【作者】 滑国华；

【导师】 杨利国；

【作者基本信息】 华中农业大学 ， 动物遗传育种与繁殖， 2009， 博士

【摘要】 牛和羊是重要的节粮型畜种,可为人类提供肉、奶、皮、毛等优质的畜产品。然而,牛是单胎动物,羊是少(寡)胎动物,自然繁殖力低是制约这两种动物生产经济效益的主要原因。因此,研究牛羊繁殖基础理论,开发提高牛羊繁殖力的育种和繁殖新技术具有重要意义。培育繁殖力高的新品种是提高牛羊繁殖力的重要手段,分析牛羊繁殖性状分子遗传规律和调节机制,开发分子标记辅助选择技术,是培育新品种的基础。牛羊繁殖性状是重要的经济性状,也是复杂的数量性状,受微效多基因控制,同时也受一个或几个主基因控制,筛选鉴定繁殖性状主基因并阐明其作用机制,研究其对卵泡发育的调控作用,对于开发提高牛羊繁殖力的新技术(如胚胎工程、繁殖控制等),建立分子育种技术(如标记辅助选择、转基因育种等),快速改良和提高牛羊繁殖性能具有重要的理论和实际意义。本研究从检测主要生殖激素、次要生殖激素和细胞因子基因多态性着手,分析多态性与产羔数、卵泡发育、精液品质之间的关系,鉴定牛和羊主要繁殖性状主基因,并利用RNA干扰技术鉴定主基因功能,探讨其作用机制,为建立高繁殖力牛羊繁殖和育种新技术奠定理论基础,提供技术支撑。1.山羊产羔和牛精液品质与卵泡发育性状主基因鉴定利用候选基因法,采用PCR-SSCP、PCR-RFLP和Forced PCR-RFLP等技术,分析了FecB、FecX(包括FecXI、FecXH、FecXB和FecXG)、与FecB和FecX连锁的5个微卫星标记(BMS2508、OARHH35、BM143、BM1329和TGLA68)、GH和INHA在6个山羊品种/群体(波尔山羊、海门山羊、黄淮山羊、马头山羊、努比山羊和波淮杂交一代羊)共计614个样本中的多态性,及候选基因或微卫星多态性对山羊产羔和生长性状的影响,鉴定山羊产羔性状主基因,筛选相关分子标记;同时采用PCR-RFLP技术研究了INHβA在荷斯坦种公牛中的多态性,及其对精液品质性状的影响,筛选分子标记,并鉴定荷斯坦种公牛精液品质性状主基因,发现:(1)绵羊繁殖性状主基因FecB(BMPR-IB突变体)、FecX(包括FecXI、FecXH、FecXB和FecXG,均为BMP15突变体)在波尔山羊、海门山羊、黄淮山羊、马头山羊、努比山羊和波淮杂交一代6个品种/群体共计614只羊中不存在多态性,即山羊BMPR-IB的突变体FecB和BMP15的突变体FecX不是山羊产羔性状主基因;(2)与FecB(BMPR-IB突变体)连锁的4个微卫星座位(BMS2508、OARHH35、BM143和BM1329)及与FecX(BMP15突变体)连锁的微卫星座位(TGLA68)在山羊中存在多态性,且不同基因型对波尔山羊产羔数性状影响显著;说明BMPR-IB和BMP15是山羊产羔性状主基因,在这两个基因中可能存在除FecB和FecX外的其他主效位点控制山羊产羔性状;(3)在波尔山羊GH基因中检测到2个SNPs(A781G和A1575G),在A781G位点检测到AA和AB两种基因型,BB基因型缺失,在A1575G位点检测到CC和CD两种基因型,DD基因型缺失;GH基因对波尔山羊的基因效应存在性别差异,对波尔山羊公羊早期生长性状调控作用显著,是波尔山羊公羊生长性状主基因,但对母羊早期生长性状调控作用不明显;波尔山羊母羊GH基因型间产羔数无统计差异(p＞0.05);GH是否对山羊产羔数有直接调控作用,尚需进一步研究;(4)在山羊抑制素α亚基基因(INHA)中共检测到11个遗传多态,分别为-506-＞G、-446C＞T、-155C＞T、-65G＞C、129G＞A、567G＞A、651A＞G、792T＞C、906C＞T、911T＞C、946A＞C,其中,911T＞C和946A＞C分别引起该处氨基酸编码改变,即299Val＞Ala和311Thr＞Pro;-446C＞T、651A＞G和946A＞C位点对不同山羊品种产羔数性状影响显著;INHA基因是山羊产羔性状主基因;(5)在荷斯坦种公牛抑制素βA亚基基因(INHβA)中发现StyⅠ-RFLP多态性,检测到AA、AB和BB三种基因型,其中AB基因型是优势基因型;性状关联分析结果表明,INHβA对荷斯坦种公牛精液品质有显著影响,不同基因型对精液品质效应表现为:AB＞AA＞BB;INHβA是控制荷斯坦种公牛精液品质的主基因。2主基因INHA对牛卵泡发育调控机制研究在候选基因研究基础上,利用RNAi技术进一步研究繁殖性状主基因抑制素α亚基基因(INHA)对牛卵泡细胞生长、凋亡和类固醇激素分泌水平的作用及作用机制,并探讨了INHA对卵泡发育和排卵的局部调节作用,发现:(1)所构建的四种重组干扰质粒(psiRNA1、psiRNA2、psiRNA3和psiRNA4)均能有效降低INHA表达量,其中psiRNA3重组质粒干扰效果最显著;(2)利用RNAi技术干扰INHA,牛颗粒细胞凋亡抑制基因bcl-2和bcl-xl表达量上调,凋亡促进基因Bax表达量下调,细胞因子IGF-Ⅰ和IGF-Ⅱ表达量上调;牛颗粒细胞凋亡减少;细胞周期变化显著,G0/G1期颗粒细胞比例减少,S期和G2/M期颗粒细胞比例增加;雌二醇和孕酮水平上调,雌二醇/孕酮比值下调;这些结果说明,抑制素可能通过调控凋亡家族(bcl-2家族)、细胞生长因子(IGF家族)相关基因表达量和类固醇激素分泌量,发挥其在卵巢内部的局部调节作用,抑制颗粒细胞增殖,促进颗粒细胞凋亡,从而抑制卵泡发育和排卵。更多还原

【Abstract】 Being as two important grain-saving livestocks,cattle and goat can provide abundant regular products for human being,such as meat,milk,fur and cashmere.However,low prolificacy(monotocous) is the main reason limiting the economic profit of these two species.To improve the fecundity,it is very important to clarify the basic mechanisms of reproduction in cattle and goat to explore the new breeding/reproductive techniques.As we all know,it is an efficient way to promote the bovine and caprine fecundity by breeding of high prolific lines or breeds.Analyzing the genetic characteristics and regulatory mechanisms of genes importment for reproduction traits and exploring the molecular marker-assisted selection(MAS) are the bases of breeding new breeds with high fecundity in