桔梗萜类合成途径PgMCS和PgHDR基因克隆与原核表达分析

Gene cloning and prokaryotic expression analysis for the PgMCS and PgHDR genes from the Platycodon grandiflorum terpenoid biosynthesis pathway

为了研究桔梗三萜类化合物生物合成途径中的关键酶,根据桔梗转录组数据中的基因序列信息,通过RT-PCR方法从桔梗根中克隆得到了2-C-甲基-D-赤藓糖醇2,4-环二磷酸合酶(2-C-methyl-D-erythritol2,4-cyclodiphosphate synthase,MCS)和1-羟基-2-甲基-2-(E)-丁烯基4-二磷酸还原酶[1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase,HDR]基因PgMCS和PgHDR,进行序列分析和原核表达分析。结果表明PgMCS和PgHDR的开放阅读框(ORF)长度分别为738和1416 bp,分别编码245和471个氨基酸,相对分子质量分别为26277.53和52984.17;PgMCS和PgHDR蛋白理论等电点分别为7.71和5.92。PgMCS具有PLN02862(MECDP合酶)家族的保守结构域,PgHDR具有PLN02821(HMBPP还原酶)家族的保守结构域。原核表达结果显示,IPTG均可成功诱导目的蛋白表达,分子质量分别为26和60 kDa。基因表达结果显示,PgMCS和PgHDR基因的表达水平在叶中最高,其次是茎和根。本文首次克隆并分析了桔梗PgMCS和PgHDR基因,成功建立原核表达体系,为进一步研究桔梗三萜类化合物生物合成的分子机制提供理论依据。

In order to identify the key enzymes in the triterpenoid biosynthesis pathway of Platycodon grandiflorum,according to the gene sequence information from the P.grandiflorum transcriptome data,genes encoding 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase(MCS)and 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase(HDR)were cloned from the roots of P.grandiflorum by RT-PCR,named as PgMCS and PgHDR,respectively.Sequence analysis and prokaryotic expression analysis were performed.The open reading frame(ORF)lengths of PgMCS and PgHDR are 738 and 1416 bp,encoding 245 and 471amino acids,and their relative molecular masses are 26277.53 and 52984.17,respectively.The theoretical isoelectric points of PgMCS and PgHDR are 7.71 and 5.92.PgMCS owns the conserved domain of PLN02862(MECDP synthase)family,and PgHDR has the conserved domain of PLN02821(HMBPP reductase)family.Prokaryotic expression results showed that IPTG induction can successfully induce the expression of the target protein,with molecular masses of 26 and 60 kDa,respectively.In this paper,the PgMCS and PgHDR genes of P.grandiflorum have been cloned and analyzed for the first time,and the prokaryotic expression system has been successfully established,which ground a theoretical basis for further research on the molecular mechanism of the biosynthesis of P.grandiflorum triterpenoids.