Cloning and Expression Analysis of CssHSP18.1 Gene in Camellia Sinensis

JIANG Junmei; FANG Yuanpeng; NING Na; CHEN Meiqing; YANG Zaifu; WANG Yong; LI Xiangyang; XIE Xin

doi:10.13305/j.cnki.jts.2020.03.004

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Journal of Tea Science ›› 2020, Vol. 40 ›› Issue (3) : 328-340. DOI: 10.13305/j.cnki.jts.2020.03.004

Cloning and Expression Analysis of CssHSP18.1 Gene in Camellia Sinensis

JIANG Junmei¹, FANG Yuanpeng¹, NING Na¹, CHEN Meiqing¹, YANG Zaifu¹, WANG Yong¹, LI Xiangyang^2,*, XIE Xin^1,*

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Abstract

The sHSPs gene family encodes a class of small molecular heat shock proteins, which are widely distributed in plants, functioned as molecular chaperones, and play an important role in plant resistance to stresses. In this study, the open reading frame (ORF) of CssHSP18.1 gene cDNA was obtained by gene cloning, which is 480 bp in length and encodes 159 amino acids. Bioinformatics analysis showed that CssHSP18.1 protein contained a typical HSP20 domain. Its molecular weight and isoelectric point are about 18.25 kDa and 5.68 respectively. Phylogenetic tree analysis showed that CssHSP18.1 has the closest relationship with quercus and apple. It was predicted that CssHSP18.1 protein was does not have signal peptide and transmembrane structure. RT-qPCR analysis showed that the expression of CssHSP18.1 under D-Mannitol treatment was lower than that in the control group. GABA could enhance the expression of CssHSP18.1 with its peak at 1 h after GABA treatment. The expression of CssHSP18.1 was upregulated upon IAA and PEG 6000 treatments, and reached the peaks at 0.5 h. Thus, GABA、IAA、PEG 6000 could induce the expression of CssHSP18.1. To obtain CssHSP18.1 soluble protein, a recombinant plasmid pET-28a-CssHSP18.1 was constructed and expressed in prokaryotic system. The expression strains, induction temperatures and induction concentrations of IPTG (isopropyl- -D-thiopyranogalactoside) were optimized. The results showed that the best expression strain of CssHSP18.1 protein was BL21 (DE3), and the best induction temperature and IPTG concentration were 30℃ and 1.2 mmol·L^-1 respectively. Finally, western blot was used to verify the expression of CssHSP18.1 protein. This study provided a basis for further study on the biological function of CssHSP18.1 gene.

Key words

CssHSP18.1 / bioinformatics analysis / Camellia sinensis / expression analysis / gene cloning / stress

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JIANG Junmei, FANG Yuanpeng, NING Na, CHEN Meiqing, YANG Zaifu, WANG Yong, LI Xiangyang, XIE Xin. Cloning and Expression Analysis of CssHSP18.1 Gene in Camellia Sinensis[J]. Journal of Tea Science. 2020, 40(3): 328-340 https://doi.org/10.13305/j.cnki.jts.2020.03.004

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