cattle and goat.The reproductive traits represent both important economic traits and complex quantitative traits,which are controlled by polygenes and one/several major genes.Therefore,it is necessary to screen and identify the major genes of reproduction and further clarify the roles and regulatory mechanisms during reproduction,such as folliculogenesis,which are very important to explore the new reproductive techniques(such as embryo engineering and reproduction control),build up molecular breeding techniques(such as MAS and transgenic breeding),to faster improve the fecundity and reproductive traits in cattle and goat.This study was designed to analyze and identify the major genes for bovine and caprine reproductive traits and further explore their roles and regulatory mechanisms in reproduction.In order to identify those major genes,we first detected the polymorphisms of several candidate genes,including reproductive hormones and some cytokines,and then analyzed the associations between the polymorphisms of these genes and the reproductive traits such as litter size,follicular development and semen quality.Several major genes were confirmed and selected for further study.RNA interference was applied to explore the functions and regulatory mechanisms of these major genes in ovary,which could provide the basic theory to explore the new techniques for improving the bovine and caprine fecundities.The main results are as follows:1.Identification of major genes associated with caprine litter size,bovine semen quality and follicular development traitsPolymorphisms of candidate genes for caprine litter size such as FecB,FecX(including FecXI,FecXH,FecXB and FecXG),5 microsatellite loci(BMS2508,OARHH35,BM143, BM1329 and TGLA68) linked with FecB and FecX,GH and INHA in 614 individuals from 6 goat breeds including Boer goat,Haimen goat,Huanghuai goat,Matou goat,Nubi goat and first generation of Boer goat crossed with Huanghuai goat were detected with PCR-SSCP,PCR-RFLP and forced PCR-RFLP.Based on the associations between those polymorphisms and caprine litter size,several molecular markers were selected for caprine MAS,and several genes were confirmed as major genes controlling caprine litter size and growth traits.Furthermore,the INHβA was identified as the major gene controlling Holstein bulls’ semen quality by polymorphisms detection and association analysis through PCR-RFLP.The major points were as follows.(1) The polymorphisms of FecB mutation in BMPR-IB gene and loci FecXI,FecXH, FecXG,FecXB in BMP15 gene were analyzed by forced PCR-RFLP in 614 individuals from 6 flocks or breeds of goats including Boer,Haimen,first generation of Boer goat crossed with Huanghuai goat,Huanghuai,Nubi and Matou goat.None of mutations were detected in these goat breeds and their crossbreed.These results suggested that fecundity of goat was not linked to the same loci in BMPR-IB and BMP15 as sheep.