【作者】 王琪；

【导师】 闫见敏；

【作者基本信息】 贵州大学 ， 园艺学， 2023， 硕士

【摘要】 质体是植物细胞内特有的一组细胞器,为植物提供必要的代谢和信号转导功能.在植物不同组织和细胞类型中存在不同类型的质体,例如,叶片中的叶绿体,果实中的有色体,根系中的白色体.然而,质体自身基因组仅编码约100个蛋白,其余大约95%(2000-2500种)的质体蛋白都由核基因编码,因此,将核编码质体蛋白翻译后导人质体对质体生物发生和生物学功能发挥是必不可少的.叶绿体外膜转运蛋白(translocon of the outer membrane of chloroplaats,TOC)是位于叶绿体外被膜表面的蛋白转运复合体,介导细胞质中叶绿体蛋白的跨膜运输.目前,有关TOC的研究集中在拟南芥和豌豆中,本研究以番茄(Solanum lycopersicum cv.Alisa Craig)为试验材料.从番茄中鉴定并分析了 10个TOC复合体组分基因;成功克隆了slToc159-2基因;实时荧光定量PCR方法分析了番茄Toc159家族基因在遮光处理下和果实成熟过程中的表达模式;利用原核表达成功获得slToc159-2保守G结构城的蛋白,并制备多克隆抗体,研究了 slToc159-2在番茄果实成熟过程中的蛋白表达模式;构建分裂泛素化酵母双杂文库.对slToc159-2相互作用蛋白进行筛选.确定了 slToc159-2的特异性转运底物,分析了 slToc159-2对不同底物的偏好性,并研究了 slToc159-2的A结构城对蛋白间相互作用的影响.主要研究结果如下:1.本研究利用生物信息学方法鉴定到番茄中的10种TOC复合体亚基组分,可分为 Toc159(slToc159-1.slToc159-2 slToc132.slToc120 slToc90)、Toc34(slToc34-1、slToc34-2)、Toc64(sIToc64-1、slToc64-2)和 Toc75(slToc75)共4个家族.进一步对鉴定到的在蛋白转运过程中具有重要作用的Toc 159家族进行表达模式分析,在遮光处理下slToc159-2表达量没有发生显著变化,slToc159-J和sIToc120的表达量呈现降低趋势;在果实成熟过程中,slToc159-2表达量呈现先上升后下降趋势,并在破色期达到最高,slToc159-1、slToc132和slToc120表达量呈逐渐下降趋势.成功克隆到slToc159-2基因,序列分析表明可分为-A、-G、-M三个结构域,G和M结构城有较好的保守性,A结构域保守性较差.2.选取slToc159-2基因N端409-745个氨基酸之间包括5个保守GTPase基序的cDNA序列,亚克隆至原核表达载体pGEX-6P-1,融合N端GST标签,并转化大肠杆菌BL21(DE3),探索不同诱导条件下目的蛋白表达.GST-slToc159-2400-745重组蛋白在1 mmol·L-1 IPTG,16℃4 h诱导表达效果最好,融合蛋白主要以包涵体形式存在.融合蛋白经过亲合纯化后作为抗原制备多克隆抗体,通过免疫印迹检测slToc159-2在番茄不同成熟度果实中的表达情况,结果显示.slToc159-2在绿熟期表达量较低,在果实成熟过程中表达量升高.3.利用Gateway技术构建了高质量的分裂泛素醇母双杂cDNA文库;首次在番茄中鉴定出41种slToc159-1和slToc159-2的特异性转运底物:并且蛋白互作强度分析表明,slToc159-2与大部分非光合作用蛋白具有较高的亲合力.4.我们比较了两种有或没有A结构域的诱饵蛋白与前体蛋白的亲合力,发现A结构域赋予slToc159-2对不同前体蛋白亲合力的特异性.A结构域的存在降低了 slToc159-2与光合前蛋白的相互作用强度,同样,A结构城的存在也改变了 slToc159对非光合作用前蛋白的亲和力.双分子荧光互补(BiFC)分析表明,A结构域具有识别前蛋白的能力.且相互作用发生在叶绿体内.此外,拟南芥原生质体A结构域的定位表明,A结构城不含叶绿体膜靶向信号,我们的数据证明了高度非保守A域的重要性,它赋予了 sIToc159转运蛋白对不同类型前体蛋白质的特异性识别能力.更多还原

