- 學位論文
利用雙價抗蟲基因轉殖甘藍與建立無篩選標誌系統之研究
Engineering Brassica with insect-resistance by stacked of trypsin inhibitor （sporamin）and chitinase genes, and building marker free system
摘要
甘藍為台灣栽培面積最大的葉菜類蔬菜，而台灣地處亞熱帶，夏季氣候高溫多濕，更加速了病蟲害的孳生，因此培育出抗病蟲害的蔬菜品種，是甘藍育種工作的重要方向。本研究主要利用實驗室可經受傷誘導啟動的啟動子pMSPOA，以實驗室現有的trypsin inhibitor（sporamin）和 chitinase gene當作防禦基因（defense genes），用gene stacking的方式轉殖到甘藍中，期望可以得到高效能的抗蟲轉殖株來減少施用農藥。以下胚軸轉殖的方法進行甘藍的農桿菌轉殖，總共得到了10個轉殖株（independent line），分別以genomic PCR及RT-PCR方式，初步確認了皆為轉殖株，之後各取其葉片分析trypsin inhibitory 生化活性，在未受傷時各轉殖株其trypsin inhibitor的表現皆較對照組植株高約20-30％不等，但個別轉殖株間表現量也有明顯的差異。在受傷誘導2小時之後各轉殖株其trypsin inhibitor的表現明顯有增加趨勢，增加約1-2倍不等。而chitinase的活性在未受傷前各植株差異甚大但皆比對照組植株強，在受傷誘導兩小時候各植株的活性約被誘導1-2倍左右。利用甘藍轉殖株葉片餵食二∼三齡的小菜蛾幼蟲，並分析轉殖株的抗蟲能力，經過2次的重複測試後，測量葉片啃食重量和蟲子存活率，以不含轉殖基因植株作為對照組，結果顯示具胰蛋白酶抑制因子和幾丁質酶活性的不同植株，與其抵抗小菜蛾幼蟲的能力呈正相關性。 為了能夠移除轉殖植株中的抗生素標誌基因，根據Two T-DNA one plasmid的方法，構築super binary vector（FM1300M），並接入pMSPOA gene stacking 的casette。利用co-transformation的方式同時進行甘藍及阿拉伯芥的轉殖。在阿拉伯芥轉殖部份，以genomic PCR及RT-PCR的方式確認得到了7個T1 transgenic line。取1個T1 line作T2世代的分析，以genomic PCR的方式篩選到了18株不含抗生素基因但含sporamin和chitinase gene的植株，取其中5株以RT-PCR的方式確認了確實不含抗生素標誌基因但有sporamin和chitinase gene的表現。
並列摘要
Cabbage（Brassica oleracea L. Capitata）is the most important leafly vegetable in Taiwan, due to it’s abundant antioxidant compounds and anticancer compounds like glucosinolate. Unfortunately, cabbage can be easily infected with or attacked by insect peats. In order to decrease the risk of cabbage attacked by insects, the goal to create transgenic cabbage by gene stacking methods is inprogress in our lab. Trypsin inhibitor（sporamin）and chitinase genes were in stack constructed using pMSPOA as promoter,. We successfully got ten independent lines through Agrobacterium tumefaciens-mediated transformation,. In trypsin inhibitor or chitinase activity assay, we found that both sporamin and chitinase activity can be increasing 1-2 times at 2 hrs after wounding. In bioassay, we found that transgenic line 4 is more effectivel to defend Diamond-back moth(Plutella xylostella) attack.. In order to alleviate public concerns on the risk of antibiotics resistance gene, recommendations have been made to eliminate all antibiotic marker genes from GM plants. Our results showed that by Agrobacterium tumefaciens-mediated co-transformation with a double T-DNA binary vector in Arabidopsis, several marker-free transformants were successfully obtained. This technology mote will further applied in the transgenic cabbage system.
參考文獻
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