小贯小绿叶蝉半胱氨酸蛋白酶基因的克隆与表达

doi:10.13305/j.cnki.jts.2018.03.006

茶叶科学 ›› 2018, Vol. 38 ›› Issue (3): 263-270.doi: 10.13305/j.cnki.jts.2018.03.006

小贯小绿叶蝉半胱氨酸蛋白酶基因的克隆与表达

于永晨^1,2,3, 肖斌^1,*, 孙晓玲^2,3,*

1. 西北农林科技大学,陕西杨凌 712100;
2. 中国农业科学院茶叶研究所,浙江杭州310008;
3. 农业部茶树生物学与资源利用重点实验室,浙江杭州 310008

收稿日期:2018-01-18 修回日期:2018-03-26 出版日期:2018-06-15 发布日期:2019-08-28
通讯作者: *1647785720@qq.com
作者简介:于永晨,男,硕士研究生,主要从事茶树害虫方面的研究。
基金资助:
公益性行业（农业）科研专项经费（201403030）、国家自然科学基金项目资助（31772180）、浙江省“151”人才工程资助项目

Molecular Cloning and Expression of a Cysteine Gene in Empoasca onukii

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

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

摘要/Abstract

摘要： 半胱氨酸蛋白酶是植食性昆虫体不可或缺的重要水解蛋白酶之一。本文从小贯小绿叶蝉的转录组数据库中获得半胱氨酸蛋白酶基因Eocyp的基因片段的转录本序列,利用RACE技术得到其cDNA全长序列,利用生物信息学技术对Eocyp所编码的理论氨基酸序列进行分析,使用qRT-PCR技术检测Eocyp在小贯小绿叶蝉不同发育阶段,雌、雄成虫不同部位以及不同温度和光周期下的表达量。结果表明,小贯小绿叶蝉Eocyp的全长cDNA序列包含一个1β656βbp的开放阅读框,编码551个氨基酸,并具有由24个氨基酸残基组成的信号肽。推测其相对分子量为62.09βkD,预测其分子式为C₂₇₈₂H₄₁₆₃N₇₃₇O₈₃₅S₂₅;理论等电点为6.02,不稳定系数为36.10。Eocyp具有半胱氨酸蛋白酶基因的典型特征,即催化三联体Cys³⁵⁷-His⁴⁹⁹-Asn⁵¹⁹,同时具有ERFNIN,GNFD和GCDGG簇等保守结构域。Eocyp编码氨基酸序列与茶翅蝽CYP基因编码序列相似度最高。表达特征分析结果表明,Eocyp在小贯小绿叶蝉的各个生长时期均有表达,在5龄若虫时期达到最高;Eocyp在小贯小绿叶蝉雌、雄成虫的腹部均有较高的表达量;然而,Eocyp的表达量对于高、低温和不同光周期均无响应。这些结果为进一步研究Eocyp功能提供了理论依据。

关键词: 小贯小绿叶蝉, 半胱氨酸蛋白酶, 克隆, 表达

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

中图分类号:

S435.711
Q52

于永晨, 肖斌, 孙晓玲. 小贯小绿叶蝉半胱氨酸蛋白酶基因的克隆与表达[J]. 茶叶科学, 2018, 38(3): 263-270. doi: 10.13305/j.cnki.jts.2018.03.006.

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. doi: 10.13305/j.cnki.jts.2018.03.006.

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小贯小绿叶蝉半胱氨酸蛋白酶基因的克隆与表达

Molecular Cloning and Expression of a Cysteine Gene in Empoasca onukii

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