尼罗罗非鱼TCP-1-beta和TCP-1-eta的分子特征及其低温诱导表达

MOLECULAR CHARACTERIZATION AND INDUCTION EXPRESSION OF TCP-1-BETA AND TCP-1-ETA UNDER LOW TEMPERATURES IN TILAPIA OREOCHROMIS NILOTICUS

为了研究尼罗罗非鱼耐寒性状的分子基础并为耐寒品种选育提供参考,研究从尼罗罗非鱼中克隆了HSP60家族TCP-1-beta和TCP-1-eta基因并对其在低温诱导下的表达特征进行了分析。尼罗罗非鱼TCP-1-beta cDNA长度为1755 bp,包括1605 bp的完整开放阅读框,编码534个氨基酸;尼罗罗非鱼TCP-1-eta cDNA长度1651 bp,包括1638 bp的完整开放阅读框,编码545个氨基酸。与其他物种同源基因的蛋白序列比对结果显示,TCP-1-beta和TCP-1-eta蛋白在物种间同源性很高,且都具有保守的ATP结合结构域等,预示其在物种间功能的保守性。实时荧光定量PCR结果表明:TCP-1-beta和TCP-1-eta在各组织中呈遍在表达,但在肌肉中表达量最高;诱导温度从22℃降至12℃,不同低温诱导48h后TCP-1-beta和TCP-1-eta均呈上调表达,在18℃时表达开始上调,随着低温胁迫程度加强,表达上调幅度增大,至12℃时表达量达到最高,TCP-1-beta和TCP-1-eta上调幅度分别达到常温的12.2倍和10.7倍。这些结果预示在尼罗罗非鱼中,TCP-1-beta和TCP-1-eta是潜在的耐寒相关基因。

Nile tilapia （Oreochromis niloticus） is one of the commercially important fish species in the southern China, but the lack of cold-resistant strains in tilapia results in a large number of deaths in winter and early spring. Some heat-shock proteins （Hsps） have been revealed to facilitate correct folding of other proteins under diverse forms of physiological stresses, such as extreme temperatures, chemical toxicity and oxidative stress. To understand the molecu- lar mechanism of cold tolerance in fish, we cloned the full-length cDNAs of TCP-l-beta and TCP-l-eta of the Hsp60 family in nile tilapia （Oreochromis niloticus）, and studied their expression patterns under normal and low temperature treatments. The TCP-l-beta cDNA consisted of 1755 bp and encoded a peptide of 534 amino acids. The TCP-l-eta cDNA consisted of 1651 bp and encoded a peptide of 545 amino acids. Multiple alignment and homological analysis revealed their evolutionary conservation and functional significance of TCP-l-beta and TCP-l-eta among eukaryotes. Significantly, the deduced amino acid sequences of TCP-l-beta and TCP-l-eta contained highly conserved amino-acid motifs and three ATP-binding sites. The tilapia TCP-l-beta was highly homologous with TCP-l-beta in other species, sharing 92% identities with Salmo salar and Danio rerio, 89% with Xenopus laevis, 88% with Mus musculus, 87% with Homo sapiens, and 73% with Drosophila melanogaster. Tilapia TCP-l-eta shared 92% identities with that of Danio rerio, 89% with Xenopus laevis, 88% with Mus musculus, 89% with Homo sapiens, and 76% with Drosophila melanogaster. Phylogenetic trees were also constructed among amino acid sequences of TCP-l-beta and TCP-l-eta in eukaryotes. The tilapia TCP-l-beta was clustered with Notothenia coriiceps TCP-l-beta, and tilapia TCP-l-eta was clustered with Danio rerio TCP-l-eta. Total RNAs were extracted from seven tissues including spleen, gut, kidney, heart, brain, muscle and hepatopancreas. Using beta-actin as internal control, their expression patterns were analyzed by real-time PCR, in which TCP-l-beta and TCP-l-eta mRNA existed in all seven tissues, and expressed mainly in muscle. To study the response of TCP-1 to low temperatures, the mRNA levels of TCP-l-beta and TCP-l-eta were analyzed in response to cold induction treatments. The experimental fishes were divided into five groups and kept at different tem- peratures （22℃, 18℃, 16℃, 14℃ and 12℃） for 48 hours. Total RNAs were extracted from hepatopancreas tissue at different temperature treatments （22℃, 18℃, 16℃, 14℃ and 12℃）. Real-time PCR analysis revealed that TCP-l-beta and TCP-l-eta mRNA increased at 18℃ and peaked at 12℃. TCP-l-beta mRNA was 2.8 fold up-regulated at 18℃ compared to normal temperature at 22℃, and then held stable until the induction temperature changed to 12℃ which induced 12.2 fold increase in mRNA levels of TCP-l-beta. The expression level of TCP-l-eta was 2 fold upregulated at 18℃ compared to normal temperature at 22℃, and then a gradual increase in the level of TCP-l-eta was observed, reaching a peak of 10.7 fold increase at 12℃. The cold-induced expression of TCP-l-beta and TCP-l-eta suggested that they might be cold tolerance-related genes in Nile tilapia. Therefore, this study established a basis for studying the mo- lecular mechanism of cold tolerance in Nile tilapia.