Cloning And Functional Analysis Of The Key Genes Associated With Anthocyanin Biosynthesis From Lycium Ruthenicum And Iris Lacteavar Chinensis

Anthocyanin is an important secondary metabolite produced by plant flavonoid biosynthesis,which are widely existed in the plant vacuole.It is the main pigments usually found in flowers,fruits and vegetables.The pericarps of Lycium ruthenicum Murr.and the petalis of Iris lactea Pall.var chinensis(Fisch.)Koidz(hereinafter referred to as I.lactea var chinensis)have high contents of anthocyanins,but their mechanisms of the regulation of the anthocyanin biosynthesis have not yet been reported.In the present research,the total RNAs were extracted from the L.ruthenicum pericarp and the I.lactea var chinensis petalis for transcriptome sequencing.The key genes associated with anthocyanin biosynthesis were screened,isolated and their bioinformatics were analyzed.The gene encoding dihydroflavonol 4-reductase(DFR)from I.lactea var chinensis was further characterized by construction of the gene into a prokaryotic expression vector and its enzymatic activity was assayed in vitro.Its substrate specificity was investigated by comparison of those DFRs from I.hollandica,Petunia×xhybrid and Nicotiana tabacum.The results are as follows:1.According to the analysis results of transcriptome sequencing,seven key genes encoding enzymes that associated with anthocyanin biosynthesis from L.ruthenicum,including chalcone synthase(CHS),chalcone isomerase(CHI),clavonone 3-hydroxylase(F3H),flavonoid 3'-hydroxylase(F3'H),flavonone 3'5'-hydroxylase(F3'5'H),dihydvroflavonol 4-reductase(DFR)and anthocyanidin synthase(ANS)(referred to as LrCHS,LrCHI,LrF3H,LrF3'H,LrF3'5'H,LrDFR and LrANS),as well as one key gene encoding DFR that associated with anthocyanin biosynthesis from I.lactea var chinensis(referred to as IlDFR)were screened,isolated and their bioinformatics were analyzed.These genes were submitted to NCBI GenBank.2.The IlDFR gene,along with three kinds of DFR genes from I.hollandica,Petunia×hybrid and Nicotiana tabacum(referred to as IhDFR,PhDFR and NtDFR)was respectively constructed into a prokaryotic expression vector pTrc-CKS(containing 6×His-Taq downstream the multicloning site)and was expressed in Escherichia coli JM109.The induced proteins(enzymes)were purified using Ni-Sepharose packed column.3.The four kinds of purified proteins were enzymatically assayed with three kinds of substrates,dihydroquercetindihydroquercetin(DHQ),dihydrokaempferol(DHK)and dihydromyricetin(DHM)in vitro respectively under nicotinamide adenine dinucleotide phosphate(NADPH),and were measured by HPLC.The IlDFR protein was found that could effectively catalyze both DHK and DHM,but could hardly catalyze DHQ,while the reduction efficiency of DHM was higher than that of DHK.The IhDFR protein could effectively catalyze DHQ,DHK or DHM,and the reduction efficiency of DHK and DHM is higher than that of DHQ.The proteins of PhDFR and NtDFR could catalyze all three substrates,DHQ,DHK and DHM.The DFRs amino acid region that determined substrate specific in IlDFR and IhDFR were "TVKRVVFTSSAGTVD VKEHQQMEYDESSWSDVEFCRRV",while those of PhDFR and NtDFR were"TVKRLVFTSSAGTLDVQEQQKLFYDQTSWSDLDFIYAK" and "TVKRLVFTSSAGTLDAQ EHQKLFYDETSWSDLDFIYAK",respectively.The third amino acid residue in all proteins were lysine(K)with negative charged,theoretically,mainly catalyze DHQ and DHM,but not DHK.However,the four proteins had different substrate preferences and reduction efficiencies,indicating that the neighbouring residues in the region are involved in the formation of the substrate-binding pocket play important roles in determining DFR substrate preferences.The research provides new gene resources and a basis for colour modification of flowers,fruits and vegetables using molecular biology and genetic engineering techniques.