Studies On Agrobacterium-Mediated Insect-Resistant Transgenic Plants Harboring Bt Cry1Ah And Cry1Ie Genes

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.