家蚕组织蛋白酶L及其特异性抑制剂基因参与微生物诱导蚕体的免疫响应研究  

Study on the Immune Response of Silkworm Cathepsin L and Its Specific Inhibitor Gene to Microorganism Induction

作　　者：刘敏敏[1] 占鹏飞 黄绍华[1] 周燕燕[1] 费建明 王华兵[1] 徐豫松[1] 

机构地区：[1]浙江大学动物科学学院,杭州310058 [2]湖州市农业科学研究院,浙江湖州313000

出　　处：《蚕业科学》2015年第4期650-657,共8页ACTA SERICOLOGICA SINICA

基　　金：国家高技术研究发展计划"863"项目(No.2013AA102507-5);湖州市科技计划(公益性技术应用研究)项目(No.2013GZ06);浙江省科技计划项目(No.2014C32076)

摘　　要：给家蚕5龄第3天幼虫分别注射藤黄微球菌(革兰阳性菌)和球孢白僵菌(真菌)进行诱导处理,利用已登录的家蚕组织蛋白酶L基因Bm Cts L(Gen Bank登录号:S77508.1)和Bm Cts L特异性抑制剂基因Bm Cts LI(Gen Bank登录号:AJ131752)的全长序列设计引物,实时荧光定量PCR检测微生物入侵后家蚕组织中Bm Cts L和Bm Cts LI基因的时空表达变化及协同反应,分析Bm Cts L酶活性变化与家蚕对微生物入侵免疫响应的相关性。家蚕的血细胞与脂肪体是微生物入侵后Bm Cts L和Bm CtsLI基因出现表达变化的主要组织,其中经藤黄微球菌诱导后,血细胞中的Bm Cts L基因响应最快,且基因mRNA转录水平极显著上调,脂肪体中的Bm Cts LI基因mRNA转录水平也极显著上调。微生物入侵后蚕体组织中Bm Cts L与Bm Cts LI基因的表达变化存在时间差异,其中Bm Cts L基因先于Bm Cts LI基因上调表达,推测Bm Cts LI基因通过对微生物入侵的协同反应调控BmCts L酶活性变化。藤黄微球菌比球孢白僵菌能更快诱导Bm Cts L和Bm Cts LI基因的表达变化;2种微生物诱导下蚕体血细胞中的Bm Cts L酶活性不仅上升快,而且明显高于其他组织。上述结果提示Bm Cts L和Bm Cts LI可能与家蚕的先天免疫相关,并且佐证了家蚕的血细胞在微生物入侵后能比其他组织更快速地产生免疫响应。In this study,Micrococcus luteus（ Gram positive bacterium） and Beauveria bassiana（ fungus） were injected into day 3 silkworm larvae of the 5th instar. With the special primers designed according to the reported full-length sequences of Bombyx mori cathepsin L（ Bm Cts L,Gen Bank accession No. S77508. 1） and Bm Cts L specific inhibitor（ BmCts LI,Gen Bank accession No. AJ131752）,the spatial and temporal expression variation and concerted reaction of BmCts L and Bm Cts LI gene in silkworm tissues were investigated by real-time fluorescent quantitative PCR（ qRT-PCR） after microorganism infection to analyze the relevance between Bm Cts L activity and the response of silkworm to microbial invasion. The results showed that expression of Bm Cts L and Bm Cts LI was mainly changed in haemocytes and fat body after microbial invasion. After induction with M. luteus,the transcriptional level of Bm Cts L mRNA in haemocytes had an extremely significant increase with a fast reaction rate,and the transcriptional level of Bm Cts LI in fatbody was increased extremely significantly as well. There existed apparent time difference between Bm Cts L and Bm Cts LI expression levels in silkworm tissues after microbial infection,and Bm Cts L reacted more quickly to microbial invasion than Bm Cts LI gene,indicating that the Bm Cts L activity changes were probably due to concerted reaction of Bm Cts LI involved in the microbial invasion. M. luteus could induce the expression variation of Bm Cts L and Bm Cts LI sooner than B. bassiana. In addition,Bm Cts L activity rise in haemocytes was not only faster but also significantly higher than that in other tissues. It is concluded that Bm Cts L and Bm Cts LI may be associated with the innate immunity response of silkworm,and haemocytes show a faster immune response than other tissues after microbial invasion.

关 键 词：家蚕 先天免疫 微生物诱导 组织蛋白酶L 组织蛋白酶L特异性抑制剂 基因表达 

分 类 号：S881.2[农业科学—特种经济动物饲养]