Molecular cloning, prokaryotic expression and characterization of a transcription factor AsWRKY62 in Aquilaria sinensis
10.7501/j.issn.0253-2670.2019.11.026
- Author:
Pei-Wen UN
1
Author Information
1. Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education & National Engineering Laboratory for Breeding of Endangered Medicinal Materials, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College
- Publication Type:Journal Article
- Keywords:
Aquilaria sinensis (Lour) Gilg;
Characteristic analysis;
Prokaryotic expression;
Transcription factor;
WRKY
- From:
Chinese Traditional and Herbal Drugs
2019;50(11):2675-2682
- CountryChina
- Language:Chinese
-
Abstract:
Objective: The molecular cloning and prokaryotic expression of the transcription factor AsWRKY62 of Aquilaria sinensis 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.