(2) There are abundant polymorphisms in 4 microsatellite loci(BMS2508,OARHH35, BM143 and BM1329) linked with FecB(BMPR-IB mutant) and locus(TGLA68) linked with FecX(BMP15 mutants) in goat.The caprine mean litter size was differed significantly among different genotypes.These results suggested that BMPR-IB and BMP15 were major genes controlling caprine litter size.There may be other loci in these genes linked with caprine litter size traits excluding FecB and FecX.(3) Two single nucleotide polymorphisms(SNPs)-A781G(Ser/Gly35) and A1575G (Leu147) were identified in gGH gene.Neither BB homozygote genotype nor DD was observed in A781G and A1575G loci,respectively.There are different effects on growth traits with different GH genotypes between Boer bucks and does,as the regulation of GH was more significantly in bucks than in does.These results implied that GH was the major gene controlling growth traits in Boer goat bucks.The litter size of Boer goat does with different genotypes was not shown a significant difference.Whether or not there is a direct regulation of GH on caprine litter size still needs to be further studied.(4) Eleven SNPs were identified in caprine INHA such as -506-＞G,-446C＞T,-155C＞T, -65G＞C,129G＞A,567G＞A,651A＞G,792T＞C,906C＞T and the non-synonymous polymorphisms 911T/C,946A/C resulting 299Val＞Ala and 311Thr＞Pro amino acid mutations,repectively.Among these 11 SNPs,3 loci including -446C＞T,651A＞G and 946A＞C were associated significantly with caprine litter size.These results strongly suggested that INHA was major gene controlling caprine litter size. (5) Sty I-RFLP was identified in INHβA in Holstein bulls with AA,AB(dominant genotype) and BB genotypes.The association analysis indicated that the bovine semen quality differed significantly among different genotypes with effects AB＞AA＞BB.Thus, INHβA was identified as major gene controlling bovine semen quality.2.Regulatory mechanism of major gene INHA on bovine follicular developmentOn the bases of the researches on candidate genes,the effects and regulatory mechanism of INHA on bovine granulose cells(GCs) proliferation,apoptosis,steroid secretion were explored,as well as the local regulatory mechanism on follicular development and ovulation.The results were as follows.(1) The relative expression of INHA in bovine GCs can be significantly reduced by RNAi with psiRNA1,psiRNA2,psiRNA3 and psiRNA4 recombined plasmids.The suppression rate of psiRNA3 was the highest among these 4 plasmids.(2) The relative expressions of bcl-2,bcl-xl,IGF-Ⅰand IGF-Ⅱwere increased, however,the relative expression of Bax was reduced by INHA RNAi in bovine GCs.In addition,the apoptosis was suppressed in GCs.The cell cycle was changed,with a decreased percentage of cells at G0/G1 stage and an increased percentage of cells at both S and G2/M stages.Besides,the secretions of estradiol and progesterone were increased with a decreased rate of estradiol/progesterone.In conclusion,these results suggested that Inhibin can function as a local regulatory mediator,promoting the apoptosis and suppressing the cell proliferation,which might be mediated by regulating the expression of bcl-2 family members and cytokines(IGFs family),and/or the secretion of steriod hormones in GCs,and finally controlling the follicluar develpopment and ovulation.更多还原