利用DD-PCR分离鳗弧菌刺激相关的牙鲆差异cDNA及表达  

Isolation and expression of differentially expressed cDNA in Paralichthys olivaceus induced by Vibrio anguillarum with DD-PCR

作　　者：范玉顶[1,2] 陈妍婕[1] 钟其旺[1] 齐洁[1] 张全启[1] 

机构地区：[1]中国海洋大学海洋生命学院,海洋生物遗传育种教育部重点实验室,山东青岛266003 [2]中国水产科学研究院长江水产研究所,湖北荆州434000

出　　处：《中国水产科学》2010年第3期431-438,共8页Journal of Fishery Sciences of China

基　　金：国家863高技术研究与发展项目(2006AA10A404);国家自然科学基金项目(30671624;30600455)资助

摘　　要：本研究采用mRNA差异显示技术(mRNA differentid display,DD-PCR)分离得到20个鳗弧菌(Vibrio anguillarum)诱导前后差异表达的牙鲆(Paralichthys olivaceus)cDNA片段。通过对差异片段进行回收、再扩增、克隆、测序、序列比对以及验证分析后,得到9个阳性片段,其中2个片段与已知基因高度同源,一个与牙鲆C3补体高度同源,同源性98%,另一个与牙鲆TEGT(睾丸增强基因转录子)基因高度同源,同源性98%;其余7个为新的cDNA片段。基因表达分析表明,这9个差异片段均在处理组、对照组的不同器官和时间段差异表达,而且多数片段经鳗弧菌诱导后都在肝脏、肾脏、脾脏等鱼类主要免疫器官中上调表达,初步推测它们都是和牙鲆免疫或鳗弧菌病相关的基因。对这些基因进一步的功能研究将对牙鲆的抗病育种及养殖业提供重要的参考。Due to the good taste and nutritional value,Japanese flounder,Paralichthys olivaceus is one of the most important marine aquaculture species and is widely cultured along northern coastal areas in China. Vibrio anguillarum is the main causative agent of vibriosis which is a highly significant disease for cultured and wild marine fish,including Japanese flounder. Vibriosis has resulted in severe economic losses,which hindered the development of aquaculture. The use of antibiotics could partially solve the problem,but bring risks of antibiotic residues in fish,environmental pollution,and antibiotic resistance development. To obtain genes associated with Vibrio anguillarum disease and provide references for molecular marker-assisted selective breeding of Japanese flounder,the mRNA differential display technique（ DD-PCR） was performed to identify and isolate differentially expressed cDNAs representing transcripts from Paralichthys olivaceus challenged by Vibrio anguillarum. Total 20 cDNA fragments were recoveried,cloned and sequenced,of which nine were confirmed to be positive fragments,after elimination of false-positives by Semi-quantitative RT-PCR（ reverse transcription polymerase chain reaction） expression studies. Among these nine positive fragments,BLAST analysis indicated that two of them shared high homology with known genes. One was homologous with complement component 3 gene（ 98% identity）,the other showed 98% identity with TEGT （testis-enhanced gene transcript） gene,while the others were new cDNA fragments and had no high homologous genes in GenBank database. RT-PCR analysis showed that these nine positive fragments differentially expressed in different organisms and times between Vibrio anguillarum treated group and control group,and expressions of most fragments increased in main fish immune organs such as liver,kidney and spleen after infection,so these fragments maybe Vibrio anguillarum-resistance associated genes of Paralichthys olivaceus. Further research on the functions of these gene

关 键 词：牙鲆 鳗弧菌 基因表达 mRAN差异显示 半定量RT-PCR 

分 类 号：Q781[生物学—分子生物学]