甘藷sporamin基因啟動子上順向作用的DNA片段之功能性分析及sporamin基因家族成員間變異性之研究

Abstract

Sporamin是甘藷塊根中含量非常豐富的儲存性蛋白質，而且其啟動子活性是可以受到受傷誘導的。因此我們希望能找出sporamin啟動子上負責受傷誘導之順向作用的DNA片段（wound-induced cis-acting element），藉此了解sporamin基因啟動子在植物遭受機械性傷害下的調控機制。利用南方墨漬法得知該基因在甘藷中是為一個基因家族。之後以genomic walking的方式，成功地延長選殖出2條sporamin基因的啟動子。將這兩條新選殖出的啟動子和我們已知的sporamin啟動子：pSPOA以Vector NTI軟體來分析其上和逆境相關之順向作用的DNA片段，我們可以發現這3條啟動子雖然序列不相同，但這些啟動子上皆包含了如NOS-like element、W-box及GCC-like box等和植物防禦機制相關之順向作用的DNA片段。因此這些sporamin基因被調控的機制很有可能是相類似的。在本實驗室之前對於pSPOA這條啟動子缺失片段的研究中發現NOS-like element是為該條啟動子和受傷誘導最為相關的DNA片段，另外SP8a及G-box這2個DNA片段可能具有加強NOS-like element的功能。因此利用合成啟動子的方式（synthetic promoter）來單純化研究NOS-like element及SP8a這兩個順向作用的DNA片段之功能，我們分別構築了含有1、2及4個同一個DNA片段及混合2種DNA片段之合成啟動子，利用轉殖阿拉伯芥的系統來檢測受傷誘導下GUS報導基因的表現模式。在GUS組織染色的結果中顯示NOS-like element及SP8a皆與受傷誘導相關，雖然於受傷誘導下所能誘導的GUS活性仍有差異，但皆可表現於受傷葉片、根、莖與未進行受傷誘導的葉片中，因此此一受傷誘導之傳遞路徑應為系統性的反應（systemic response）；另外如果進行受傷誘導之植株已開始抽花絮，該部分組織中的GUS表現量也會增加。 另一方面利用構築甘藷塊根之cDNA library來選殖出更多的sporamin 基因，以期能分析該基因家族成員間之變異性。我們成功的從甘藷塊根之cDNA library中選殖到19條sporamin A以及1條sporamin B。以Vector NTI軟體分析後發現同一群之基因成員間相似度高達90％，兩群基因之相似度也高達80％。而根據現有的序列可再將A群基因細分為兩個sub-family，其相似度則高達95％。而這些sporamin序列上的差異多是來自於5’及３’-UTR之變異，此結果與Hattori於1989年所提出之理論相類似。另一方面如果將sporamin基因以軟體預測轉譯成氨基酸序列後，發現其活性區域組成之氨基酸序列並無改變，因此這些蛋白質的活性強度可能是相類似的。而以電腦軟體分析我們sporamin基因於序列上可能發生同義與非同義之取代位置，可得到該基因家族KA/KS＜1.0，因此在演化上應是屬於purifying evolution。

Sporamin is a storage protein of sweet potato tuber, and the expression of sporamin gene in leaves is wound-inducible. In order to define the wound-response cis-acting elements of the sporamin promoter, the genomic walking method was used to clone upstream promoter regions. Three segments of promoter region of this gene family were obtained. Although the sequences of these promoters are various, they all contain almost the same cis-acting elements, such as W-box and GCC-box. Therefore, the expression pattern of each gene member may be quite similar. Our previous data demonstrated that a sporamin promoter : SP1 (pSPOA) is wounding-induced in the transgenic tobacco by promoter-GUS fusion assay. The GUS staining results indicate that the sequences containing the NOS-like element confer the most wound-induced activity, the SP8a and G-box may contribute to enhance the effect of NOS-like element. So we use the synthetic promoter method to identify each cis-acting element. Constructions with one, two and four copies of each element were tested for its wounding-induced activity by promoter-GUS fusion assays. We also tested the constructs with mixed motifs in transgenic Arabidopsis. The GUS staining and GUS quantitative results showed that both the NOS-like element and the SP8a motif contribute to the GUS expression under mechanical wounding in transgenic Arabidopsis. The NOS-like element and SP8a motif indeed confer wounding-induced activity when working alone, but the expression level of the reporter gene is higher when the number of motif increased. Although the induced level of GUS proteins was different between these constructs, the expression patterns were quite similar. The induced GUS proteins were expressed in wounded leaf, root, stem, and unwounded leaves. So the wounding response might be a systemic effect. We also constructed a cDNA library of sweet potato tuberous root to clone more sporamin genes, because we intend to investigate the polymorphisms among these genes. Twenty sporamin genes were cloned, 19 of these genes belong to the sporamin A and only one gene belong to the sporamin B. The homology between these two groups is 80％, and the homology within each group is about 90％. We also divide the sporamin A into two sub-family, and the homology within each sub-family is 95％. The sequence polymorphisms are taking place almost in the 5’, and 3’-UTR, these findings are resemble with the theory proposed by Hattori in 1989. We found that the active domain within amino acid sequences of these sporamin genes were identical, so the activities of these proteins might be the same. And the evolution prediction of this gene family tends to purifying evolution pathway.