Bt杀虫基因的结构改造与功能研究
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摘要
自从1987年报道了关于转Bt基因烟草和蕃茄中以来，国内外已经研制成功200种以上的转基因植物，转基因棉花便是其中重要的一种。我国是继美国之后第二个研制成功转基因抗虫棉的国家，转基因棉花种植面积占棉花种植总面积的80%以上。随着转Bt基因抗虫棉的问世及不断发展，其体现出来的巨大社会、经济价值得到了广泛认可，但是在转基因抗虫棉分子育种过程中仍然存在一些问题有待解决：第一、转基因抗虫棉的大面积种植，对靶标害虫群体造成巨大的生态选择压，为昆虫不断进化而产生抗性后代提供了极大的概率。第二、杀虫蛋白在不同抗虫棉品种之间和转基因棉花整个生长发育期表达不稳定，导致抗虫棉抗虫能力降低。第三、棉花遗传转化方法主要是农杆菌介导法和花粉管通道法，方法繁琐、条件苛刻、受体材料范围狭窄，不能满足分子育种需求。本论文基于以上内容分别展开研究取得的结果如下：
1）对本实验室自主知识产权的Bt（GFM Cry1A）基因进行缺失Domain I第一个α螺旋改造。已有的研究结果表明棉铃虫对Bt蛋白产生的抗性主要是由于钙粘蛋白等受体发生突变引起的，通过将3D-Cry基因Domain I的第一个α螺旋缺失后，杀虫蛋白能够不依赖钙粘蛋白受体即对棉铃虫具有高致死率，甚至对钙粘蛋白受体发生突变的抗性棉铃虫具有高效毒杀作用。利用生物信息软件分析实验室GFM Cry1A蛋白二级结构和三维结构，获得了缺失第一个α螺旋的BtΔα基因，并与Cpti融合获得了融合基因FBCΔα，分别构建了原核表达载体和植物表达载体进行表达研究。原核表达表明：BtΔα和FBCΔα蛋白对于敏感棉铃虫分别比对照GFM Cry1A蛋白的杀虫效率提高了36%和38%；BtΔα和FBCΔα蛋白能够高效杀死抗性棉铃虫，LC_(50)分别为491.04ng/g和448.41ng/g，而对照蛋白在6400ng/g杀虫蛋白浓度范围内不能有效杀死抗性棉铃虫。同时烟草表达也证明BtΔα蛋白、FBCΔα都能够在烟草中表达且具有高效杀虫活性。
2）研究细胞器定位对提高转基因植物中Bt（GFM Cry1A）蛋白含量的作用。据报道Bt Cry蛋白在细胞质内过量表达对棉花细胞产生一定的毒害作用，影响转基因棉花的生长，降低转基因棉花的抗虫性。依据线粒体、叶绿体等细胞器具有双层膜组成相对独立封闭的环境、蛋白质合成具有半自主性和较强的物质承受能力等特点，本研究将拟南芥ALDH线粒体靶向肽AtMTP基因序列和棉花EPSPS叶绿体靶向肽GhCTP基因序列分别与GFP基因和Bt基因融合并转化烟草。荧光显微镜观察GFP蛋白信号表明两个信号肽都具有亚细胞定位活性。对PCR、Bt试纸条检测获得的转基因烟草进行Southern blot分析，证明了Bt Cry基因已经整合于烟草染色体上。选择实时荧光定量PCR相对定量值分别为1.2、1.3、1.5、1.7的pGBI4AAtMTPBt、pGBI4AGhCTPBt和pGBI4AB三个类型的单株进行ELISA检测Bt Cry蛋白含量，结果表明pGBI4AAtMTPBt类型植株Bt蛋白含量比对照pGBI4AB类型植株提高32.3~104.8%，pGBI4AGhCTPBt类型植株Bt Cry蛋白含量比对照植株提高41.2~140%。
3）建立了一套农杆菌喷花法遗传转化体系。转化方法为将含有目的基因的农杆菌培养至OD600=0.8左右，收集菌体用含有10%蔗糖和0.05%的SilWet L-77溶液重悬至OD600=0.4左右，上午散粉前将农杆菌悬浮液喷雾于柱头及花药上，待种子成熟后进行播种筛选阳性植株。与棉花农杆菌介导遗传转化法相比，该方法操作简单、无受体材料遗传背景限制、不需要组织培养再生过程直接得到转化获得的受体材料的种子、获得转基因植株时间短、得到后代材料变异率低；与棉花花粉管通道遗传转化法相比，该方法对花器官损伤小、得种率高、转化方法简便、转化效率高，按照转化花朵数算转化效率达到了9%。利用该方法转化转化融合抗虫基因BtCpti，共获得了32株T_0代转基因植株，经过连续4代筛选获得了5个抗草苷膦、表型优良的单株材料，分子检测证明融合抗虫基因稳定整合于棉花染色体上且能够高效表达。
本论文通过研究Bt Cry蛋白的结构改造提高对抗性棉铃虫的杀虫效率；通过研究Bt Cry蛋白表达后细胞器定位来提高转基因植物组织中杀虫蛋白的含量；通过研究棉花农杆菌喷花法转化方法提高棉花遗传转化效率，为棉花转基因工程新体系奠定理论基础。
Swedish scientists firstly introduced Bacillus thuringiensis (Bt) crystal toxin gene into tobacco andpotato through transgenic approaches in1987. After this Landmarker event, many genetical modification(GM) crops, carrying novel traits such as insect-resistant and herbicide-tolerance, have been developed invarious plants. Bt cotton is one of most widely commercialized GM crops across the world. Following theUnited States, Chinese scientists successfully developed the Bt cotton with independent intellectual propertyright in1997. The Bt cotton has been widely and rapidly adopted by farmer in China because Bt cottondisplayed good performances such as insecticide-saving, high-yield and environment-friendly. Bt cottonaccounts for more than80%of the total planting cotton in2010. With the advantage of Bt cotton, its socialand economic benefits have been widely recognized by the governments and peoples. However, there stillhave some underlying limitations and problems remaining to be resolved in current cotton molecularbreeding: Firstly, with the massive use of Bt cotton, insect pests will quickly develop resistance to cotton withBt toxin eventually resulting in the potential environmental risks. Secondly, the dosage of Bt toxin is still lowin reproductive stage of cotton leading to Bt cotton reduce the function of insect-resistant at late stage ofcotton. Thirdly, the cotton genetic transformation methods mainly depend on tissue culture basedAgrobacterium-mediated approach and this method is strongly depended upon the cotton genotype, which isseverely restricted the cotton improvement through genetically modification. According to the aboveproblems, this paper achieved some new methods and strategies to resolve them:
