胡瓜及苦瓜轉殖表達外源質脂結合蛋白基因之研究

Abstract

為了瞭解苦瓜 (Momordica charantia L.) 質脂結合蛋白 (plastid lipid-associated protein, PAP) McPAP1基因之功能，將McPAP1基因之過量表現質體轉殖至胡瓜 (Cucumis sativus L.) 及苦瓜 (Momordica charantia L.) 。於胡瓜轉殖株之驗證方面，經選取具有GUS活性反應之擬轉殖株，進行聚合酶連鎖反應 (polymerase chain reaction, PCR) 分析，可偵測到報導基因GUS及McPAP1目標基因，確認為轉殖株。進一步以反轉錄聚合酶連鎖反應 (reverse transcription PCR, RT-PCR)偵測轉殖株內McPAP1基因表現情形，顯示McPAP1基因能夠於轉殖株中進行穩定表現。為了瞭解過量表現McPAP1對轉殖胡瓜中花性相關基因表現之影響，針對PAP、ACS 合成酶ACS2、Aux/IAA轉錄因子基因IAA2、乙烯受體相關基因ETR1及逆境相關氧化酶基因AOX2之表現量，以即時定量RT-PCR (real-time quantitive RT-PCR) 進行分析，結果顯示，PAP 之 RNA 累積量高的植株中，IAA2的表現較高，ACS2及AOX2表現量則較低，ETR1表現量皆高於未轉殖株，但與所檢測之其他基因表現類型皆不相同。檢測轉殖株之開花習性，過量表現McPAP1基因造成植株出現雌花之節位提早。以苦瓜葉片作為培植體，培養於添加0.5 mg．L-1 NAA及2 mg．L-1 TDZ之MSB5 培養基，具較高之癒傷組織誘導效率。葉片培植體經過3天之預培養，使用培養至OD600 0.8之農桿菌，與葉片培植體經超音波震盪5秒，再於添加100 μΜ 乙醯丁香酮之培養基中，感染30分鐘，接著共培養72小時後，以添加200 mg．L-1 kanamycin之癒傷組織誘導培養基進行篩選。葉片培植體經轉殖90天後生成之癒傷組織，經GUS活性組織化學染色分析，呈藍色反應。

To understand the function of plastid lipid-associated protein McPAP1 gene of bitter gourd, plasmid construct of McPAP1 gene overexpression was transformed into the monoecious plants such as Cucumis sativus and Momordica charantia. The putative transgenic cucumber plants after GUS histochemical assay, were analysed by and both polymerase chain reaction (PCR) and both reporter gene GUS and target gene McPAP1 were detectable. Gene expression of McPAPA1 gene was confirmed by reverse-transcription PCR (RT-PCR). Real-time quantitive RT-PCR were performed to realize the effect of overexpression McPAP1 in transgenic cucumber on expression of several flower sex-related genes, such as ACC synthase gene ACS2, Aux/IAA transcription factor gene IAA2, ethylene receptor gene ETR1 and stress-induced alternative oxidase gene AOX2. High expression level of PAP was consisted of high expression of IAA2 , but in contrast with ACS2 and AOX2. The expression pattern of ETR1was totally different with others gene. The appearance of female flowers was earlier in cucumber overexpression lines than untransformant. On the other hand calli were induced from leaf disc of bitter gourd on MSB5 medium with 0.5 mg．L-1 NAA and 2 mg．L-1 TDZ. Agrobacterium strain LBA4404 harboring McPAP1 gene overexpression vector was used for Agrobacterium-mediated gene transformation using leaf disc of bitter gourd as explant. Explants were pre-cultured for three days and then treated by sonication 5 sec with Agrobacterium solution achieved OD600 0.8. Infection was performed in medium with 100 μΜ acetosyringone for 30 min and then co-culturing for 3 days. Finally, transformed leaf discs were selected on the medium with 200 mg．L-1 kanamycin. GUS activity was detected in survival calli after transformation ninety days. Key words: Gene overexpression, flower sexuality, plant genetic transformation