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-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.
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