刘飞玲,廖珍,洪喜雄,彭睿智,肖花美,李飞,2022,家蚕Piwi亚家族基因的克隆、表达及进化分析[J].环境昆虫学报,(4):838-849
家蚕Piwi亚家族基因的克隆、表达及进化分析
Cloning, expression and phylogenetic analysis of Piwi subfamily gene in silkworm
  
DOI:
中文关键词:  家蚕  Piwi亚家族蛋白  分子克隆  进化分析  表达谱
英文关键词:Bombyx mori  Piwi subfamily protein gene  molecular cloning  phylogenetic analysis  expression profile
基金项目:国家自然科学基金(31760514);江西省自然科学基金青年基金(20181BAB214012)
作者单位
刘飞玲,廖珍,洪喜雄,彭睿智,肖花美,李飞 1. 宜春学院生命科学与资源环境学院江西省作物生长发育调控重点实验室江西宜春 3360002. 浙江大学昆虫科学研究所杭州 3100583. 南京农业大学植物保护学院南京 210095 
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中文摘要:
      为克隆家蚕Bombyx mori Piwi亚家族蛋白基因cDNA全长序列,分析其分子特征和表达模式,探究Piwi亚家族蛋白在家蚕中的生理功能,本研究利用已知物种的Piwi亚家族蛋白搜索家蚕基因组,预测获得家蚕Piwi亚家族蛋白基因siwi1和siwi2,采用RACE技术克隆siwi1和siwi2的全长cDNA序列,利用ORFfinder、Gene-Explorer、InterPro等分析其分子特征;其次,利用已知的所有物种Piwi蛋白及其类似物Piwil构建系统发育树;最后,通过荧光定量PCR技术检测了siwi1和siwi2在丝腺、马氏管、中肠、头部、卵巢和精巢以及不同发育时期(卵、1~5龄幼虫、蛹、成虫)的表达水平,结果显示,克隆获得了siwi1 cDNA全长3 277 bp,包含部分5′UTR、完整的开放阅读框ORF和3′UTR,获得了siwi2的部分序列,其中siwi1对应BmPiwi,siwi2对应BmAgo3。系统发育结果显示,家蚕Piwi亚家族蛋白与乳草长蝽Oncopeltus fasciatus、黑腹果蝇Drosophila melanogaster、橘小实蝇Bactrocera dorsalis、优雅蝈螽Gampsocleis gratiosa、斯氏按蚊Anopheles stephensi、褐飞虱Nilaparvata lugens的PIWI蛋白以及东亚飞蝗Locusta migratoria的PIWI2和PIWI3蛋白亲缘关系最近,和三疣梭子蟹Portunus trituberculatus的PIWI2和PIWI3同为一支。家蚕AGO3蛋白与小菜蛾Plutella xylostella的AGO3亲缘关系最近,与西方玉米根虫Diabrotica virgifera virgifera、白蜡窄吉丁Agrilus planipennis的AGO3及东亚飞蝗Locusta migratoria的PIWI1同属一支,与橘小实蝇Bactrocera dorsalis的AGO3同属一总支。组织表达结果表明BmPiwi在精巢表达水平显著高于其他组织,其次是卵巢及头部,而BmAgo3在头部表达水平显著高于其他组织。BmPiwi和BmAgo3在卵期表达量最高,但在初孵幼虫中表达量迅速下降,并在整个幼虫时期都维持低表达状态,直至蛹期开始表达量出现大幅回升,并维持高水平表达至成虫期。本研究发现BmPiwi和BmAgo3在家蚕中的表达具有明显的时空特异性,在生殖组织以及需要进行大量细胞活动的时期,家蚕Piwi亚家族蛋白基因的表达均发生了明显的变化,为进一步探测家蚕Piwi亚家族的生理功能提供了研究方向和理论基础。
英文摘要:
      To explore the physiological function of the Piwi subfamily protein in the silkworm, the full-length cDNA of Bombyx mori Piwi subfamily protein gene was cloned, and the molecular characteristics and expression patterns were analyzed. First, we searched the B. mori genome using the Piwi subfamily proteins of known species and obtained the B. mori Piwi subfamily protein genes siwi1 and siwi2. The full-length cDNA sequences of siwi1 and siwi2 was cloned using RACE technology. Then the molecular characteristics were analyzed using ORFfinder, Gene-Explorer, InterPro etc. Secondly, the phylogenetic tree was constructed using all known species Piwi protein and the analogue Piwil. Finally, the expression levels of siwi1 and siwi2 in silk gland, malpighian tubes, midgut, head, ovary and testis were detected by quantitative real-time PCR technology, as well as the expression levels of siwi1 and siwi2 at different developmental stages (eggs, 1~5 instar larvae, pupae, and adult). The full length of siwi1 was 3 277 bp, containing part of 5′UTR, complete open reading frame (ORF) and 3′UTR, but we just got a partial of siwi2 with 823 bp, and siwi1 corresponded to BmPiwi, siwi2 corresponded to BmAgo3. Phylogenetic trees showed that the silkworm Piwi subfamily protein was closely related to the PIWI proteins of Oncopeltus fasciatus, Drosophila melanogaster, Bactrocera dorsalis, Gampsocleis gratiosa, Anopheles stephensi, Nilaparvata lugens, as well as the PIWI2 and PIWI3 proteins of Locusta migratoria, and was clustered to one clade with PIWI2 and PIWI3 of Portunus trituberculatus. The BmAgo3 protein had the closest relationship with the Plutella xylostella AGO3, and clustered to one clade with AGO3 of Diabrotica virgifera virgifera, Agrilus planipennis, and PIWI1 of Locusta migratoria. And clustered to a general clade with AGO3 of Bactrocera dorsalis. The expression level of BmPiwi in testis was significantly higher than other tissues, followed by ovaries and head, while the expression level of BmAgo3 in head was significantly higher than other tissues, followed by testes and ovaries. The expression levels of BmPiwi and BmAgo3 were the highest in the egg stage, but the expression level in the newly hatched larvae decreased rapidly and maintained a low expression level state during the whole larval period, until the pupa stage, the expression level raised sharply and maintained a high level expression to the adult stage. The expression of BmPiwi and BmAgo3 in B. mori had obvious spatial and temporal specificity. During the period of reproductive tissues and the period of a lot of cell activities, the expression of Piwi subfamily gene in B. mori changed a lot. Our research provided research direction and theoretical basis for further detecting the physiological function of the B. mori Piwi subfamily.
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