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目的:克隆褐飞虱钾通道蛋白KCTD3基因，并研究其序列特征和时空表达情况，以更好地了解褐飞虱KCTD3在褐飞虱生长发育中的作用，为防治褐飞虱提供理论依据。方法:通过PCR扩增褐飞虱KCTD3基因，运用生物信息学方法对褐飞虱KCTD3基因序列进行分析；之后采用荧光定量PCR(qPCR)检测KCTD3基因在褐飞虱不同生长发育时期(1～5龄若虫和1～9日龄雌成虫)和不同组织(胸部、卵巢、脂肪体、头部和肠道)中的表达情况。结果:褐飞虱KCTD3基因开放阅读框长度为2 139 bp,编码712个氨基酸残基，预测蛋白相对分子质量为78 200 u,等电点为7.6,包含有一个BTB保守结构域，无信号肽。系统进化树显示，褐飞虱KCTD3与半翅目茶翅蝽Halyomorpha halys和绿盲蝽Apolygus lucorum的KCTD3亲缘关系最近。qPCR结果显示，褐飞虱KCTD3在褐飞虱发育的所有阶段均有表达且在雌成虫中高水平表达，KCTD3在若虫时期表达量较为稳定，而在3日龄成虫中的表达量最高；组织表达分析表明，褐飞虱KCTD3在各组织中均有表达，且在卵巢中的表达量明显高于其他组织，在脂肪体中的表达量最低。结论:本研究成功克隆了褐飞虱KCTD3基因，明确了KCTD3的序列特征和时空表达模式，结果暗示KCTD3在褐飞虱的生长发育和生殖中起重要作用。
Abstract：Aims: This paper aims to clone the Nilaparvata lugens potassium channel tetramerization domain-containing protein 3(NlKCTD3) gene and study its sequence characteristics and spatio-temporal expression, so as to better understand the function of NlKCTD3 in the growth and development of Nilaparvata lugens and provide a theoretical basis for the control of Nilaparvata lugens. Methods: The NlKCTD3 gene was amplified by PCR; and the sequence of NlKCTD3 gene was analyzed by the bioinformatics method. Then the expression levels of NlKCTD3 gene at different growth and development stages of Nilaparvata lugens(1st to 5th instar nymphs and one-to nine-days old female adults) and in different tissues(thorax, ovary, fat body, head and gut) were detected by fluorescence quantitative PCR. Results: The open reading frame(ORF) length of NlKCTD3 gene was 2139 bp that encoded 712 amino acids. The molecular weight of NlKCTD3 was 78 200 u and the isoelectric point was 7.6. NlKCTD3 contained a BTB conserved domain and had no signal peptide. The phylogenetic tree analysis showed that KCTD3 from Nilaparvata lugens was most closely related to that of Halyomorpha halys and Apolygus lucorum. qPCR results showed that NlKCTD3 was expressed at all developmental stages of Nilaparvata lugens; and there was a high expression level in female adults. The expression of NlKCTD3 was stable at the nymph stage and was highest in three-day old female adults at the adult stage. The tissue-specific expression analysis showed that NlKCTD3 was expressed in all tissues; and the expression in ovary was significantly higher than that in other tissues, while the expression of NlKCTD3 in fat body was the lowest. Conclusions: The NlKCTD3 gene was successfully cloned; and its sequence characteristics and spatio-temporal expression pattern were clarified. Results imply that NlKCTD3 may play an important role in the development and reproduction of Nilaparvata lugens.
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基本信息:
DOI：
中图分类号:S435.112.3
引用信息:
[1]姬金亮,张瑞娟,李雅彬等.褐飞虱KCTD3基因克隆、序列分析及其时空表达研究[J].中国计量大学学报,2022,33(03):423-431.
基金信息:
国家自然科学基金项目(No.31871961,U21A20223,31501632); 浙江省自然科学基金项目(No.LY22C140007); 浙江省重点研发计划项目(No.2019C02015,2022C02047); 浙江省属高校基本科研业务费专项资金项目(No.2020YW14)