农杆菌介导转Bt cry1Ah和cry1Ie基因抗虫植物的研究
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摘要
Bt cry1Ah基因是中国农业科学院植物保护研究所张杰课题组从国内苏云金芽胞杆菌（Bacillus thuringiensis，Bt）菌株BT8中分离克隆的一个新型杀虫蛋白基因。Cry1Ah基因与cry1Ac基因的氨基酸序列相似性最高，为82%。Cry1Ah蛋白对鳞翅目害虫具有高毒力，对棉铃虫、水稻二化螟的杀虫活性强于Cry1Ac蛋白；对亚洲玉米螟的杀虫活性强于Cry1Ac、Cry1Ab蛋白。Bt cry1Ie基因是张杰课题组从Bt菌株Btc007中分离克隆的另一个具有自主知识产权的杀虫蛋白基因。Cry1Ie基因与cry1A类基因相似性很低，只有30%，并且它们之间无交互抗性。其编码的蛋白对抗性和敏感玉米螟都有一定的毒力。将cry1Ah和cry1Ie基因同时构建到一个表达载体中，能够有效地克服基因种类单一、同源性高以及昆虫对Bt产生抗性等一系列问题，同时也有望筛选到抗虫性能更高的转基因作物。
对于一个来源于原核生物的基因，其编码区序列所使用的密码子通常在植物中使用的频率较低，这会使外源基因在植物中仅有少量的积累。而将外源基因根据植物密码子偏好进行优化后可以显著地提高外源基因在植物中的表达。根据植物密码子偏好性，本实验室早期工作对cry1Ah基因进行过两次改造以及对cry1Ie基因进行过改造，本研究将cry1Ah基因进行了第三次密码子优化。三次改造的cry1Ah基因(m1-cry1Ah，m2-cry1Ah，m3-cry1Ah)与原始cry1Ah基因进行比对：GC含量由37%分别提高到48%，55%和63%。优化后的cry1Ie基因(mcry1Ie)的GC含量提高到55%。
在转基因作物研究中，提高转基因表达的另一个主要策略是将外源蛋白进行亚细胞定位表达。亚细胞定位主要依赖于信号肽的作用，其中定位于叶绿体和内质网的研究比较多也比较成功。本研究将m3-cry1Ah基因的N端连接一段来源于玉米RuBP羧化酶的叶绿体定位信号肽序列，命名为CPT-m3-cry1Ah；另外将m3-cry1Ah基因的N端连接内质网定位信号肽，在C端连接内质网滞留信号KDEL，命名为ER-m3-cry1Ah；将不连接任何信号肽的m3-cry1Ah基因命名为NK-m3-cry1Ah。依次构建了三个植物表达载体pMhNK、pMhCTP和pMhER，并且都是以bar基因作为筛选标记基因。
本研究首先利用农杆菌介导法将实验室保存的pMhGM (m1-cry1Ah)，pMAhb (m2-cry1Ah)以及本研究构建的pMhNK (NK-m3-cry1Ah)，pMhCTP (CPT-m3-cry1Ah)共计四个植物表达载体进行烟草转化。通过对模式生物烟草的研究确定出最优的cry1Ah基因密码子使用形式以及研究蛋白叶绿体定位对基因表达的影响。一共侵染320个烟草叶片外植体，共获得84株烟草再生苗，每个载体分别获得21、28、20和15株。通过PCR检测确定了每个载体的阳性植株数分别为7、5、6和5株；通过Southern blot结果表明外源基因已整合到烟草基因组中；实时荧光定量PCR (quantitative RT-PCR，qRT-PCR)检测结果表明pMhCTP烟草植株在四个载体的植株中表现出最高的转录水平，并且pMAhb、pMhNK和pMhCTP烟草植株的转录水平分别是pMhGM植株的4倍、9.5倍和12.5倍；ELISA检测结果表明pMhCTP烟草植株中Cry1Ah蛋白表达量最高，并且pMAhb、pMhNK和pMhCTP烟草植株的Cry1Ah蛋白表达量分别是pMhGM植株的4倍、6倍和10倍；通过生物活性检测结果表明喂食pMhGM、pMAhb、pMhNK和pMhCTP烟草叶片的棉铃虫幼虫致死率分别为63%、82%、93%和100%，这四个载体烟草叶片对棉铃虫的抗性等级分别为2.57、1.8、1.33和1级。通过以上实验结果表明cry1Ah基因的三次密码子优化和叶绿体定位表达逐步提高了cry1Ah基因在转基因烟草中的表达。
