马蓝IGPS基因的克隆、表达分析及原核表达

Cloning,Expression Analysis and Prokaryotic Expression of IGPS from Baphicacanthus cusia

吲哚-3-甘油磷酸合酶(indole-3-glycerol phosphate synthase,IGPS)是广泛参与生物体内色氨酸、生长素等吲哚化合物合成途径中重要的关键酶之一。为了研究BcIGPS在马蓝吲哚类生物碱合成中的作用,基于马蓝转录组数据,通过RT-PCR技术从马蓝中克隆得到IGPS基因序列,命名为BcIGPS;利用生物信息学分析BcIGPS序列特性;运用qPCR分析BcIGPS在马蓝不同器官及外源诱导处理下的时空表达情况;构建pET-32a-BcIGPS原核表达载体,并优化诱导表达条件。结果表明:BcIGPS(GenBank登录号:MT210517)全长为1176 bp,包含1个开放阅读框(ORF),编码392个氨基酸,具丝氨酸(Ser)、苏氨酸(Thr)、酪氨酸(Tyr)磷酸化位点31个,无跨膜结构,无信号肽,亚细胞定位于叶绿体中。BcIGPS含有product(indole)活性结构域和IGPS、TrpC特异性位点。qPCR分析结果显示,BcIGPS基因在不同器官的相对表达丰度依次为:叶>茎>花>根;其响应茉莉酸甲酯(MeJA)、脱落酸(ABA)、水杨酸(SA)、乙烯利(ETH)外源诱导信号的诱导,经MeJA和ETH处理后,分别在24 h和36 h最高,为初始水平的5.53、6.87倍;在SA处理下,BcIGPS表达响应最为强烈,呈先骤升后骤降的变化趋势,在12 h最高达初始水平的33.13倍;ABA处理后,其表达量变化不显著。所构建的pET-32a-BcIGPS原核表达载体在大肠杆菌BL21中表达,其最适条件为37℃、0.4 mmol/L IPTG培养3 h,BcIGPS重组蛋白主要以包涵体形式存在,且蛋白分子量与预测相符。

Indole-3-glycerol phosphate synthase(IGPS) is one of the most key enzymes involved in the synthesis of indole compounds such as tryptophine and auxin in organisms. In order to study the function of BcIGPS in the synthesis of the indole alkaloids from Baphicacanthus cusia, based on the transcriptome data of B. cusia, the gene sequence of IGPS was obtained by RT-PCR, the BcIGPS sequence characteristics was analyzed by bioinformatics. qPCR analysis was used to analyze the spatiotemporal expression of B. cusia in different organs and exogenous induction. The prokaryotic expression vector of pET-32 a-BcIGPS was constructed and the conditions were optimized for inducing expression. Results showed that BcIGPS(GenBank login number: MT210517) contained an open reading frame(ORF) of 1176 bp, encoded 392 amino acids without transmembrane structure and signal peptide. There were 31 phosphorylation sites for Ser, Thr and Tyr. Subcellular localization prediction showed that BcIGPS was located on the chloroplasts. And BcIGPS contained product(indole) active structure domain and specific sites of IGPS and TrpC. The results of qPCR showed that the relative expression abundance of BcIGPS in different organs was leaf>stem>flower>root. It responsed to the induction of exogenous MeJA, SA, ETH. After the treatment of MeJA and ETH, the maximum value was up to 5.53, 6.87 times at 24 h and 36 h respectively. BcIGPS showed the strongest response to SA treatment, the expression level increased quickly and then decreased sharply, more 33.13 times than the initial level at 12 h. After ABA treatment, there was no significant change in the expression level. The prokaryote expression of pET-32 a-BcIGPS was constructed successfully and the optimal conditions for expression in E. coli BL21 was 37 ℃, 0.4 mmol/L IPTG and cultured for 3 h. The recombinant BcIGPS protein mainly existed in the form of inclusion body, and the molecular weight of BcIGPS was consistent with the prediction.