Construction And Application Of Virus-induced Gene Silencing Vector For Water Dropwort（Oenanthe Javanica）

Water dropwort（Oenanthe javanica）,a perennial aquatic herb of the genus Oenanthe in Umbelliferous family is one of the most important aquatic vegetables in China and is mainly distributed in the southern region of China.Because of containing a variety of mineral nutrients,water dropwort is very popular among consumers.At present,the genetic transformation system,gene silencing system and gene knockout system of water dropwort have not been established,and there is no corresponding technical support for gene function research.VIGS has the advantages of simple operation,short research period,and no need for genetic transformation,which has been widely used in many monocotyledonous and dicotyledonous plants.In order to carry out the study of water dropwort,we established a gene silencing vector based on cucumber mosaic virus.The main results of the paper are as follows:1.Detection and identification of CMV in water dropwortUsing ’Fql’ as experimental material,RT-PCR and western blot techniques were used to detect cucumber mosaic virus（CMV）in water dropwort plants.The 547 bp fragment was amplified.And the blastn alignment analysis and phylogenetic analysis not only showed that it shares 99%similarity with the CMV CP of many plants such as kidney bean（KT270563.1）,tomato（KC847076.1）and watermelon（KC878465.1）,but also had close genetic relationship with the Egyptian geranium isolate（JQ013954.1）and the Egyptian beet isolate（MT491996.1）,all of which belonged to subgroup IA.Among 22 water dropwort samples taken from Yangzhou,the detection rate of CMV was up to 59.1%,indicating that the virus can infect water dropwort under natural conditions.2.Construction of CMV VIGS in water dropwortBased on the CMV-Fny infectious clone,the 2b deletion mutant vector pCB301-Fny2-2b del was constructed to realize the transformation of the CMV-Fny infectious clone of water dropwort.The modified CMV VIGS vector was inoculated respectively into Nicotiana benthamiana and O.javanica,both RT-PCR and western blot can detect the target bands,indicating that the modified CMV VIGS vector is usable.The modified CMV VIGS vector was used to silence the PDS gene of N.benthamiana and O.javanica,respectively.The target bands could be detected by RT-PCR,western blot and semi-quantitative analysis,and the expression level of PDS gene was significantly lower than the control,showed that the CMV VIGS vector of water dropwort constructed in this study can be used to study the gene function of water dropwort.3.Application of CMV VIGS in water dropwortThe CMV VIGS vector was used to silence the expression of GID1.After infected,the silenced plants exhibited slender leaves and shorter height.Meanwhile,the expression level of GID1 was significantly decreased in the silenced plants as expected.Using the water dropwort CMV VIGS vector,miR319 and miR96 were respectively silenced.The water dropwort plants exhibited dwarf phenotype,and the leaves of which silenced miR319 were significantly smaller than the control.Through fluorescence quantitative detection,the results showed that the expression of miR319 and miR396 was reduced respectively,while the expression of TCP2 and TCP 4 which were target genes of miR319,growth-regulating factor 9-like which was the target gene of miR396 was significantly higher than the control.