【作者】 杨洁；

【导师】 吴信生； 秦枫；

【作者基本信息】 扬州大学 ， 农业硕士（专业学位）， 2025， 硕士

【摘要】 近年来,家兔养殖的规模不断提高,但部分养殖场母兔的繁殖效率仍然较低,导致养殖场所获得的经济效益并不理想。产仔数是动物繁殖性状的一个重要经济性状,是评价畜禽生产效益的重要指标。产仔数与卵巢卵泡发育密切相关,高水平的促卵泡激素和雌激素会使动物排卵数增加,这可能会提高动物产仔数。家兔的繁殖性能是影响养殖效率与产业经济效益的重要因素,本研究旨在筛选与产仔性状显著相关的SNP位点作为新西兰白兔产仔性状的选育标记,探究SNP位点对MTHFR基因的m RNA表达的影响以及转录因子对MTHFR的表达调控,为后续遗传改良家兔繁殖性能提供必要的理论依据和经济效益。主要结果如下:1.通过在线软件绘制了MTHFR的基因结构图,结果显示MTHFR基因存在11个外显子,10个内含子。为寻找MTHFR基因上潜在的遗传标记位点,采集125只新西兰白兔母兔的耳样组织提取DNA,对新西兰白兔MTHFR基因的外显子和核心启动子区多态性检测,结果发现MTHFR基因的外显子区不存在SNP位点,可能外显子区相对保守;而在核心启动子区检测出4个SNP位点,分别是g.2231867G>A、g.2231821A>T、g.2231574G>A、g.2231512C>A,均符合哈代温伯格定律(P>0.05)。将这4个SNP位点与新西兰白兔的繁殖性能相关数据进行关联性分析,结果发现只有g.2231512C>A位点的CC型在胎总产仔数、初生窝重、5周龄平均窝仔数显著高于AA基因型(P<0.05),3周龄平均窝仔数极显著高于AA基因型(P<0.01),其它3个SNP位点与新西兰白兔的繁殖性能没有显著关联(P>0.05)。对以上SNP位点进行连锁不平衡分析,结果表明g.2231867G>A、g.2231821A>T存在强连锁不平衡,并且存在3种单倍型,分别是TA、AG、TG。由此组合成的单倍型组合即:TTAA、ATGA、AAGG分别与新西兰白兔繁殖性能相关联分析,结果都没有显著关联(P>0.05)。2.进一步探究转录因子ZNF300对MTHFR启动子活性及表达调控研究。对MTHFR基因启动子区进行核心位点预测,潜在核心位点为-400bp。MTHFR基因的启动子区划分为4段,分别构建启动子区缺失载体,通过双荧光素酶报告系统检测,MTHFR基因的启动子区-300bp～-800bp的双荧光素酶含量最高,与预测结果一致。转录因子预测显示ZNF300位于启动子核心区域。荧光定量结果显示,过表达ZNF300后可以极显著下调MTHFR的m RNA表达水平(P<0.01)。构建g.2231512C>A的野生型和突变型质粒并与过表达ZNF300共转染,通过双荧光素酶检测发现,过表达ZNF300可以极显著降低MTHFR的启动子活性(P<0.01)。Western Blot结果表明,ZNF300蛋白可以负向调控MTHFR启动子活性,最终下调MTHFR的基因表达。3.为探究MTHFR基因的生物学功能,克隆了MTHFR基因的CDS序列,将过表达和敲低MTHFR转染到卵巢颗粒细胞中,荧光定量结果显示过表达MTHFR可以极显著提高MTHFR基因的m RNA表达量(P<0.01),敲低MTHFR可以显著降低MTHFR基因的m RNA表达量(P<0.05)。荧光定量和Western Blot检测过表达和敲低MTHFR后能够调节HSD17B1、TIMP1等与卵巢卵泡发育相关的基因。进一步检测MTHFR基因对卵巢颗粒细胞增殖和凋亡的影响,酶标仪及流式细胞仪检测结果显示,过表达MTHFR基因可以极显著抑制卵巢颗粒细胞增殖,促进颗粒细胞凋亡(P<0.01);敲低MTHFR基因可以极显著促进卵巢颗粒细胞增殖,抑制颗粒细胞凋亡(P<0.01)。更多还原

