调控山羊乳腺脂肪酸代谢miRNAs筛选及相关pri-miRNAs克隆验证

microRNA(miRNA)是调控脂类代谢的重要分子。本研究采用数据库预测和自由能分析法,筛选与山羊(Capra hirus)乳腺脂肪酸代谢相关的miRNA,并对预测得到的miRNA进行克隆验证。预测结果表明,通过MicroCosm、TargertScan和PicTar3个在线数据库预测与山羊脂肪酸代谢相关的30个基因相对应的miRNA,预测得到50条miRNA,其中3个数据库预测一致的miRNA为13条,2个数据库预测一致的为37条。结果显示,脂肪酸合酶基因(FASN)对应4个不同的miRNA,嗜乳脂蛋白基因(BTN1A1)对应2个,而磷酸甘油酰基转移酶6基因(AGPAT6)没有预测到miRNA靶位点;FASN与BTN1A1的3’UTR上miR-103的结合位点分别有3个和2个。通过MFOLD软件对预测所得的50条miRNA靶位点两侧序列的自由能(ΔG>-20kcal/mol)进行分析,9种miRNA与调控脂肪酸代谢基因相关度较高,分别为miR-103/BTN1A1、miR-15/FASN、miR-23/LPL(脂蛋白酯酶)、miR-27/PPARγ(过氧化物酶体增殖物激活受体)、miR-29/GPR41(短链脂肪酸受体)、miR-146/BTN1A1、miR-195/FASN、miR-200/SCD(硬脂酰辅酶A去饱和酶)和miR-497/GPR41,其中miR-103/27/195分别位于BTN1A1、PPARγ和FASN的靶位点与已知物种间同源性较高,增加了这些miRNA/mRNA结合的可能性。此外,靶位点单侧和双侧ΔG小于阈值的miRNA/mRNA分别为14对和9对,其中miR-27/mRNA预测的靶基因为4个,依次为FASN、ACOX1(乙酰辅酶氧化酶基因1)、LPL和PPARγ,miR-103和miR-15的靶基因同为FASN、BTN1A1和ACOX1。以西农萨能奶山羊(Capra hirus)基因组DNA为模板,可扩增出与5条miRNA(miR-103-1/23a/27a/146b/200a)分别相对应的初级miRNA(pri-miRNA);通过序列分析和二级结构分析表明,5条pri-miRNA均包含完整的pre-miRNA序列,同时具有典型的茎环结构,能够产生相应的miRNA。与牛的同源性分析表明,除pre-miR-27a同源性为98%外(山羊pre-miR-27a3’端第11个碱基为G,而牛为A),其余4条pre-miRNA同源性均为100%。因此,本研究筛选的9种miRNA可能调控山羊乳腺脂肪酸代谢,克隆的5条pri-miRNA为其功能研究提供基础。

Screening miRNAs Regulating Fatty Acid Metabolism in Goat(Capra hirus)Mammary Gland and Cloning Determination of Related Pri-miRNAs

microRNA(miRNA) plays an important role for lipid metabolism.To find the potential miRNAs regulating fatty acid metabolism of mammary gland of goat,Databases and MFOLD were used for predicting miRNA and analyzing ΔG,respectively.And pri-miRNA were cloned for further study.Prediction results showed that 50 miRNAs including 13 identical miRNAs predicted by 3 databases and 37 identical miRNAs predicted by 2 databases were obtained through predicting 30 genes regulating fatty acid metabolism of goat by MicroCosm,TargertScan and PicTar on line.There were 4 and 2 different miRNA predicted targeting FASN(fatty acid synthase) and BTN1A1(goat butyrophilin,subfamily 1,member A1),respectively.And no miRNA binding sites was predicted on 3’UTR of AGPAT6(1-acylaglycerol-3-phosphate O-acyltransferase 6).There were 3 and 2 numbers of different location respectively on 3’UTR of FASN and BTN1A1 targeting by miR-103.The results of ΔG analysis(ΔG >-20 kcal/mol) of flanking regions of 50 miRNAs binding sites showed that 9 miRNA were related to fatty acid metabolism,miR-103/BTN1A1,miR-15/FASN,miR-23/LPL(lipoprotein lipase),miR-27/PPARγ(peroxisome proliferator-activated receptor γ),miR-29/GPR41(orphan g protein-coupled receptor family,member 41),miR-146/BTN1A1,miR-195/FASN,miR-200/SCD(Stearoyl-CoA desaturase) and miR-497/GPR41.And there were highly homology on the binding sites of miR-103/BTN1A1,miR-27/PPARγ,miR-195/FASN among species,which sthrengthened the possibility of the connection between miRNA and their targets.Also,the numbers of one side and two sides of flanking regions of miRNA/mRNA binding sites whose ΔG were smaller than threshold were 14 and 9,respectively.Among all the predicted targets,FASN,ACOX1(acyl-CoA oxidase 1),LPL and PPARγ were targets of miR-27.FASN,BTN1A1 and ACOX1 were both targets of miR-103 and miR-15.5 pri-miRNA(pri-miR-103-1/23a/27a/146b/200a) were cloned from DNA template from blood of Xinong Saanen dairy goat(Capra hirus).The sequence and secondary structure analysis demonstrated that all the 5 primary miRNA(pri-miRNA) comprising completed pre-miRNA and the stem loop structure could generate functional miRNA in vivo.The results of homology analysis showed that the homology of 4 pre-miRNA of goat were 100 % with cow except for pre-miR-27a whose homology was 98 %(There was a G located in the 11 base from 3’ end of pre-miR-27a of goat instead of A of cow).In conclusion,those 9 miRNAs can regulate fatty acid metabolism in goat mammary gland,and cloning of 5 pri-miRNAs provides a basis for their function studying.