鲤脑组织cDNA文库的构建及脑室管膜素基因鉴定

Construction and identification of cDNA library from common carp(Cyprinus carpio Linnaeus)brain

用Gubler-Hoffman法构建了低温下鲤(Cyprinus carpio Linnaeus)脑组织cDNA文库,经测定库容量为5.7×105CFU/mL,文库的重组率接近95%,平均插入片段长度约为1.5kb,符合文库保存和筛选实验的要求。同时,根据鲤与低温相关消减cDNA文库中低温下特异表达基因片段序列,设计PCR引物在此cDNA文库中进行PCR检测和序列测定,使用BLAST软件将测序的10个cDNA序列同GenBank等数据库进行比对,结果显示8个阳性克隆有相关同源性,2个克隆为功能未知的基因,全长率近75%;以上结果说明本实验所构建的cDNA文库质量较高,可作为筛选与鲤低温性状相关的功能基因的重要资源。同时对其中一个与低温相关的基因脑室管膜素基因构建了表达载体,为进一步验证该基因在鱼类低温适应中所发挥的作用及其作用机理奠定了基础。

Encephalic cDNA library of common carp（Cyprinus carpio Linnaeus）was constructed by Gubler-Hoffman technique. First,total RNA of brain was extracted by QIAzol reagent and determined by agarose electrophoresis and UV meter. The result showed that the purity and integrity of total RNA were both good. mRNA was isolated with a column of oligo（dT）30 cellulose and then was reversely transcripted to the first strand cDNA by using a modified oligo（dT） primer（contained Not I site）. Second,strand cDNA was produced by employing replacement synthesis technique. After adding EcoR I adaptor and digesting with Not I,double strand cDNAs were fractionated with Sepharose CL- 2B gel filtration medium and sample fractions（〉400 bp）were collected. Finally,ds cDNAs were ligated directionally into pAP3neo vectors and the recombinant vectors were electroperated into E.coli DH10B. A plasmid cDNA library with common carp brain was constructed. The capacity of library reaches 5.7×10^5 CFU/mL in which the percentage of recombinant clones was 95% and the average size of inserts was 1.2 kb approximately. Ten positive clones were sequenced and aligned on Internet,which showed higher identities among 8 clones and 2 clones maybe unknown genes and the percentage of complete cDNA sequences was 75%.These results indicated the encephalic cDNA library of common carp has excellent quality. This library provided a useful resource for the functional genomic research related to cold tolerant traits of common carp. Meanwhile,eukaryotic expression vector about one of the genes was constructed. This study will further the benefit verification of the function and mechanism of the gene in the cold acclimation process of fish.