【摘要】：質(zhì)體編碼的聚合酶(PEP)是葉綠體中主要的聚合酶,負(fù)責(zé)絕大部分葉綠體基因的轉(zhuǎn)錄。PEP是由葉綠體編碼的核心亞基以及細(xì)胞核編碼的外周蛋白組成。pTAC15/mTERF8是PEP復(fù)合物(也稱(chēng)TAC復(fù)合物)成員之一。本文研究了pTAC15在質(zhì)體基因表達(dá)過(guò)程中的作用。pTAC15含有8個(gè)保守的線粒體轉(zhuǎn)錄終止因子(mTERF)結(jié)構(gòu)域,屬于線粒體轉(zhuǎn)錄終止因子(mTERF)家族的成員。煙草體內(nèi)瞬時(shí)表達(dá)結(jié)果表明,pTAC15:GFP融合蛋白定位于葉綠體中。進(jìn)一步利用pTAC15:MYC轉(zhuǎn)基因植株進(jìn)行免疫實(shí)驗(yàn)分析表明,pTAC15是一個(gè)類(lèi)囊體定位的葉綠體蛋白。結(jié)合BN-PAGE,2-D電泳和免疫雜交的實(shí)驗(yàn),pTAC15:MYC出現(xiàn)在660kD的超分子復(fù)合物中,同PEP復(fù)合物核心亞基rpoB共遷移。酵母雙雜交實(shí)驗(yàn)表明,pTAC15在PEP復(fù)合物中與另一個(gè)成員pTAC10相互作用。為了分析pTAC15在葉綠體基因表達(dá)過(guò)程中的作用,分離鑒定到ptac15的突變體。qRT-PCR分析表明,突變體中絕大部分葉綠體基因的表達(dá)水平與野生型相比沒(méi)有明顯差異,只有psbJ的轉(zhuǎn)錄水平上出現(xiàn)了上調(diào)。環(huán)式PCR的實(shí)驗(yàn)結(jié)果表明,野生型中葉綠體psbJ的轉(zhuǎn)錄終止位于終止密碼子后第94和95的核苷酸位置,而ptac15突變體中psbJ的轉(zhuǎn)錄終止除了位于終止密碼子后第94和95的核苷酸位置外,還出現(xiàn)了其他異常的轉(zhuǎn)錄終止位點(diǎn)。這些結(jié)果表明,pTAC15的缺失干擾了葉綠體基因組中psbJ的轉(zhuǎn)錄終止。凝膠阻滯(EMSA)實(shí)驗(yàn)以及體內(nèi)的免疫共沉淀產(chǎn)物qPCR分析表明,pTAC15能夠特異性結(jié)合質(zhì)體基因psbJ的3’端序列。進(jìn)一步的體外生化實(shí)驗(yàn)結(jié)果表明,pTAC15蛋白具有轉(zhuǎn)錄終止的作用。這些研究結(jié)果表明,擬南芥葉綠體蛋白pTAC15作為PEP復(fù)合物中的一員,能夠特異性結(jié)合葉綠體基因psbJ的3'末端,參與該基因轉(zhuǎn)錄終止的作用。
[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.
【學(xué)位授予單位】：上海師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：Q943.2

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