文章摘要
鲍宝龙.抑制差减杂交法克隆牙鲆变态早期差异表达的基因[J].水产学报,2006,30(2):
抑制差减杂交法克隆牙鲆变态早期差异表达的基因
Differential expression genes cloned in pro-metamorphosing Paralichthys ofivaceus by suppression subtractive hybridization
投稿时间:2014-03-08  修订日期:2014-03-08
DOI:
中文关键词: 抑制差减杂交 牙鲆 变态早期 变态前 差异表达 相对定量RT-PCR
英文关键词: suppression subtractive hybridization ( SSH) Paralichthys olivaceus pro-metamorphos pre-metamorphosis differential expression relative quantative RT-PCR
基金项目:上海市教委重点学科项目(Y1101);农业部“948”引进项目(2004-Z44);上海市教委发展基金项目
作者单位地址
鲍宝龙 上海水产大学生命科学与技术学院 上海水产大学生命科学与技术学院
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中文摘要:
      为了克隆变态早期牙鲆头部差异表达的基因,采用抑制差减杂交法,以变态前的仔鱼头部表达的RNA作为驱动RNA,建立了牙鲆变态早期的差减cDNA文库,并利用相对定量RT-PCR对其进行了筛选。差减库的平均插入片段558bp左右,阳性率为81.8%。随机测定的45个克隆,在剔除假阳性克隆后,共得到22个不同的基因,其中13个为已知cDNA的同源基因,而另外9个为已知蛋白的同源基因,分别为prolactin receptor-like,COL1A1,M3-muscarinic receptor,Sfrs3,tropomyosin,ribosomal protein L27,ribosomal [rpteom S12,GAPDH,COL1A3。在所有22个基因中,COL1A1基因在差减库中的分布频率最高,为22.2%。RT-PCR检测结果表明,在所检测的11个基因中,所有基因在右眼移动前后的牙鲆头部均有表达,只是体现在表达水平上的不同。
英文摘要:
      To clone.the differential expressional genes between the pre-metamorphosis (17 day post hatching, DPH) and prometamorphosis stage (23 DPH), we used suppression subtracfive hybridization (SSH) to construct a cDNA library of prometamorphosis flounder Paralichthys olivaceus, when the RNA from pre-metamorphosis larvae was used as the driver. The lengths of inserted cDNA fragments in the library ranged from 215 to 774 bp, and the average length was about 558bp, which is much close to the cut frequency of restrict endonuclease Rsa Ⅰ. The percentage of positive clones in the SSH library was 81.8%. Total 45 clones Were sequenced randomly. Using relative quantafive RT-PCR to check the gene expressional level, we thought that the positive clones should express'at higher level in pro-metamorphosis flounder than that in pre-metamorphosis larvae, otherwise, they should be regarded as false positive clones. Total22 genes represent positive clones including 13 homologue genes with unknown proteins, of which most have high similar sequences with other organisms, and 9 homologue genes with known proteins like prolactin receptorlike, COL1A1,M3-muscarinic receptor, Sfrs3, tropomyosin, ribosomal protein L27, ribosomal protein S12, GAPDH, COL1A3. Among all the 22 positive genes, the frequency of COL1A1 clone was highest as 22.2%, the prolactin receptor-like was next to the highest as 8.9%, most genes distributed with lowest frequency 2.2% in SSH cDNA library. The results of RT-PCR showed that the 11 representative genes all expressed in the heads of pre-metamorphosis and pro-metamorphosis flounder larvae.
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