本研究随后利用农杆菌介导法将七个植物表达载体转化玉米自交系综31幼胚进行抗虫玉米的研究。这七个植物表达载体包括实验室保存的四个植物表达载体pMhGM (m1-cry1Ah)，pMAhb (m2-cry1Ah)，pMIeb (mcry1Ie)和pMAhIeb (含m2-cry1Ah和mcry1Ie)以及本研究构建的三个植物表达载体pMhNK (NK-m3-cry1Ah)， pMhCTP (CPT-m3-cry1Ah)和pMhER(ER-m3-cry1Ah)。前期将植物表达载体pMhGM经农杆菌介导法转化玉米自交系综31未成熟幼胚，一共转化500个幼胚，共得到48株T0代玉米再生植株。PCR检测表明有23株为PCR阳性植株；然后通过各种分子检测和生物活性检测确定出两个高抗虫玉米事件1-4和1-5；Southern blot检测结果表明这两个高抗虫玉米事件中的外源基因均以单拷贝形式插入玉米基因组中；通过对这两个抗虫事件的T1~T6代植株的Cry1Ah蛋白的表达和遗传稳定性进行研究，结果表明Cry1Ah蛋白在每一代的玉米植株中都能正常稳定的表达，并且植株在田间对玉米螟的抗性也是稳定遗传的。随后我们将以上七个植物表达载体对玉米自交系综31幼胚进行大规模转化，一共转化20070个玉米未成熟幼胚，获得7745块抗性愈伤，共得到T0代玉米转化植株1764株。通过PCR检测确定1360株为PCR阳性植株；然后通过分子检测和生物活性检测确定了转cry1Ah单基因以及转cry1Ah和cry1Ie双基因的抗虫玉米事件数分别是29个和14个，其中表现为高抗虫的玉米事件数分别为8个和3个；通过对这43个抗虫事件的T1~T2代植株的Cry1Ah蛋白的表达和遗传稳定性进行研究，结果表明Cry1Ah蛋白在T1~T2代的玉米植株中都能正常稳定的表达，并且植株在田间对玉米螟的抗性也是稳定遗传的。获得的抗虫转基因玉米材料具有很好的应用价值，可以作为候选材料进行下一步Bt抗虫玉米的育种工作。
此外，我们对抗虫性表现非常突出的两个双基因抗虫玉米事件pMAhIeb60和pMAhIeb186的T2代植株的苞叶、叶片和花丝进行了Cry1Ah蛋白量的分析，结果表明事件pMAhIeb60的植株中Cry1Ah蛋白表达量都比较高，由高到低的表达部位分别是苞叶、叶片和花丝，每鲜克重表达Cry1Ah蛋白量分别是4.5μg/g fresh weight (f.w.)、3.5μg/g (f.w)和2.5μg/g(f.w)；事件pMAhIeb186植株的苞叶、叶片中表达Cry1Ah蛋白量较高，都为3.5μg/g (f.w)，而花丝中表达量为0.8μg/g (f.w)。Cry1Ah蛋白在叶片中的高表达可以有效防治玉米螟初期危害，而在苞叶和花丝中的高表达可以有效防治玉米螟后期危害，这两个高抗虫事件有望走向产业化。
The Bt cry1Ah gene is a novel insecticidal gene，which was cloned from the Bacillusthuringiensis (Bt) isolate BT8．The toxic fragment of Cry1Ah has82%similarity to Cry1Ac．TheCry1Ah protein is highly toxic to lepidopteran insects：this protein is more toxic than Cry1Ac toHelicoverpa armigera，Ostrinia furnacalis and Chilo suppressalis，and it is more toxic thanCry1Ab to O．furnacalis．The Bt cry1Ie gene is another insecticidal gene，and it was cloned fromthe Bacillus thuringiensis (Bt) isolate Btc007．Its encoding protein has toxicity not only to thesensitive O．furnacalis but also to the resistant O．furnacalis．Cry1Ie has no cross-resistance withCry1A protein，thus combining cry1Ah and cry1Ie to the same expression vector can moreeffectively overcome the problems such as the gene’s high homology，single species，pestresistance to Bt protein and so on，at the same time the insect-resistant transgenic plants withhigher toxicity are expected to be obtained.
