叉角厉蝽毒液丝氨酸蛋白酶基因EfSP2克隆、表达及捕食功能分析

扫码查看

原文链接

国家科技期刊平台
NETL
NSTL
万方数据
维普

中文摘要：[目的]通过对叉角厉蝽[Eocanthecona furcellata(Wolff)]毒液丝氨酸蛋白酶(Serine protease，SP)基因EfSP2的克隆、序列分析、表达谱分析及RNA干扰(RNA interference，RNAi)，解析EfSP2蛋白的功能，为EfSP2基因的分子特征研究及功能注释提供科学依据。[方法]从叉角厉蝽唾液腺转录组中获得EfSP2基因序列，利用PCR技术扩增EfSP2基因完整开放阅读框(Open reading frame，ORF)序列，对其进行生物信息学分析;利用实时荧光定量PCR方法分析EfSP2基因在叉角厉蝽不同发育阶段(卵、1～5龄若虫和雌雄成虫)唾液腺及雌成虫不同组织(马氏管、唾液腺、脂肪体、卵巢、中肠、头)中的表达情况;通过RNAi技术检测其对叉角厉蝽雌雄成虫存活及捕食的影响。[结果]克隆获得EfSP2基因ORF序列长861 bp，编码286个氨基酸;EfSP2氨基酸序列中具有1个完整的Tryp-SPc结构域，含有胰蛋白酶N末端保守的起始氨基酸序列(IVGG);含有保守的SP三联体催化活性中心，属于丝氨酸蛋白酶家族的类胰蛋白酶亚家族;与稻绿蝽(Nezara viridula)SP同源性最高，氨基酸序列一致性为44。69%。EfSP2基因在叉角厉蝽雌雄成虫和唾液腺中有极高的表达量。注射dsEfSP2能显著抑制靶标基因的表达(P<0。05，下同)，且显著降低叉角厉蝽雌雄成虫的存活率和捕食量。[结论]成功克隆了叉角厉蝽毒液丝氨酸蛋白酶EfSP2基因。时空表达谱及RNAi结果表明，EfSP2可能是叉角厉蝽生长发育、捕食和消化过程中的重要蛋白之一。

外文标题：Cloning,expression and predation function analysis of serine protease EfSP2 gene from Eocanthecona furcellata(Wolff)venom

外文摘要：[Objective]The purpose of the study was to analyze the function of EfSP2 protein by cloning,sequence analysis,expression profile analysis and RNA interference(RNAi)of serine protease(SP)gene EfSP2 from the venom of Eocanthecona furcellata(Wolff),and to provide a scientific basis for the molecular characterization and functional an-notation of the EfSP2 gene.[Method]The EfSP2 gene sequence was obtained from the salivary gland transcriptome of E.furcellata.The complete open reading frame(ORF)sequence of the EfSP2 gene was amplified by PCR technology and analyzed by bioinformatics.Real-time fluorescence quantitative PCR was used to analyze the expression of EfSP2 gene in different developmental stages(eggs,1st-5th instar nymphs,male and female adults)salivary gland and different tissues(malpighian tube,salivary gland,fat body,ovary,gut,head)of female adults.At the same time,RNAi technology was used to detect its effects on the survival and predation of male and female E.furcellata adults.[Result]The ORF sequence of EfSP2 gene obtained from cloning was 861 bp,encoding 286 amino acids.The amino acid sequence of EfSP2 con-tained a complete Tryp-SPc domain and a conserved initial amino acid sequence(IVGG)at the N-terminus of trypsin.It contained a conserved SP triplet catalytic activity center and belonged to the tryptase subfamily of the serine protease fami-ly.It had the highest homology with Nezara viridula SP,with an amino acid sequence identity of 44.69%.The EfSP2 gene was highly expressed in male and female adults and salivary glands of E.furcellata.The injection of dsEfSP2 signifi-cantly inhibited the expression of target genes(P<0.05,the same below),and significantly reduced the survival rate and predation of male and female E.furcellata adults.[Conclusion]The E.furcellata venom serine protease EfSP2 gene is suc-cessfully cloned.The spatiotemporal expression profile and RNAi results show that EfSP2 is likely to be one of the impor-tant proteins in the processes of growth,development,predation and digestion of E.furcellata.

外文关键词：

Eocanthecona furcellata(Wolff)salivary glandserine proteasegene cloningexpression patternRNA interference

作者：

张曼、刘炳顺、陈晓、岳茂婷、朱国渊、田永明、宁婧怡、秦得强、吴国星、高熹

展开 >

作者单位：

云南农业大学植物保护学院,云南昆明 650201

禄丰市农业技术推广中心,云南楚雄 651200

关键词：

叉角厉蝽唾液腺丝氨酸蛋白酶基因克隆表达模式 RNA干扰

基金：

云南省农业基础研究联合专项云南省农业基础研究联合专项云南省中青年学术和技术带头人后备人才项目云南省烟草公司科技计划项目

项目编号：

202301BD070001-137202101BD070001-050202205AC1600772023530000241012

出版年：

2024

DOI：

10.3969/j.issn.2095-1191.2024.02.019

南方农业学报

广西壮族自治区农业科学院

南方农业学报

CSTPCD北大核心

影响因子：0.83

ISSN：2095-1191

年,卷(期)：2024.55(2)

参考文献量43