1) Recent report discovered that deletion of Cry1Ac α-helix domain is able to enhance the toxicityof Bt toxin. We therefore deleted the first α-helix domain, containing129bp nucleotide acids, oflaboratory storage Bt (GFM Cry1A) gene. The Bt protein truncation constructs: BtΔα was designed andmade. Furthermore, BtΔα fusing with Cpti, another protein toxin, FBCΔα was also successfullydeveloped. Bt and Bt:Cpti fusion genes were set as control. Prokaryotic expression vocter PT7: BtΔαand PT7: FBCΔα based on PET-28a vector backbone has been constructed and introduced into E.coliBL21strain. The protein BtΔα and FBCΔα have been expressed and purified for toxin activity assay.The results showed that the LC50of sensitive bollworm and resistant bollworm. For sensitive bollworm:Bt protein LC50was161.93ng/g, BtΔα protein LC50was104.08ng/g, FBC protein LC50was of148.85ng/g, FBCΔα protein LC50was100.81ng/g. For5times Bt toxin resistant bollworm: Btprotein LC50was>6400ng/g, BtΔα protein LC50was491.04ng/g, FBC protein LC50was>6400ng/g, FBCΔα protein LC50was448.41ng/g. Additionally, the plant expression vocter P35s: BtΔα, P35S:FBCΔα, P35s: Bt, P35S: Cpti, have been constructed and transformed into tobacco. Through analysisthe obtained transgenic tobacco by the PCR, ELISA, bioassay and field pick insects experimental, theresults shows that BtΔα protein, FBCΔα are able to expressed in tobacco and with insecticidal activity.
2) Recent studies showed that mitochondrial targeting peptide and chloroplast targeting peptideenable to significantly increase the dosage of exogenous protein in the cells. We therefore isolated anArabidopsis ALDH gene containing mitochondrial targeting peptide nucleic acid sequences AtMTP anda cotton EPSPS gene containing chloroplast targeting peptides nucleic acid sequences GhCTP, respectively. After fused them with the Bt gene and the GFP gene,respectively, then transformed intotobacco. The GFP signal has been detected by Fluorescence microscopy in the transgenic tobacco,indicating that two signal peptide has the expected signaling targeting activity. The results suggestedthat the dosage of Bt protein can be increased double times in transgenic tobacco through fusing Btprotein with mitochondria and chloroplasts targeting peptides.
3) Established flower spraying Agrobacterium method for cotton genetic transformation. Basically,spinning and collection the OD600=0.8agrobacteria containing the target constructs; re-suspension thepellet in10%sucrose and0.05%SilWet L-77solution; spraying the mixture solution on the stigma andanther of cotton unopened flower in the morning. The spraying tool is a little aerosol spray bottle. Byusing the method,42T0transgenic cotton were obtained via this approach. The transformat efficiency isabout9%. Five independent transgenic lines were selected for further analysis by Southern blot andElisa. The results suggested that the exogenous genes were integrated into cotton genome and can bestably passed down from T1generation to T3generation.
