抑制差减杂交法克隆牙鲆变态早期差异表达的基因

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。

Differential expression genes cloned in pro-metamorphosing Paralichthys olivaceus by suppression subtractive hybridization

To clone the differential expressional genes between the pre-metamorphosis(17 day post hatching,DPH) and pro-metamorphosis stage(23 DPH),we used suppression subtractive hybridization(SSH) to construct a cDNA library of pro-metamorphosis 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 quantative 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.Total 22 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 receptor-like,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.