基于双元转基因CRISPR/Cas9系统的家蚕pax3基因功能分析  

Functional analysis of pax3 gene in Bombyx mori by the binary transgenic CRISPR/Cas9 system

作　　者：蒲尚昆 王磊[1] 谭安江 魏国清[1] PU Shang-Kun;WANG Lei;TAN An-Jiang;WEI Guo-Qing(School of Life Sciences,Anhui Agricultural University,Hefei 230036,China;School of Biotechnology,Jiangsu University of Science and Technology,Zhenjiang,Jiangsu 212100,China;Sericultural Research Institute,Chinese Academy of Agricultural Sciences,Zhenjiang,Jiangsu 212100,China)

机构地区：[1]安徽农业大学生命科学学院,合肥230036 [2]江苏科技大学生物技术学院,江苏镇江212100 [3]中国农业科学院蚕业研究所,江苏镇江212100

出　　处：《昆虫学报》2022年第12期1598-1605,共8页Acta Entomologica Sinica

基　　金：国家自然科学基金项目(31472147);安徽省自然科学基金项目(2208085MC46);安徽省高等学校科研计划项目(2022AH050888);安徽农业大学高层次人才团队项目(ANRC2019028)。

摘　　要：【目的】本研究旨在以家蚕Bombyx mori为研究模型探索pax3基因在鳞翅目昆虫中的生物学功能。【方法】利用PCR扩增验证家蚕Bmpax3外显子序列;利用qRT-PCR检测Bmpax3在5龄第3天家蚕幼虫头、表皮、脂肪体、中肠、马氏管、前部丝腺、中部丝腺、后部丝腺和生殖腺(包括精巢和卵巢)中的表达谱;利用双元转基因CRISPR/Cas9系统构建Bmpax3敲除突变体,分析Bmpax3突变对家蚕幼虫存活、体节分化及性别差异的影响。【结果】Bmpax3在家蚕5龄第3天幼虫头、中肠和丝腺中均有表达,其中在前部丝腺表达量最高。Bmpax3突变体的卵孵化率约为90%,但约有80%的突变体在1龄幼虫期死亡,有将近10%的突变个体能幸存并发育到成虫阶段,并且存活成虫数存在性别差异,雄性显著多于雌性。在幸存的成虫中,约有将近1/2的个体腹部末端体节分节异常,表皮条纹混乱,腹节腹板部分缺失,生殖器官及其周围的其他辅助器官出现发育缺陷。【结论】Bmpax3发生突变后会对家蚕的生存及形态发育产生较大的影响,提示Bmpax3可能参与了家蚕的生长发育过程。【Aim】This study aims to investigate the biological functions of pax3 gene in Lepidoptera insects using the silkworm,Bombyx mori,as a model.【Methods】The exon sequence of Bmpax3 from B.mori was verified by PCR amplification.The expression profile of Bmpax3 in the head,cuticle,fat body,midgut,Malpighian tubules,anterior silk gland,middle silk gland,posterior silk gland and gonads(including testis and ovary)of the day-35th instar larvae of B.mori was detected by qRT-PCR.The Bmpax3 knockout mutants were constructed using the binary transgenic CRISPR/cas9 system to analyze the effects of Bmpax3 mutation on the larval survival,somite differentiation and gender difference of B.mori.【Results】Bmpax3 was expressed in the head,midgut and silk gland of the day-35th instar larvae of B.mori,with its highest expression level in the anterior silk gland.The egg hatching rate of the Bmpax3 mutants was about 90%,but about 80%of mutants died in the 1st instar larval stage,and only about 10%of the mutated individuals survived to the adult stage.And there was gender difference in the number of surviving adults,in which the number of male survivors was significantly higher than that of female survivors.About half of the surviving adults showed abnormal differentiation in distal abdominal somites,disorder of cuticle stripes,deficiency of partial abdominal sternum and developmental defects in reproductive organs and other auxiliary organs around them.【Conclusion】Bmpax3 mutation has a great impact on the survival and morphological development of B.mori,suggesting that Bmpax3 may participate in the growth and development processes in B.mori.

关 键 词：家蚕 Bmpax3 CRISPR-Cas9 存活率 体节分化 

分 类 号：Q966[生物学—昆虫学]