Abstract：
The cotton boll weevil (CBW, Anthonomus grandis) stands as one of the most significant threats to cotton crops (Gossypium hirsutum). Despite substantial efforts, the development of a commercially viable transgenic cotton event for effective open-field control of CBW has remained elusive. This study describes a detailed characterization of the insecticidal toxins Cry23Aa and Cry37Aa against CBW. Our findings reveal that CBW larvae fed on artificial diets supplemented exclusively with Cry23Aa decreased larval survival by roughly by 69%, while supplementation with Cry37Aa alone displayed no statistical difference compared to the control. However, the combined provision of both toxins in the artificial diet led to mortality rates approaching 100% among CBW larvae (LC50 equal to 0.26 PPM). Additionally, we engineered transgenic cotton plants by introducing cry23Aa and cry37Aa genes under control of the flower bud-specific pGhFS4 and pGhFS1 promoters, respectively. Seven transgenic cotton events expressing high levels of Cry23Aa and Cry37Aa toxins in flower buds were selected for greenhouse bioassays, and the mortality rate of CBW larvae feeding on their T0 and T1 generations ranged from 75% to 100%. Our in silico analyses unveiled that Cry23Aa displays all the hallmark characteristics of β-pore-forming toxins (β-PFTs) that bind to sugar moieties in glycoproteins. Intriguingly, we also discovered a distinctive zinc-binding site within Cry23Aa, which appears to be involved in protein-protein interactions. Finally, we discuss the major structural features of Cry23Aa that likely play a role in the toxin\'s mechanism of action. In view of the low LC50 for CBW larvae and the significant accumulation of these toxins in the flower buds of both T0 and T1 plants, we anticipate that through successive generations of these transgenic lines, cotton plants engineered to overexpress cry23Aa and cry37Aa hold promise for effectively managing CBW infestations in cotton crops.
摘要：
棉铃象鼻虫(CBW，Anthonomusgrandis）是对棉花作物（陆地棉）的最重大威胁之一。尽管付出了巨大的努力，用于CBW的有效露地控制的商业上可行的转基因棉花事件的开发仍然难以捉摸。这项研究描述了杀虫毒素Cry23Aa和Cry37Aa对CBW的详细表征。我们的发现表明，仅使用补充了Cry37Aa的人工饮食喂养的CBW幼虫与对照组相比没有统计学差异。相反，当只暴露于Cry23Aa时，幼虫存活率下降了约69％。然而,在人工饮食中同时提供Cry23Aa和Cry37Aa导致CBW幼虫的死亡率接近100％（LC50等于0.26PPM）。此外,我们通过在花芽特异性pGhFS4和pGhFS1启动子的调控下引入cry23Aa和cry37Aa基因来改造转基因棉花植物，分别。在确认了45个转基因棉花事件后，我们选择了在花蕾中表现出Cry23Aa和Cry37Aa毒素表达水平升高的前七个事件，70%,用于温室生物测定。在T0和T1代转基因棉花植株上取食的CBW幼虫的死亡率为75％至100％。我们的计算分析揭示了Cry23Aa具有β-孔形成毒素（β-PFT）的所有标志特征，特异性结合糖蛋白中的糖成分。有趣的是，我们的研究还在Cry23Aa中发现了一个独特的锌结合位点，似乎参与了蛋白质-蛋白质的相互作用。最终,我们的讨论集中在Cry23Aa的关键结构属性上，这些属性可能在毒素的作用机制中起作用。由于观察到CBW的LC50较低，并且这些毒素在T0和T1植物的花蕾中都有显着积累，我们预计在这些转基因品系的连续世代中，设计用于过度表达cry23Aa和cry37Aa的棉花植物有望有效管理棉花作物中的CBW侵染。