Most codon of prokaryotic genes′coding sequence are rare codons in plants，and they maylead to low expression of the gene in plants．Codon optimization can significantly improve foreigngenes’ expression in plants．According to the plant codon bias，the cry1Ah gene was modifiedtwice and cry1Ie gene was modified once in the previous work of our lab．In the present study，we modified the cry1Ah gene for the third time．The GC content of the three modified cry1Ahgenes (m1-cry1Ah，m2-cry1Ah，m3-cry1Ah) were48%，55%and63%，respectively，and theGC content of modified cry1Ie gene (mcry1Ie) was55%.
Targeting foreign proteins to specific subcellular locations is another strategy to enhance theexpression of the transgene in the study of genetically modified crops(GMC)． Subcellularlocalization rely mainly on the role of signal peptide，and the study of targeting to chloroplasts orendoplasmic reticulum is more successful．In this study，the m3-cry1Ah gene was linked with achloroplast transit peptide sequence from maize RuBP carboxylase，and this gene was designatedctp-m3-cry1Ah．Additionally，the m3-cry1Ah gene was linked with a endoplasmic reticulum signalpeptide，and this gene was designated ER-m3-cry1Ah，and the m3-cry1Ah gene with no signalpeptide was designated NK-m3-cry1Ah．We construct pMhNK、pMhCTP and pMhER plantexpression vectors，and the bar gene was used as a selectable maker gene.
Four vectors pMhGM (m1-cry1Ah)，pMAhb (m2-cry1Ah)，pMhNK (NK-m3-cry1Ah) andpMhCTP (CPT-m3-cry1Ah) were transferred into tobacco leaves by Agrobacterium-mediatedtransformation．Based on the research of the model organism of tobacco to determine the optimalcodon pattern of the cry1Ah gene and to study the effect of chloroplast-targeted on transgene expression levels．A total of320tobacco leaf explants were infected，84regenerated tobacco plantswere obtained，21，28，20and15plants for each vector．Through PCR detection，7，5，6and5PCR positive lines were obtained for each vector；Southern hybridization indicated that thecry1Ah gene was integrated into the tobacco genome； the quantitative RT-PCR resultsdemonstrated that the pMhCTP plants showed the highest cry1Ah transcript level of the fourdifferent transgenic constructs，the cry1Ah transcript level of pMAhb，pMhNK and pMhCTP plantswere higher than that of pMhGM plants by approximately4-，9.5-and12.5-fold；ELISA analysisrevealed that the pMhCTP plants showed the highest protein expression levels，the proteinexpression levels of pMAhb，pMhNK and pMhCTP were higher than that of pMhGM plants byapproximately4-，6-and10-fold．The bioassays revealed that the mortality rates of larvae feedingon pMhGM，pMAhb，pMhNK and pMhCTP plants were63%，82%，93%and100%，respectively，and the resistance rating levels were2.57，1.8，1.33and1，respectively．Our resultsdemonstrated that combining the codon optimization of cry1Ah gene with the targeting of Cry1Ahprotein to the chloroplasts conferred a high level of protein expression.
