M.A. Aitkhozhin Institute of Molecular Biology and Biochemistry, Almaty 050012, Kazakhstan.
Faculty of Biology and Biotechnology, Al-Farabi Kazakh National University, Almaty 050040, Kazakhstan.
Int J Mol Sci. 2022 Jul 20;23(14):8020. doi: 10.3390/ijms23148020.
Tissue culture methods enable virus elimination from vegetatively propagated crop plants but cannot prevent new infections. Here we used a tissue culture transgenic approach for curing field cultivars of through the stimulation of RNA interference (RNAi)-based antiviral defenses. Expression cassettes carrying inverted repeats of potato virus S (PVS, genus ) movement or coat protein sequences were used for the transformation of potato cultivars naturally infected with PVS and/or a related carlavirus potato virus M (PVM), without or with potato virus Y (PVY, genus ). A high proportion of transformants PCR-positive for transgenes were cured from both carlaviruses and PVY. After 3-year field trials, 22 transgenic lines representing seven cultivars remained free of any virus or became infected only with PVY. Vegetative progenies of the transgenic lines of cultivar Zeren (initially coinfected with PVS, PVM, and PVY), sampled after in vitro propagation or field trials, and other field cultivars accumulated transgene-derived 21, 22, and 24 nt small interfering (si)RNAs almost exclusively from the PVS inverted repeats. Additionally, some field progenies accumulated 21-22 nt siRNAs from the entire PVY genome, confirming PVY infection. Taken together, transgenic RNAi is effective for virus elimination from naturally infected potato cultivars and their sequence-specific immunization against new infections.
组织培养方法可用于消除无性繁殖作物中的病毒，但无法预防新的感染。在这里，我们使用组织培养转基因方法，通过刺激基于 RNA 干扰（RNAi）的抗病毒防御来治愈田间品种。携带马铃薯 Y 病毒（PVY，属）运动或外壳蛋白序列的马铃薯病毒 S（PVS，属）反向重复表达盒被用于转化自然感染 PVS 和/或相关的马铃薯卷叶病毒（PVM）的马铃薯品种，而无需或带有马铃薯病毒 Y（PVY）。用于转化的转化体中，高比例的 PCR 阳性转化体对两种卷叶病毒和 PVY 均被治愈。经过 3 年的田间试验，22 个代表 7 个品种的转基因系仍然没有任何病毒，或仅感染 PVY。在体外繁殖或田间试验后，从品种泽伦（最初同时感染 PVS、PVM 和 PVY）的转基因系无性后代中，以及其他田间品种中，几乎仅从 PVS 反向重复序列积累了由转基因衍生的 21、22 和 24 个核苷酸小干扰（si）RNA。此外，一些田间后代还积累了来自整个 PVY 基因组的 21-22 个核苷酸 siRNA，证实了 PVY 的感染。总之，转基因 RNAi 可有效消除自然感染的马铃薯品种中的病毒，并对新的感染具有序列特异性的免疫作用。