In short, through exploration of optimization of Bt protein； mitochondria and chloroplastsubcellular localizated expression of Bt protein in the tobacco and improvement of genetictransformation methond in cotton, we developed an improved method for cotton genetic transfomation,increasing the dosage of Bt toxin in plant cell, optimization the Bt protein structure and subsequentlyincreasing the toxicity of Bt protein in transgenic cotton in this study.
1）对本实验室自主知识产权的Bt（GFM Cry1A）基因进行缺失Domain I第一个α螺旋改造。已有的研究结果表明棉铃虫对Bt蛋白产生的抗性主要是由于钙粘蛋白等受体发生突变引起的，通过将3D-Cry基因Domain I的第一个α螺旋缺失后，杀虫蛋白能够不依赖钙粘蛋白受体即对棉铃虫具有高致死率，甚至对钙粘蛋白受体发生突变的抗性棉铃虫具有高效毒杀作用。利用生物信息软件分析实验室GFM Cry1A蛋白二级结构和三维结构，获得了缺失第一个α螺旋的BtΔα基因，并与Cpti融合获得了融合基因FBCΔα，分别构建了原核表达载体和植物表达载体进行表达研究。原核表达表明：BtΔα和FBCΔα蛋白对于敏感棉铃虫分别比对照GFM Cry1A蛋白的杀虫效率提高了36%和38%；BtΔα和FBCΔα蛋白能够高效杀死抗性棉铃虫，LC_(50)分别为491.04ng/g和448.41ng/g，而对照蛋白在6400ng/g杀虫蛋白浓度范围内不能有效杀死抗性棉铃虫。同时烟草表达也证明BtΔα蛋白、FBCΔα都能够在烟草中表达且具有高效杀虫活性。
2）研究细胞器定位对提高转基因植物中Bt（GFM Cry1A）蛋白含量的作用。据报道Bt Cry蛋白在细胞质内过量表达对棉花细胞产生一定的毒害作用，影响转基因棉花的生长，降低转基因棉花的抗虫性。依据线粒体、叶绿体等细胞器具有双层膜组成相对独立封闭的环境、蛋白质合成具有半自主性和较强的物质承受能力等特点，本研究将拟南芥ALDH线粒体靶向肽AtMTP基因序列和棉花EPSPS叶绿体靶向肽GhCTP基因序列分别与GFP基因和Bt基因融合并转化烟草。荧光显微镜观察GFP蛋白信号表明两个信号肽都具有亚细胞定位活性。对PCR、Bt试纸条检测获得的转基因烟草进行Southern blot分析，证明了Bt Cry基因已经整合于烟草染色体上。选择实时荧光定量PCR相对定量值分别为1.2、1.3、1.5、1.7的pGBI4AAtMTPBt、pGBI4AGhCTPBt和pGBI4AB三个类型的单株进行ELISA检测Bt Cry蛋白含量，结果表明pGBI4AAtMTPBt类型植株Bt蛋白含量比对照pGBI4AB类型植株提高32.3~104.8%，pGBI4AGhCTPBt类型植株Bt Cry蛋白含量比对照植株提高41.2~140%。
3）建立了一套农杆菌喷花法遗传转化体系。转化方法为将含有目的基因的农杆菌培养至OD600=0.8左右，收集菌体用含有10%蔗糖和0.05%的SilWet L-77溶液重悬至OD600=0.4左右，上午散粉前将农杆菌悬浮液喷雾于柱头及花药上，待种子成熟后进行播种筛选阳性植株。与棉花农杆菌介导遗传转化法相比，该方法操作简单、无受体材料遗传背景限制、不需要组织培养再生过程直接得到转化获得的受体材料的种子、获得转基因植株时间短、得到后代材料变异率低；与棉花花粉管通道遗传转化法相比，该方法对花器官损伤小、得种率高、转化方法简便、转化效率高，按照转化花朵数算转化效率达到了9%。利用该方法转化转化融合抗虫基因BtCpti，共获得了32株T_0代转基因植株，经过连续4代筛选获得了5个抗草苷膦、表型优良的单株材料，分子检测证明融合抗虫基因稳定整合于棉花染色体上且能够高效表达。
本论文通过研究Bt Cry蛋白的结构改造提高对抗性棉铃虫的杀虫效率；通过研究Bt Cry蛋白表达后细胞器定位来提高转基因植物组织中杀虫蛋白的含量；通过研究棉花农杆菌喷花法转化方法提高棉花遗传转化效率，为棉花转基因工程新体系奠定理论基础。
Swedish scientists firstly introduced Bacillus thuringiensis (Bt) crystal toxin gene into tobacco andpotato through transgenic approaches in1987. After this Landmarker event, many genetical modification(GM) crops, carrying novel traits such as insect-resistant and herbicide-tolerance, have been developed invarious plants. Bt cotton is one of most widely commercialized GM crops across the world. Following theUnited States, Chinese scientists successfully developed the Bt cotton with independent intellectual propertyright in1997. The Bt cotton has been widely and rapidly adopted by farmer in China because Bt cottondisplayed good performances such as insecticide-saving, high-yield and environment-friendly. Bt cottonaccounts for more than80%of the total planting cotton in2010. With the advantage of Bt cotton, its socialand economic benefits have been widely recognized by the governments and peoples. However, there stillhave some underlying limitations and problems remaining to be resolved in current cotton molecularbreeding: Firstly, with the massive use of Bt cotton, insect pests will quickly develop resistance to cotton withBt toxin eventually resulting in the potential environmental risks. Secondly, the dosage of Bt toxin is still lowin reproductive stage of cotton leading to Bt cotton reduce the function of insect-resistant at late stage ofcotton. Thirdly, the cotton genetic transformation methods mainly depend on tissue culture basedAgrobacterium-mediated approach and this method is strongly depended upon the cotton genotype, which isseverely restricted the cotton improvement through genetically modification. According to the aboveproblems, this paper achieved some new methods and strategies to resolve them:
