【作者】 邓效禹；

【导师】 杨焕民；

【作者基本信息】 黑龙江八一农垦大学 ， 基础兽医学， 2010， 硕士

【摘要】 为深入研究籽鹅垂体调控生殖生理的分子机理,以籽鹅垂体组织为试验材料,用SMART(switching mechanism at 5′end of RNA transcript)方法构建了籽鹅垂体组织的全长cDNA文库。随机挑取11个噬菌斑,用载体克隆位点两端的通用引物进行扩增,以检测所构建的文库的质量。将文库的λTriplEx2噬菌体载体转化到pTriplEx2质粒载体,随机挑取217个克隆,用T7和SP6通用引物进行PCR鉴定,并选取不同长度的基因143个测序。取得结果如下:1.利用λTriplEx2噬菌体构建了籽鹅垂体组织cDNA文库,并对文库质量进行鉴定结果显示,该文库的初始滴度为6×105 pfu/mL,重组率为94%,插入片段长度范围为0.5～2.0 kb。结果表明,成功构建了籽鹅垂体组织cDNA文库。2.选取不同长度143个克隆测序,完成测序140个,去掉冗余序列后共有108个序列,unigene比率为87%。测定的cDNA序列与BLASTn进行比较,其中73个与鸡具有较高的同源性,可认定为已知序列EST,其中1个与大雁的催乳素基因的相似性高达99%;1个与大雁的生长激素基因的相似性高达100%,还有1个与原鸡的铁蛋白重链基因的相似性达到90%。获得35个未注册基因。本研究为进一步从分子水平探讨籽鹅垂体调控生殖生理的分子机理奠定了基础。更多还原

【Abstract】 In order to further study the molecular mechanism of reproductive physiology controlled by pituitary of Zi-goose. The experiment was conducted to constructe the full-length cDNA library from the pituitary gland of the Zi-goose (Anser anser) by SMART (switching mechanism at 5′end of RNA Transcript) technique, which usedλTriplEx2 as a vector. Thenλphage packaging reaction and library amplification were performed. Eleven plaques were randomly picked and tested using PCR method with universal primers derived from the sequence flanking the vector. Then convertingλTriplEx2 to pTriplEx2, 217 clones were randomly picked and tested using PCR with universal primers T7 and SP6, 143 clones were chosen at randomly for sequencing.The results were as follows:1. The full-length cDNA library of pituitary gland from Zi-goose was constructive withλTriplEx2 vector and the qualities of original cDNA library were strictly checked by conventional titer ditermination. The results showed that the titration of the cDNA library constructed was 6×105 pfu/mL, the rate of recombination was 94 %, and the fragment length of inserted DNA ranged mainly from 0.5 to 2.0kb. The library titer, recombination rate and average inserted size all match quality requirments for cDNA library and this indicated that cDNA library of pituitary gland was successfully constructed.2. 143 clones were chosen at randomly for sequencing from the cDNA library, and 140 clones were sequenced successfully, 108 unigene weregained after unigene clusters. The ratio of unigene obtained was up to 87%. The BLAST sequence analysis indicated that 73 clones were known ESTs, and 35 clones were novel ESTs, and after BLASTn analysis of cDNAs, there possible function were predict. Through cross-species genenomic comparison, one of these are similar to the prolaction(PRL) gene of Anser anser ,homology was up to 99%; one of these are similar to the growth hormone(GH) of Anser anser, homology was up to 100%. one of these are similar to the ferritin heavy chain(FTH)of Gallus gallus, homology was up to 90%. This stady laid the foundation for further research on the mechanism of reproductive physiology controlled by pituitary of Zi-goose.更多还原