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首页> 美国卫生研究院文献>Viruses >Engineered Disease Resistance in Cotton Using RNA-Interference to Knock down Cotton leaf curl Kokhran virus-Burewala and Cotton leaf curl Multan betasatellite Expression
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Engineered Disease Resistance in Cotton Using RNA-Interference to Knock down Cotton leaf curl Kokhran virus-Burewala and Cotton leaf curl Multan betasatellite Expression

机译：利用RNA干扰敲低棉花叶片卷曲Kokhran病毒-Burewala和棉花叶片卷曲Multanβ卫星表达的棉花工程抗病性

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Cotton leaf curl virus disease (CLCuD) is caused by a suite of whitefly-transmitted begomovirus species and strains, resulting in extensive losses annually in India and Pakistan. RNA-interference (RNAi) is a proven technology used for knockdown of gene expression in higher organisms and viruses. In this study, a small interfering RNA (siRNA) construct was designed to target the AC1 gene of Cotton leaf curl Kokhran virus-Burewala (CLCuKoV-Bu) and the βC1 gene and satellite conserved region of the Cotton leaf curl Multan betasatellite (CLCuMB). The AC1 gene and CLCuMB coding and non-coding regions function in replication initiation and suppression of the plant host defense pathway, respectively. The construct, Vβ, was transformed into cotton plants using the Agrobacterium-mediated embryo shoot apex cut method. Results from fluorescence in situ hybridization and karyotyping assays indicated that six of the 11 T1 plants harbored a single copy of the Vβ transgene. Transgenic cotton plants and non-transgenic (susceptible) test plants included as the positive control were challenge-inoculated using the viruliferous whitefly vector to transmit the CLCuKoV-Bu/CLCuMB complex. Among the test plants, plant Vβ-6 was asymptomatic, had the lowest amount of detectable virus, and harbored a single copy of the transgene on chromosome six. Absence of characteristic leaf curl symptom development in transgenic Vβ-6 cotton plants, and significantly reduced begomoviral-betasatellite accumulation based on real-time polymerase chain reaction, indicated the successful knockdown of CLCuKoV-Bu and CLCuMB expression, resulting in leaf curl resistant plants.
机译：棉卷叶病毒病（CLCuD）是由一系列由粉虱传播的贝莫病毒种类和株引起的，在印度和巴基斯坦每年造成大量损失。 RNA干扰（RNAi）是一种成熟的技术，可用于抑制高等生物和病毒中的基因表达。在这项研究中，设计了一个小的干扰RNA（siRNA）构建体，以棉卷毛柯克兰病毒-Burewala（CLCuKoV-Bu）的AC1基因和棉卷毛木尔坦β卫星（CLCuMB）的βC1基因和卫星保守区为靶标。 。 AC1基因和CLCuMB编码区和非编码区分别在复制启动和植物宿主防御途径的抑制中起作用。使用农杆菌介导的胚芽顶切法将构建体Vβ转化到棉花植物中。荧光原位杂交和核型分析的结果表明，11株T1植物中有6株具有单拷贝的Vβ转基因。使用有毒的粉虱载体传播CLCuKoV-Bu / CLCuMB复合体，挑战接种了转基因棉花植物和非转基因（易感）试验植物（作为阳性对照）。在测试植物中，植物Vβ-6无症状，可检测病毒量最低，并且在第六号染色体上具有单拷贝的转基因。转基因Vβ-6棉花植株中缺乏特征性的叶片卷曲症状发展，并且基于实时聚合酶链反应显着降低了Bemovoviral-β卫星积累，表明成功敲除CLCuKoV-Bu和CLCuMB表达，从而产生了抗叶片卷曲的植物。

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