptional termination of the gene.
【学位授予单位】：上海师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2
[Abstract]:Plastid encoded polymerase (PEP) is the main polymerase in chloroplasts. PEP, which is responsible for the transcription of most chloroplast genes, is composed of the core subunit encoded by chloroplast and the peripheral protein encoded by nucleus. PTAC15 / mTERF8 is one of the members of PEP complex (also called TAC complex). The role of pTAC15 in plastid gene expression. PTAC15 contains eight conserved mitochondrial transcriptional termination factor (mTERF) domains and belongs to the mitochondrial transcriptional termination factor (mTERF) family. Transient expression in tobacco indicated that pTAC15: GFP fusion protein was located in chloroplast. The further immunoassay of pTAC15: MYC transgenic plants showed that pTAC15 was a thylakoid located chloroplast protein. In combination with BN-PAGEG 2-D electrophoresis and immuno-hybridization, pTAC15: MYC appeared in 660kD supramolecular complex and co-migrated with the core subunit rpoB of PEP complex. Yeast two-hybrid experiments showed that pTAC15 interacted with another member pTAC10 in PEP complex. In order to analyze the role of pTAC15 in chloroplast gene expression, the ptac15 mutant. QRT-PCR analysis showed that there was no significant difference in the expression level of most chloroplast genes between the mutant and wild type. Only psbJ transcriptional levels were up-regulated. The results of cyclic PCR showed that the transcriptional termination of chloroplast PSBJ in wild type was located at the 94th and 95th nucleotides after the termination of codon, while in the ptac15 mutant, the transcriptional termination of PSBJ was at the nucleotide position of 94th and 95th after the termination of codon. There are also other abnormal transcriptional termination sites. These results suggest that the deletion of pTAC15 interferes with the transcriptional termination of psbJ in chloroplast genome. Gel block assay (EMSA) and qPCR analysis of immunoprecipitation products showed that pTAC15 could specifically bind to the 3'terminal sequence of plastid gene psbJ. Further in vitro biochemical results showed that pTAC 15 protein had transcriptional termination. These results suggest that the chloroplast protein pTAC15, as a member of the PEP complex, can specifically bind to the 3'terminal of the chloroplast gene psbJ and play a role in the transcriptional termination of the gene.
【学位授予单位】：上海师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2
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