過去研究指出，重組活化基因( recombination activating genes, RAG1, RAG2)引導的V(D)J重組在後天免疫的發育過程中造就了淋巴細胞表面受體的多樣性，並得以對抗各種不同的抗原。因此，本研究選殖龍膽石斑重組活化基因(RAG1, RAG2) 和T細胞表面受體α鏈 ( T cell receptor α chain, TCR-α) 作為龍膽石斑早期後天免疫發育的代表性標記，進行特性分析。藉由即時聚合酶連鎖反應偵測發現RAG1, RAG2及 TCR-α在開翅期(12、14天)及收翅期(30天)表現量有上升趨勢，顯示魚苗型態變化期間同時也是後天免疫發展的重要時期。為了探究龍膽石斑T細胞受體之多樣性，本研究在TCR-α之Vα-Jα交界處進行多重序列比對，結果發現Vα-Jα交界處具有序列多樣性，使的TCR-α能夠辨識不同抗原。利用及時聚合酶連鎖反應快速檢驗及定量，並使用螢光原位雜交技術觀察DNA上TCR-α之V(D)J重組現象，發現龍膽石斑幼苗的胸腺大量進行 TCR-α之V(D)J重組事件，顯示胸腺可能是T細胞發育的重要器官。本研究提供龍膽石斑幼苗後天免疫發育之時間參考點，確認龍膽石斑中TCR-α的V(D)J重組現象，並證實胸腺可能是T細胞發育的主要器官。這些結果有助於了解龍膽石斑抵禦外來抗原的機制，希望減少疾病對水產養殖業的損失。

During the early stages of T lymphocytes and B lymphocytes, the diversity of functional T cell receptors and immunoglobulins is accomplished through V(D)J recombination, and is regulated by the recombination activating genes, RAG1 and RAG2. Therefore, this research characterizes the recombination activating genes and T lymphocytes at an early stage in giant grouper. The results of real-time PCR show that RAG1, RAG2 and TCR-α exhibit high expression at the kitting stage, which is about 12 to 14 days, and the diskitting stage at about 30 days, which is indicated that the period of morphology changes is also an important period for the development of adaptive immunity in early stage of giant grouper. To investigate the diversity of T cell receptors in giant grouper, multiple sequence alignment is performed on the Vα-Jα junction of ggTCR-α. The results show that the sequences of Vα-Jα junction are variable, which can result in ggTCR-α diversity allows recognition of different antigens. In addition, the results of a qPCR-based V(D)J recombination assay and DNA-fiber fluorescence in situ hybridization, show that the V(D)J recombination event is highly expressed in the thymus, which is indicated that thymus is an important organ for T cell development. These results provide a reference time point for the development of adaptive immunity in giant grouper, verified the V(D)J recombination event of TCR-α in giant grouper, and demonstrated that the thymus could be an important organ for the development of T lymphocytes. It is expected to contribute to the research on the resistance of early stage giant grouper to foreign pathogens and efforts to reduce losses in the aquaculture industry from diseases.

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