Yin Qin, Peng Jin-Xia, Cui Liang, Xie Da-Xiang, Wang Zhi-Wei, Li Kui, Chen Xiao-Han
Guangxi Fishery Institute, Nanning 530021, China.
Yi Chuan. 2011 Feb;33(2):168-74. doi: 10.3724/sp.j.1005.2011.00168.
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.
为研究凡纳滨对虾耐寒性的分子机制，我们从凡纳滨对虾中克隆了TCP-1-eta同源基因，并研究了其与耐寒性的关系。基于电子克隆获得的序列设计了一对引物，通过RT-PCR获得了一条1705 bp的cDNA。该cDNA包含1629 bp的开放阅读框（ORF），编码一个542个氨基酸的肽段。实时荧光定量PCR分析组织表达模式显示，TCP-1-eta mRNA主要存在于肌肉组织中。不同温度的冷诱导实验表明，在15℃处理36 h后，TCP-1-eta mRNA表达开始增加，在13℃处理后增加显著。对于在13℃处理不同时间，在前36 h表达模式几乎没有变化，但处理36 h后显著增加。在ORF的731 bp处发现一个单核苷酸多态性（SNP）位点，通过PCR-RFLP确定了216只凡纳滨对虾的基因型。方差分析表明，SNP基因型与耐寒性参数冷却度小时数（CDH）显著相关，凡纳滨对虾CC型的CDH长于TT型。
