【作者】 淮亚红；

【导师】 许尚忠；

【作者基本信息】 西北农林科技大学 ， 动物遗传育种与繁殖， 2008， 博士

【摘要】 繁殖性状是一个重要的经济性状,其中产仔性能是影响生产效率的最主要因素,受多基因控制,具有加性-显性基因作用模式,其遗传力很低,牛的群体双胎遗传力和排卵遗传力分别为0.03和0.07。目前普遍的方法是通过一些实践技术(如激素诱导、激素免疫和胚胎移植等)提高双胎率,但其存在遗传选择进展慢,手段繁琐、成本高等一系列问题,所以借助分子生物技术手段来提高产仔数是人们研究的另一个方向。公牛的精液品质是影响母牛受胎率及种公牛利用效率的重要因素,也是影响生产效益的关键因素。克隆精液品质相关基因并探讨其相关功能,这对于揭示动物繁殖性状调控机制及加快动物育种进展有重要作用。因此,本研究利用生物信息学和分子生物学相关技术,选取了与精液品质有关的ACTN1、Dmrt7、Mina53、Trf2、Tbxa2r、CyclinA1、GDNF和CIB1共8个基因作为候选基因,进行cDNA克隆、组织表达谱和SNPs检测及与母牛产仔数性状关联分析,为进一步研究这些基因与种公牛精液品质奠定了理论基础。取得了以下结果:基因的cDNA克隆方面1.利用EST拼接和GenScan技术,结合RT-PCR方法,得到了牛ACTN1、Dmrt7、Mina53、Trf2、Tbxa2r、CyclinA1、GDNF和CIB1共8个候选基因完整的CDS区序列并进行了生物信息学分析。2.通过3’RACE的方法,得到了牛Dmrt7基因完整的3’UTR区序列。向GenBank数据库递交了ACTN1、Dmrt7和Trf2基因的cDNA序列,分别获取登录号EF512630、EF534775和EU140625。组织表达方面3.用RT-PCR的方法对其中的5个候选基因在肝脏、睾丸、肺、瘤胃、子宫、小肠、脾脏、心脏、脂肪、卵巢、肾脏和肌肉共12个组织中的表达情况进行了研究,结果发现,除Dmrt7基因只在睾丸组织中表达外,其余4个基因都有比较广泛的组织表达。4.以SYBR Green I与dsDNA结合后检测荧光强度的方法进行定量。对在睾丸组织中表达量比较高的Trf2和Mina53基因在肝脏、睾丸、肺、瘤胃、子宫、小肠、脾脏、心脏、脂肪、卵巢、肾脏和肌肉共12个组织中表达情况进行了研究。结果发现,Trf2基因在睾丸组织中表达量最高,其次是肝脏和子宫,表达最低的是胃和脾脏。Mina53在睾丸组织中表达最高,其次是肝脏和子宫,表达最低的是卵巢。SNPs检测及关联分析方面5.采用PCR-SSCP方法和测序的方法对Dmrt7基因第1内含子、第2外显子和第2内含子;第4内含子、第5外显子和第5内含子;第7内含子,第8外显子和第8内含子进行了SNP扫描,发现在牛Dmrt7基因第2内含子存在G/A和A/T、第4内含子存在C/G和C/T、第7内含子存在C/G共5个单碱基突变位点。在鲁西单胎牛,西门塔尔牛,水牛,晋南牛和海福特牛群体中,对该基因的第4内含子的C/G突变的分布情况进行了研究,结果发现:CC基因型个体在群体中占绝对优势;在水牛和西门塔尔牛群体中没有发现多态现象,其他3个群体均处于中度多态。6.采用PCR-RFLP方法和测序的方法对ACTN1基因的第8外显子和第9内含子,第9外显子和第10内含子,第10外显子和第11内含子,第12外显子和第13内含子,第14外显子和第15内含子进行SNPs扫描,发现在牛ACTN1基因的第9内含子存在G/A,第10内含子存在A/G和G/A,第11内含子存在G/C,13内含子存在A/G,在第15内含子存在G/A和A/G共7个单碱基突变位点。以下是对6个地方品种(鲁西单胎牛,鲁西双胎牛,南阳牛,晋南牛,中国西门塔尔牛和荷斯坦牛群体)的群体进行检测多态性并且运用SAS 9.1软件,采用GLM对其基因多态性与产犊数进行了最小二乘均值差异显著性检验。对第10内含子的A/G突变位点用ApaⅠ限制性内切酶在6个群体中进行群体遗传学分析及其与产犊数进行关联分析。结果表明:所有群体在该位点都处于低度多态。在内含子10的ApaⅠ酶切位点,AG基因型的产犊数的最小二乘均数极显著(P<0.01)高于基因型GG基因型。用HaeⅢ限制性内切酶对13内含子的A/G突变位点在6个群体中进行群体遗传学分析及关联分析。结果表明:在内含子13的HaeⅢ酶切位点,AA基因型个体与AG基因型个体间的产犊数差异不显著(P>0.05)。对第15内含子的G/A突变用RsaⅠ限制性内切酶在6个群体中进行群体遗传学分析及其与产犊数进行了关联分析。结果表明:所有群体在该位点都处于中度多态。在内含子15的RsaⅠ酶切位点,产犊数性状在3种基因型间的差异均不显著(P>0.05)。Dmrt7基因原核表达7.对在睾丸组织中惟一表达的Dmrt7基因进行构建了Dmrt7基因的pET28a+原核表达载体,并成功地实现了对Dmrt7蛋白的融合表达。探讨其对雄性发育的重要作用。更多还原

