改造Cry1Ac蛋白C端對轉(zhuǎn)基因水稻抗二化螟蟲的影響
發(fā)布時間：2018-11-07 17:02
【摘要】：二化螟蟲的泛濫嚴重影響水稻的產(chǎn)量,cry1Ac基因是目前世界上應(yīng)用最廣泛和最高效的抗蟲基因之一,但二化螟蟲通過進化會逐漸對其產(chǎn)生抗性。通過改變蛋白結(jié)構(gòu)來構(gòu)建新的Cry蛋白是解決這一問題的有效途徑之一。為分析改造Cry1Ac蛋白C端能否對轉(zhuǎn)基因作物抗蟲性產(chǎn)生影響,本研究采用Cry1Ja蛋白的C端替換Cry1Ac蛋白的C端,重組獲得CryFLAc蛋白。將cry1Ac基因和編碼CryFLAc蛋白的cryFLAc基因分別構(gòu)建到pTF101.1-ubi植物表達載體上,利用農(nóng)桿菌介導(dǎo)方法轉(zhuǎn)化到吉林省水稻品種吉粳88中;采用PCR、RT-PCR、免疫檢測試紙條檢測的方法確定cry1Ac、cryFLAc基因整合和表達情況;通過室內(nèi)和田間抗蟲性測試評價CryFLAc和Cry1Ac蛋白對T1代轉(zhuǎn)基因水稻抗蟲性的影響。研究發(fā)現(xiàn):cry1Ac、cryFLAc基因均成功整合到水稻基因組中,并穩(wěn)定表達;轉(zhuǎn)cryFLAc基因水稻抗二化螟水平達到抗性級別,轉(zhuǎn)cry1Ac基因水稻達到高抗級別;轉(zhuǎn)cry1Ac基因水稻二化螟抗性高于轉(zhuǎn)cryFLAc基因水稻。結(jié)果表明:Cry1Ja蛋白的C端替換Cry1Ac蛋白的C端不會提高轉(zhuǎn)基因水稻的抗蟲性。上述研究為人工設(shè)計合理化改造Cry蛋白提供了新的理論依據(jù)。
[Abstract]:Cry1Ac gene is one of the most widely used and highly effective insect-resistant genes in the world, but the resistance of Chilo suppressalis is gradually produced through evolution. One of the effective ways to solve this problem is to construct a new Cry protein by changing its structure. In order to analyze whether the C terminal of modified Cry1Ac protein can affect the insecticidal resistance of transgenic crops, the C terminal of Cry1Ja protein was replaced by C terminal of Cry1Ac protein, and the CryFLAc protein was obtained. Cry1Ac gene and cryFLAc gene encoding CryFLAc protein were constructed into pTF101.1-ubi plant expression vector, and transformed into Jilin rice variety Jijing 88 by Agrobacterium tumefaciens. The integration and expression of cry1Ac,cryFLAc gene were determined by PCR,RT-PCR, immunoassay, and the effects of CryFLAc and Cry1Ac proteins on the insecticidal resistance of T1 generation transgenic rice were evaluated by indoor and field insecticidal tests. The results showed that cry1Ac,cryFLAc gene was successfully integrated into rice genome and expressed stably, and transgenic rice with cryFLAc gene reached resistance level, and transgenic rice with cry1Ac gene reached high resistance level. The resistance of transgenic rice with cry1Ac gene was higher than that of transgenic rice with cryFLAc gene. The results showed that the C terminal substitution of Cry1Ja protein for Cry1Ac protein did not improve the insecticidal resistance of transgenic rice. These studies provide a new theoretical basis for the rationalization of artificial design and modification of Cry protein.
【作者單位】： 吉林省農(nóng)業(yè)科學(xué)院農(nóng)業(yè)生物技術(shù)研究所吉林省農(nóng)業(yè)生物技術(shù)重點實驗室;東北師范大學(xué);哈爾濱師范大學(xué)生命科學(xué)與技術(shù)學(xué)院;吉林農(nóng)業(yè)大學(xué)生命科學(xué)學(xué)院;
【基金】：吉林省農(nóng)業(yè)科技創(chuàng)新工程(吉財教[2012]1072) 吉林省科技發(fā)展計劃項目(20160520060JH);吉林省科技發(fā)展計劃項目(20160204033NY);吉林省科技發(fā)展計劃項目(20140204014NY)共同資助
【分類號】：S435.112.1
[Abstract]:Cry1Ac gene is one of the most widely used and highly effective insect-resistant genes in the world, but the resistance of Chilo suppressalis is gradually produced through evolution. One of the effective ways to solve this problem is to construct a new Cry protein by changing its structure. In order to analyze whether the C terminal of modified Cry1Ac protein can affect the insecticidal resistance of transgenic crops, the C terminal of Cry1Ja protein was replaced by C terminal of Cry1Ac protein, and the CryFLAc protein was obtained. Cry1Ac gene and cryFLAc gene encoding CryFLAc protein were constructed into pTF101.1-ubi plant expression vector, and transformed into Jilin rice variety Jijing 88 by Agrobacterium tumefaciens. The integration and expression of cry1Ac,cryFLAc gene were determined by PCR,RT-PCR, immunoassay, and the effects of CryFLAc and Cry1Ac proteins on the insecticidal resistance of T1 generation transgenic rice were evaluated by indoor and field insecticidal tests. The results showed that cry1Ac,cryFLAc gene was successfully integrated into rice genome and expressed stably, and transgenic rice with cryFLAc gene reached resistance level, and transgenic rice with cry1Ac gene reached high resistance level. The resistance of transgenic rice with cry1Ac gene was higher than that of transgenic rice with cryFLAc gene. The results showed that the C terminal substitution of Cry1Ja protein for Cry1Ac protein did not improve the insecticidal resistance of transgenic rice. These studies provide a new theoretical basis for the rationalization of artificial design and modification of Cry protein.
【作者單位】： 吉林省農(nóng)業(yè)科學(xué)院農(nóng)業(yè)生物技術(shù)研究所吉林省農(nóng)業(yè)生物技術(shù)重點實驗室;東北師范大學(xué);哈爾濱師范大學(xué)生命科學(xué)與技術(shù)學(xué)院;吉林農(nóng)業(yè)大學(xué)生命科學(xué)學(xué)院;
【基金】：吉林省農(nóng)業(yè)科技創(chuàng)新工程(吉財教[2012]1072) 吉林省科技發(fā)展計劃項目(20160520060JH);吉林省科技發(fā)展計劃項目(20160204033NY);吉林省科技發(fā)展計劃項目(20140204014NY)共同資助
【分類號】：S435.112.1
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