旨在利用抑制消减杂交技术构建鹅就巢与产蛋cDNA基因文库,筛选就巢母鹅上调与下调表达的基因.以就巢期和产蛋期鹅卵巢组织互为检测子和驱动子,进行正反双向消减杂交,获得鹅卵巢差异表达基因文库.从双向文库随机挑选单菌落进行PCR验证,结果表明文库质量良好.选取654个阳性克隆进行测序分析,获得641条表达序列标签(ESTs).对ESTs进行去除载体序列、质量检测、聚类及拼接,经BLASTn比对,有166条ESTs找到与之匹配的同源序列,其中92个是功能基因.比较就巢与产蛋SSH文库,发现就巢SSH库中参与细胞凋亡、信号转导及细胞结构的基因出现频率较高；产蛋特异性基因文库中参与物质能量代谢、细胞防御的基因较多；尚有许多差异片段属于未知功能蛋白或理论推定蛋白.结果提示,催乳素受体、抗苗勒管激素以及雌激素受体基因在就巢期上调表达可能与鹅就巢行为的启动与维持有关.该结果为进一步研究鹅就巢行为分子调控机制奠定基础.%The aim of this study was to construct broodiness and laying geese cDNA libraries with SSH, and to identify differentially expressed genes between laying and broody phases. Ovary in broody and laying phases was ascribed to tester and driver for reciprocal SSH procedures. cDNA libraries of specifically or highly expressed genes in broodiness and laying were obtained after two-step hybridization. Positive clones from libraries were randomly selected to validate with PCR. Sequently, 654 positive clones were selected to sequence, qualify, cluster, assemble and compare in GenBank using BLASTn. 166 differentially expressed gene fragments from two libraries showed homology to other known sequences, including 92 functional genes. The expression profiles showed some broodiness-specified genes invovled in apoptosis, cell component, hormones-related signal transduction, and some genes involved in metabolism and cell defense in the laying-specified genes library; some ESTs belonged to unknown or hypothetical protein. The result indicate that three genes(prolactin receptor, estrogen receptor α and anti-mullerian hormone receptor Ⅱ) in broodiness-specified library are associated to trigger or maintain broodiness in geese based on biological function and signal transduction. The result provide the basis for future revealing the mechanism on broody behavior.
展开▼
