RNA-seq-based Analysis Of Indigo CYP450 Gene Family And Preliminary Study On Genes Related To Glucosinolate Synthesis

Isatis indigotica is a kind of biennial herbaceous plant belongs to Isatis of Cruciferae,containing indigo,indirubin,epigoitrin,trytanthrin,sitosterol,adenosine,glucosinolate and other active ingredients.Glucosinolate(GS)is an important secondary metabolite in Isatis indigotica,which has anti-tumor activity,and mainly involvs in plant defensive reactions.CYP79s and CYP83s were the key genes in the glucosinolate synthesis pathway which belongs to P450 family.In this study,Illumina RNA-seq platform was implemented to construct a transcriptome database from the leaves of Isatis indigotica treated with different elicitors(MeJA,YE,Ag+).Cytochromes P450(CYP450)family genes of Isatis indigotica were developed base on the transcriptome database.Four CYP450 family genes involved in glucosinolate synthesis,including IiCYP79B2,IiCYP79F1,IiCYP83A1 and IiCYP83B1 were cloned.In order to explore the biological function of these genes,the bioinformatic analysis,expression patterns and prokaryotic expression were also carried out.The obtained results will provide theoretical basis to understand the functional diversity and evolutionary status of the CYP450 genes in Isatis indigoticas and aslo provide molecular basis to study the glucosinolate metabolic pathways.The main results were as follows:(1)The transcriptome of Isatis indigotica was investigated with Illumina HiSeq2500.A total of 58,920 unigenes were produced from a 42.21 Gb transcriptome data.There are 108 CYP450 unigenes identified.All the CYP450 unigenes were screened against COG,GO,NR,KEGG,and 72,105,107,and 53 annotations were found,respectively.The coding sequence(CDS)prediction results showed that,42 CYP450 unigenes had complete CDS,and belonged to 23 families and 31 subfamilies.42 CYP450 CDS encoded 458-580 amino acid residues.Furthermore,the molecular weight of 42 CYP450 proteins ranged from 52 to 65 kDa,and most of them located in the endoplasmic reticulum membrane.Protelin sequence alignment revealed the typical conserved CYP450 motifs,including the heme binding motif,K-helix,I-helix and PERF motif.(2)The IiCYP79B2(Genbank accession number:KY774688),IiCYP79F1(Genbank accession number:KY774689),IiCYP83A1(Genbank accession number:KX528206)and IiCYP83B1(Genbank accession number:KX259478)involved in glucosinolate synthesis had been cloned from Isatis indigotica.Sequence analysis of IiCYP79B2 indicated that the ORF was 1,629 bp,and encoded 542 amino acids with molecular weight of 61.58 kDa and isoelectric point(pI)of 8.61.IiCYP79B2 was mainly located in the chloroplast thylakoid membrane,and had a close relationship with Sinapis alba(081345.1).Sequence analysis of IiCYP79F1 indicated that the ORF was 1,626 bp,and encoded 541 amino acids with molecular weight of 61.41 kDa and isoelectric point(pI)of 8.80.IiCYP79F1 was mainly located in the endoplasmic reticulum membrane or plasma membrane,and had a close relationship with Eruca saliva(AGS49166.1).Sequence analysis of IiCYP83A1 indicated that the ORF was 1,512 bp,and encoded 503 amino acids with molecular weight of 57.64 kDa and isoelectric point(pI)of 8.09.IiCYP83A1 was mainly located in the endoplasmic reticulum membrane,and had a close relationship with Brassica oleracea(AIK28472.1).Sequence analysis of IiCYP83B1 indicated that the ORF was 1,500 bp,and encoded 499 amino acids with molecular weight of 56.88 kDa and isoelectric point(pI)of 8.79.IiCYP83B1 was mainly located in the endoplasmic reticulum membrane,and had a close relationship with Raphanus sativus(AHB11193.1).(3)Quantitative real-time PCR(qRT-PCR)were used to analysis the expression levels of glucosinolate synthesis genes(IiCYP79B2?IiCYP79F1?RCYP83A1 and IiCYP83B1)in different organs,different development stages and treated with different elicitors.The results indicated that the four glucosinolate synthesis genes were expressed in all test tissues but showed different expression levels in the highest in leaf and stem,followed by flower,fruit and root.The four glucosinolate synthesis genes were expressed throughout leaf development with different levels.They had relatively higher expression levels during the seedling,vegetative growth and flowering period,compared with the germination period.The profiles of the four glucosinolate synthesis genes under the different elicitors induction showed that they were regulated at the transcription levels.The four glucosinolate synthesis genes transcription level were effectively up-regulated by MeJA(500 ?M),glucose(90%,m/v)and mechanical wound,but significant down-regulated by SA(300 ?M)and cold(4?).Moreover,the expression of IiCYP79B2 and IiCYP79F1 were induced by Ag+,while the expression of IiCYP83A1 and IiCYP83B1 were initial inhibited and later induced by Ag+.(4)The prokaryotic expression of IiCYP79B2,IiCYP79F1?IiCYP83A1 and IiCYP83B1 were also discussed.They were linked with the expressive vector pET-28a,and carried out in E.coli BL21(DE3),respectively.The research on optimizing the expressive condition of pET-28a-IiCYP83A1 showed that the optimal induction conditions were as followes:the concentration of 0.5 mM for IPTG,temperature of 30?and time of 6 h.Furthermore,the pET-28a-IiCYP83A1 and pET-28a-IiCYP83B1 could express their target proteins under the optimal conditions,and the recombinant protein existed as inclusion bodies.Nevertheless,pET-28a-IiC17P79B2 and pET-28a-IiCYP79F1 recombinant protein were not detected almost in BL21(DE3).It could be that IiCYP7982 and IiCYP79F1 contained a high proportion of rare codons,and influenced the normal expression of the recombinant protein.