MADS-box基因（SPMADS1）表現與甘藷塊根發育期間之生理功能研究

Abstract

本實驗室利用cDNA-AFLP的技術，篩選與甘藷塊根發育相關及可能誘發塊根形成之誘發基因（trigger gene）的片段，作為選殖全長基因之基礎，本論文即是針對其中一片段TDF26-1所對應之基因進行更深入的探討。TDF26-1約150 bp經序列的分析比對顯示此片段與MADS-box基因有60?70％的相似性。為瞭解此基因於甘藷植株中的表現特性及生理功能，本論文利用RACE（rapid amplification of cDNA ends）的方式選殖出全長之cDNA基因（命名為SPMADS1），共1022bp，可轉譯出長度為208個胺基酸之蛋白質，經DNA及胺基酸序列的比對分析，顯示SPMADS1的結構與已發表之MADS-box基因，同樣具有MADS-box與K-box兩個保留性高的區域，且SPMADS1基因與其他物種於營養器官中特定表現之MADS-box基因如：nmhC5、AGL17、ANR1等基因，具有較高的相似性。北方雜合分析（Northern hybridization）的結果顯示此基因主要是於甘藷初膨大之塊根中表現，並隨塊根膨大，該基因表現量明顯遞減，此外於葉部及鬚根組織中亦有微量表現。本實驗進一步以SPMADS1與antisense SPMADS1轉殖至野生型阿拉伯芥中，藉觀察阿拉伯芥轉殖株外表型之改變情形，推測SPMADS1基因於植株中扮演之生理功能。利用RT-PCR檢測結果顯示，轉殖株可充分轉錄SPMADS1及antisense SPMADS1基因，利用西方墨漬分析亦測得SPMADS1蛋白質的表現，但阿拉伯芥轉殖株卻無法觀察到外部形態明顯的改變，故推測SPMADS1主要的生理功能可能與甘藷塊根之發育有關。

A tuberization-related transcript-derived fragment (TDF26-1) was obtained from previous study by use of cDNA-AFLP. It showed 60-70% homology with MADS-box genes, which belonged to a homeotic gene family published earlier by several groups. The complete cDNA gene of 1022 bp (SPMADS1) was further isolated from sweet potato tuberous root by the cDNA RACE method. The deduced amino acids sequence of SPMADS1 cDNA encoded 208 amino acids, including the MADS-box domain (56 amino acids)and the K-box domain (68 amino acids). SPMADS1 showed significant homology to orphan group of MADS-box proteins, such as nmhC5, AGL17, ANR1, which might play important roles in root development. High level of SPMADS1 RNA was spatially expressed in small size of tuberous root and a little was observed in flower, leaf, root and large size tuber. The functional study of SPMADS1 was performed by transforming Arabidopsis with sense and antisense SPMADS1 gene. However, phenotypic alternation was not observed from Arabidopsis transformants. It is possible that SPMADS1 is a tuber-trigger gene. It specifically acts at early step of tuberization, While acts no function on other organs or tissues.