Seven vectors pMhGM (m1-cry1Ah)，pMAhb (m2-cry1Ah)，pMIeb (mcry1Ie)，pMAhIeb(m2-cry1Ah and mcry1Ie)，pMhNK (NK-m3-cry1Ah)，pMhCTP(CPT-m3-cry1Ah) and pMhER(ER-m3-cry1Ah) were transferred into maize immature embryos by Agrobacterium-mediated．Atthe beginning，the pMhGM were transferred into maize embryos，a total of500maize embryoswere infected and48regenerated maize plants were obtained．A total of23PCR positive eventswere obtained；the molecular detections and bioassay results showed that events1-4and1-5exhibited high resistance to the O．furnacalis；Southern blot analyses suggested that a single copyof the cry1Ah gene was successfully integrated into the maize genome．The results of T1~T6detection of two insect-resistant events indicated that the cry1Ah was expressed stably at highlevels in maize and could be inherited stably over generations，the transgenic plants were highlytoxic to the O．furnacalis and that their resistance could be inherited stably from generation togeneration．Afterwards，the seven vectors mentioned above were transferred into maize embryosby Agrobacterium-mediated．A total of20070maize embryos were infected and7745resistantcallus tissue were obtained，1764regenerated maize plants were obtained．PCR detection showedthat1360PCR positive events were obtained； the molecular detections and bioassayresults showed that the insect-resistant maize plants harboring cry1Ah gene were29events，harboring cry1Ah and cry1Ie genes were14events．The highly insect-resistant maize plants were8and3events，respectively．The results of T1~T2detection of the43insect-resistant eventsindicated that the cry1Ah was expressed stably at high levels in maize and could be inheritedstably over generations，the transgenic plants were highly toxic to the O．furnacalis and that theirresistance could be inherited stably from generation to generation．The obained insect-resistantmaize materials have good application value，and they could be potential candidates for thebreeding of Bt insect-resistant transgenic maize.
In addition，the highly insect-resistant maize events pMAhIeb60and pMAhIeb186were further detected．The Cry1Ah expression of husk，leaf and silk of the two events’ T2plants weredetected by ELISA．The ELISA indicated that the Cry1Ah expressions of husk，leaf and silk inpMAhIeb60were4.5，3.5and2.5μg/g fresh weight (f.w.)，respectively，and those in pMAhIeb186were3.5，3.5and0.8μg/g (f.w) respectively．The high Cry1Ah expressions in leaves caneffectively prevent the early period damage of O．furnacalis，and the high Cry1Ah expression inhusk and silk can effectively prevent the later period damage of O．furnacalis．The two highlyinsect-resistant events are expected to industrialization.
