鸡miR-1666前体区SNP对其成熟体生成和其靶基因调控的影响

发布时间：2018-05-30 16:02

本文选题：鸡 + miRNA　；参考：《河南农业大学》2015年硕士论文

【摘要】：Micro RNA(mi RNA)是近年来鉴定的具有重要转录后调控功能的非编码RNA,由于每个mi RNA的靶基因众多,而位于mi RNA基因上的单核甘酸多态性(Single Nucleotide Polymorphisms,SNP)能够改变其加工过程或其对靶基因的选择进而影响该mi RNA功能的发挥,因此鉴定与揭示mi RNA SNPs的功能与作用显得尤为重要。本研究的主要目的是探究位于mi R-1666前体区的SNP是否影响mi RNA成熟体的生成,揭示mi R-1666的生物学功能以及该SNP对mi R-1666功能的影响。该研究用生物信息学方法筛选出鸡mi R-1666前体区存在的SNP,以河南省家禽种质资源创新工程研究中心构建的固始鸡-安卡鸡F2代资源群为研究材料,DNA测序和PCR-RFLP的方法鉴定出位于资源群mi R-1666基因的SNP并进行基因分型,与资源群的肉质性状、生长性状、屠体性状等经济性状进行关联分析初步揭示该SNP的功能;通过构建mi R-1666不同等位基因的p EGFP-N1表达载体并转染DF1细胞,用荧光定量的方法检测该SNP对成熟mi RNA生成的影响;用双荧光素酶报告系统结合荧光定量的方法鉴定和验证mi R-1666的靶基因,并探究该SNP对mi R-1666靶基因调控的影响。本研究主要得到以下结果:1.生物信息学结合测序鉴定的方法在固始鸡和安卡鸡F2代资源群中检测到唯一一个位于mi R-1666前体区上的突变位点即rs14120863(C/G)。与F2代资源群经济性状关联分析表明,该SNP与鸡的半净膛重、全净膛重、胸肌重、腿肌重及部分体尺性状包括胫围、胸深、胸骨长和体斜长存在显著关联,且CC型个体显著优于GG型。2.M-fold软件预测显示该SNP能够引起碱基的错配进而产生一个新的凸起且该SNP改变了mi R-1666二级结构的最低自由能,自由能由G等位基因的-37.5kcal/mol改变为C等位基因的-35.2kcal/mol,二级结构的稳定性降低。荧光定量结果显示固始-安卡鸡F2代资源群中GG基因型个体胸肌组织样中成熟mi R-1666的表达量显著高于CC基因型;通过构建mi R-1666不同等位基因的p EGFP-N1表达载体并转染DF1细胞,荧光定量检测表明G等位基因成熟mi R-1666的表达显著高于C等位基因,与上述结果相符。结果表明mi R-1666前体区的rs14120863 SNP能够影响成熟mi R-1666的生成。3.生物信息学的方法结合荧光定量技术筛选出ARF6,CBFB和TAB2为mi R-1666的候选靶基因,进而通过构建上述三个基因的psi-Check2双荧光素酶报告载体,确定CBFB为mi R-1666的一个靶基因,而ARF6和TAB2并非其靶基因。最终通过荧光定量检测mi R-1666不同等位基因对靶基因CBFB的调控作用,结果显示该SNP能够影响mi R-1666对CBFB的调控能力。由以上试验结果,可得出如下结论:Mi R-1666前体区的SNP能够改变成熟mi R-1666的表达量,进而影响mi R-1666对靶基因CBFB的调控作用,由此可能导致该SNP与鸡的体尺性状显著相关,而在鸡的生长发育过程中扮演重要的角色。
[Abstract]:Micro RNA(mi RNAs are noncoding RNAs with important posttranscriptional regulation functions identified in recent years, because of the large number of target genes in each mi RNA. The single Nucleotide polymorphism located on the mi RNA gene can change the processing process or the selection of target gene, thus affecting the function of the mi RNA. Therefore, it is very important to identify and reveal the function and function of mi RNA SNPs. The main purpose of this study was to investigate whether the SNP located in the precursor region of mi R-1666 affects the production of the mature body of mi R-1666, and to reveal the biological function of mi R-1666 and the effect of the SNP on the function of the R-1666. In this study, we used bioinformatics method to screen out the SNPs in the precursor region of chicken mi R-1666. Using the F _ 2 generation resource population of Gushi Chicken and Anka Chicken constructed by Henan Poultry Germplasm Resources Innovation Engineering Research Center as the research material, we sequenced the DNA and PCR-RFLP of the F _ 2 generation of Gushi Chicken and Anka Chicken. The SNP located in the resource group mi R-1666 was identified and genotyped. The function of SNP was preliminarily revealed by correlation analysis with the fleshy traits, growth traits and carcass traits of the resource group, and the expression vector of p EGFP-N1 of different alleles of mi R-1666 was constructed and transfected into DF1 cells. The effect of SNP on the production of mature mi RNA was detected by fluorescence quantitative method, and the target gene of mi R 1666 was identified and verified by double luciferase report system combined with fluorescence quantitative method, and the effect of this SNP on the regulation of target gene of mi R 1666 was investigated. The main results of this study are as follows: 1. Bioinformatics combined with sequencing method detected the only mutation site in F _ 2 resource population of Gushi chicken and Anka chicken in the precursor region of mi R-1666, which was rs14120863 / C / GG. The correlation analysis with F2 resource group showed that there were significant correlations between the SNP and the half weight, the whole weight, the breast muscle weight, the leg muscle weight and some body size traits, including tibia circumference, chest depth, sternum length and body oblique length. Moreover, CC type is superior to GG type .2.M-fold software in predicting that the SNP can cause base mismatch and then produce a new bulge, and the SNP changes the minimum free energy of the secondary structure of mi R-1666. The free energy changed from -37.5 kcal / mol of G allele to -35.2kcal / mol of C allele, and the stability of secondary structure decreased. The results of fluorescence quantitative analysis showed that the expression of mature miR-1666 in GG genotype individuals was significantly higher than that in CC genotype, and the expression vector of p EGFP-N1 with different alleles of miR-1666 was constructed and transfected into DF1 cells. Fluorescence quantitative analysis showed that the expression of mature mi R-1666 of G allele was significantly higher than that of C allele, which was consistent with the above results. The results showed that the rs14120863 SNP of the precursor region of mi R-1666 could affect the formation of mature mi R-1666. The candidate target genes of miR-1666 were screened by bioinformatics combined with fluorescence quantitative technique. Then, by constructing psi-Check2 double luciferase report vector of the three genes, CBFB was identified as a target gene of miR-1666. ARF6 and TAB2 are not their target genes. Finally, the regulatory effects of different alleles of miR-1666 on target gene CBFB were detected by fluorescence quantitative analysis. The results showed that the SNP could affect the regulatory ability of mi R-1666 to CBFB. From the above results, it can be concluded that the SNP of the precursor region of 1: Mi R-1666 can change the expression of mature mi R-1666, and then affect the regulation of the target gene CBFB by mi R-1666, which may lead to a significant correlation between the SNP and the body size traits of chicken. And play an important role in the growth and development of chickens.
【学位授予单位】：河南农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S831

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