本文選題：京海黃雞　切入點(diǎn)：產(chǎn)蛋數(shù)　出處：《揚(yáng)州大學(xué)》2017年碩士論文

【摘要】：京海黃雞擁有小型、優(yōu)質(zhì)、早熟、抗逆性強(qiáng)四大特點(diǎn)。從生產(chǎn)記錄中發(fā)現(xiàn)其群體中存在300日齡產(chǎn)蛋數(shù)高和低的特殊個(gè)體。產(chǎn)蛋數(shù)作為數(shù)量性狀,其不僅易受環(huán)境的影響,同時(shí)受到微效多基因的控制。本文著眼于這兩點(diǎn),選擇了可能對(duì)京海黃雞產(chǎn)蛋數(shù)具有影響的4個(gè)基因(ZP2基因、Stat5b基因、AMH基因、MDH1基因),采用qRT-PCR的方法研究了四個(gè)基因在300日齡京海黃雞個(gè)體組織里的表達(dá)規(guī)律,重點(diǎn)剖析了卵巢組織里的表達(dá)狀況,并通過雙熒光素酶基因報(bào)告系統(tǒng)對(duì)ZP2基因核心啟動(dòng)子區(qū)域進(jìn)行了研究,本文還采用BSP(Bisulfite Sequencing PCR)測(cè)序的方法,對(duì)卵巢組織中ZP2基因啟動(dòng)子區(qū)CpG島的甲基化進(jìn)行了定量檢測(cè),同時(shí)分析了重要甲基化位點(diǎn)對(duì)基因mRNA表達(dá)量的調(diào)控作用,結(jié)合啟動(dòng)子區(qū)潛在的轉(zhuǎn)錄因子結(jié)合位點(diǎn)的預(yù)測(cè),分析甲基化位點(diǎn)所在區(qū)域,進(jìn)一步探討ZP2基因的分子調(diào)控機(jī)制。主要實(shí)驗(yàn)結(jié)果如下:1.通過實(shí)時(shí)熒光定量檢測(cè)4個(gè)基因在300日齡京海黃雞9個(gè)組織中的mRNA的表達(dá)量發(fā)現(xiàn),ZP2基因在卵巢中的表達(dá)豐度最高,其次是腎臟;Stat5b基因在腿肌中的表達(dá)豐度最高,其次是胸肌,在卵巢中的表達(dá)量較低;AMH基因在卵巢中的表達(dá)豐度最高,其它組織中的表達(dá)量較低;MDH1基因在心臟中的表達(dá)豐度最高,其次是腿肌,在卵巢中表達(dá)量較低。ZP2基因在高產(chǎn)組中的表達(dá)量極顯著高于低產(chǎn)組(P0.01),Stat5b基因和AMH基因在高產(chǎn)組的表達(dá)量低于低產(chǎn)組,但沒有明顯差異,MDH1基因在兩組間的表達(dá)量幾乎相同。以上結(jié)果說明,ZP2基因表達(dá)上調(diào)對(duì)京海黃雞的產(chǎn)蛋數(shù)有一定影響,Stat5b基因和MDH1基因?qū)┖｜S雞的產(chǎn)蛋數(shù)可能沒有直接影響,AMH基因在卵巢中具有較高的表達(dá)量,但高低產(chǎn)組的表達(dá)差異不顯著,其在京海黃雞卵巢中發(fā)揮的作用還需進(jìn)一步研究。2.京海黃雞ZP2基因啟動(dòng)子系列缺失片段在DF-1細(xì)胞中具有不同的啟動(dòng)子活性,重組質(zhì)粒pGL3-P6的活性最高,pGL3-P5的活性明顯下降,說明ZP2基因啟動(dòng)子的核心區(qū)域在-1552～-1348之間,與Neural Network Promoter Predietion在線網(wǎng)站預(yù)測(cè)的結(jié)果一致。3.通過軟件預(yù)測(cè)發(fā)現(xiàn)ZP2基因啟動(dòng)子區(qū)共有4個(gè)CpG島,利用BSP技術(shù)檢測(cè)啟動(dòng)子區(qū)CpG島的甲基化狀態(tài)。ZP2-1擴(kuò)增片段總體甲基化程度與mRNA表達(dá)量有一定程度的負(fù)相關(guān)(R=-0.197,P=0.672),所有單個(gè)位點(diǎn)的甲基化程度與mRNA表達(dá)量的相關(guān)性都沒有達(dá)到顯著水平;ZP2-2擴(kuò)增片段總體的甲基化程度與mRNA的表達(dá)量也有一定程度的負(fù)相關(guān)(R=-0.264,P=0.567),其中mC-9位點(diǎn)的甲基化程度與mRNA存在極顯著的負(fù)相關(guān)(P0.01),但其并沒有位于轉(zhuǎn)錄因子結(jié)合位點(diǎn)內(nèi),mC-20和mC-21位點(diǎn)的甲基化程度與mRNA存在顯著的負(fù)相關(guān)(P0.05),且都位于Sp1轉(zhuǎn)錄因子結(jié)合位點(diǎn)上,推測(cè)上述位點(diǎn)可能是調(diào)控轉(zhuǎn)錄的主要位點(diǎn),且可能通過抑制Sp1和DNA的結(jié)合,從而抑制ZP2基因的轉(zhuǎn)錄。
[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.
【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S831

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