对于一个来源于原核生物的基因，其编码区序列所使用的密码子通常在植物中使用的频率较低，这会使外源基因在植物中仅有少量的积累。而将外源基因根据植物密码子偏好进行优化后可以显著地提高外源基因在植物中的表达。根据植物密码子偏好性，本实验室早期工作对cry1Ah基因进行过两次改造以及对cry1Ie基因进行过改造，本研究将cry1Ah基因进行了第三次密码子优化。三次改造的cry1Ah基因(m1-cry1Ah，m2-cry1Ah，m3-cry1Ah)与原始cry1Ah基因进行比对：GC含量由37%分别提高到48%，55%和63%。优化后的cry1Ie基因(mcry1Ie)的GC含量提高到55%。
在转基因作物研究中，提高转基因表达的另一个主要策略是将外源蛋白进行亚细胞定位表达。亚细胞定位主要依赖于信号肽的作用，其中定位于叶绿体和内质网的研究比较多也比较成功。本研究将m3-cry1Ah基因的N端连接一段来源于玉米RuBP羧化酶的叶绿体定位信号肽序列，命名为CPT-m3-cry1Ah；另外将m3-cry1Ah基因的N端连接内质网定位信号肽，在C端连接内质网滞留信号KDEL，命名为ER-m3-cry1Ah；将不连接任何信号肽的m3-cry1Ah基因命名为NK-m3-cry1Ah。依次构建了三个植物表达载体pMhNK、pMhCTP和pMhER，并且都是以bar基因作为筛选标记基因。
本研究首先利用农杆菌介导法将实验室保存的pMhGM (m1-cry1Ah)，pMAhb (m2-cry1Ah)以及本研究构建的pMhNK (NK-m3-cry1Ah)，pMhCTP (CPT-m3-cry1Ah)共计四个植物表达载体进行烟草转化。通过对模式生物烟草的研究确定出最优的cry1Ah基因密码子使用形式以及研究蛋白叶绿体定位对基因表达的影响。一共侵染320个烟草叶片外植体，共获得84株烟草再生苗，每个载体分别获得21、28、20和15株。通过PCR检测确定了每个载体的阳性植株数分别为7、5、6和5株；通过Southern blot结果表明外源基因已整合到烟草基因组中；实时荧光定量PCR (quantitative RT-PCR，qRT-PCR)检测结果表明pMhCTP烟草植株在四个载体的植株中表现出最高的转录水平，并且pMAhb、pMhNK和pMhCTP烟草植株的转录水平分别是pMhGM植株的4倍、9.5倍和12.5倍；ELISA检测结果表明pMhCTP烟草植株中Cry1Ah蛋白表达量最高，并且pMAhb、pMhNK和pMhCTP烟草植株的Cry1Ah蛋白表达量分别是pMhGM植株的4倍、6倍和10倍；通过生物活性检测结果表明喂食pMhGM、pMAhb、pMhNK和pMhCTP烟草叶片的棉铃虫幼虫致死率分别为63%、82%、93%和100%，这四个载体烟草叶片对棉铃虫的抗性等级分别为2.57、1.8、1.33和1级。通过以上实验结果表明cry1Ah基因的三次密码子优化和叶绿体定位表达逐步提高了cry1Ah基因在转基因烟草中的表达。
本研究随后利用农杆菌介导法将七个植物表达载体转化玉米自交系综31幼胚进行抗虫玉米的研究。这七个植物表达载体包括实验室保存的四个植物表达载体pMhGM (m1-cry1Ah)，pMAhb (m2-cry1Ah)，pMIeb (mcry1Ie)和pMAhIeb (含m2-cry1Ah和mcry1Ie)以及本研究构建的三个植物表达载体pMhNK (NK-m3-cry1Ah)， pMhCTP (CPT-m3-cry1Ah)和pMhER(ER-m3-cry1Ah)。前期将植物表达载体pMhGM经农杆菌介导法转化玉米自交系综31未成熟幼胚，一共转化500个幼胚，共得到48株T0代玉米再生植株。PCR检测表明有23株为PCR阳性植株；然后通过各种分子检测和生物活性检测确定出两个高抗虫玉米事件1-4和1-5；Southern blot检测结果表明这两个高抗虫玉米事件中的外源基因均以单拷贝形式插入玉米基因组中；通过对这两个抗虫事件的T1~T6代植株的Cry1Ah蛋白的表达和遗传稳定性进行研究，结果表明Cry1Ah蛋白在每一代的玉米植株中都能正常稳定的表达，并且植株在田间对玉米螟的抗性也是稳定遗传的。随后我们将以上七个植物表达载体对玉米自交系综31幼胚进行大规模转化，一共转化20070个玉米未成熟幼胚，获得7745块抗性愈伤，共得到T0代玉米转化植株1764株。通过PCR检测确定1360株为PCR阳性植株；然后通过分子检测和生物活性检测确定了转cry1Ah单基因以及转cry1Ah和cry1Ie双基因的抗虫玉米事件数分别是29个和14个，其中表现为高抗虫的玉米事件数分别为8个和3个；通过对这43个抗虫事件的T1~T2代植株的Cry1Ah蛋白的表达和遗传稳定性进行研究，结果表明Cry1Ah蛋白在T1~T2代的玉米植株中都能正常稳定的表达，并且植株在田间对玉米螟的抗性也是稳定遗传的。获得的抗虫转基因玉米材料具有很好的应用价值，可以作为候选材料进行下一步Bt抗虫玉米的育种工作。
此外，我们对抗虫性表现非常突出的两个双基因抗虫玉米事件pMAhIeb60和pMAhIeb186的T2代植株的苞叶、叶片和花丝进行了Cry1Ah蛋白量的分析，结果表明事件pMAhIeb60的植株中Cry1Ah蛋白表达量都比较高，由高到低的表达部位分别是苞叶、叶片和花丝，每鲜克重表达Cry1Ah蛋白量分别是4.5μg/g fresh weight (f.w.)、3.5μg/g (f.w)和2.5μg/g(f.w)；事件pMAhIeb186植株的苞叶、叶片中表达Cry1Ah蛋白量较高，都为3.5μg/g (f.w)，而花丝中表达量为0.8μg/g (f.w)。Cry1Ah蛋白在叶片中的高表达可以有效防治玉米螟初期危害，而在苞叶和花丝中的高表达可以有效防治玉米螟后期危害，这两个高抗虫事件有望走向产业化。
