家蚕丝腺发育相关基因Bmsage缺失突变体的RNA-seq分析  

RNA-seq Analysis on Loss-of-function Mutation in Bmsage Gene Related to Silk Gland Development in Silkworm,Bombyx mori

作　　者：辛虎虎 张登攀[1] 矫鹏[1] 陈瑞婷[1] 陆喦[1] 缪云根[1] 

机构地区：[1]浙江大学动物科学学院蚕蜂研究所,杭州310058

出　　处：《蚕业科学》2015年第5期854-863,共10页ACTA SERICOLOGICA SINICA

基　　金：国家重点基础研究发展计划"973"项目(No.2012CB1146-01);国家自然科学基金项目(No.31372374/C1703)

摘　　要：Bmsage是在家蚕丝腺中特异表达的基因,之前的研究明确其对丝素重链基因fib-H转录具有调控作用。在开展Bmsage功能的研究中,运用CRISPR/Cas9基因敲除系统,获得了Bmsage缺失的纯合突变体家蚕,其表型特征是幼虫的丝腺异常发育,缺失中部丝腺和后部丝腺。以Bmsage缺失型家蚕及正常型家蚕产下后5 d的卵为材料,应用RNA-seq测序技术在转录水平上获得194个差异表达基因。对Bmsage缺失型家蚕突变体胚胎期的这些差异表达基因进行GO功能注释,结果显示:Bmsage基因缺失后,对家蚕生理功能的影响主要集中在一些细胞组分,如与肌肉功能相关的肌球蛋白基因和肌钙蛋白基因都明显上调表达,与碳水化合物代谢相关的基因如多种凝集素基因和糖苷酶基因,除个别下调表达外,其余均上调表达;排在前10的GO term中,出现较多的是与几丁质分解代谢相关的几丁质酶基因和多种丝氨酸蛋白酶抑制剂基因。KEGG信号通路分析显示:差异基因主要与一些糖类、脂类和氨基酸的代谢的信号通路相关,其中又以糖类代谢为主,如半乳糖代谢、糖胺聚糖降解及氨基糖和核苷酸糖代谢,所以Bmsage可能参与了多个糖类代谢途径;在差异基因中,与丝腺发育关系最大的是几丁质酶基因、β-N-乙酰葡糖胺糖苷酶基因和丝氨酸蛋白酶抑制剂基因,以及一些转运RNA基因(如TRNAE-CUC、TRNAN-GUU、TRNADGUC和TRNAV-GAC)。因此推测Bmsage可能通过复杂的调控网络,参与了丝腺组织的分化与形成过程。Bmsage is a gene specially expressed in silk gland of silkworm（ Bombyx mori）. It is involved in transcription regulation of fibroin heavy-chain gene fib-H. In the study of Bmsage function,we successfully obtained homozygous mutant silkworm of Bmsage deletion using CRISPR/Cas9 genome editing technology. Its larva phenotype is characterized by abnormal development of silk gland,missing the middle silk gland and posterior silk gland. In this paper,the transcriptional level of Bmsage knockout silkworm and wild-type silkworm was detected by RNA-seq sequencing technology using eggs at day 5 after oviposition as material,and total 194 differentially expressed genes were obtained. GO analysis of differentially expressed genes in Bmsage loss-of-function mutant at the embryonic stage indicated that lose-of-function in Bmsage mainly affected some cellular component related genes. For instance,the myosin and troponin genes associated with muscle function showed obvious up-regulation,and most of lectin and glycosidase genes associated with carbohydratemetabolic process also showed obvious upregulation except some down-regulated genes. Moreover,in the top 10 GO terms,most of differentially expressed genes were chitinase genes associated with chitin degradation and several serine protease inhibitor genes. KEGG pathway analysis showed that the differentially expressed genes were mainly related to the signal pathways of carbohydrate,lipid and amino acid metabolism. Among them,most genes were involved in carbohydrate metabolism,such as galactose,glycosaminoglycan,amino sugar and nucleotide sugar metabolism. In these differentially expressed genes,chitinase,beta-N-acetylglucosaminidase,serine protease inhibitor genes and some tRNA genes such as TRNAECUC,TRNAN-GUU,TRNAD-GUC and TRNAV-GAC are probably the most relevant to silk gland development in silkworm. Therefore,Bmsage is perhaps involved in the differentiation and formation of silk gland through a complex regulatory network.

关 键 词：家蚕 丝腺 分化与发育 Bmsage基因 基因敲除 RNA-seq测序 

分 类 号：Q78[生物学—分子生物学] S881.2[农业科学—特种经济动物饲养]