山羊与牛乳腺差异表达基因的筛选及半定量RT-PCR分析

Screening and semi-quantitative RT-PCR analysis of differentially expressed genes in mammary glands between goat and cow

山羊和奶牛具有非常相似的泌乳过程,但在产乳量和乳成分(脂肪和蛋白含量)之间存在很大的差异,而且山羊奶还具有特殊的膻味。本研究根据山羊和牛在基因编码序列上具有非常高的保守性原理,利用抑制消减杂交技术对3只西农萨能奶山羊和3头荷斯坦奶牛泌乳末期的乳腺组织进行差异表达基因分析。分别提取山羊和牛乳腺组织总RNA,分离mRNA并合成cDNA,以山羊乳腺组织cDNA作为测试,牛的乳腺组织cDNA作为对照。cDNA经RsaⅠ酶切后将测试组cDNA分成两组,分别衔接两种不同接头,再与对照组cDNA进行两次消减杂交及两次PCR反应,产物与T/A克隆载体连接,构建cDNA消减文库,并转染大肠杆菌进行文库扩增。随机挑选克隆经PCR鉴定发现有150个克隆具有200bp-1000bp插入片段,挑选50个差异片段不等的克隆进行测序及同源性分析,结果得到5个已知乳蛋白(αs2酪蛋白、β酪蛋白、К酪蛋白、α乳清白蛋白和β乳球蛋白)基因的编码序列和6个新的EST序列,EST序列已登录GenBank,登录号分别为EG588067、EG588068、EG588069、EG588070、EG588071和EG588072。通过半定量RT-PCR分析,发现β酪蛋白基因mRNA表达量在山羊乳腺中显著高于其在牛乳腺中的表达量,К酪蛋白在乳腺中的mRNA含量与其所表达的蛋白质在乳中含量存在很大差异,表明К酪蛋白虽然和其他酪蛋白具有相同的调控机制,但其在分泌和运输机制上与其他酪蛋白不同。

Although the effect of lactation stage is similar, milk yield and composition （fat and protein contents） differ markedly between goats and cows, and goat milk also has a distinctive flavor. In the present study, because goats and cows exhibit very high homology in their gene coding sequences, the mammary glands in late lactation of 3 Xinong Saanen goats and 3 Holstein cows were chosen to isolate differentially expressed genes in the mammary gland of goat, and a forward suppression subtractive hybridization （SSH） cDNA library was constructed. SSH was performed with eDNA from late lactation mammary glands of cows as the ＂driver＂ and eDNA from late lactation mammary glands of goats as the ＂tester.＂ A total of 50 clones in the SSH library were sequenced. Blastn results revealed that 40 cDNAs had significant sequence homologies with known sequences in the NCBI database, the identified cDNAs only including 5 milk protein genes （os2, β and K-casein, α-lactnlbumin and β-lactoglobnlin）, the other 10 sequences represent 6 new EST sequences and have been registered in GenBank, the accession nos. are EG588067, EG588068, EG588069, EG588070, EG588071 and EG588072. These five milk protein genes and asl-casein gene were chosen to examinetheir expressions through semi-quantitative RT-PCR analysis in mammary gland of goats and cows. The expressions of these genes were all higher in goat mammary gland except αsl-casein. There were significant differences in β casein in the mammary glands between goat and cow. The mRNA abundance of K-casein was very high in mammary glands of the goat and cow, but in milk casein micelles only included 10% K-casein. It is suggested that K-casein had different secretion and transportation mechanisms from other caseins, though they shared similar regulation mechanism .