Plant Transformation Lab, Centre of Excellence in Molecular Biology (CEMB), University of the Punjab Lahore, Lahore, 53700, Pakistan.
Institute of Molecular Biology and Biotechnology (IMBB), Centre for Research in Molecular Medicine (CRiMM), The University of Lahore, 1 Km Defence Road, Lahore, 54500, Pakistan.
Plant Cell Rep. 2021 Apr;40(4):707-721. doi: 10.1007/s00299-021-02669-6. Epub 2021 Feb 25.
Second generation Bt insecticidal toxin in comibination with Allium sativum leaf agglutinin gene has been successfully expressed in cotton to develop sustainable resistance against major chewing and sucking insects. The first evidence of using the Second-generation Bt gene in combination with Allium sativum plant lectin to develop sustainable resistance against chewing and sucking insects has been successfully addressed in the current study. Excessive use of Bt δ-endotoxins in the field is delimiting its insecticidal potential. Second-generation Bt Vip3Aa could be the possible alternative because it does not share midgut receptor sites with any known cry proteins. Insecticidal potential of plant lectins against whitefly remains to be evaluated. In this study, codon-optimized synthetic Bt Vip3Aa gene under CaMV35S promoter and Allium sativum leaf agglutinin gene under phloem-specific promoter were transformed in a local cotton variety. Initial screening of putative transgenic cotton plants was done through amplification, histochemical staining and immunostrip assay. The mRNA expression of Vip3Aa gene was increased to be ninefold in transgenic cotton line LP than non-transgenic control while ASAL expression was found to be fivefold higher in transgenic line LP as compared to non-transgenic control. The maximum Vip3Aa concentration was observed in transgenic line LP. Two copy numbers in homozygous form at chromosome number 9 and one copy number in hemizygous form at chromosome number 10 was observed in transgenic line LP through fluorescent in situ hybridization. Significant variation was observed in transgenic cotton lines for morphological characteristics, whereas physiological parameters of plants and fiber characteristics (as assessed by scanning electron microscopic) remained comparable in transgenic and non-transgenic cotton lines. Leaf-detach bioassay showed that all the transgenic lines were significantly resistant to Helicoverpa armigera showing mortality rates between 78% and 100%. Similarly, up to 95% mortality of whiteflies was observed in transgenic cotton lines when compared with non-transgenic control lines.
第二代 Bt 杀虫毒素与大蒜叶凝集素基因的组合已成功在棉花中表达，以开发针对主要咀嚼和吮吸昆虫的可持续抗性。本研究首次成功利用第二代 Bt 基因与大蒜植物凝集素组合开发针对咀嚼和吮吸昆虫的可持续抗性。田间过度使用 Bt δ-内毒素限制了其杀虫潜力。第二代 Bt Vip3Aa 可能是一种替代方法，因为它与任何已知的 Cry 蛋白都没有共享中肠受体位点。植物凝集素对粉虱的杀虫潜力仍有待评估。在这项研究中，在 CaMV35S 启动子下优化了合成的 Bt Vip3Aa 基因，在韧皮部特异性启动子下优化了大蒜叶凝集素基因，并将其转化为当地棉花品种。通过扩增、组织化学染色和免疫条带分析对潜在的转基因棉花植物进行了初步筛选。与非转基因对照相比，在转基因棉花系 LP 中，Vip3Aa 基因的 mRNA 表达增加了 9 倍，而在转基因系 LP 中，ASAL 的表达比非转基因对照高 5 倍。在转基因系 LP 中观察到最大的 Vip3Aa 浓度。通过荧光原位杂交，在转基因系 LP 中观察到在染色体 9 上以纯合形式存在的两个拷贝数和在染色体 10 上以半合子形式存在的一个拷贝数。在转基因棉花系中观察到形态特征的显著变化，而植物的生理参数和纤维特征（通过扫描电子显微镜评估）在转基因和非转基因棉花系中保持相似。叶片脱落生物测定表明，所有转基因系对棉铃虫均表现出显著抗性，死亡率在 78%至 100%之间。同样，与非转基因对照系相比，在转基因棉花系中观察到粉虱的死亡率高达 95%。
