家蚕BmlncR2036调控P25基因启动子活性及参与中肠发育调控  

BmlncR2036 regulates the P25 promoter activity and is involved in the regulation of midgut development in the silkworm,Bombyx mori

作　　者：霍春月 程浩 付裕 常美玲 王艺 阚云超[1] 李丹丹[1] HUO Chun-Yue;CHENG Hao;FU Yu;CHANG Mei-Ling;WANG Yi;KAN Yun-Chao;LI Dan-Dan(Henan Key Laboratory of Insect Biology in Funiu Mountain, Nanyang Normal University, Nanyang, Henan 473061, China)

机构地区：[1]南阳师范学院,河南省伏牛山昆虫生物学重点实验室,河南南阳473061

出　　处：《昆虫学报》2021年第8期897-907,共11页Acta Entomologica Sinica

基　　金：国家自然科学基金项目(31970480);中国科协青年人才托举工程项目(YESS20150026);南阳师范学院校级青年骨干教师培养计划项目(nynuxjqg-2019-03)。

摘　　要：【目的】长链非编码RNA(long non-coding RNA,lncRNA)对家蚕Bombyx mori发育具有重要调控作用。我们在前期研究中发现一个位于家蚕丝素蛋白基因P25附近的lncRNA BmlncR2036。本研究旨在进一步探索BmlncR2036调控家蚕P25基因表达的分子机制。【方法】qPCR检测BmlncR2036在5龄第3天家蚕幼虫不同组织(体壁、脑、神经、精巢、卵巢、丝腺、马氏管、血淋巴、脂肪体和中肠)中和不同发育阶段前丝腺、中丝腺和后丝腺中的表达谱。预测能够同时靶向P25和BmlncR2036的miRNA,利用荧光素酶检测法验证miRNA与P25互作关系。荧光素酶检测法测定BmlncR2036对P25基因启动子转录活性的影响。在家蚕5龄第3天幼虫中注射dsRNA敲低BmlncR2036的表达,观察丝腺及中肠表型的变化。【结果】qPCR结果显示,BmlncR2036在家蚕5龄第3天幼虫和5龄熟蚕的后丝腺中高表达,与P25基因呈现一致的表达趋势;BmlncR2036在家蚕5龄第3天幼虫的精巢和中肠中高表达,在其他组织中表达量较低,暗示其功能的多样性。miR-2739和miR-279a既能够与BmlncR2036匹配,也可能靶向P25基因的3′UTR。但荧光素酶检测法结果显示P25不是miR-2739和miR-279a的真实靶标。BmlncR2036负调控P25启动子活性,共转染pcDNA3.1(+)[BmlncR2036]和pGL3-Enhancer[P25-promoter]载体后,细胞荧光素酶活性极显著下降了52%。在家蚕5龄第3天幼虫中敲低BmlncR2036表达后出现体色变暗,中肠残留大量内容物,直至死亡的现象,表明BmlncR2036可能参与家蚕中肠的发育调控。【结论】发现lncRNA BmlncR2036可调控P25基因启动子活性,还可能参与调控家蚕中肠的发育。本研究为进一步探索家蚕lncRNA的功能提供了实验依据。【Aim】Long non-coding RNAs(lncRNAs)play important roles in the regulation of development of the silkworm,Bombyx mori.In our previous study,a lncRNA BmlncR2036 was found near the silk fibroin gene P25 of B.mori.This study aims to further explore the molecular mechanism of BmlncR2036 regulating the expression of P25.【Methods】qPCR was used to detect the expression profiles of BmlncR2036 in different tissues(cuticle,brain,nerve,testis,ovary,silk gland,Malpighian tubules,hemolymph,fat body and midgut)of the day-35th instar larvae of B.mori and in the anterior silk gland,middle silk gland and posterior silk gland during different developmental stages of B.mori.miRNAs that can target both P25 and BmlncR2036 were predicted.The interaction between miRNA and P25 was verified by luciferase assay.Luciferase assay was used to assess the effect of BmlncR2036 on the transcription activity of P25 promoter.Finally,the expression of BmlncR2036 was knocked down in the day-35th instar larvae of B.mori by dsRNA injection,and phenotype changes of their silk glands and midgut were observed.【Results】The qPCR results showed that BmlncR2036 was highly expressed in the posterior silk glands of the day-35th instar larvae and mature 5th instar larvae,showing a expression pattern consistent with that of P25 gene.Meanwhile,BmlncR2036 was also highly expressed in the testis and midgut of the day-35th instar larvae of B.mori,while lowly expressed in other tissues,suggesting the diverse roles of BmlncR2036.miR-2739 and miR-279a can not only match with BmlncR2036,but also target the 3′UTR of P25 gene.Luciferase assay results showed that P25,however,was not the real target of miR-2739 and miR-279a,and BmlncR2036 had a negative effect on the promoter activity of P25,extremely significantly decreasing the luciferase activity by 52%after the co-transfection with pcDNA3.1(+)[BmlncR2036]and pGL3-Enhancer[P25-promoter]vector.After knocking down the expression of BmlncR2036 in the day-35th instar larvae of B.mori,the body color darkened,and a

关 键 词：家蚕 长链非编码RNA 丝素蛋白 丝腺 变态发育 中肠 

分 类 号：Q522[生物学—生物化学]