甘藷傷害誘導互補DNA之分離

Abstract

經過長久以來的演化，植物已經發展出良好的防禦機制以抵抗昆蟲、草食性動物和病原菌的侵害。傷害誘導產生的防禦機制一直是科學家注意的對象。 本實驗目的是甘藷傷害誘導基因的分離，實驗初步在於分離甘藷之傷害誘導基因的訊息核糖核酸，我們將植物材料分成兩組：A組是未受任何傷害的甘藷植株，B組則是以鑷子尖端夾傷的傷害處理甘藷植株，所採用的材料都是由頂芽往後算起的第4、5、6、7片完全開展的幼嫩葉片。在B組植株以鑷子傷害處理2小時後，同時摘取這兩組植株葉片，抽取其total RNA。 經過3次未受傷害基因之互補DNA與傷害誘導基因之訊息RNA之間的相減雜合反應，留下傷害誘導基因特有的訊息核糖核酸，再經反轉錄反應和聚合酵素連鎖反應產生傷害誘導基因之互補DNA後，將此傷害誘導基因之互補DNA構築於質體pTZ-18U上，建構成相減式基因庫。 由此相減式基因庫中選取18個含有cDNA的質體，經PCR將此段cDNA以α-35S-dATP標定為探針，進行北方墨點法實驗，由結果得知，有3個探針：No15，No23A，No24B是經傷害誘導產生，另有2個探針：No1和No10B為持續性表現。同時亦證明瞭No15可由斜紋夜盜蛾幼蟲咬傷引發產生。由於No15和No1探針有較強的訊號表現，故選定No15和No1這兩個探針，對甘藷的總DNA做南方墨點法實驗，結果都獲得了5個分子量大小不同的訊號，最後，將相對應的DNA bands由膠體上萃取出來，與質體pTZ-18U進行黏合反應，構築這兩個探針的subgenomic library，並以原先的探針，經圓點墨點法證實我們得到的菌體轉殖株具有與No15和No1互補DNA同源的基因後，再將轉植株的總DNA定序，希望能獲得完整的基因甚至其啟動子。

After millions of years of evolution, plants already develop many defensive mechanisms to protect themselves against the invasion of insects, herbivores and pathogens. Although some defensive systems are constitutively expressed, scientists are much interested in the inducible defense mechanisms. The purpose of this study is to isolate the wound-induced genes from sweet potato, and further to analyze the defense mechanisms of sweet potato. In order to isolate the wound-induced messenger RNAs from sweet potato, the sweet potato plants were divided into two groups: the group A is the unwounded plants and the group B is the plants wounded by clips. Two hours later, the 4th, 5th, 6th and 7th leaves were harvested from the apex of the two groups and their total RNA were purified. Through three times subtractive hybridization between the wounded mRNAs and the unwounded cDNAs, some mRNAs that can't bind with unwounded cDNAs were isolated and considered to be wound-induced mRNAs. After the reverse transcription and polymerase chain reaction to produce the wounded specific cDNAs, the specific cDNAs were ligated to the vector pTZ-18U digested at Xba I and Hind III sites, and a subtraction library was constructed. Eighteen transformants contained cDNA inserts selected from the subtraction library were used as the probes for northern blot analysis. According to the results of northern blotting, three cDNA probes (No15, No23A and No24B) are wound-induced and two cDNA probes (No1 and No10B) are constitutively expressed. And No15 is induced by chewing of Spodoptera litura larvaes. Both No15 and No1 showed better expression than other probes, and were selected to proceed the further southern blotting. As the results of southern blotting, both No15 and No1 probes resulted in 5 signals with different molecular weight. Corresponding DNA bands were cut, eluted from the gel and ligated to vector pTZ-18UIEcoR I and constructed the subgenomic libraries. The transformants were further identified by dot blotting to conform that they are homologous to probes No1 and No15. Hopefully, the promoters and intact structural genes can be obtained to perform the further study.