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一种提高甲醇产量和双 Bt 基因的组合方法,用于转基因棉的广谱抗虫性。

A Combinational Approach of Enhanced Methanol Production and Double Bt Genes for Broad Spectrum Insect Resistance in Transgenic Cotton.

机构信息

Centre of Excellence in Molecular Biology, University of the Punjab, 87-West Canal Bank Road, Lahore, 53700, Pakistan.

出版信息

Mol Biotechnol. 2019 Sep;61(9):663-673. doi: 10.1007/s12033-019-00192-4.

Abstract

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.

摘要

棉虫对 Bt 毒素的抗药性日益严重,这促使人们产生了一种想法,即通过果胶甲酯酶提高细胞壁果胶的脱甲基化作用,从而过量生产甲醇,甲醇对棉铃虫等害虫具有毒性。将 AtPME 和 AnPME 片段连接到 pCAMBIA1301 载体上,通过 EcoR1 和 BamH1 进行限制酶切进行了确认。从 11850bp 载体上切除 3363bp 片段,证实了两个片段都连接到了 pCAMBIA1301 载体上。将拟南芥和黑曲霉的果胶甲酯酶产生基因 AtPME 和 AnPME 克隆到植物表达载体 pCAMBIA1301 中,在 35S 启动子的控制下,转化到分别携带两个 Bt 基因 Cry1Ac 和 Cry2A 的棉花品种 CEMB-33 中,采用芽尖切割的农杆菌介导的转化方法进行。最初在 MS 培养基上添加潮霉素筛选植物。通过基因特异性引物分别获得了 412bp 和 543bp 的扩增产物,证实了 pCAMBIA AtPME 和 AnPME 基因已成功导入棉花品种 CEMB33。通过实时 PCR 确定 AtPME 和 AnPME 基因的相对表达水平,不同转基因棉花品系的基因表达水平在 3 到 3.5 倍之间,甲醇的产量在 0.8 到 0.9%的最大值之间,在 0.5%到 0.6%的最小值之间,但在非转基因对照棉花植物中没有表达,甲醇的含量最低,为 0.1%。在离体叶片生物测定中,棉铃虫的死亡率几乎达到 100%,在生长的转基因棉铃上对粉红棉铃虫(Pectinophora gossypiella)的死亡率为 63%,而对含有双 Bt 基因的阳性对照转基因棉花的死亡率分别为 82%和 50%,而非转基因对照植物的死亡率为 0%。

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