羊绒细度候选基因靶标miRNAs筛选和鉴定

Screening and Identification of the Candidate Gene Target miRNAs for Cashmere Fineness

为筛选和验证辽宁绒山羊粗型和细型个体羊绒细度相关候选基因ALX4、NT-3、POLD2和AKT1表达调控潜在靶标miRNAs及其表达差异,提取辽宁绒山羊细型和粗型皮肤组织DNA和RNA,先采集辽宁绒山羊粗型和细型肩胛部皮肤组织,根据NCBI上的序列克隆获得ALX4、NT-3、POLD2和AKT1基因的3'UTR,利用Primer 5.0设计并合成功能引物,通过常规PCR扩增ALX4、NT-3、POLD2和AKT1基因的3'UTR序列,在miRBase数据库中,筛选羊绒细度候选靶基因3'UTR靶标miRNAs,然后与辽宁绒山羊(LCG)和内蒙古绒山羊(MCG)品种间皮肤miRNAs高通量测序结果共聚焦,筛选和验证羊绒细度相关候选基因表达调控潜在靶标miRNAs,在NCBI中检索到miRNAs的前体序列,应用RNAfold获得二级结构图及势能图,通过qPCR验证细型辽宁绒山羊(Fine type LCG)和粗型辽宁绒山羊(Coarse type LCG)皮肤组织中羊绒细度候选基因ALX4、NT-3、POLD2和AKT1靶标miRNAs的表达量。结果表明:通过miRBase数据库预测,羊绒细度候选基因ALX4、NT-3、POLD2和AKT1分别获得104,27,19和32个靶标miRNAs,与绒山羊品种间皮肤miRNAs高通量测序结果聚焦分析,ALX4基因聚焦到3个靶标miR-m13,miR-433-5p和miR-671,其表达量是56,2和27;NT-3基因聚焦到1个靶标miR-1296,表达量是41;POLD2基因聚焦到1个靶标miR-130b,表达量是177;AKT1基因聚焦到2个靶标miR-345-3p和miR-2331-3p,其表达量是52和5。其中前体序列的二级结构表明miR-2331-3p的结构最不稳定,miR-1296最小自由能的绝对值最大,结构最为稳定。用qPCR验证miR-m13,miR-433-5p,miR-671,miR-1296,miR-345-3p和miR-2331-3p在辽宁绒山羊细型皮肤中的表达量都显著高于粗型皮肤中的表达量,可能对羊绒细度候选基因发挥重要的正调控作用,而miR-130b在LCG粗型的表达量高于LCG细型表达量,可能miR-130b对羊绒细度相关候选基因起负调控作用。

The purpose of this study was to screen and verify the expression and regulation of the potential target miRNAs and its expression difference between coarse and fine individual cashmere fineness related candidate genes ALX4,NT-3,POLD2 and AKT1 in Liaoning Cashmere Goat.DNA and RNA were extracted from fine and coarse skin tissue of Liaoning Cashmere Goat.The coarse and fine scapular skin tissues of Liaoning Cashmere Goat were collected firstly.According to the sequence of NCBI,the 3'UTRs of ALX4,NT-3,POLD2 and AKT1 genes were obtained,and functional primers were designed and synthesized by Primer 5.0.3'UTR sequences of ALX4,NT-3,POLD2 and AKT1 genes were amplified by conventional PCR.In miRBase database,the target miRNAs was screened by 3'UTR of cashmere fineness candidate gene and then confocal with high-throughput miRNAs sequencing results of Liaoning Cashmere Goat(LCG)and Inner Mongolia Cashmere Goat(MCG).Screening and Verification of Cashmere fineness related candidate Gene expression Regulation potential Target miRNAs,the precursor sequence of miRNAs was found in NCBI.The secondary structure diagram and potential energy map were obtained by using RNAfold.The expression of candidate gene ALX4,NT-3,POLD2 and AKT1 target miRNAs in fine Liaoning cashmere goat(Fine type LCG)and coarse Liaoning cashmere goat(Coarse type LCG)skin was detected by qPCR.The results showed that the candidate gene of cashmere fineness,ALX4,NT-3,POLD2 and AKT1,were predicted by miRBase to obtain 104,27,19 and 32 target miRNAs.Focusing analysis of high-throughput sequencing results of skin miRNAs between cashmere goat breeds,ALX4 gene focused on three targets miR-m13,miR-433-5p and miR-671,the expression levels were 56,2 and 27;The miR-1296,expression of NT-3 gene focused on one target was 41.The expression of POLD2 gene focused on one target miR-130b,was 177.The AKT1 gene was focused on two targets,miR-345-3p and miR-2331-3p,and the expression levels were 52 and 5.The centroid secondary structure of the precursor sequence indicates that the structure of miR-2331-3p is the most unstable,and the absolute value of the minimum free energy of miR-1296 is the largest,and the structure is the most stable.The expression of miR-m13,miR-433-5p,miR-671,miR-1296,miR-345-3p and miR-2331-3p in fine skin of Liaoning cashmere goat was significantly higher than that in coarse skin by qPCR.It may play an important role in positive regulation of cashmere fineness candidate genes,while the expression of miR-130b in coarse type of LCG is higher than that in fine type of LCG.It may be that miR-130b plays a negative role in controlling candidate genes related to cashmere fineness.