凡纳滨对虾TCP-1-Beta基因的克隆及其与耐寒性状的相关性  

MOLECULAR CLONING OF LITOPENAEUS VANNAMEI TCP-1-BETA GENE AND ANALYSIS ON ITS RELATIONSHIP WITH COLD TOLERANCE

作　　者：彭金霞[1] 殷勤[1,2] 崔亮[1] 王志伟[2] 李奎[2] 陈晓汉[1] 

机构地区：[1]广西壮族自治区水产研究所,南宁530021 [2]中国农业科学院北京畜牧兽医研究所,北京100094

出　　处：《水生生物学报》2011年第4期604-609,共6页Acta Hydrobiologica Sinica

基　　金：国家科技支撑计划项目（2008BADB9B03）; 现代农业产业技术体系建设专项资金（编号：nycytx-46）; 广西科学研究与技术开发计划（桂科攻10100007-2）; 广西壮族自治区直属公益性科研院所基本科研业务费（编号：2060302GXIF-2010-04）资助

摘　　要：为研究凡纳滨对虾耐寒性状的分子机理,研究克隆了凡纳滨对虾耐寒相关基因TCP-1-Beta并对其与耐寒性状的关系进行了研究。根据电子克隆所得序列设计引物,采用RT-PCR方法克隆得到长1691 bp的凡纳滨对虾TCP-1-Beta基因序列,其中包括1608 bp的完整开放阅读框,编码535个氨基酸残基。然后,运用荧光定量PCR对TCP-1-Beta基因进行时空表达谱的分析：组织表达谱的结果显示,该基因在凡纳滨对虾肝胰腺组织中表达量最高;不同低温处理下的表达结果显示,在28℃和15℃处理下基因表达量无显著变化,13℃开始呈上调表达,11℃时表达量升高;13℃低温处理发现该基因在24h内表达量无显著变化,但36h后表达量显著升高。采用PCR-RFLP方法对216尾凡纳滨对虾TCP-1-Beta基因进行了SNP多态性检测,并将其与耐寒性状进行了关联分析。在该基因编码区第420碱基上发现G/A突变,方差分析结果表明该位点的不同基因型与耐寒力指标CDH值相关（P〈0.05）,其中GG基因型的耐寒能力比AA基因型强。Litopenaeus vannamei,native to the Western Pacific Coast of Latin America,was introduced to China in 1988,and now contributes over 80% of China’s shrimp production.However,cold wave weather during recent winters of southern China led heavy losses to the culture industry of Litopenaeus vannamei.So,breeding of cold-tolerant variety is an urgent task,and molecular marker assisted selecting is an effective method for selective breeding.To study the mo-lecular mechanism of cold tolerance,we cloned TCP-1-Beta homolog gene of Litopenaeus vannamei,and studied its relationship with cold tolerance.Based on the sequence from electronic cloning,a pair of primers were designed,and a 1691 bp cDNA was obtained by RT-PCR.The cDNA contained 1608 bp ORF which encoding a peptide of 535aa.Tissue expression pattern by real-time PCR revealed that TCP-1-Beta mRNA mainly existed in hepatopancreas tissue.Cold induction by different temperatures revealed that TCP-1-Beta mRNA expression started to increase after 13℃ treatment for 24 hours,and the increase was remarkable after 11℃ treatment.As for 13℃ treatment for different times,the ex-pression pattern showed no changes during the first 24 hours,but significantly increased after 36 hours treatment.A SNP（single nucleotide polymorphism） site was found in 420 bp of ORF,and Genetype of 216 Litopenaeus vannamei were determined by PCR-RFLP.Variance analysis revealed that the SNP Genotype was significantly related with cold toler-ance parameter CDH（cooling degree hours）,and CDH for GG type Litopenaeus vannamei was longer than AA type,which provided a potential marker for selective breeding of cold tolerance variety.

关 键 词：凡纳滨对虾 TCP-1-Beta SNP PCR-RFLP 耐寒力指标CDH值 

分 类 号：Q781[生物学—分子生物学]