| 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. |
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