水稻類鉬輔因子硫化酶基因之表現及功能分析

Abstract

離層酸(abscisic acid; ABA)被認為是種重要的逆境賀爾蒙，其生合成途徑最後一個步驟需要乙醛氧化酶(aldehyde oxidase; AO)的催化。乙醛氧化酶為需要鉬輔因子協助之酵素，且其複合物(complex)上之鉬輔因子需經硫化過程，才能促使乙醛氧化酶活化。因此負責硫化鉬輔因子之鉬輔因子硫化酶(molybdenum cofactor sulfurase; MCSU)即為調控ABA生合成的主要因子之一。經水稻基因組序列之比較、分析，得知水稻具有三個molybdenum cofactor sulfurase like (MCSUL)基因，分別命名為OsMCSUL1、OsMCSUL2、OsMCSUL3。OsMCSUL3基因和阿拉伯芥MCSU基因(ABA3)之cDNA序列及胺基酸序列互相比較均具有約60％相似度。OsMCSUL1和OsMCSUL2之胺基酸預測序列僅具有阿拉伯芥MCSU蛋白後段(carboxyl terminal end)之功能區域(function domain)。本論文主要針對水稻，OsMCSUL家族基因在鹽害逆境下之表現情形進行分析，並將OsMCSUL基因轉入阿拉伯芥，以探討OsMCSUL之功能。利用real-time RT-PCR分析基因表現的結果得知，OsMCSUL1在葉部組織的表現可明顯地都受到鹽分逆境的促進，並且受到ABA的正向調控。在根部組織中則是三個基因表現均會受NaCl及ABA所促進。藉由將OsMCSUL2基因轉殖入阿拉伯芥aba3突變體之轉殖株分析，推測OsMCSUL2具有類似阿拉伯芥MCSU蛋白質的功能，即其可能亦能活化AO進而促使ABA生合成。

ABA is considered as an important stress hormone. The last step of ABA biosynthetic pathway is catalyzed by aldehyde oxidase (AO). The combination of AO with a sulfurized molybdenum cofactor (Mo-cofactor) is necessary for enzyme activity. Therefore Mo-cofactor sulfurase (MCSU), the enzyme in charge of the process of Mo-cofactor sulfuration, is one of the important factor for controlling of ABA biosynthesis. Base on the search on rice genome sequence, three molybdenum cofactor sulfurase-like (MCSUL) genes, named OsMCSUL1, OsMCSUL2 and OsMCSUL3, are presented on rice genome. The putative OsMCSUL3 amino acid sequence shared approximate 60％ homology with Arabidopsis MCSU. According to the protein functional domain analysis, the N-terminal end of OsMCSUL3 contains a putative Nifs domain. The C-terminal end of OsMCSUL3 is the MCSU conserved region. On the other hand although there is no Nifs function domain found on OsMCSUL1 and OsMCSUL2, the full length of Osmcsul1 and OsMCSUL2 have high homology with C-terminal of Arabidopsis MCSU. In this study, an effort was made to determine gene expression of OsMCSUL family genes in salt-stress rice seedling. Base on real-time RT-PCR analysis OsMCSUL1 expression was up-regulated by salt stress and ABA in both leaf and root tissues. OsMCSUL2 gene expression almost could not be affected by NaCl in leaves and roots, however, it’s expression was enhanced by ABA. OsMCSUL3 mRNA level was in salt-stress root samples, but not in leaves. In addition, ABA was a positive on regulator for OsMCSUL3 expression in roots, but not in leaves. To determine the function of OsMCSUL genes, the genes were transformed into Arabidopsis mcsu mutant (aba3 mutant), the result suggested that OsMCSUL2 was likely to share identical (or similar) functions of the protein of the Arabidopsis MCSU, which can active AO than ABA biosynthesis.