Molecular Cloning and Expression of a Cysteine Gene in Empoasca onukii

YU Yongchen; XIAO Bin; SUN Xiaoling

doi:10.13305/j.cnki.jts.2018.03.006

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Journal of Tea Science ›› 2018, Vol. 38 ›› Issue (3) : 263-270. DOI: 10.13305/j.cnki.jts.2018.03.006

Molecular Cloning and Expression of a Cysteine Gene in Empoasca onukii

YU Yongchen^1,2,3, XIAO Bin^1,*, SUN Xiaoling^2,3,*

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Abstract

Cysteine proteinases are important proteolytic enzymes for herbivorous insects. In this paper, the transcript sequence of Eocyp was obtained from the transcriptome database of Empoasca onukii. The full-length cDNA sequence of Eocyp was then cloned by RACE. The theoretical amino acid sequence of Eocyp was analyzed by bioinformatics. The expression levels of Eocyp in different developmental stages, different parts of female and male adults of E. onukii and nymphs under different temperatures and photoperiods were detected by qRT-PCR. The full-length cDNA sequence of Eocyp contained an open reading frame of 1β656βbp, which encoded 551 amino acids and had a signal peptide consisting of 24 amino acid residues. Its molecular formula was predicted to be C₂₇₈₂H₄₁₆₃N₇₃₇O₈₃₅S₂₅. Its relative molecular weight and the theoretical isoelectric point were estimated to be 62.09βkD and 6.02, and the instability coefficient of Eocyp was 36.10. Eocyp had the typical feature of cysteine protease, a catalytic triad Cys³⁵⁷-His⁴⁹⁹-Asn⁵¹⁹, and the conserved domains such as ERFNIN, GNFD amino acid residues and the GCDGG clusters. The amino acid sequence of Eocyp had a high similarity with the CYP gene of Halyomorpha halys. Expression analysis showed that Eocyp was expressed in all developmental stages of E. onukii with its peak in the fifth instar nymphs. Higher expression of Eocyp was observed in the abdomen of adult E. onukii, irrespective of female and male. However, the expression level of Eocyp did not respond to the variation of temperatures and photoperiods. These results provided a theoretical basis for further study of Eocyp.

Key words

Empoasca onukii / clone / cysteine proteinase / expression profile

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YU Yongchen, XIAO Bin, SUN Xiaoling. Molecular Cloning and Expression of a Cysteine Gene in Empoasca onukii[J]. Journal of Tea Science. 2018, 38(3): 263-270 https://doi.org/10.13305/j.cnki.jts.2018.03.006

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