【作者】 李艳华；

【导师】 孙东晓；

【作者基本信息】 中国农业大学 ， 动物遗传育种与繁殖， 2019， 博士

【摘要】 本课题组前期针对具有极端高低乳蛋白率、乳脂率的泌乳期荷斯坦牛乳腺上皮组织进行了转录组测序(RNA-Seq)研究,,鉴定到31个奶牛乳蛋白和乳脂性状候选基因。本研究选择其中4个功能基因(DDIT3、RPL23A、SESN2和NR4A1)作为研究对象,旨在分析其对中国荷斯坦牛产奶性状的遗传效应,并寻找潜在致因突变。基于北京三元绿荷的中国荷斯坦母牛群体,选择1093头系谱完整、DHI记录规范的母牛为试验群体,首先利用40头荷斯坦公牛(1093头母牛的父亲)的基因组DNA,采用基因组DNA混池测序方法对上述4个候选基因的全部编码区及上、下游调控区各3000 bp进行分段PCR扩增和测序。根据混池DNA测序结果,共发现38个突变位点(35个SNPs、3个indels),包括DDIT3基因3个、RPL23A基因12个、SESN2基因16个、NR4A1基因7个,其中SESN2基因的2个突变g.125714860 AGCGGGGTGGGGG>-和g.125714806 CCCC>A为本研究新发现。进一步通过飞行时间质谱技术对1093头母牛进行个体基因型检测,单标记关联分析结果显示:35个SNPs和3个indels均至少与一个产奶性状关联显著(P≤0.0001～0.0493)。此外,通过连锁不平衡分析,分别在DDIT3、SESN2和NR4A1基因中发现了 1、1、3和1个单倍型块。关联分析结果显示:部分单倍型与产奶性状关联显著(P≤0.0001～0.0461)。通过JASPAR在线软件分别对DDIT3、RPL23A、SESN2和NR4A1基因的5’调控区突变位点进行转录因子结合位点预测,结果表明11个SNPs和3个indels位点分别改变了一些特异性转录因子的结合位点(TFBSs),提示这些突变位点可能影响了基因的转录活性和表达水平。综上所述,本研究进一步验证了课题组前期RNA-seq研究鉴定的4个候选功能基因的遗传效应,首次确定了DDIT3、R L23A、SESN2和NR4A1基因与中国荷斯坦牛产奶量和乳成分性状显著关联;DDIT3基因的2个突变位点(g.56283814 C>T和g.56284880 C>T)、RPL23A基因的1个位点(g.20702122C>G)、SESN2基因的4个突变位点(g.125714723 A>G、g.125714806 CCCC>A、g.125716120 C>T 和 g.125716884 A>G)和NR4A1基因的1个突变位点(g.27992897 C>T)可能改变了转录因子结合位点,下一步将深入验证其调控功能。更多还原

【Abstract】 Our previous RNA-seq study with the mammary glands of four lactating Holstein cows with extremely high and low phenotypic values of milk protein and fat percentages identified 31 candidate genes,Out of them,in this study,we selected four candidate genes,including DDIT3、RPL23A、SESN2 and NR4A1,to investigate whether they have significant genetic effects on on milk production traits in Chinese Holstein.Through re-sequencing the entire coding regions and 3000 bp of both upstream and downstream regulatory regions of the four genes by using the pooled genomic DNA of 40 Holstein bulls,a total of 35 SNPs and three indels were identified,including three in DDIT3,12 in RPL23A,16 in SESN2,and seven in NR4A1;moreover,two indels,g.125714860 AGCGGGGTGGGGG>-and g.125714806 CCCC>A in SESN2 were novel and have not been reported previously.A total of 1093 Chinese Holstein cows were genotyped for these identified SNPs using MALDI-TOF-MS.Subsequent single SNP analyses revealed multiple significant association to all 35 SNPs and three indels regressed against the dairy production traits(P<0.0001-0.0493).In addition,with linkage disequilibrium(LD)analysis,we found one,one,three,and one haplotype blocks in DDIT3,RPL23A,SESN2,and NR4A1 genes,respectively.Haplotype-based association analyses revealed some haplotypes were also significantly associated with milk production traits(P ≤ 0.0001～0.0461).We predicted the impacts of the alleles on each SNP in the 5’promoter region of DDIT3,RPL23A,SESN2,and NR4A1 genes on potential binding sites using JASPAR software,and found that two,two,nine and one SNPs changed the transcription factor binding sites on the promoter region of the above four genes,respectively.We also found that 11 SNPs and three indels(two in DDIT3,two in RPL23A,nine in SESN2,and one in NR4A1)altered the specific transcription factor binding sites(TFBS)in the promoter thereby regulating the promoter activity,suggesting that they might be the promising potential functional mutations for the milk traits.Together,our findings first indicated the DDIT3,RPL23A,SESN2,and NR4A1 genes had significant genetic effects on milk production traits in Chinese Holstein and verified the results of our previous RNA-seq study.Several SNPs,including g.56283814 C>T and g.56284880 C>T in DDIT3 gene,g.20702122 C>G in RPL23A gene,g.125714723 A>G,g.125714806 CCCC>A,g.125716120 C>T,and g.125716884 A>G in SESN2 gene,and g.27992897 C>T in NR4A1 gene,are potential regulatory mutations for the exprsssion of the four genes and are needed to perform in-depth validation investigation.更多还原