西农萨能羊泌乳高峰期和初期乳腺组织差异表达基因研究

Studies on Differentially Expressed Genes in Mammary Gland of Xinong Saanen Goat at Peak and Early Lactation Stages

利用抑制性削减杂交和实时定量PCR研究西农萨能羊泌乳高峰期差异表达基因，结果表明成功构建泌乳高峰期和泌乳初期乳腺组织差异表达削减eDNA文库，以GAPDH为指标检测文库削减效率为2^5倍，共获得78个阳性克隆，PCR检测插入片段主要分布在150～1000bp，挑选插入片段不等的30个克隆测序，获得25个有效序列，代表18个基因。对文库中所包含的血清淀粉样蛋白A3（SAA3），ATP结合盒亚家族G成员2（ABCG2），心脏型脂肪酸结合蛋白（H-FABP）和黄嘌呤脱氢酶（XDH）进行实时定量PCR检测，发现上述4个基因在泌乳高峰期乳腺组织中的表达水平分别是泌乳初期的17．0，7．7，16．3和1．7倍。结论：构建的消减文库可用于筛选泌乳高峰期差异基因，已鉴定的4个基因很可能是调控产奶量和乳成分变化的候选基因。

Suppression subtractive hybridization technology and real-time quantitative PCR were used to isolate differential expressed genes in peak lactation stage. Results showed that peak to early subtraction library was successfully built with cDNA from mammary gland of Xinong Saanen goat at the two lactation stages, and the subtraction efficiency was approximately 2^5 fold indicated by GAPDH; PCR analysis of the total 78 positive clones demonstrated that the cDNA inserts range largely from 150 bp to 1 000 bp; 25 cDNA sequences were identified from 30 clones sequenced which represent 18 genes from the peak to early SSH library; Moreover four genes namely serum amyloid A protein 3 （SAA3）, ATP-binding cassette sub-family G member 2 （AB- CG2）, heart fatty acid-binding protein （H-FABP） and xanthine dehydrogenase （XDH） were chosen to do real-time quantitative PCR to confirm the expression differentiation, and found they were 17.0, 7. 7, 16.3 and 1.7 fold higher in mammary gland of peak lactation than that of early lactation, respectively. Conclusion： the constructed subtraction library could be used to identify different expression genes in peak lactation stage, and the 4 genes verified by real-time quantitative PCR might become candidate genes which had function in regulating the change of milk yield and composition.