苦瓜植物生長素輸入運送蛋白基因之選殖與啟動子活性分析

Abstract

為選殖苦瓜（Momordica charantia）植物生長素輸入運送蛋白基因McLAX1，以pMAIC11 cDNA為探針，篩選苦瓜基因組庫，選取基因組選殖系λMAIC1，進行次選殖及基因全長定序分析，得到基因序列4,346 bp及其啟動子序列2,106 bp。McLAX1基因具有八個顯子和七個隱子，顯子與隱子連接部位之核苷酸序列除第一個隱子外均符合GT-AG rule，轉譯起始密碼位於第一個顯子，共可解碼469個胺基酸，預測之分子量為52.70 kDa，pI值為8.28。McLAX1與pMAIC11 cDNA的核苷酸序列相似度為99.3％，所演繹之胺基酸序列相似度為99.1％，推測其可能為等位基因，而McLAX1與其他作物之植物生長素輸入運送蛋白之胺基酸序列相比，同源性介於61％到82％之間。依啟動子序列比對分析結果顯示，McLAX1基因啟動子具有植物生長素、離層酸、茉莉酸、水楊酸、乙烯、光、低溫、創傷等保守性cis-acting elements。 為了進一步分析McLAX基因啟動子的活性，將McLAX2之啟動子片段構築於報導基因GUS上游形成表達質體，傳送至蝴蝶蘭花瓣與苦瓜幼葉、成熟葉圓片、雄花花瓣及雌花花瓣等組織器官，進行啟動子活性暫時性表現分析，試驗結果顯示McLAX2之啟動子活性，於苦瓜雌花花瓣中，較高於CaMV35S啟動子的活性。另外，經由農桿菌媒介法，進行阿拉伯芥與菸草之穩定性轉殖，以南方氏雜交分析確認轉殖株後，進行GUS活性組織化學染色分析，顯示其於阿拉伯芥的葉片、花梗、小花序、雌蕊、雄蕊、果莢，與菸草的根部、葉片均具有活性。

To obtain auxin influx carrier gene McLAX1 from bitter gourd（Momordica charantia）, pMAIC11 cDNA was used as the probe for screening the genomic library. Based on restriction map and Southern analysis, λMAIC1 was delegated to be subcloned and sequenced. The McLAX1 gene in λMAIC1 is consisted of 4,346 base pairs with eight exons and seven introns. The splice junctions between introns and extrons of McLAX1 are according to the GT/AG rule except the first intron. The start codon locates in the first exon. McLAX1 encodes a polypeptide containing 469 amino acids with a calculated molecular mass of 52.70 kDa and a predicted isoelectric point of 8.28. The identity of nucleotide sequences and amino acid sequences between λMAIC1 and pMAIC11 cDNA is 99.3% and 99.1%, respectively, therefore the gene in λMAIC1 was supposed to be an allele of McLAX1. The homology of the amino acid sequences of McLAX1 with AUX1 and AUX1-like proteins is 61%∼82%. According to the result of analysis for upstream promoter sequence of McLAX1, several predicted responsive elements related to auxin, abscisic acid, Methyl jasmonate, salicylic acid, ethylene, light, low-temperature, and wounding were found. To analyze the promoter activity of McLAX, the plasmid 20P12 containing McLAX2::GUS had been constructed. The expression plasmid was transformed into petal discs of Phalaenopsis, young leaf, mature leaf, male flower, and female flower of bitter gourd via particle bombardment. The result of transient expression analysis indicated that the promoter activity of McLAX2 in female flower of bitter gourd is higher than CaMV 35S promoter. The stable expression analysis in Arabidopsis and tobacco was performed by Agrobacterium-mediated transformation. Southern analysis had verified the transfomants. The results of GUS histochemical staining indicated that the promoter activity of McLAX2 exists in the leaf, inflorescence, stamen, pistil and pod of Arabidopsis, and in the leaf and root of tobacco.