鸡mir-1658前体基因多态性分析

【目的】研究鸡gga-mir-1658前体区基因遗传变异/单倍型及其在品种间的分布,分析其对micro RNA二级茎环结构和靶基因选择的影响,旨在筛选其中具有潜在生物学功能的变异位点,为进一步揭示其对gga-mir-1658基因表达调控的影响及表型效应奠定基础。【方法】根据鸡gga-mir-1658基因组序列(Gen Bank登录号:NR_035151.1)设计一对特异性引物,采用PCR产物直接测序的方法,对太行鸡(95只)、北京油鸡(83只)和来航鸡(42只)3个鸡种220只个体的gga-mir-1658基因前体区进行多态性检测。使用DNAman、MEGA和mfold软件进行pre-gga-mir-1658基因组序列的比对分析和二级结构模拟。通过SHEsis软件和Haploview软件进行配对连锁不平衡分析和单倍型分析。使用mi Randa软件对gga-mir-1658的靶基因及其复合物自由能变化进行预测分析。【结果】在pre-gga-mir-1658基因共发现6个变异位点,其中次要等位基因频率≥5%位点有g.28 CG、g.31CT、g.70 GA和g.71 G–,4个变异位点均定位于gga-mir-1658基因成熟体的种子区。遗传变异特征分析显示,g.70 GA位点表现为低度多态(PIC0.25),其余3个位点均表现为中度多态(0.25PIC0.50)。适合性检验表明,除了来航鸡的g.31 CT、g.71 G–位点、太行鸡的g.71 G–位点以及北京油鸡的g.70 GA位点之外,其他变异位点在各鸡种均处于哈代-温伯格平衡状态(P0.05)。连锁不平衡和单倍型分析表明,各突变位点之间存在弱连锁平衡;从3个品种鸡中共检测到11种单倍型,其中H1(C C G–)和H11(G T G G)是群体的优势单倍型,频率均大于25%。生物信息学分析表明,种子区的突变可影响gga-mir-1658基因前体二级结构的空间构型和自由能,其中H6单倍型突变体的自由能最高(41.00 kcal·mol-1),H2和H5单倍型突变体的自由能最低(35.70 kcal·mol-1);群体的优势单倍型H1和H11突变体的自由能分别为-36.10和40.04 kcal·mol-1。gga-mir-1658基因不同单倍型成熟体种子区序列存在差异,在gga-mir-1658-5p存在"AUACCAU"、"AUACCAC"2种种子区序列,gga-mir-1658-3p共有"AACUCUG"、"AGCUGUG"、"AACUGUG"和"AGCUCUG"4种种子区序列。针对gga-mir-1658的预测靶基因的生物信息学分析显示,它们主要在基因表达调控、细胞凋亡、免疫系统的发育和B细胞激活等基本生物学过程中显著富集;此外,种子区变化可以影响gga-mir-1658成熟体对靶基因的选择。【结论】(1)gga-mir-1658基因种子区存在4个具有潜在生物学功能和表型效应的突变位点,可组成11种单倍型,其中H1(C C G–)和H11(G T G G)在北京油鸡、太行鸡和来航鸡为优势单倍型。(2)种子区突变影响gga-mir-1658基因前体二级结构的稳定性和靶基因的选择,可能是具有潜在表型效应的重要功能位点。

Polymorphism of Pre-microRNA-1658 Gene in Chicken

【Objective】 To investigate chicken gga-mir-1658 precursor area gene genetic variations/haplotypes and its distribution among all varieties. To analyze its effect on microRNA secondary stem-loop structure and target gene selection. The purpose was to screen the mutation sites as having potential biological function, which might lay the foundation to further reveal its influence on gga-mir-1658 gene expression regulation and phenotypic effects. 【Method】 A pair of specific primers were designed according to the chicken gga-mir-1658 genome sequence (GenBank accession number: NR_035151.1). The polymorphism detection was performed on gga-mir-1658 gene precursor area of 220 individuals including three chicken breeds: Taihang chicken (95), Beijing Fatty Chicken (83) and Leghorn chicken (42) by the PCR products direct sequencing. The comparison analysis and secondary structural simulation of gga-mir-1658 genome sequences were performed using DNAman, MEGA and mfold software. The matching linkage disequilibrium analysis and haplotype analysis were performed by SHEsis and Haploview software. The predictive parsing on the free energy changes of gga-mir-1658 target genes and their complexes was performed by miRanda software. 【Result】There were 6 mutation sites in pre-gga-mir-1658 gene. Among them, secondary allele frequency ≥5% included g.28 C＞G, g.31 C＞T, g.70G＞A and g.71G＞-. Four variable sites were positioned in the seed zone of gga-mir-1658 gene mature body. The genetic variation analysis showed that g.70G＞A site manifested low polymorphism (PIC＜0.25), and the other three sites manifested moderate polymorphism (0.25＜PIC＜0.50). The compatibility test showed that the other variation sites were in a Hardy-Weinberg equilibrium state in all breeds except for Leghorn chicken g.31 C＞T, g.71G＞-, Taihang chicken g.71G＞- and Beijing Fatty Chicken g.70G＞A site (P＞0.05). Linkage disequilibrium and haplotype analysis showed that there was a weak linkage equilibrium between mutated sites; 11 haplotypes were detected among the three breeds. Of which, H1 (C C G -) and H11 (G T G G) were the dominant haplotypes. The frequencies were greater than 25%. Bioinformatics analysis showed that the mutation in the seed zone could influence the space configuration and free energy of gga-mir-1658 gene precursor secondary structure. Of which, H6 haplotype mutants was highest (41.00 kcal·mol^-1). H2 and H5 haplotype mutants were lowest (35.70 kcal·mol^-1); The predominant haplotype H1 and H11 mutants were -36.10 kcal·mol^-1 and 40.04 kcal·mol^-1. Different haplotype mature seed zone sequences of gga-mir-1658 gene were different. The gga-mir-1658-5p included two kinds of seed sequences: “AUACCAU” and “AUACCAC”. The gga-mir-1658-3p included four seed zone sequences: “AACUCUG”, “AGCUGUG”, “AACUGUG” and “AGCUCUG”. The bioinformatics analysis of gga-mir-1658 predictable target gene showed that seed zone could affect the selection of gga-mir-1658 mature body on the target gene. They were mainly enriched in gene expression regulation, cell apoptosis, immune system development and B cell activation, other basic biological processes. 【Conclusion】 (1) There were four mutation sites having potential biological function and phenotypic effects in gga-mir-1658 gene seed zone. They could constitute 11 haplotypes. Of which, H1 (C C G -) and H11 (G T G G) were the dominant haplotypes in Beijing Fatty Chicken, Taihang chicken and Leghorn chicken. (2) The seed zone mutation could affect the stability of gga-mir-1658 gene precursor secondary structure and the selection of target genes, which might be the important functional site having potential phenotype effect.