【作者】 谢岩；

【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， 2013， 博士

【摘要】 课题组前期开展的中国荷斯坦牛产奶性状全基因组关联分析利用传递不平衡(L1-TDT)和回归分析(MMRA)两种统计分析方法同时检测到38个显著SNP位点。本研究旨在基于该GWAS结果进一步筛选和鉴定奶牛产奶性状关键基因并进行功能验证。(1)位置候选基因鉴定及功能注释：基于牛基因组序列草图(Btau4.2),采用生物信息学和比较基因组学方法进行了上述38个显著SNPs的位置候选基因筛查和功能预测,最终鉴定到24个位置候选基因并确定了其物理位置、基因类型及功能注释；基于此,进一步从该24个位置候选基因中挑选了10个基因,利用实时荧光定量RT-PCR技术进行了组织表达谱分析,结果发现EEF1D和PDE9A基因在泌乳期中国荷斯坦牛乳腺组织中的mRNA表达量均明显高于其他7个组织(心肌,子宫,肾,肝,肺,卵巢和小肠)。结合前期GWAS结果,初步说明这2个基因与奶牛泌乳功能相关,将之确定为奶牛产奶性状候选基因,进行细胞水平功能验证。(2)EEF1D和PDE9A基因可变剪接鉴定：通过NCBI和UCSC数据库检索发现EEF1D和PDE9A基因的mRNA序列不完整并且多次改变,同时人和小鼠EEF1D和PDE9A基因mRNA序列的5’端在存在可变剪接,可形成不同转录本,因此推测奶牛的EEF1D和PDE9A基因存在可变剪接可能性较大。通过5’RACE实验,发现EEF1D基因在泌乳期中国荷斯坦牛乳腺组织中确实存在2种转录本,命名为EEF1Da和EEF1D6；EEF1Da(1202bp)和EEF1Db(2195bp)包含不同的第1外显子(exon1a:294bp；exonlb:1287bp),这2个第1外显子没有重叠,其余7个外显子序列和位置一致；利用实时荧光定量RT-PCR技术检测了不同转录本在奶牛乳腺组织的表达水平,结果显示EEF1Da为主要表达的转录本；8个组织的基因表达谱分析表明：EEF1Da在乳腺组织的表达量明显高于EEFIDb并且与EEF1D基因的表达趋势一致。另外,实验结果显不PDE9A基因在荷斯坦牛乳腺组织中只存在一种转录本,通过与已知序列拼接,获得了完整的PDE9A基因mRNA序列。(3)乳腺上皮细胞体外培养：采集健康的泌乳期中国荷斯坦母牛乳腺组织,通过组织块培养法和差酶消化法,获得了纯化的体外培养的乳腺上皮细胞。同时,采用乳汁分离法从收集的泌乳中后期荷斯坦母牛的乳汁中也获得了具有增殖能力的乳腺上皮细胞。经验证,培养的细胞具有生长和增殖能力,可表达上皮特有的角蛋白18基因、部分乳蛋白基因、产奶性状主效基因DAGT1和GHR以及EEF1D和PDE9A基因,适用于目的基因的功能验证。(4)RNA表达量干扰分析：基于EEF1D基因的完整mRNA序列,设计了4条siRNA序列,瞬时转染到奶牛成纤维细胞中,通过实时荧光定量RT-PCR技术检测EEF1D mRNA表达量,结果表明,3条siRNA显著降低EEF1D基因表达量,基于其中2条有效siRNA构建了相应的shRNA载体pGPU6-GFP-NEO-E1357和pGPU6-GFP-NEO-E1893,同时构建了相应shRNA慢病毒表达载体；由于PDE9A基因在牛成纤维细胞中表达量低,未作RNA干扰；另外,人工合成EEF1Da和PDE9A基因的CDS序列并连接到过表达载体,瞬时转染到人293FT细胞,RT-PCR技术检测到了EEF1Da和PDE9A基因表达,说明所构建的载体可用,同时也构建了含EEF1Da和PDE9A基因的CDS序列的慢病毒过表达载体。上述研究结果为深入进行EEF1Da和PDE9A基因的功能验证和奶牛产奶性状关键基因鉴定提供了前期试验基础。更多还原

