Adkar-Purushothama Charith Raj, Kasai Atsushi, Sugawara Kohei, Yamamoto Hideki, Yamazaki Yuto, He Ying-Hong, Takada Nobuyuki, Goto Hideki, Shindo Sahori, Harada Takeo, Sano Teruo
Faculty of Agriculture and Life Science, Hirosaki University, Bunkyo-cho 3, Hirosaki 036-8561, Japan.
Sci Rep. 2015 Dec 14;5:17949. doi: 10.1038/srep17949.
Previous attempts to develop RNAi-mediated viroid-resistant transgenic plants using nearly full-length Potato spindle tuber viroid (PSTVd) hairpin RNA (hpRNA) were successful; however unusual phenotypes resembling viroid infection occurred. Therefore, in the present work, transgenic Nicotiana benthamiana lines expressing both partial and truncated versions of PSTVd hpRNA were developed. Specifically, seven partial or truncated versions of PSTVd sequences were selected according to the hotspots of both PSTVd-sRNAs and functional domains of the PSTVd. A total of 21 transgenic lines Nicotiana benthamiana were developed under the control of either the CaMV-35S or the CoYMV promoters. All of the transgenic lines established here were monitored for the induction of phenotypic changes, for PSTVd-sRNA expression and for the resistance against PSTVd infection. Additionally, this study demonstrates the use of inverted repeat construct sequences as short as 26- to -49 nucleotides for both the efficient expression of the PSTVd-sRNA and the inhibition of PSTVd infection.
此前利用近乎全长的马铃薯纺锤块茎类病毒（PSTVd）发夹RNA（hpRNA）培育RNA干扰介导的抗类病毒转基因植物的尝试取得了成功；然而，出现了类似类病毒感染的异常表型。因此，在本研究中，培育了表达PSTVd hpRNA部分和截短版本的转基因本氏烟草株系。具体而言，根据PSTVd小干扰RNA（sRNA）的热点区域和PSTVd的功能域，选择了7个PSTVd序列的部分或截短版本。在花椰菜花叶病毒35S启动子（CaMV-35S）或木薯叶脉花叶病毒启动子（CoYMV）的控制下，共培育了21个转基因本氏烟草株系。对这里建立的所有转基因株系进行了监测，观察其表型变化的诱导情况、PSTVd-sRNA的表达情况以及对PSTVd感染的抗性。此外，本研究证明了使用短至26至49个核苷酸的反向重复构建序列可有效表达PSTVd-sRNA并抑制PSTVd感染。