The Bt cry1Ah gene is a novel insecticidal gene，which was cloned from the Bacillusthuringiensis (Bt) isolate BT8．The toxic fragment of Cry1Ah has82%similarity to Cry1Ac．TheCry1Ah protein is highly toxic to lepidopteran insects：this protein is more toxic than Cry1Ac toHelicoverpa armigera，Ostrinia furnacalis and Chilo suppressalis，and it is more toxic thanCry1Ab to O．furnacalis．The Bt cry1Ie gene is another insecticidal gene，and it was cloned fromthe Bacillus thuringiensis (Bt) isolate Btc007．Its encoding protein has toxicity not only to thesensitive O．furnacalis but also to the resistant O．furnacalis．Cry1Ie has no cross-resistance withCry1A protein，thus combining cry1Ah and cry1Ie to the same expression vector can moreeffectively overcome the problems such as the gene’s high homology，single species，pestresistance to Bt protein and so on，at the same time the insect-resistant transgenic plants withhigher toxicity are expected to be obtained.
Most codon of prokaryotic genes′coding sequence are rare codons in plants，and they maylead to low expression of the gene in plants．Codon optimization can significantly improve foreigngenes’ expression in plants．According to the plant codon bias，the cry1Ah gene was modifiedtwice and cry1Ie gene was modified once in the previous work of our lab．In the present study，we modified the cry1Ah gene for the third time．The GC content of the three modified cry1Ahgenes (m1-cry1Ah，m2-cry1Ah，m3-cry1Ah) were48%，55%and63%，respectively，and theGC content of modified cry1Ie gene (mcry1Ie) was55%.
Targeting foreign proteins to specific subcellular locations is another strategy to enhance theexpression of the transgene in the study of genetically modified crops(GMC)． Subcellularlocalization rely mainly on the role of signal peptide，and the study of targeting to chloroplasts orendoplasmic reticulum is more successful．In this study，the m3-cry1Ah gene was linked with achloroplast transit peptide sequence from maize RuBP carboxylase，and this gene was designatedctp-m3-cry1Ah．Additionally，the m3-cry1Ah gene was linked with a endoplasmic reticulum signalpeptide，and this gene was designated ER-m3-cry1Ah，and the m3-cry1Ah gene with no signalpeptide was designated NK-m3-cry1Ah．We construct pMhNK、pMhCTP and pMhER plantexpression vectors，and the bar gene was used as a selectable maker gene.