【Abstract】 Reproduction traits are an important economic trait. Calving trait, which is controlled by polygene, is a mostly factor affecting production efficiency. This trait was affected by add-apparent gene model and it has low heredity. The heredity of twinning and ovary are 0.03 and 0.07, respectively. At present, the rate of twinning is increased by hormone inducement, hormone immunity and embryo transfer, but there are some defects, such as slow choose evolve, numerous means and high cost.How to choose some genes which can improve the sperm quality and/or increase the number of calving is a goog aspect. To open out mechanism of reproduction trait and quicken the course of animal breeding, the genes were cloned about sperm quality. Therefore, 8 genes including ACTN1、Dmrt7、Mina53、Trf2、Tbxa2r、CyclinA1、GDNF and CIB1 were served as candidate genes, theses genes were cloned by RT-PCR technology and bioinformatics means. Tissue expression was employed by RT-PCR technique and real-time PCR .SNP was detected by sequencing and association between the SNPs and the number of calving.1. The complete CDS sequence of ACTN1, Dmrt7, Mina53, Trf2, Tbxa2r, CyclinA1, GDNF and CIB1 cDNA were cloned by RT-PCR, EST, Genescan and analyzed by bioinformatics methods.2. The entire 3’UTR sequence of Dmrt7 gene was cloned by 3’rapid amplified .And the CDS sequence of ACTN1, Dmrt7 and Trf2 genes were submitted to Genbank, the accession numbers were Ef512630, EF634775 and EU140625, respectively.3. The 5 candidate genes were detected in by RT-PCR and the results showed that 4 candidate genes have expression in various tissues except that Dmrt7 gene in specific expressed in testicle.4. The syntheses of the first-strand cDNA was followed by PCR using SYBR Green I was combined with dsDNA , twelve different tissues including liver, testicle, lung, rumen, womb, small intestine, spleen, heart, fat ,ovary, kidney and muscle were collected from a mature Simmental bull and cow for genes expression study of Trf2 and Mina53. SYBR Green I analysis was performed to determine the relative mRNA level of Trf2 and Mina53 in various tissues. The housekeeping geneβ-actin was used for endogenous control. Expression data demonstrate that the bovine Trf2 gene expressed in all 12 tissues of interest, it is expressed predominantly in testis, and very low rumen and spleen. Mina53 gene expressed in 12 tissues of interest, it is expressed predominantly in testis, and very low in ovary.5. Identification for mutation in the amplified fragments of Dmrt7gene was performed in intron1, exon 2 and intron2, intron4, intron5 and exon5, intron7, exon8 and intron8 area. 5 SNPs including G/A, A/T, C/G/, C/T, C/G mutation in different introns were detected by sequencing. Allele frequencies of 1 SNP that can be detected by PCR-SSCP were analyzed among different bovine population (Luxi cattle, Simmental, buffalo, Jinnan cattle and Hereford) and the results showed that the CC genotype frequencies were high in all population exception Simmental group; 3 populations were located in middle polymorphism.6. Identification for mutation in the amplified fragments of ACTN1 gene was performed in exon8 and intron9, exon9 and intron10, exon10 and intron11, exon12 and intron13, exon14 and intron15 area. 7 SNPs including G/A, A/G, G/A, G/C, A/G, G/A, A/G mutation sites in different introns were detected by sequencing. These some domestic breed including Luxi cattle, Luxi twinning cattle, Nanyang cattle, Jinnan cattle, Chinese Simmental and Holstein were used to dectect SNPs. The analysis of least square was used to employ to association between these SNPs and the number of calving by SAS 9.1 software. A/G mutation site in intron10 was detected by ApaⅠrestrition enzyme in domestic breeds. The result showed that, all population was located in low polymorphism. intron10-3124 nt was significant association between the SNPs and the number of calving(P<0.01).A/G mutation in intron13 was detected by HaeⅢrestriction enzyme in domestic breeds. The result showed that there were no association between intron 13-669 SNP and the number of calving (P>0.05).G /A mutation in intron15 was detected by RsaⅠrestriction enzyme in domestic breeds. The result showed that all population was located in middle polymorphism. There were no association between intron15 -227 SNP and the number of calving (P>0.05).7. The prokaryotic expression vector of Dmrt7 genes which was expressed only in testicle was structured using RT-PCR and pET28a+vector, and the Dmrt7 fusion protein was expressed in the BL21 strain.更多还原