京海黄鸡ZP2基因启动子区甲基化对基因表达及产蛋数的影响分析

发布时间：2018-03-30 09:46

本文选题：京海黄鸡　切入点：产蛋数　出处：《扬州大学》2017年硕士论文

【摘要】：京海黄鸡拥有小型、优质、早熟、抗逆性强四大特点。从生产记录中发现其群体中存在300日龄产蛋数高和低的特殊个体。产蛋数作为数量性状,其不仅易受环境的影响,同时受到微效多基因的控制。本文着眼于这两点,选择了可能对京海黄鸡产蛋数具有影响的4个基因(ZP2基因、Stat5b基因、AMH基因、MDH1基因),采用qRT-PCR的方法研究了四个基因在300日龄京海黄鸡个体组织里的表达规律,重点剖析了卵巢组织里的表达状况,并通过双荧光素酶基因报告系统对ZP2基因核心启动子区域进行了研究,本文还采用BSP(Bisulfite Sequencing PCR)测序的方法,对卵巢组织中ZP2基因启动子区CpG岛的甲基化进行了定量检测,同时分析了重要甲基化位点对基因mRNA表达量的调控作用,结合启动子区潜在的转录因子结合位点的预测,分析甲基化位点所在区域,进一步探讨ZP2基因的分子调控机制。主要实验结果如下:1.通过实时荧光定量检测4个基因在300日龄京海黄鸡9个组织中的mRNA的表达量发现,ZP2基因在卵巢中的表达丰度最高,其次是肾脏;Stat5b基因在腿肌中的表达丰度最高,其次是胸肌,在卵巢中的表达量较低;AMH基因在卵巢中的表达丰度最高,其它组织中的表达量较低;MDH1基因在心脏中的表达丰度最高,其次是腿肌,在卵巢中表达量较低。ZP2基因在高产组中的表达量极显著高于低产组(P0.01),Stat5b基因和AMH基因在高产组的表达量低于低产组,但没有明显差异,MDH1基因在两组间的表达量几乎相同。以上结果说明,ZP2基因表达上调对京海黄鸡的产蛋数有一定影响,Stat5b基因和MDH1基因对京海黄鸡的产蛋数可能没有直接影响,AMH基因在卵巢中具有较高的表达量,但高低产组的表达差异不显著,其在京海黄鸡卵巢中发挥的作用还需进一步研究。2.京海黄鸡ZP2基因启动子系列缺失片段在DF-1细胞中具有不同的启动子活性,重组质粒pGL3-P6的活性最高,pGL3-P5的活性明显下降,说明ZP2基因启动子的核心区域在-1552～-1348之间,与Neural Network Promoter Predietion在线网站预测的结果一致。3.通过软件预测发现ZP2基因启动子区共有4个CpG岛,利用BSP技术检测启动子区CpG岛的甲基化状态。ZP2-1扩增片段总体甲基化程度与mRNA表达量有一定程度的负相关(R=-0.197,P=0.672),所有单个位点的甲基化程度与mRNA表达量的相关性都没有达到显著水平;ZP2-2扩增片段总体的甲基化程度与mRNA的表达量也有一定程度的负相关(R=-0.264,P=0.567),其中mC-9位点的甲基化程度与mRNA存在极显著的负相关(P0.01),但其并没有位于转录因子结合位点内,mC-20和mC-21位点的甲基化程度与mRNA存在显著的负相关(P0.05),且都位于Sp1转录因子结合位点上,推测上述位点可能是调控转录的主要位点,且可能通过抑制Sp1和DNA的结合,从而抑制ZP2基因的转录。
[Abstract]:Jinghai Yellow Chicken has four characteristics: small, high quality, early maturing and strong resistance to stress. It is found from the production records that there are special individuals with high and low egg number at 300 days of age in their population. As a quantitative trait, the egg number is not only easily affected by the environment, but also affected by the environment. It's also controlled by multiple genes with minimal effects. This paper focuses on these two points. Four genes, ZP2 gene, Stat5b gene, AMH gene and MDH1 gene, which may have an effect on egg number of Jinghai yellow chicken were selected. The expression of four genes in the individual tissues of 300 day old Jinghai yellow chicken was studied by qRT-PCR method. The expression of ZP2 gene in ovarian tissues was analyzed, and the core promoter region of ZP2 gene was studied by double luciferase gene reporting system. The method of BSP(Bisulfite Sequencing PCR sequencing was also used in this paper. The methylation of CpG islands in the promoter region of ZP2 gene was quantitatively detected in ovarian tissues. The regulation of important methylation sites on the expression of gene mRNA was analyzed, and the prediction of the potential transcription factor binding sites in the promoter region was also analyzed. Analysis of the region where the methylation site is located, The main results were as follows: 1. By real-time fluorescence quantitative detection of mRNA expression in 9 tissues of 300-day-old Jinghai Yellow Chicken, it was found that ZP2 gene was the most abundant in the ovary. The second was the highest expression abundance of Stat5b gene in the leg muscle, followed by the pectoralis muscle, the highest expression of AMH gene in ovary, and the highest expression of MDH1 gene in heart in other tissues. The lower expression of ZP2 gene in the ovary was significantly higher than that in the low yield group and the expression of AMH gene in the low yield group was lower than that in the low yield group. However, there was no significant difference in the expression of MDH1 gene between the two groups. The above results indicate that the up-regulation of ZP2 gene expression may have some effect on egg production of Jinghai Yellow Chicken. The results suggest that the number of eggs produced in Jinghai Yellow Chicken may not be affected by MDH1 gene and Stat5b gene. The expression of AMH gene in ovary was directly affected by the expression of AMH gene. However, there was no significant difference in expression between high and low birth groups, and its role in the ovary of Jinghai Yellow Chicken needed to be further studied. 2.The deletion fragment of ZP2 promoter series of Jinghai Yellow chicken had different promoter activity in DF-1 cells. The activity of pGL3-P5 was the highest in the recombinant plasmid pGL3-P6, indicating that the core region of the promoter of ZP2 gene was between -1552 and 1348, which was consistent with the results predicted by the Neural Network Promoter Predietion online website. The results of software prediction showed that there were four CpG islands in the promoter region of ZP2 gene. BSP technique was used to detect methylation status of CpG island in promoter region. ZP2-1 total methylation level was negatively correlated with mRNA expression to some extent. There was no correlation between methylation level of all single loci and mRNA expression level. There was also a negative correlation between the total methylation of ZP2-2 amplified fragment and the expression of mRNA. The methylation degree of mC-9 locus was negatively correlated with mRNA, but it was not located in transcription factor. The methylation of mC-20 and mC-21 sites in binding sites was negatively correlated with mRNA, and they were both located at Sp1 transcription factor binding sites. It is suggested that these sites may be the main sites regulating transcription and may inhibit the transcription of ZP2 gene by inhibiting the binding of Sp1 and DNA.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S831

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