Four vectors pMhGM (m1-cry1Ah)，pMAhb (m2-cry1Ah)，pMhNK (NK-m3-cry1Ah) andpMhCTP (CPT-m3-cry1Ah) were transferred into tobacco leaves by Agrobacterium-mediatedtransformation．Based on the research of the model organism of tobacco to determine the optimalcodon pattern of the cry1Ah gene and to study the effect of chloroplast-targeted on transgene expression levels．A total of320tobacco leaf explants were infected，84regenerated tobacco plantswere obtained，21，28，20and15plants for each vector．Through PCR detection，7，5，6and5PCR positive lines were obtained for each vector；Southern hybridization indicated that thecry1Ah gene was integrated into the tobacco genome； the quantitative RT-PCR resultsdemonstrated that the pMhCTP plants showed the highest cry1Ah transcript level of the fourdifferent transgenic constructs，the cry1Ah transcript level of pMAhb，pMhNK and pMhCTP plantswere higher than that of pMhGM plants by approximately4-，9.5-and12.5-fold；ELISA analysisrevealed that the pMhCTP plants showed the highest protein expression levels，the proteinexpression levels of pMAhb，pMhNK and pMhCTP were higher than that of pMhGM plants byapproximately4-，6-and10-fold．The bioassays revealed that the mortality rates of larvae feedingon pMhGM，pMAhb，pMhNK and pMhCTP plants were63%，82%，93%and100%，respectively，and the resistance rating levels were2.57，1.8，1.33and1，respectively．Our resultsdemonstrated that combining the codon optimization of cry1Ah gene with the targeting of Cry1Ahprotein to the chloroplasts conferred a high level of protein expression.
Seven vectors pMhGM (m1-cry1Ah)，pMAhb (m2-cry1Ah)，pMIeb (mcry1Ie)，pMAhIeb(m2-cry1Ah and mcry1Ie)，pMhNK (NK-m3-cry1Ah)，pMhCTP(CPT-m3-cry1Ah) and pMhER(ER-m3-cry1Ah) were transferred into maize immature embryos by Agrobacterium-mediated．Atthe beginning，the pMhGM were transferred into maize embryos，a total of500maize embryoswere infected and48regenerated maize plants were obtained．A total of23PCR positive eventswere obtained；the molecular detections and bioassay results showed that events1-4and1-5exhibited high resistance to the O．furnacalis；Southern blot analyses suggested that a single copyof the cry1Ah gene was successfully integrated into the maize genome．The results of T1~T6detection of two insect-resistant events indicated that the cry1Ah was expressed stably at highlevels in maize and could be inherited stably over generations，the transgenic plants were highlytoxic to the O．furnacalis and that their resistance could be inherited stably from generation togeneration．Afterwards，the seven vectors mentioned above were transferred into maize embryosby Agrobacterium-mediated．A total of20070maize embryos were infected and7745resistantcallus tissue were obtained，1764regenerated maize plants were obtained．PCR detection showedthat1360PCR positive events were obtained； the molecular detections and bioassayresults showed that the insect-resistant maize plants harboring cry1Ah gene were29events，harboring cry1Ah and cry1Ie genes were14events．The highly insect-resistant maize plants were8and3events，respectively．The results of T1~T2detection of the43insect-resistant eventsindicated that the cry1Ah was expressed stably at high levels in maize and could be inheritedstably over generations，the transgenic plants were highly toxic to the O．furnacalis and that theirresistance could be inherited stably from generation to generation．The obained insect-resistantmaize materials have good application value，and they could be potential candidates for thebreeding of Bt insect-resistant transgenic maize.
In addition，the highly insect-resistant maize events pMAhIeb60and pMAhIeb186were further detected．The Cry1Ah expression of husk，leaf and silk of the two events’ T2plants weredetected by ELISA．The ELISA indicated that the Cry1Ah expressions of husk，leaf and silk inpMAhIeb60were4.5，3.5and2.5μg/g fresh weight (f.w.)，respectively，and those in pMAhIeb186were3.5，3.5and0.8μg/g (f.w) respectively．The high Cry1Ah expressions in leaves caneffectively prevent the early period damage of O．furnacalis，and the high Cry1Ah expression inhusk and silk can effectively prevent the later period damage of O．furnacalis．The two highlyinsect-resistant events are expected to industrialization.
引文
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