【Abstract】 Plastids are a peculiar group of organelles within plant cells that provide essential metabolic and signal transduction functions to plants.There ate different types of plastids present in different tissues and cell types of plants,e.g.,chloroplasts in leaves,colored bodies in fruits,white bodies in roots.However,the plastid’s own genome encodes only 100 proteins,and the remaining 95%(2000～2500)of plastid proteins are all encoded by nuclear genes.PostttanslationaL import of nuclear encoded plastid proteins into plastids is therefore essential for plastid biogenesis and biological function exertion.Chloroplast outer membrane translocons(TOC)are protein transport complexes located on the outer envelope surface of chloroplasts that mediate the transmembrane transport of chloroplast proteins in the cytoplasm.Currently,studies on TOC focus on Arabidopsis and pea.In this study,10 TOC complex component genes were first identified and analyzed from tomato(Solatium lycopenicum cv.Alisa Craig);The slToc 159-2 gene was successfully cloned;Real time PCR was used to analyze the expression patterns of tomato Toc159 family genes under shade treatment and during fruit ripening;Polyclonal antibodies were generated by prokaryotic expression of a protein containing the conserved G domain of slToc 159-2,which was used as an antigen,and we finally investigated the protein expression pattern of slToc159-2 during tomato fruit ripening;A split ubiquitinated yeast two hybrid library was constructed to screen for slToc 159-2 interacting proteins,specific transport substrates of slToc 159-2 were identified,the preference of slToc 159-2 for different substrates was analyzed and the effect of the A domain of slToc 159-2 on protein-protein interactions was investigated.The main results are as follows:1.In this study,we used bio-informatics to identify 10 TOC complex subunit components in tomato,which can be divided into four families:Toc159(slToc 159-1,slToc159-2,slToc132,slToc120,slToc90),Toc34(slToc34-1,slToc34-2),Toc64(stToc64-1,slToc64-2),and Toc75(siToc75).Further analysis of the expression pattern of the identified Toc159 family,which has an important role in protein transport,showed no significant change in slToc 159-2 expression under shade treatment and a trend towards lower expression of slToc 159-1 and slToc120;During fruit ripening,slToc159-2 expression showed an initial increase followed by a decrease and reached its highest level at the breaker stage,while slToc159-1,stToc132,and slToc120 showed a gradual decrease.slToc159-2 was successfully cloned,and sequence analysis showed that it can be divided into three domains-A,G,and-M,with relatively good conservation of the G and M domains and poor conservation of the A domain.2.The cDNA sequence including five conserved GTPase motifs between N-terminal 409-745 amino acids of slTocl59-2 gene was selected,subcloned into prokaryotic expression vector pGEX-6p-1,fused with N-terminal GST tag,and transformed into E.coli BL21(DE3)to explore the target protein expression under different induction conditions.The GST-SlToc159-2409-745 recombinant protein induced the best expression at 1 mmol·L-1 IFTG,16℃ for 4 h,with the fusion protein mainly in the form of inclusion bodies.The fusion protein was affinity purified and used as an antigen to produce polyclonal antibody.The expression of slToc159-2 in tomato fruits of different ripening stages was detected by immunoblotting.The results showed(hat slToc 159-2 was expressed at a low level during green ripening and increased during fruit ripening.3.A high-quality split ubiquitin yeast two hybrid cDNA library was constructed using gateway technology;41 specific transport substrates of slToc 159-1 and slTocl59-2 were first identified in tomato;And protein interaction intensity analysis showed that slToc 159-2 had high affinity with most of the non-photosynthetic proteins.4.We compared the avidity of two bait proteins with or without the A-domain with that of the precursor protein and found that the A-domain confers specificity of fie avidity of slToc 159-2 for different precursor proteins.The presence of the A-domain decreases the strength of interaction of slToc159-2 with photosynthetic pre proteins.Similarly,the presence of the A-domain also changes the affinity of slToc159 for non-photosynthetic preproteins.Bimolecular fluorescence comptementation(BiFC)analysis showed that the A domain has the ability to recognize preproteins and that the interaction occurs within chloroplasts Furthermone,the localization of the A domain of Arabidopsis protoplasts shows that the A domain does not contain a chloroplast membrane targeting signal.Our data demonstrate the importance of the highly non conserved A-domain which comferm the specific recognition capability of slToc159 transporters for different ypes of precursor proteins.更多还原