| A group of four genes (cpcS, cpcT, cpcU, and cpcV) had been identified inSynechococcus sp. PCC7002that code for lyases that attach PCB to cysteine-84(consensus numbering) of the β-subunits of phycobiliprotein by Shen G in2004. Inthis study, the gene sll0853had highly homology with cpcS, it was found byhomology analysis software. In this thesis, the function of the gene sll0853in thechromosome of Synechocystis sp. PCC6803were studied. It includes:1) The gene sll0853was screened and amplified by PCR., then the sll0853genewas connected with cloning vector, pMD18-T and a prokaryotic expression vector,pET-30a. The protein structure of gene sll0853was analysed by bioinformatics. Inorder to improve the expression level of sll0853in E.coli BL21(DE3), optimalconditions for expression of fusion protein were analyzed by SDS-PAGE at thedifferent concentration, temperature and time of IPTG.2) The cpcB gene was cloned from Synechocystis sp. PCC6803, then two genessll0853and cpcB connected to co-expression vector pACYC-Duet, recombinantvector pACYC-cpcB-0853was build successfully. The gene hol and pcyA geneco-express in the E. coli and PCB was synthesized in the E. coli, pET-hol-pcyA whichwas kept in our laboratory. In order to improve the expression level of the vitroreconstitution system, optimize conditions for expression of pACYC-cpcB-0853andpET-ho1-pcyA were carried out.3) PACYC-cpcB-0853and pET-ho1-pcyA were co-transformed into E.coli, Theco-expression products of vivo recombination system in E. coli was studied bySDS-PAGE protein electrophoresis and fluorescence spectrum.4) In order to study the function of gene sll0853, sll0853gene fragment wasdigested and replaced with a kanamycin resistance gene fragment, then mutantplasmid was transferred to Synechocystis sp. PCC6803by natural transform. Positivetransformants obtained by kanamycin resistance screening. Though cultivated, themutant PCC6803was successfully constructed. Mutant and wild type were studied bydifferent light intensity and the same BG11medium.The gene sll0853was found from NCBI and function of the gene was unknown.According to the above research, we can conclude: CDD’s analysis showed that thesll0853amino acid’s sequence from12to180had highly conservation with the CpeSprotein family, the protein of sll0853probably had nature of CpeS through theprediction of bioinformatics. Optimal conditions for expression of sll0853protein were analyzed by SDS-PAGE at different concentration, temperature and time ofIPTG. The results showed that the fusion protein expression of recombinant plasmidin E.coli BL21(DE3) was the best at0.2mM of IPTG for6h at28℃. The SDS-PAGEresults showed that the protein expression of recombinant plasmidpACYC-cpcB-0853in E.coli BL21(DE3) was the best at0.2mM of IPTG for5h at22℃. The protein expression levels of the recombinant plasmid pET-hol-pcyA wasbest at0.6mM IPTG at22℃in E.coli BL21(DE3). Electrophoresis also showed thatthe protein expression levels of pET-hol-pcyA were the same at different times ofprotein expression levels. Optimize conditions for expression of pACYC-cpcB-0853and pET-ho1-pcyA were innovation. The expression products of in vivorecombination system in E. coli were studied by SDS-PAGE protein electrophoresisand fluorescence spectrum, the electrophoresis result of SDS-PAGE had no band inthe corresponding position, fluorescence spectrum also didn’t have any response at620nm, so the results showed that the gene sll0853had no lyase function. In order tostudy the function of gene sll0853, mutant was build. OD value of mutant and wildstrains were tested in BG11medium and different light intensity, the results showedthat the growth of the mutant was significantly slower than the wild-type growth inthe same BG11medium, and the growth rate of the mutant strain was significantlyslower than the wild-type growth rate in the same lighting conditions. |
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