Akhtar Sohail, Tahir Muhammad Nouman, Amin Imran, Mansoor Shahid
Molecular Virology and Gene Silencing Laboratory, National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Postal code 38000 Pakistan.
Present Address: Sub-Campus Burewala, University of Agriculture, Faisalabad, Postal code 61010 Pakistan.
3 Biotech. 2021 Jun;11(6):256. doi: 10.1007/s13205-021-02816-6. Epub 2021 May 8.
Cotton leaf curl disease (CLCuD) is one of the major limiting factors affecting cotton production in Pakistan for the last three decades. The disease is caused by begomoviruses of the family . RNA interference (RNAi) is a promising tool that has been proved effective against several pathogens. Using RNAi, different genomic regions of geminiviruses have been targeted to attain sustainable resistance. However, the silencing of the transgene upon virus infection is a limiting factor. Here, we have developed for the first time an amplicon-based RNAi construct to target βC1 gene of betasatellite associated with cotton leaf curl begomoviruses. In addition to producing short interfering (si) RNAs, Rep-based activation or looping out of the construct induced upon virus infection produces multiple copies of transgene that results in accumulation of defective molecules of betasatellite. Subsequent transcription gives rise to increased number of siRNAs that gives enhanced resistance. Transgenic plants having RCβ (RNAi construct for betasatellite) were challenged against (CLCuKV) and Cotton leaf curl Multan betasatellite (CLCuMB). Reduced titer of the virus and betasatellite were detected through Southern blot hybridization. Significance of the study has been discussed.
The online version contains supplementary material available at 10.1007/s13205-021-02816-6.
棉叶卷曲病（CLCuD）是过去三十年来影响巴基斯坦棉花生产的主要限制因素之一。该病害由双生病毒科的病毒引起。RNA干扰（RNAi）是一种很有前景的工具，已被证明对多种病原体有效。利用RNAi技术，已针对双生病毒的不同基因组区域来获得可持续抗性。然而，病毒感染后转基因的沉默是一个限制因素。在此，我们首次开发了一种基于扩增子的RNAi构建体，以靶向与棉花叶卷曲双生病毒相关的β卫星的βC1基因。除了产生短干扰（si）RNA外，病毒感染后基于复制酶（Rep）的构建体激活或环出会产生转基因的多个拷贝，从而导致β卫星缺陷分子的积累。随后的转录会产生更多的siRNA，从而增强抗性。用含有RCβ（β卫星RNAi构建体）的转基因植物对抗棉花叶卷曲卡纳尔病毒（CLCuKV）和棉花叶卷曲木尔坦β卫星（CLCuMB）。通过Southern杂交检测到病毒和β卫星的滴度降低。已讨论了该研究的意义。
在线版本包含可在10.1007/s13205-021-02816-6获取的补充材料。
