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

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Molecular Cloning and Expression of a Cysteine Gene in Empoasca onukii

YU Yongchen1,2,3, XIAO Bin1,*, SUN Xiaoling2,3,*   

  1. 1. Northwest A&F University, Yangling 712100, China;
    2. Tea Research Institute of Chinese Academy of Agricultural Sciences, Hangzhou 310008, China;
    3. Key Laboratory of Tea Biology and Resource Utilization of Ministry of Agriculture, Hangzhou 310008, China
  • Received:2018-01-18 Revised:2018-03-26 Online:2018-06-15 Published:2019-08-28

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, cysteine proteinase, clone, expression profile

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