家蚕幼虫血细胞密度变化成因及血细胞密度与耐高温性的关系  

Reasons for changes of hemocyte densities and the relationship between hemocyte density and high temperature resistance of Bombyx mori larvae

作　　者：李田[1,2] 刘昊宇 王隔敏 李亚腾 余华献 严东生 郭雨 张太云 陈萍 LI Tian;LIU Hao-Yu;WANG Ge-Min;LI Ya-Teng;YU Hua-Xian;YAN Dong-Sheng;GUO Yu;ZHANG Tai-Yun;CHEN Ping(College of Sericulture, Textile and Biomass Sciences, Southwest University, Chongqing 400715, China;Drug Discovery Research Center, Southwest Medical University, Luzhou, Sichuan 646000, China;Chongqing Sericulture General Station, Chongqing 400020, China;Guide Station for the Development of Economic Crops in Hechuan District of Chongqing, Chongqing 401520, China)

机构地区：[1]西南大学蚕桑纺织与生物质科学学院,重庆400715 [2]西南医科大学药物研究中心,四川泸州646000 [3]重庆市蚕业管理总站,重庆400020 [4]重庆市合川区经济作物发展指导站,重庆401520

出　　处：《昆虫学报》2022年第2期130-143,共14页Acta Entomologica Sinica

基　　金：重庆市自然科学基金重点项目(cstc2018jcyjAX0075);重庆市现代山地特色效益农业(蚕桑)产业技术体系专项(202109);重庆市2021年度茧丝绸发展专项资金项目(20210607223136209)。

摘　　要：【目的】血细胞是昆虫血淋巴免疫的主导者。调查家蚕Bombyx mori幼虫血细胞密度变化和成因、血细胞密度与家蚕抗性的关系,是研究家蚕血细胞相关的免疫调控和抗性育种的重要组成。【方法】用细胞计数板统计家蚕品种大造不同龄期(4龄第1-4天、5龄第1-8天和上蔟期)幼虫10μL血淋巴中的血细胞数目并计算血细胞密度,利用ImageJ软件估算横截面成像图面积来代表造血器官相对体积;用细胞计数板统计不同家蚕品种(菁松、皓月、两广二号、932、芙蓉、7532和湘辉)5龄第1天幼虫血淋巴中的血细胞数目;用qRT-PCR分析皓月和两广二号5龄第1天幼虫血淋巴中造血调控因子基因BmBCFI,BmUsh,BmGATA和BmLz的相对表达量;大肠杆菌Escherichia coli侵染大造5龄第1和3天幼虫不同时间后,用细胞计数板统计血淋巴中血细胞数目,用qRT-PCR检测血淋巴中上述造血调控因子基因的表达量;BmBCFI,BmUsh和BmLz被RNAi干扰不同时间后,qRT-PCR检测大造5龄幼虫血淋巴中靶基因的相对表达量;BmBCFI,BmUsh和BmLz被RNAi干扰48 h后大肠杆菌侵染6 h时用qRT-PCR检测大造5龄幼虫血淋巴中靶基因和免疫相关基因BmRelish,BmCactus,BmSpzl,BmPGRP-LE的相对表达量,用细胞计数板统计血淋巴中不同类型血细胞数目;35℃高温饲养12个实用家蚕品种5龄幼虫,分析血细胞数目和生存力之间的关联性。【结果】4龄期大造幼虫血细胞数目随生长逐渐上升,到5龄第1天达到顶峰,5龄第2天开始下降,直至上蔟期出现小幅升高;造血器官在5龄前中期随幼虫生长逐渐增大,5龄第6天体积达到最大,随后显著变小直至完全解离,其大小变化与血细胞数目变化时间和趋势不一致。血细胞数目多的两广二号中造血调控因子基因BmBCFI,BmUsh和BmLz的表达量均高于在血细胞数目少的皓月中,在大肠杆菌侵染后大造5龄幼虫血细胞数目达到高峰时BmBCFI,BmUsh和BmLz被显著上调表达�【Aim】Hemocyte plays a leading role in insect hemolymph immunity.Analyzing the changes of hemocyte density of the silkworm,Bombyx mori larvae and their causes,and the relationship between hemocyte density and B.mori resistance,is an important part of hemocyte immune regulation and resistance breeding of silkworm.【Methods】Hemocyte density(hemocyte number)in 10μL hemolymph of the silkworm variety Dazao at different instars(the day-1-44th instar,the day-1-85th instar and the wandering larval stage)was evaluated by a hemocytometer.The cross-sectional image area was estimated by ImageJ software to represent the relative size of hematopoietic organs.The numbers of hemocytes in the hemolymph of the day-15th instar larvae of different silkworm varieties including Jingsong,Haoyue,Liangguang 2,932,Furong,7532 and Xianghui were measured by a hemocytometer.The relative expression levels of hematopoietic regulation factor genes BmBCFI,BmUsh,BmGATA and BmLz in the hemolymph of the day-15th instar larvae of Haoyue and Liangguang 2 were detected by qRT-PCR.After the day-1 and-35th instar larvae of Dazao were infected by Escherichia coli for different time,the hemocyte numbers in the hemolymph were measured by a hemocytometer and the relative expression levels of the above hematopoietic regulation factor genes in the hemolymph were detected by qRT-PCR.After RNAi of BmBCFI,BmUsh and BmLz for different time,the relative expression levels of target genes in the hemolymph of the 5th instar larvae of Dazao were detected by qRT-PCR.At 6 h after the 5th instar larvae of Dazao were infected by E.coli following RNAi of BmBCFI,BmUsh and BmLz for 48 h,the relative expression levels of target genes and immune-related genes BmRelish,BmCactus,BmSpzl and BmPGRP-LE in the hemolymph were detected by qRT-PCR,and the numbers of different types of hemocytes in the hemolymph were counted by a hemocytometer.The correlations between the hemocyte number and viability of the 5th instar larvae at high temperature of 35℃were analyzed for 12 pract

关 键 词：家蚕 血细胞数目 造血器官 造血调控因子 生存力 

分 类 号：S881.2[农业科学—特种经济动物饲养]