1) Recent report discovered that deletion of Cry1Ac α-helix domain is able to enhance the toxicityof Bt toxin. We therefore deleted the first α-helix domain, containing129bp nucleotide acids, oflaboratory storage Bt (GFM Cry1A) gene. The Bt protein truncation constructs: BtΔα was designed andmade. Furthermore, BtΔα fusing with Cpti, another protein toxin, FBCΔα was also successfullydeveloped. Bt and Bt:Cpti fusion genes were set as control. Prokaryotic expression vocter PT7: BtΔαand PT7: FBCΔα based on PET-28a vector backbone has been constructed and introduced into E.coliBL21strain. The protein BtΔα and FBCΔα have been expressed and purified for toxin activity assay.The results showed that the LC50of sensitive bollworm and resistant bollworm. For sensitive bollworm:Bt protein LC50was161.93ng/g, BtΔα protein LC50was104.08ng/g, FBC protein LC50was of148.85ng/g, FBCΔα protein LC50was100.81ng/g. For5times Bt toxin resistant bollworm: Btprotein LC50was>6400ng/g, BtΔα protein LC50was491.04ng/g, FBC protein LC50was>6400ng/g, FBCΔα protein LC50was448.41ng/g. Additionally, the plant expression vocter P35s: BtΔα, P35S:FBCΔα, P35s: Bt, P35S: Cpti, have been constructed and transformed into tobacco. Through analysisthe obtained transgenic tobacco by the PCR, ELISA, bioassay and field pick insects experimental, theresults shows that BtΔα protein, FBCΔα are able to expressed in tobacco and with insecticidal activity.
2) Recent studies showed that mitochondrial targeting peptide and chloroplast targeting peptideenable to significantly increase the dosage of exogenous protein in the cells. We therefore isolated anArabidopsis ALDH gene containing mitochondrial targeting peptide nucleic acid sequences AtMTP anda cotton EPSPS gene containing chloroplast targeting peptides nucleic acid sequences GhCTP, respectively. After fused them with the Bt gene and the GFP gene,respectively, then transformed intotobacco. The GFP signal has been detected by Fluorescence microscopy in the transgenic tobacco,indicating that two signal peptide has the expected signaling targeting activity. The results suggestedthat the dosage of Bt protein can be increased double times in transgenic tobacco through fusing Btprotein with mitochondria and chloroplasts targeting peptides.
3) Established flower spraying Agrobacterium method for cotton genetic transformation. Basically,spinning and collection the OD600=0.8agrobacteria containing the target constructs; re-suspension thepellet in10%sucrose and0.05%SilWet L-77solution; spraying the mixture solution on the stigma andanther of cotton unopened flower in the morning. The spraying tool is a little aerosol spray bottle. Byusing the method,42T0transgenic cotton were obtained via this approach. The transformat efficiency isabout9%. Five independent transgenic lines were selected for further analysis by Southern blot andElisa. The results suggested that the exogenous genes were integrated into cotton genome and can bestably passed down from T1generation to T3generation.
In short, through exploration of optimization of Bt protein； mitochondria and chloroplastsubcellular localizated expression of Bt protein in the tobacco and improvement of genetictransformation methond in cotton, we developed an improved method for cotton genetic transfomation,increasing the dosage of Bt toxin in plant cell, optimization the Bt protein structure and subsequentlyincreasing the toxicity of Bt protein in transgenic cotton in this study.
引文
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