香港牡蛎淋巴因子基因tal的克隆与功能

为了研究淋巴因子基因(T-cell acute lymphocytic leukemia,tal)在香港牡蛎中的免疫功能,实验采用c DNA末端快速扩增技术(rapid-amplification of cDNA ends,RACE)克隆了香港牡蛎tal的c DNA全长序列,命名为Chtal,该基因全长812 bp,其中5?非编码区17 bp,3?非编码区135 bp,开放阅读框(open reading frame,ORF)660 bp,编码219个氨基酸,相对分子质量25.11 ku,理论等电点5.80。多序列比对和系统进化树结果显示,Chtal与长牡蛎、美洲牡蛎和海蜗牛tal同源性较高,证明了该基因是软体动物tal家族的一个成员。荧光定量PCR结果显示,Chtal在香港牡蛎血淋巴细胞中表达量最高,其次是心脏、鳃和消化腺。在溶藻弧菌和溶血性葡萄球菌刺激后,Chtal表达量显著上调,分别在24和12 h达到最高水平,随着时间的增加该基因的表达量逐渐下降。亚细胞定位结果显示,Chtal在细胞核中表达。活体注射重组TAL蛋白能够显著提高淋巴细胞的数量,通过Edu增殖实验进一步证实了tal具有促进香港牡蛎血淋巴细胞再生的功能。本研究初步揭示了香港牡蛎tal参与了淋巴细胞再生,为深入研究该基因在牡蛎抗病中的功能提供了依据。

Cloning and functions of the lymphogenic tal gene of Crassostrea hongkongensis

To study the immune function of T-cell acute lymphocytic leukemia (tal) in Crassostrea hongkongensis. In this study, RACE technique was used to clone the full-length cDNA sequence of the (T-cell acute lymphocytic leukemia) tal gene of Crassostrea hongkongensis, named Chtal. The open reading frame (ORF) is 660 bp, encoding 219 amino acids, with the relative molecular weight is 25.11 ku. Multiple sequence alignment and phylogenetic tree results showed that the Chtal gene has high homology with C. gigas, C. virginica and A. californica, which proves that it is a member of the tal family of mollusks. Fluorescence quantitative PCR results showed that Chtal was the most expressed in haemolytic lymphocytes of oyster in C. hongkongensis, followed by heart, gill and digestive gland, suggesting its function in lymphogenesis. After the stimulation of V. alginolyticus and S. haemolyticus, the expression levels of Chtal were significantly up-regulated, reaching the highest levels at 24 h and 12 h, respectively, and the expression of the gene gradually decreased with time. The results of subcellular localization showed that Chtal was expressed in the nucleus. In vivo injection of recombinant TAL can significantly increase the number of hemocytes. Edu proliferation assay have further confirmed that Chtal can promote the regeneration of hemocytes in C. hongkongensis. In summary, this study revealed that Chtal was involved in hemocytes regeneration, providing a basis for further research on the function of this gene in immune response of oysters.