丹参分支酸变位酶基因的克隆和生物信息学分析

Cloning and bioinformatics analysis of chorismate mutase gene from Salvia miltiorrhiza

应用BlastX检索丹参毛状根的EST数据库,发现1条与分支酸变位酶(chorismate mutase,CM)高度同源的序列,克隆得到其全长cDNA,命名为SmCM1,Genbank登录号为KC784342。SmCM1全长948 bp,理论pI 6.41,与矮牵牛Petunia×hybrida、拟南芥Arabidopsis thaliana和毛果杨Populus trichocarpa的CM1分别具有70%,72%,64%的相似性。实时荧光定量PCR(real time quantitative PCR,QRT-PCR)分析表明SmCM1在丹参叶中的含量较高,其次是茎,在根中的含量相对较低。酵母诱导子(YE)和离子(Ag+)联合诱导丹参毛状根后,SmCM1与莽草酸途径的关键酶3-脱氧-D-阿拉伯-庚酮糖-7-磷酸合酶(3-deoxy-7-phosphoheptulonate synthase,DAHPS)和分支酸合酶(chorismate synthase,CS)基因表达趋势呈协同变化。YE+Ag+处理后3个基因皆上调表达,8 h达到表达高峰,分别是对照的7.9,5.5,9.8倍,随后下调,36 h时下降至对照水平以下,说明诱导处理之后糖代谢中间产物经莽草酸和分支酸途径合成苯丙氨酸和酪氨酸从而引起酚酸类化合物的过量积累。

Chorismate mutase catalyzes the conversion of chorismate to prephenate that is the first committed step in the biosynthesis of the aromatic amino acids phenylalanine and tyrosine. A chorismate mutase gene, designated SmCM1, was isolated from Salvia miltiorrhiza by using RT-PCR. The full length of SmCM1 cDNA consists of 948 nucleotides and has an open reading frame of 765 bp. The deduced amino acid sequence of SmCM1 has 255 amino acid residues which forms a 36.0 kD polypeptide with calculated pI of 6.41 as expected. The putative polypeptide contains a CM_2 super family function domain. Blast W results showed that SmCM1 had 70% of the similarity with Petunia×hybrid CM, 72% of the similarity with Arabidopsis thaliana CM, and 64% of similarity with Populus trichocarpa CM. The transcription level of SmCM1 in root, stem and leaf was analysed by realtime quantitative PCR. The results showed the expression level of the SmCM1 in leaf was highest, and lowest in root. Yeast extract and silver ion joint induction could markedly stimulate the increase of mRNA expression of SmCM1 and its upstream 3-deoxy-7- phosphoheptulonate synthase （DAHPS） and chorismate synthase （CS）. It was 7.9, 5.5 and 9.8 times of control on 8 h after induction, respectively.