| Phycocyanin, a kind of chromoprotein, from Arthrospira platensis and otheralgae can absorb and transfer light energy. It has high application value not onlybecause it has antioxidate and anti-tumor properties, but also it is a natural pigment tobe widely used in medicine, food, cosmetics and other industries.Heme oxygenase1is a key enzyme in the expression of Phycocyanin with optical activity. It catalyzesheme to generate biliverdin, and then biliverdin is converted to natural chromophore-phycocyanobilin, which is attached to apoprotein to produce the Phycocyanin withoptical activity under the catalyzation of the heterodimeric lyase. Therefore, toexpress of fluorescent Phycocyanin in heterologous hosts, cloning hox1gene andstuding its fuction should be carried out at first.In this study, hox1gene is firstly cloned form Athrospira platensis FACHB314by Genome Walking.By analysis of open reading frame, the complete hox1gene is729bp, which coding242amino acids,with the molecular weight of27.7kD and pI5.54. There are108hydrophobic amino acids,67polar amino acids,33basic aminoacids and34acidic amino acids in the deduced amino acid sequence.-helix is themajor part of the secondary structure of Hox1, accounting for48.76%, mainly in themiddle and the C-terminal by analysis of SMART software. By analysis of Blastp, itis found that Hox1contains conserved domains of heme oxygenase, including eightconserved sites making up the binding pocket of the heme oxygenase. By usingMAGA5.0, Phylogenetic tree (NJ tree) of Hox1was constructed, and the highestsimilarity of amino acid sequence is found up to97%between Arthrospira maximaCS-328reported in GenBank and Athrospira platensis FACHB314and they cluster inone branch.On the base of cloning of hox1gene, one expression vector pET-hox1(314)-pcyA is constructed.The other is pACYCDuet-ussBcpcBA-cpcE-cpcF,containing gene ofphycocyanin subunit and lyase.The two expression vector were transformed into E.coli BL21(DE3) collectively. Positive clone of E.coli is blue-green and is detected tocontain the characteristic peak of phycocyanin at635nm by fluorescence spectroscopy.It is indicated that the gene cloned from Athrospira platensis FACHB314couldexpress heme oxygenase, which can catalyze heme to biliverdin and futher producephycocyanobilin.In order to deply study the founction of Hox1，it is needed to establish theefficient and stable heterologous expression and treatment conditions of phycocyanin.Therefore，in this experiment，the impacts of different solvent conditions and differentexpression conditions for recombinant strains on fluorescence spectra were studied.The results show that0.01mol/L PBS（pH7.0) is the best solvent for fluorescencedetection of phycocyanin at635nm.After single factor pre-experiment of temperature,IPTG concentration and time, orthogonal experiment including three factors and threelevels were designed: temperature (20℃,28℃,37℃), the IPTG (0.1mmol/L,1mmol/L,10mmol/L) and time (2h,6h,10h). By detection the expression production andfluorescence intensity of phycocyanin, the best inducement condition for phycocyaninis in0.1mmol/L IPTG at37℃for2hours.On this basis, in order to discover and analysis the activity sites of hox1genefrom Athrospira platensis FACHB314, a series of sites of hox1gene were mutated:methionine (M, a non-polar amino acid) at146amino acid site was changed tothreonine (T, polar amino acids), phenylalanine (F, non-polar amino acids) at156amino acid site was replaced by tyrosine (Y, polar amino acids).The mutant strainsalmost can not exhibit blue-green color. It is indicated that146and156amino acidsites are important activity sites of Hox1and mutation of the two amino acids canseverely affected the function of Hox1. In addition, in the experiment, tworecombinant expression strains of E.coli/uBAEF (314), E.coli/uBA (314) wereconstructed containing hox1gene from Arthrospira platensis FACHB314.Recombinant expression strains of E.coli/uBAEF(6803) and Ecoli/uBA(6803) wereconstructed containing hox1gene from Synechocystis sp. PCC6803in previous work of my lab. The fluorescence intensity of phycocyanin between the fourrecombinant expression strains are significant different. Phycocyanin fluorescenceintensity of E.coli/uBAEF (314) is23.0%higher than that of E.coli/uBAEF(6803).And E.coli/uBA(314) is55.9%higher than that of E.coli/uBA(6803). It is indicatedthat the activity of Hox1from Arthrospira platensis FACHB314is higer than thatfrom Synechocystis sp. PCC6803in recombinant synthesis of fluorescentphycocyanin of Athrospira platensis FACHB314.This paper first cloned heme oxygenase gene (hox1) from Arthrospira platensisFACHB314, which was proved to have the function in expression of opticalphycocyanin in E.coli. This research laid an important foundation for the heterologousexpression and application of phycocyanin with optical activity. |
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