| 张曼，高平，陈晓，梁晨，汤永玉，秦得强，吴国星，高熹,2024,叉角厉蝽丝氨酸蛋白酶及抑制剂基因的克隆与表达分析[J].环境昆虫学报,(3):659-669 |
| 叉角厉蝽丝氨酸蛋白酶及抑制剂基因的克隆与表达分析 |
| Gene cloning and expression of the serine protease and serine protease inhibitor in Eocanthecona furcellata (Wolff) |
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| DOI： |
| 中文关键词: 叉角厉蝽 丝氨酸蛋白酶 丝氨酸蛋白酶抑制剂 表达特征 |
| 英文关键词:Eocanthecona furcellata (Wolff) serine protease serine protease inhibitor expression pattern |
| 基金项目:云南省农业联合专项（202301BD070001-137，202101BD070001-050）；云南省中青年学术和技术带头人后备人才项目（202205AC160077）；云南省教育厅科学研究基金（2023Y1051） |
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| 中文摘要: |
| 为明确叉角厉蝽Eocanthecona furcellata (Wolff)丝氨酸蛋白酶基因EfSP1及抑制剂基因EfSPI20的基因序列特征和时空转录特征，为其生理功能研究奠定基础。利用PCR克隆技术获得叉角厉蝽唾液腺EfSPI20和EfSP1的完整开放阅读框(Open reading frame, ORF)序列，使用生物信息学软件进行序列分析以及系统进化分析，采用实时荧光定量PCR (Real time quantitativate PCR，RT-qPCR)分析两个基因分别在叉角厉蝽不同发育时期和组织中的表达特征。结果表明，EfSPI20与EfSP1基因完整开放阅读框长度分别为378 bp和921 bp，分别编码125个氨基酸和306个氨基酸，预测均为亲水蛋白质，理论分子量分别为13.48 kDa和33.82 kDa，等电点分别为6.68和5.80，分别有30个和23个氨基酸残基的信号肽序列，EfSPI20有跨膜结构域，EfSP1无跨膜结构域。序列比对显示叉角厉蝽EfSPI20与茶翅蝽Halyomorpha halys PPI同源性最高，氨基酸序列一致性达58%；EfSP1与稻绿蝽Nezara viridula SP同源性最高，氨基酸序列一致性达66%；系统发育树显示叉角厉蝽与同为蝽科的茶翅蝽和稻绿蝽物种亲缘关系近。EfSPI20基因在雌雄成虫和唾液腺中高表达，推测EfSPI20可能具有抑制胰蛋白酶活性的功能和与叉角厉蝽的捕食消化相关；EfSP1基因在卵期、卵巢和肠道中高表达，推测EfSP1可能与叉角厉蝽的生殖功能和蛋白消化相关。 |
| 英文摘要: |
| In order to clarify the gene sequence characteristics and spatio-temporal transcription characteristics of serine protease gene EfSP1 and inhibitor gene EfSPI20 in Eocanthecona furcellata (Wolff), and lay a foundation for its physiological function research， the complete open reading frame (ORF) sequences of EfSPI20 and EfSP1 in the salivary gland of E. furcellata were obtained by PCR cloning, and sequence analysis and phylogenetic analysis were carried out by bioinformatics software. The expression characteristics of EfSPI20 and EfSP1 in different developmental stages and tissues were analyzed by real-time quantitative PCR (RT-qPCR). The results showed that the CDS of EfSPI20 and EfSP1 were 378 bp and 921 bp, encoding 125 amino acids and 306 amino acids. They were both hydrophilic proteins, the theoretical molecular weights were 13.48 kDa and 33.82 kDa. The isoelectric points were 6.68 and 5.80. There were signal peptide sequences of 30 and 23 amino acid residues, EfSPI20 had a transmembrane domain, EfSP1 had no transmembrane domain. Sequence alignment showed that EfSPI20 had the highest homology with Halyomorpha halys PPI, and the amino acid sequence identity was 58%. EfSP1 had the highest homology with Nezara viridula SP, and the amino acid sequence identity was 66%. The phylogenetic tree showed that E. furcellata was closely related to the species of H. halys and N. viridula in the Pentatomidae. The EfSPI20 gene was highly expressed in male and female adults and salivary glands, it was speculated that EfSPI20 may have the function of inhibiting trypsin activity and be related to the predation and digestion of E. furcellata. The EfSP1 gene was highly expressed in the egg stage, ovary and intestine, suggesting that EfSP1 may be related to the reproductive function and protein digestion of E. furcellata. |
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