【Abstract】 In recent years,the scale of rabbit farming has continuously expanded,but the reproductive efficiency of female rabbits in some farms remains low,leading to unsatisfactory economic benefits.Litter size is a critical economic trait in animal reproductive performance and an important indicator for evaluating the production efficiency of livestock and poultry.Litter size is closely associated with ovarian follicle development,as high levels of follicle-stimulating hormone and estrogen increase ovulation,potentially enhancing litter size.The reproductive performance of rabbits is a key factor affecting farming efficiency and industrial economic benefits,with litter traits serving as important economic indicators.This study aimed to screen SNP loci significantly associated with litter traits as selection markers for litter traits in New Zealand white rabbits,explore the effects of SNP loci on MTHFR m RNA expression,and investigate the regulatory role of transcription factors on MTHFR expression,providing essential theoretical and economic foundations for subsequent genetic improvement of rabbit reproductive performance.The main results are as follows:1.The gene structure of MTHFR was mapped using online software,revealing 11exons and 10 introns.To identify potential genetic marker loci in the MTHFR gene,DNA was extracted from ear tissue samples of 125 New Zealand White female rabbits,and polymorphisms in the exons and core promoter region of MTHFR gene were analyzed.No SNP loci were detected in the exons,suggesting that exon regions are relatively conserved.In contrast,four SNP loci(g.2231867G>A,g.2231821A>T,g.2231574G>A,and g.2231512C>A)were identified in the core promoter region,all conforming to Hardy-Weinberg equilibrium(P>0.05).Association analysis between these SNPs and reproductive performance traits demonstrated that the CC genotype of g.2231512C>A exhibited significantly higher total litter size at birth,birth litter weight,and average litter size at 5 weeks(P<0.05),as well as a highly significant increase in average litter size at 3 weeks(P<0.01)compared to the AA genotype.The other three SNPs showed no significant association with reproductive traits(P>0.05).Linkage disequilibrium(LD)analysis revealed strong LD between g.2231867G>A and g.2231821A>T,with three haplotypes identified:TA,AG,and TG.Subsequent haplotype combination analyses(TTAA,ATGA,AAGG)showed no significant correlations with reproductive performance in New Zealand White rabbits(P>0.05).2.Further Investigation into the Regulation of MTHFR Promoter Activity and Expression by the Transcription Factor ZNF300.Potential core regulatory regions within the promoter of the MTHFR(methylenetetrahydrofolate reductase)gene were predicted,identifying a key site at approximately-400 bp.The MTHFR promoter region was divided into four segments,and corresponding deletion constructs were generated.Dual-luciferase reporter assays revealed that the segment spanning-300 bp to-800 bp exhibited the highest luciferase activity,consistent with the prediction.Transcription factor binding site prediction indicated that ZNF300 binds within this core promoter region.Quantitative real-time PCR(q RT-PCR)analysis demonstrated that overexpression of ZNF300 significantly downregulated MTHFR m RNA expression levels(P<0.01).Wild-type and mutant(g.2231512C>A)promoter reporter plasmids were constructed and co-transfected with a ZNF300 overexpression vector.Dual-luciferase assays showed that overexpression of ZNF300 significantly reduced MTHFR promoter activity(P<0.01).Furthermore,Western Blot analysis confirmed that the ZNF300 protein negatively regulates MTHFR promoter activity,ultimately leading to the downregulation of MTHFR gene expression.3.To investigate the biological functions of the MTHFR gene,the Coding sequence(CDS)of the MTHFR gene was cloned.Overexpression and knockdown vectors of MTHFR were transfected into ovarian granulosa cells.q RT-PCR results showed that overexpression significantly increased the m RNA expression level of the MTHFR gene(P<0.01),while knockdown significantly decreased it(P<0.05).Both q RT-PCR and Western blot analyses demonstrated that overexpression and knockdown of MTHFR could regulate the expression of genes related to ovarian follicle development,such as HSD17B1 and TIMP1.Further examination of the effects of the MTHFR gene on the proliferation and apoptosis of ovarian granulosa cells was conducted using a microplate reader and flow cytometry.The results showed that overexpression of the MTHFR gene significantly inhibited the proliferation and promoted the apoptosis of ovarian granulosa cells(P<0.01),whereas knockdown of the MTHFR gene significantly promoted cell proliferation and inhibited apoptosis(P<0.01).更多还原