Xie Daxiang

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

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庀P-1-eta并对其-9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1705bp的陀P—1—eta基因序列，其中包括1629bp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对形P-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36h内呈小幅上调表达，但36h后表达量显著升高，48h表达量达到0h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-1-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°C for 36 h, and the increase was remarkable after treatment at 13°C. As for treatment at 13°C 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 TT type.