轉CmTreⅡ基因水稻RNAi效應的室內和田間抗性及遺傳性鑒定

王爽　杜娟　李尚偉

摘要：本研究采用農桿菌介導的遺傳轉化技術，將CmTreⅡ （膜結合型海藻糖酶基因一個靶位點）導入粳稻中花 11 號中，利用 CmTreⅡ-dsRNA （含有膜結合型海藻糖酶基因一個靶位點的雙鏈RNA）的沉默效應，沉默稻縱卷葉螟的膜結合型海藻糖酶，最終獲得一批抗蟲性較好的轉基因純合株系。研究中進一步檢測了這些株系的室外卷葉率、白葉率以及室內抗蟲性。采用實時熒光定量 PCR（RT-qPCR）檢測轉基因水稻室內抗蟲性 CmTre2 基因的相對表達量。通過 RT-PCR 技術檢測了轉 CmTreⅡ 基因水稻的遺傳性。結果顯示：在室外抗蟲性試驗中，稻縱卷葉螟對水稻的取食量明顯下降，轉 CmTreⅡ 基因水稻的卷葉率和白葉率分別為 1340% 和 1247%，相比對照組分別下降了 1132% 和 1010%；在室內抗蟲性試驗中，RT-qPCR 檢測結果表明，稻縱卷葉螟在取食轉 CmTreⅡ 基因水稻的 96h后，CmTre2 基因的相對表達量比對照組下降了 2900%；7d后，實驗組的死亡率為 5667%，相比對照組增加了 4667%。在遺傳性試驗中，檢測發現轉 CmTreⅡ 基因水稻的遺傳性良好。因此本研究成功鑒定了轉 CmTreⅡ 基因水稻對稻縱卷葉螟的抗蟲性以及遺傳性。

關鍵詞：稻縱卷葉螟；轉基因；抗蟲性；膜結合型海藻糖酶；RNA干擾

中圖分類號：Q9581；S4433（273）

文獻標識碼：A

文章編號：1008-0457（2017）02-0040-06國際DOI編碼：10.15958/j.cnki.sdnyswxb.2017.02.008

Abstract：The CmTre2 gene， CmTreII， was introduced into the rice （Chinese Flower 11） by agrobacterium-mediated genetic transformation technology. A number of good insect resistance transgenic plants were obtained by using the silencing effect of CmTre II – dsRNAn to silent the membrane - bound trehalase of Cnaphalocrocis medinalis. The rate of white leaves and the identification of insect resistance were further investigated. The relative expression of CmTre2 gene in transgenic rice was detected by real-time fluorescence quantitative PCR （RT-qPCR）. At the same time， RT-PCR technology was adopted to detect the genetic characteristics of transgenic CmTreⅡ gene rice. The results showed that in the outdoor insect resistance experiments， the feeding amount by C. medinalis of transgenic CmTreⅡ gene rice decreased significantly. The leaf roll rate and white leaf rate of transgenic CmTreⅡ gene rice were 1340% and 1247%， respectively， which were a decrease of 1132% and 1010%， respectively， compared to the control group. In the indoor experiment of insect resistance， RT-qPCR results showed that， after 96 hours of C. medinalis feeding on transgenic CmTreⅡ gene rice， the relative expression of CmTre2 was decreased by 29% of the control group. After 7 days of feeding on transgenic CmTreⅡ gene rice， the mortality rate of insects was 5667%， an increase of 46.67% compared to the control group. In the genetic test， the genetic characteristics of the transgenic CmTreⅡ gene rice was detected. This study successfully identified the pest resistance and genetics of transgenic CmTreⅡ gene rice.

Key words：Cnaphalocrocis medinalis ； transgenosis ； insect resistance； the membrane - bound trehalase； RNAi

水稻是世界上最重要的糧食作物之一，全球近半數人以稻米為食，而我國又是世界上最大的稻米生產國和消費國之一，全國有 65%以上的人口以稻米為主食。因此，水稻高產豐收對我國糧食安全至關重要。稻縱卷葉螟（Cnaphalocrocis medinalis）俗稱卷葉蟲、刮青蟲、苞葉蟲等，屬于鱗翅目（Lepidoptera）螟蛾科（Pyralidae），是對水稻危害較為嚴重的主要螟蟲之一[1-3]，是一種典型的遷飛性害蟲，在我國和國外都有廣泛的分布[1]。……