本文關(guān)鍵詞： 水稻 抗蟲性 cryNGc基因 CryNGc蛋白　出處：《東北師范大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

【摘要】：水稻(Oryzasativa)是全球近一半人口的主要食物來源,也是我國重要的糧食作物之一。近年來水稻病蟲害呈偏重發(fā)生態(tài)勢,蟲害以螟蟲為主,嚴重影響了水稻的產(chǎn)量,制約農(nóng)業(yè)的穩(wěn)定發(fā)展。傳統(tǒng)的化學(xué)防治方法有成本高、污染環(huán)境和害蟲抗藥性增加等缺點。蘇云金芽胞桿菌(Bacillus thuringiensis,簡稱Bt)為一種革蘭氏陽性菌,其產(chǎn)生的殺蟲晶體蛋白(Insecticidal crystal proteins,ICP)對各類害蟲具有特異的毒性,其中來源于Bt的Cry蛋白家族殺蟲活性最強。隨著轉(zhuǎn)基因技術(shù)的發(fā)展,編碼這種殺蟲晶體蛋白的基因被轉(zhuǎn)入作物中,為害蟲的防治提供了新的策略�？墒请S著研究的不斷深入,人們發(fā)現(xiàn)許多害蟲對Bt蛋白產(chǎn)生了抗性。因此,如何提高轉(zhuǎn)Bt作物的抗蟲性和擴大抗蟲譜是目前轉(zhuǎn)基因抗蟲作物面臨的主要問題。本課題組前期利用基因篩選、分子改造等方法,選取Cry1Ab蛋白作為主要供體,其具有明確抗鱗翅目(Lepidoptera)昆蟲的特點。又選取Cry1Gc蛋白,同樣具有抗鱗翅目昆蟲的活性。將Cry1Gc的結(jié)構(gòu)域Ⅲ與Cry1Ab的結(jié)構(gòu)域Ⅲ進行互換,得到重組蛋白CryNGc。期望兩種不同殺蟲蛋白之間構(gòu)建的重組蛋白可以提高作物抗蟲性和擴大抗蟲譜。作為課題組系統(tǒng)研究的一部分,為了驗證殺蟲蛋白CryNGc能否在作物中成功表達,并且對鱗翅目昆蟲產(chǎn)生抗性。本研究將重組蛋白基因cryNGc轉(zhuǎn)入吉林省粳稻品種吉粳88中。通過對轉(zhuǎn)基因水稻進行核酸檢測、蛋白定量和抗蟲性鑒定等研究,獲得抗二化螟轉(zhuǎn)基因水稻新材料。主要研究結(jié)果如下:1.構(gòu)建植物表達載體pTF101.1-ubi-NGc。采用農(nóng)桿菌介導(dǎo)法,將PTF101.1-ubi-NGc入水稻吉粳88中,經(jīng)篩選獲得15個抗性植株。2.PCR、Southern雜交和RT-PCR檢測表明:外源基因cryNGc已單拷貝整合到水稻基因組中,且能正常轉(zhuǎn)錄。3.ELISA結(jié)果表明:T1代轉(zhuǎn)cryNGc基因水稻在分蘗期和灌漿期的根、莖和葉中都能檢測到Bt蛋白,且濃度都明顯高于非轉(zhuǎn)基因材料。不同器官在同一生長時期以及同一器官在不同生長時期Bt蛋白的表達量均存在差異,其中葉中Bt蛋白的表達量最高。4.cryNGc基因表達分析結(jié)果表明:T1代轉(zhuǎn)基因水稻中cryNGc基因已成功表達。室內(nèi)二化螟抗性鑒定中,T1代轉(zhuǎn)cryNGc基因水稻植株的莖稈和葉片沒有明顯的蟲害癥狀。轉(zhuǎn)基因水稻植株葉片接蟲后,二化螟的平均校正死亡率96.16%;莖稈接蟲后二化螟的平均校正死亡率為95.12%。田間二化螟抗性實驗中,T1代轉(zhuǎn)基因水稻植株正常生長,沒有明顯的蟲害癥狀。感蟲率為3.95%,明顯低于非轉(zhuǎn)基因植株。根據(jù)抗蟲評價標準可以得出結(jié)論:轉(zhuǎn)cryNGc水稻對二化螟的抗性級別為高抗。綜上,實驗結(jié)果表明:外源基因cryNGc以單拷貝形式轉(zhuǎn)入到水稻中,能夠正常轉(zhuǎn)錄且成功表達,并且對二化螟表現(xiàn)為高抗。
[Abstract]:Rice Oryza sativa (Oryza sativa) is the main food source for nearly half of the world's population and one of the most important food crops in China. It seriously affects the yield of rice and restricts the stable development of agriculture. Traditional chemical control methods have high cost. Bacillus thuringiensis (BT) is a Gram-positive bacteria. The insecticidal crystal proteinsus produced by ICP has specific toxicity to all kinds of pests. The Cry protein family derived from BT had the strongest insecticidal activity. With the development of transgenic technology, the gene encoding the insecticidal crystal protein was transferred into crops. It provides a new strategy for pest control. However, with the development of research, it has been found that many pests have resistance to BT protein. How to improve the insecticidal resistance of transgenic BT crops and expand the insect-resistant spectrum are the main problems faced by transgenic insect-resistant crops at present. Cry1Ab protein was selected as the main donor, which has the characteristics of anti-Lepidoptera insects and Cry1Gc protein. Cry1Gc domain 鈪，

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