凡纳滨对虾TCP-1-eta基因的克隆及与耐寒性状的相关性  

Molecular cloning of Litopenaeus vannamei TCP-1-eta gene and analysis on its relationship with cold tolerance

作　　者：殷勤[1,2] 彭金霞[1] 崔亮[1] 谢达祥[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,北京100193

出　　处：《遗传》2011年第2期168-174,共7页Hereditas（Beijing)

基　　金：国家科技支撑计划项目(编号:2008BADB9B03;2007BAD29B01-6);国家虾产业技术体系岗位科学家经费(编号:nycytx-46);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-2008-02;2060302GXIF-2010-04)

摘　　要：为研究凡纳滨对虾耐寒性状的分子机理,文章克隆了凡纳滨对虾的耐寒相关基因TCP-1-eta并对其与耐寒性状的关系进行了研究。首先,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1 705 bp的TCP-1-eta基因序列,其中包括1 629 bp的完整开放阅读框,编码542个氨基酸残基。然后,运用荧光定量PCR对TCP-1-eta基因进行时空表达谱的分析:(1)组织表达分析结果显示,该基因在凡纳滨对虾肌肉组织中表达量最高;(2)不同低温处理下的表达结果显示,该基因在15℃开始呈上调表达,13℃时表达量最高;(3)13℃低温处理的表达结果显示,该基因在36 h内呈小幅上调表达,但36 h后表达量显著升高,48 h表达量达到0 h的150倍。进一步采用PCR-RFLP方法对216只凡纳滨对虾TCP-1-eta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731碱基上发现C/T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(Cooling-degree hours)值相关(P<0.05),其中CC基因型的耐寒能力比TT基因型强。To study the molecular mechanism of cold tolerance, we cloned TCP-l-eta homolog gene from Litopenaeus vannamei and studied its relationship with cold tolerance. Based on the sequence from electronic cloning, a pair of primers were designed, and a 1 705 bp cDNA was obtained by RT-PCR. The cDNA contains 1 629 bp ORF, which encodes a peptide of 542 aa. Tissue expression pattern by real-time PCR analysis revealed that TCP-1-eta mRNA mainly existed in muscle tissue. Cold induction by different temperatures revealed that TCP-1-eta mRNA expression started to increase after treatment at 15℃ for 36 h, and the increase was remarkable after treatment at 13℃. As for treatment at 13℃ for different times, the expression pattern was almost not changed during the first 36 h, but significantly increased after 36 h of treatment. A SNP （single nucleotide polymorphism） site was found in 731 bp of ORF, and the genotypes of 216 Litopenaeus vannamei were determined by PCR-RFLP. Variance analysis indicated that the SNP genotype was significantly related with cold tolerance parameter Cooling-Degree Hours （CDH）, and CDH for CC type of Litopenaeus vannamei was longer than TTtype.

关 键 词：凡纳滨对虾 TCP-1-eta SNP 耐寒性状 

分 类 号：S917.4[农业科学—水产科学]