轉殖茄子之抗壞血酸過氧化酶增加稻米耐淹水之研究

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Title:	轉殖茄子之抗壞血酸過氧化酶增加稻米耐淹水之研究
Authors:	施尚汶
Contributors:	生物科技研究所
Keywords:	茄子定量即時聚合酶鏈鎖反應水稻阿拉伯芥農桿菌轉殖抗壞血酸過氧化酶
Date:	2010
Issue Date:	2011-10-27 14:16:50 (UTC+8)
Abstract:	台灣夏季作物栽培常因颱風造成淹水而導致品質不良及產量銳減，因此部分農民使用嫁接方式，以茄子當作砧本番茄做為接穗來解決問題。我們先前發現造成茄子耐淹水主要原因之一是其根部抗壞血酸過氧化酶 (ascorbate peroxidase , APX)的活性增加，因此選殖出全長APX基因。本研究之目的是將’屏東長茄’ EG117茄子APX基因經由農桿菌 (EHA105以及LBA4404)轉殖至模式作物水稻及阿拉伯芥，分析轉殖株水稻APX基因的表達量以及測定APX的活性。經由與未轉殖植株分析比較後，發現6株T0水稻轉殖株 (21、23-2、24-1、24-3、24-4)比未轉殖水稻有10倍以上的APX1基因表達。透過酵素活性的測定得知目標基因APX1轉殖株因具較高之APX活性而有耐淹水逆境的效果存在。而在使用不同的農桿菌進行阿拉伯芥花粉管感染，發現LBA4404對於阿拉伯芥感染在第一周篩選具有較高存活率 (0.9％)，並且在加入25µM acetosyringone可將第一周存活率提高至8％，但獲得種子總數量則從2201顆下降至1393顆種子，由此可知在阿拉伯芥的發粉管感染中，使用含有acetosyringone的轉殖LBA4404菌液效果比EHA105更好。 Flooding is one of the most serious environmental stresses that affect plant growth and productivity. Flooding from large rainfalls is a major risk to fresh market tomato production in Taiwan. To overcome this problem, some tomato growers use tomato seedlings on selected eggplant rootstocks to reduce the loss of tomatoes. However, the cost of this grafting procedure is prohibitive. Previously, we found that flooding leads to an increase in APX enzyme activity in the roots of eggplants, followed by cloning the full length of APX gene. The objective this study was to transfer the eggplant ‘EG117’ APX gene via Agrobacterium (EHA105 and LBA4404) to the model crops -- rice and arabidopsis. APX gene of the transgenic plants was determined by extracting total RNA from the T0 plantsusing reverse transcriptase into cDNA, followed by using polymerase chain reaction (PCR) to amplify APX DNA. Real-time qPCR analysis was ultilized for APX gene expression. APX activity was also carried out for comparisons between transgenic plants and untranformants. The results show that APX1 gene expression in the transgenic rice plants (21、23-2、24-1、24-3、24-4) were 10-fold times higher than that in wild type. The transgenic rice plants might be tolerant to flood stress due to containing higher APX activity compared to wild type. In addition, LBA4404 had better infection to pollen tube of Arabidopsis using acetesyringone compared to EHA105
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