The prevalence of insect resistance against Bt toxins has led to the idea of enhancing demethylation from cell wall pectin by pectin methylesterase enzyme for overproduction of methanol which is toxic to insects pests. The AtPME and AnPME fragments ligated into pCAMBIA1301 vector were confirmed through restriction digestion with EcoR1 and BamH1. Excision of 3363 bp fragment from 11,850 bp vector confirmed the ligation of both fragments into pCAMBIA1301 vector. Transformation of pectin methylesterase-producing genes, i.e., AtPME and AnPME from Arabidopsis thaliana and Aspergillus niger cloned in plant expression vector pCAMBIA1301 under 35S promoter into cotton variety CEMB-33 harboring two Bt genes Cry1Ac and Cry2A, respectively, was done by using shoot apex-cut Agrobacterium-mediated transformation method. The plantlets were screened on MS medium supplemented with hygromycin on initial basis. Amplification of 412 and 543 bp, respectively, through gene-specific primer has been obtained which confirmed the successful introduction of pCAMBIA AtPME and AnPME genes into cotton variety CEMB 33. Relative expression of AtPME and AnPME genes through real-time PCR determined the expression level of both gene ranges between 3- and 3.5-fold in different transgenic cotton lines along with quantity of methanol ranging from 0.8 to 0.9% of maximum while 0.5% to 0.6% of minimum but no expression was obtained in negative non-transgenic control cotton plant with least quantity of methanol, i.e., 0.1%. Almost 100% mortality was observed in insect bioassay for Helicoverpa armigera on detached leaves bioassay and 63% for Pink Bollworm (Pectinophora gossypiella) on growing transgenic cotton bolls as compared to positive control transgenic cotton with double Bt genes where mortality was found to be 82% for H. armigera and 50% for P. gossypiella while 0% in negative control non-transgenic plants.

中文翻译：

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转基因棉花中增强甲醇生产和双Bt基因用于广谱昆虫抗性的组合方法。

昆虫对Bt毒素的抗药性盛行，导致了通过果胶甲基酯酶增强细胞壁果胶脱甲基作用的想法，以过量生产对害虫有害的甲醇。通过用EcoR1和BamH1的限制性消化来确认连接到pCAMBIA1301载体中的AtPME和AnPME片段。从11,850 bp载体中切下3363 bp片段，证实两个片段均已连接到pCAMBIA1301载体中。利用茎尖将35S启动子下克隆到植物表达载体pCAMBIA1301中的拟南芥和黑曲霉的果胶甲基酯酶生产基因AtPME和AnPME转化为分别带有两个Bt基因Cry1Ac和Cry2A的棉种CEMB-33。根癌农杆菌介导的转化方法。最初，在补充有潮霉素的MS培养基上筛选幼苗。已通过基因特异性引物分别扩增了412和543 bp，这证实将pCAMBIA AtPME和AnPME基因成功导入了棉种CEMB 33中。通过实时PCR确定AtPME和AnPME基因的相对表达确定了表达水平基因在不同转基因棉花品系中的3-倍和3.5倍之间的差异，以及甲醇的量为最大的0.8％至0.9％，最小的为0.5％至0.6％，但在阴性非转基因对照棉花中未获得表达用最少的甲醇，即0.1％。