利用长链非编码RNAs与mRNAs联合分析半番鸭及番鸭胚胎期性腺发育差异  

Combined analysis of embryonic gonadal development differences of mulard duck and muscovy duck using long non-coding RNAs and mRNAs

作　　者：李丽[1,2] 章琳俐 辛清武[1,2] 缪中纬 朱志明[1,2] 邱君志 郝晓娜[4] 黄勤楼 郑嫩珠[1,2] LI Li;ZHANG Linli;XIN Qingwu;MIAO Zhongwei;ZHU Zhiming;QIU Junzhi;HAO Xiaona;HUANG Qinlou;ZHENG Nenzhu(Institute of Animal Husbandry and Veterinary Medicine,Fujian Academy of Agricultural Sciences,Fuzhou 350013,Fujian,China;Fujian Key Laboratory of Animal Genetics and Breeding,Fuzhou 350013,Fujian,China;College of Life Sciences,Fujian Agriculture and Forestry University,Fuzhou 350002,Fujian,China;College of Animal Sciences(College of Bee Science),Fujian Agriculture and Forestry University,Fuzhou 350002,Fujian,China)

机构地区：[1]福建省农业科学院畜牧兽医研究所,福建福州350013 [2]福建省畜禽遗传育种重点实验室,福建福州350013 [3]福建农林大学生命科学学院,福建福州350002 [4]福建农林大学动物科学学院(蜂学学院),福建福州350002

出　　处：《浙江大学学报（农业与生命科学版）》2023年第5期729-743,共15页Journal of Zhejiang University:Agriculture and Life Sciences

基　　金：福建省自然科学基金项目(2021J01483);福建省省属公益类项目(2020R10260016);福建省农业科学院英才项目(YC20210046);福建省农业科学院科技创新团队项目(CXTD2021006-2);福建省现代家禽产业技术体系建设专项(2019—2022);福建省“5511”协同创新工程项目(XTCXGC2021008)。

摘　　要：本研究旨在筛选影响鸭胚胎期性腺发育的关键mRNAs和长链非编码RNAs(long non-coding RNAs,lncRNAs),科学解释半番鸭性腺发育缺陷问题。采集半番鸭(BF_(1)、BF_(2)、BF_(3))和番鸭(F_(1)、F_(2)、F_(3))各3个雄性胚胎性腺组织并提取其RNA,通过高通量测序筛选差异基因和lncRNAs,预测靶基因并进行功能注释,最后应用实时荧光定量聚合酶链反应(real-time fluorescence quantitative polymerase chain reaction,qRT-PCR)对测序结果进行验证。结果显示:从半番鸭和番鸭性腺组织中共筛选出1109个差异基因;相对于番鸭,半番鸭中上调表达基因共857个,下调表达基因共252个,其中,醛酮还原酶家族1成员D1基因(aldo-keto reductase family 1 member D1gene,AKR1D1)、17β-羟基类固醇脱氢酶3基因(17β-hydroxysteroid dehydrogenase 3 gene,17β-HSD3)和胆固醇侧链裂解酶基因(cholesterol side-chain cleavage enzyme gene,P450scc)等可能与半番鸭性腺分化发育相关。同时,获得了733个显著差异表达的lncRNAs;相对于番鸭,半番鸭中显著上调的lncRNAs共660个,显著下调的lncRNAs共73个。靶基因预测分析显示,136个下调的lncRNAs和893个上调的lncRNAs可能参与差异基因表达并存在潜在的调控关系,其中,TCONS_00246198靶向17β-HSD3,TCONS_00229529靶向四次穿膜蛋白2基因(tetraspanin-2 gene,TSPAN2),说明以上lncRNAs可能通过靶向关键基因来参与鸭胚胎期性腺发育。qRT-PCR结果显示,差异基因和lncRNAs表达水平与转录组测序中的表达趋势一致,表明通过高通量测序获得的数据较为可靠。RNA结合蛋白免疫沉淀(RNA binding protein immunoprecipitation,RIP)试验结果发现,与IgG相比,TCONS_00246198富集水平达71.51倍,表明TCONS_00246198与17β-HSD3蛋白存在直接或间接的相互作用,即两者可能存在靶向关系。综上所述,本研究获得了一批可能影响鸭胚胎期性腺发育的关键mRNAs和lncRNAs,推测差异lncRNAs可以调控差异基因的表达,为了解The purpose of this study was to screen key mRNAs and long non-coding RNAs(lncRNAs)that affect gonadal development in duck embryos and to explain scientifically gonadal development defects in the mulard duck.Three male embryonic gonadal tissues of the mulard duck(BF_(1),BF_(2),BF_(3))and the muscovy duck(F_(1),F_(2),F_(3))were collected to extract RNA and perform high-throughput sequencing,and differential genes and lncRNAs were screened to predict target genes and perform functional annotations.Finally,the sequencing data were verified by real-time fluorescence quantitative polymerase chain reaction(qRT-PCR).The results showed that a total of 1109 differentially expressed genes were screened from the gonadal tissues of the mulard duck and the muscovy duck.Compared with the muscovy duck,857 genes were up-regulated and 252 genes were down-regulated in the mulard duck.Among them,the aldo-keto reductase family 1 member D1 gene(AKR1D1),17β-hydroxysteroid dehydrogenase 3 gene(17β-HSD3),and cholesterol side-chain cleavage enzyme gene(P450scc)may be related to gonadal differentiation and development in the mulard duck.Meanwhile,733 significantly differentially expressed lncRNAs were obtained.Compared with the muscovy duck,a total of 660 lncRNAs were significantly up-regulated and 73 lncRNAs were significantly down-regulated in the mulard duck.Target gene prediction analysis showed that a total of 136 down-regulated lncRNAs and 893 up-regulated lncRNAs may be involved in differential gene expression and had potential regulatory relationships,among which,TCONS_00246198 targeted 17β-HSD3,and TCONS_00229529 targeted tetraspanin-2 gene(TSPAN2),suggesting that the above lncRNAs may participate in duck embryonic gonadal development by targeting key genes.The qRT-PCR results showed that the expression levels of differential genes and lncRNAs were consistent with the expression trends in transcriptome sequencing,indicating that the data obtained by high-throughput sequencing are relatively reliable.RNA binding protein immunop

关 键 词：半番鸭 番鸭 性腺 不育 长链非编码RNAs MRNAS 

分 类 号：S834.89[农业科学—畜牧学]