Cloning, Sequence Analysis and Prokaryotic Expression of CmWRKY from Castanea mollissima BL

doi:10.7668/hbnxb.201751408

Abstract

Abstract: The EST sequence of WRKY gene was obtained based on the transcriptome database of Castanea mollissima BL, and the cDNA(CmWRKY)sequence of WRKY transcription factor in chestnut was cloned by RT-PCR. The information of CmWRKY gene and its encoding protein were analyzed and the gene expression in the prokaryotic cell was carried out. The results showed that CmWRKY gene was 1 437 bp in length, encoding 479 amino acids with a calculated molecular weight of 52.128 96 ku and theoretical isoelectric point of 9.15. It had two WRKY conserved domains and two C2H2 zinc finger domains, belonging to Group Ⅰ of the WRKY family, and its gene accession number was KY312850.1. At the nucleotide level, the similarity of the CmWRKY gene with the corresponding sequence of Prunus persica was over 80%, and the highest similarity with the Qurcus suber WRKY gene was 97%. Homology modeling indicated that the tertiary structure of CmWRKY protein was similar to WRKY1 protein of Arabidopsis thaliana, suggesting that it might have the similar regulatory function as AtWRKY1 did. Molecular evolution analysis showed that chestnut CmWRKY was closely related to the WRKY of Qurcus suber and Juglans regia. SDS-PAGE analysis showed that the optimal expression condition of CmWRKY protein was 0.2 mmol/L IPTG induced at 30℃ for 10 h, and the molecular weight of the protein was about 56 ku, mainly in the form of soluble protein. The results laid the foundation for further studying the biological function and application of CmWRKY gene.

Key words: Castanea mollissima BL, CmWRKY, Gene cloning, Sequence analysis, Prokaryotic expression

CLC Number:

LIU Yufeng, ZHU Tianhui, LIU Yinggao, QIAO Tianmin, LI Shujiang, LONG Xumei, HAN Shan. Cloning, Sequence Analysis and Prokaryotic Expression of CmWRKY from Castanea mollissima BL[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2019, 34(4): 37-45. doi: 10.7668/hbnxb.201751408.

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