[关键词]
[摘要]
目的 对白木香Aquilaria sinensis AsWRKY62转录因子进行分子克隆、原核表达,并对其进行生物信息学分析和表达特性分析,为深入研究AsWRKY62在白木香生长发育、沉香形成等方面的功能奠定基础。方法 以白木香愈伤组织总RNA反转录的cDNA为模板,采用RT-PCR及PCR技术克隆基因编码序列(CDS)全长;构建pET-21a-AsWRKY62原核表达载体,转化大肠杆菌BL21 (DE3) 感受态进行原核诱导表达;并通过生物信息学软件预测蛋白的理化性质、结构域和亚细胞定位等特性;用软件DNAMAN和MEGA 5分别进行氨基酸多序列比对和进化关系分析,通过组织特异性表达分析不同组织中的基因表达模式。结果 AsWRKY62(GenBank注册号MH925301)基因CDS全长为1 581 bp,编码一条由526个氨基酸组成的多肽,结构域分析表明其属于WRKY group I类蛋白;密码子优化后的pET-21a-AsWRKY62在大肠杆菌BL21 (DE3) 中成功诱导的较适条件为0.5 mmol/L IPTG 37℃连续培养4 h;AsWRKY62基因具有组织特异性,其在根、茎中表达量最高,其次是沉香层和花中。结论 本研究首次在白木香中克隆AsWRKY62,并进行原核表达和生物特性分析,表明其可能与沉香的形成有关,也为进一步研究其生物学功能提供了理论依据。
[Key word]
[Abstract]
Objective The molecular cloning and prokaryotic expression of the transcription factor AsWRKY62 of Aquilariasinensis were carried out, at the same time, the bioinformatics analysis and expression pattern analysis were also performed. The purpose of this study was to lay a foundation for further study on the role of AsWRKY62 in the growth and development of A. sinensis and the formation of agarwood. Methods With the cDNA isolated from A. sinensis callus as template, the full-length coding sequence (CDS) of AsWRKY62 was amplified using RT-PCR and PCR method. The recombinant vector pET-21a-AsWRKY62 which was built and verified by gene recombination technique was transformed into E. coli BL21 (DE3) for prokaryotic expression and purification. The characteristics of physiochemical properties, conserved domains and subcellular localization of AsWRKY62 were calculated by a series of bioinformatics tools. The analyses of multiple sequence alignment of amino acid and phylogenetic tree were performed using DNAMAN and MEGA 5.0, respectively. The gene expression pattern in different tissues was detected by RNA-seq data. Results The full length CDS of AsWRKY62 (GenBank accession MH925301) was 1581 bp, encoding a 526-aa protein which belongs to WRKY group I. The optimized induction conditions of recombinant pET-21a-AsWRKY62 were 0.5 mmol/L IPTG at 37℃ for 4 h. According to the tissue-specific expression pattern analysis, the AsWRKY62 gene in A. sinensis is mainly expressed in roots and stems, followed by agarwood and flowers. Conclusion Cloning, expression and characterization of the AsWRKY62 gene for the first time indicated that it may be related to the formation of agarwood, which provided a theoretical basis for further study of its biological function.
[中图分类号]
[基金项目]
国家自然科学基金资助项目(81673549);国家自然科学基金资助项目(81573525);海南省重大科技专项(ZDKJ2016004);中组部“万人计划”(99950534)