【Abstract】 Our previous genome-wide association studies (GWAS) have identified38significant SNPs associated with one or multiple milk production traits in a Chinese Holstein population. The purpose of this study was to determine potential genes affecting milk yield and milk composition and function annotation based on such GWAS results and to validate there biological functions.(1) Based on the bovine genome sequence (Btau4.2), bioinformation and comparative analysis were conducted to determine potential genes and predict their functions. As a result, totally24genes corresponding to the aforementioned38SNPs were identified. Then,10out of such24genes were selected for further investigation. The mRNA expression patterns of10genes in8kinds of tissues of Holstein cows showed that both EEF1D and PDE9A genes were highly expressed in the mammary gland of lactating cows than in other7tiusses (cardiac muscle, uterus, kidney, liver, lung, ovary and small intestine). Combined with our previously GWAS results, EEF1D and PDE9A may be related to lactation capacity in Holstein cows. Determined the two genes as candidate genes for milk production traits of cows, and verified their functions in cellular level.(2) The mRNA sequences of both EEF1D and PDE9A genes were found to be not complete and kept changable in NCBI database, and alternative splicing of these genes exist in the mouse and human genes, so that variant splicing is likely to existed in cow. In the present study, we isolated the full-length mRNA sequences of EEF1D to determine if alternative splice variants existed. With rapid amplification of5’cDNA end (5’RACE) analysis, two novel alternative spliced transcript variants were observed in the mammary gland of lactating Holstein cow with length of1202bp and2195bp, named EEF1Da and EEF1Db. Such two variants contained the different first exon from each other (exon1a vs exon1b:294bp versus1287bp) with no overlap between the two types of exon1and the same remaining7exons. With quantitative real-time RT-PCR, the expression patterns of EEF1Da and EEF1Db were investigated across8kinds of tissues of lactating Chinese Holstein cows. It was found that the mRNA expression of EEF1Da was significantly higher than that of EEF1Db and similar to the overall mRNA level of EEF1D in the mammary gland. The PDE9A only exist1transcription in the breast tissue, connecting with the known sequence, get complete mRNA sequence.(3) Based on fresh mammary gland samples lactating Holstein cows from slaughterhouse, we separated the mammary epithelial cells and purified epithelial cells by enzyme digestion differences. Meanwhile, mammary epithelial cells can also be separated from the milk which was collected from Holstein cows in middle or late lactation. It was found that the celles have growth and proliferation ability and were able to express keratin18,some milk protein genes, DAGT1, GHR, EEF1D and PDE9A genes, so that the cell line can be used for gene function validation.(4) Based the complete mRNA sequence of EEF1D, we designed4siRNA fragments for RNAi and transfected them into bovine fibroblasts for12hours. Through real-time quantitative RT-PCR, EEF1D mRNA expression levels were detected. The result showed that3out of4siRNA can significantly reduced EEFID gene expression, of them2siRNA were used to construct shRNA vectors pGPU6-GFP-NEO-E1357and pGPU6-GFP-NEO-E1893, and also constructed the corresponding lentiviral vectors of shRNA. We did not conduct RNAi analysis for PDE9A due to its low expression level in such cell line. On the other hand, we synthesized the CDS sequences of both PDE9A and EEF1Da and connected them to the expression vectors pEGFP-Cl and pIRES-EGFP. Through vector transfection into293FT cells for48h, EEF1Da and PDE9A cDNAs were amplified with RT-PCR method. Result revealed that such vectors were available. In addition, the CDS sequences, we also had connected them to lentiviral vectors.Together, our results provided basis for further systematic function validation of EEF1Da and PDE9A genes and regulation on the formation of milk production traits.更多还原