利用cDNA-AFLP技術進行甘藷中受甲基化/去甲基化調控及塊根發育相關基因之研究

Abstract

cDNA-AFLP是近年來發展出的一項關於RNA fingerprinting的新技術。本研究即是利用此項技術，觀察甘藷根部經去甲基化藥劑處理後及塊根發育時期的相關基因表現情形，並進一步分離出其基因片段，進行序列分析後以cDNA-PCR及北方氏雜合反應確認其差異性表現。首先，抽取經5-azacytidine（5-azaC）處理及未經處理的甘藷水耕苗根部RNA，合成cDNA，經TaqI及AseI限制酵素切割後，接上相對應的adaptors，並利用adaptors及限制酵素切位鄰近的序列設計引子，藉由引子3'端上兩個鹽基的延伸組合及PCR反應，選擇性地放大某些基因片段。經由DNA序列電泳分析，可觀察到一群不同長度的基因片段，此即為cDNA-AFLP圖譜，比較經5-azaC處理及未處理的甘藷cDNA-AFLP圖譜，我們分離出五個基因片段（TDFs: transcript-derived fragments），但經cDNA-PCR的結果確認後，僅pA11 clone會受5-azaC誘導而表現。另一方面，亦藉由甘藷葉、莖、根部及不同大小塊根的cDNA-AFLP圖譜，觀察並分離出六個根部或塊根特有的TDFs。經由北方氏雜合反應分析，得到四個可能與塊根發育有關的TDFs。而由序列分析及比對，發現其中一個TDF，pT26-1 clone，和許多具MADS-box的基因有60-70％的相同性（identity），而其他TDFs和目前已知的基因則無明顯的相同性。本研究結果顯示，cDNA-AFLP除提供RNA fingerpringting外，亦可進一步應用於研究基因的差異性表現，特別是研究某一生長發育時期基因群的表現及其基因之選殖。

Using a powerful technique for RNA fingerprinting, cDNA-AFLP (amplified fragment length polymorphism), transcriptional changes of sweet potato (Ipomoea patatas) roots treated with 5-azacytidine (5-azaC), a demethylation agent, were analyzed. After we recovered certain transcript-derived fragments (TDFs) from polyacrylamide sequencing gel followed by PCR amplification and cloning, five clones assumed to be induced by 5-azaC were obtained. However, the results of cDNA-PCR indicated that only one clone, pA11, was clearly responsed to 5-azaC treatment. cDNA-AFLP is efficient for acquiring differentially expressed genes, especially for identifying devlopmentally regulated genes. Therefore, we investigated tuberization-related genes by cDNA-AFLP with cDNAs derived from mRNAs of leaves, stems, roots and tuberous roots with various sizes. Six clones of root- and tuberous root-specific TDFs were obtained from polyacrylamide gel followed by PCR amplification and cloning. The results of Northern blot analysis indicated that four of these clones could be tuberization-related genes. One clone, pT26-1, with 202 bp in length showed 60-70% identity in about 150 bp overlaped region with several published MADS-box genes, which belonged to a homeotic gene family. The other three clones may belong to novel genes since there were little identity with known genes. These results demonstrate that cDNA-AFLP is applicable for differential display of genes induced by specific treatments and for cloning a set of genes which are involved in a certain developmental stage.