长春花茉莉酸-异亮氨酸合成酶CrJAR1生物信息学分析与原核表达

Bioinformatics analysis and prokaryotic expression of jasmonic acid-isoleucine synthase CrJAR1 from Catharanthus roseus

关键词:  长春花, 茉莉酸-异亮氨酸合成酶, 序列分析, 三级结构预测, 原核表达

Key words:   Catharanthus roseus, jasmonic acid-isoleucine synthase(JAR1), sequence analysis, prediction of tertiary structure, prokaryotic expression

摘要:

长春花(Catharanthus roseus)可以产生多种萜类吲哚生物碱,其中包括多种天然的抗癌药物,但合成含量很低,茉莉酸信号通路可以调控这些萜类吲哚生物碱的生物合成,茉莉酸-异亮氨酸合成酶为茉莉酸信号通路中的关键元件。为了研究其功能,该文以长春花叶片为材料,分析了CrJAR1基因所编码的氨基酸序列,并进行原核表达。结果表明:CrJAR1基因编码了585个氨基酸,该蛋白不存在跨膜区域,定位于细胞质中,且该蛋白不含有信号肽; 进一步系统进化树分析表明, 长春花CrJAR1与笋瓜和番木瓜的JAR1同源性最高; 对二级、三级结构进行预测,发现CrJAR1蛋白主要由α-螺旋构成; 同时,成功构建了pET-30b-CrJAR1重组表达质粒,并经IPTG诱导后在大肠杆菌BL21中异源表达,经16、37 ℃分别诱导至16 h后,均显示出最高的表达量。综上结果,对长春花中CrJAR1蛋白进行生物信息学分析,并成功在大肠杆菌中进行异源表达,这对体外该蛋白功能的研究具有深远影响,为调节茉莉酸信号通路甚至调控长春花中次级代谢产物的生物合成提供了指导

Abstract:

Catharanthus roseus can produce a variety of terpeniod indole alkaloids, including a variety of natural anti-cancer, but the biosynthesis content is very low. The jasmonic acid signaling pathway regulates the biosynthesis of these terpeniod indole alkaloids. The jasmonic acid-isoleucine synthase is a key component in the jasmonic acid signaling pathway. In order to study its function, the amino acid sequence of CrJAR1 was analyzed, and prokaryotic expression was performed. Results had shown that the CrJAR1 gene encoded 585 amino acids, and the protein did not contain transmembrane structure. The subcellular localization analysis showed that the protein might be localized in the cytoplasm, and the protein did not contain signal peptide. Further phylogenetic tree analysis showed that the CrJAR1 had the highest homology with the JAR1 of the Cucurbita maxima and Carica papaya. The secondary and tertiary structures were predicted, and it was found that the CrJAR1 protein was mainly composed of α-helix. In addition, the recombinant expression plasmid pET-30b-CrJAR1 was constructed and successfully expressed in Escherichia coli BL21 after induction by IPTG. Induction at 16 and 37 ℃ after 16 hours, both showed the highest expression levels. In this paper, the bioinformatics analysis of CrJAR1 protein in Catharanthus roseus was successfully carried out and heterologously expressed in Escherichia coli. The study will have deep effect on research of CrJAR1 protein function in vitro, and provide instructive revelation for regulation of jasmonic acid signaling pathway, and even the regulation of the biosynthesis of secondary metabolites in Catharanthus roseus.