miRNASNPs调控鸡miRNA生物合成及功能的机制

MicroRNAs (miRNAs) are recently discovered a type of small non-coding RNA that perform their function in the posttranscriptional gene regulation. MiRNAs not only regulate complex phenotype in animal, but miR-SNPs may also affect miRNA dysfunction, resulting in biological phenotypic traits variation. In this study, we used bioinformatics method to select and identify SNP sites in chicken miRNAs.For each of these variants,Northern blotting and real time were performed to validate the effects of the SNP on processing of miRNAs, and Luciferase repoter was adopt to detect the effects on miRNAs target gene expression. The resource population generated from Gushi (G) chicken and Anka (A) broilers was used as research materials, we analyzed the genetic effect between genotypes and traits, studied the relationship of SNP genotypes which were highly relevant to the production performance with gene expression , protein expression differences and miRNAs expression differences. The purpose of the study was to clarify the role of miRNA-SNP to the formation of chicken growth and meat traits. Reveal how miRNA-SNP affects miRNAs synthesis, mature, and the regulation role to secondary structure of the precursor miRNA and target gene expression, providing evidence for the chicken traits formation molecular mechanism.

miRNA是近几年发现的基因表达转录后调控的小分子，不仅参与动物复杂性状表型形成的分子调控过程，而且miRNA自身或靶基因结合位点发生突变（简称miRNASNP），还可能导致miRNA功能异常，进而引起生物表型性状的变异。本课题对鸡miRNA基因和miRNA靶基因结合位点的SNP筛选和鉴定，采用qPCR或northern blot检测SNP对miRNA生物合成和功能的影响；荧光报告素酶基因检测SNP对miRNA靶基因选择的影响；对目的的miRNASNP以固始鸡资源群体为素材，进行SNP与性状遗传效应分析；对与生产性能高度相关的SNP不同基因型的基因表达、蛋白表达和miRNA表达差异间关系分析。明确miRNASNP对鸡肉质和生长等性状形成的作用，揭示miRNASNP影响miRNA合成、成熟，以及对前体二级结构的影响和对靶基因表达的调控，为研究鸡性状形成的的分子机理提供理论依据。

利用在线软件对预测到88个miRNASNP， 8 个SNP位点在种子区； 9个SNP在miRNA成熟体， 40个SNP有宿主基因。对88个SNP位点，采用混合DNA池法，在固始鸡F2资源群体中检测到 53个SNP，真实存在的SNP有34个，新发现19个，与35 miRNA有关。采用PCR-RFLP和采用飞行质谱方法对31个miRNASNP在F2代资源群的860只个体中进行基因分型，并与生长、屠体等经济性状关联分析。筛选到8个miRNA SNP位点与体重显著关联； 8个miRNA SNP等位点与屠宰性能显著关联， 4个miRNA SNP与胸肌肌纤维直径显著相关，这些标记有望作为分子标记在育种中使用。采用M-fold软件预测25个miRNASNP不同基因型对二级结构的自由能和二级结构的影响，其中位于miR-1666等13个位点即改变形成二级结构的自由能也改变二级结构，位于miR-1749等7个位点仅改变自由能但不改变二级结构，miR-1816等5个位点不改变自由能和二级结构。qPCR检测在不同基因型的固始鸡资源群腿肌中成熟miR-1666、miR-1606、miR-1704 miRNA表达量差异显著（P<0.05）；体外试验，构建表达载体转染DF1细胞，qPCR结果表明：不同基因型转染后df1细胞后成熟miR-1666、miR-1606、miR-1749、miR-1704的表达量差异显著。miR-1816不同基因型成熟miR-1816的表达量差异不显著。通过构建靶基因psiCHECK2载体，分别与构建的miRNA过表达载体或者miRNA的Mimics转染细胞，双荧光报告系统结果表明：CBFB是miR-1666靶基因、IRS2可能是miR-1704的靶基因，TNRC6B是miRNA-1749靶基因， miR-1606的mimics转染试验，但是不影响PDGFB、SCL25A1和COG5的表达量，暗示该这三个基因不是miR-1606的靶基因。miR-1666不同等位基因的表达载体转染DF1细胞后，荧光定量检测靶基因CBFB的表达，结果显示miR-1666表达载体对CBFB有更强的抑制作用，且两组间差异显著(P<0.05)，结果提示miR-1666前体区的SNP能够影响miR-1666对靶基因CBFB的调控作用。但miR-1704前体的SNP不影响IRS2的表达量。