Cloning And Functional Identification Of Viral Host Factor Genes StTOM1-A、stTOM1-B And StTOM3from Potato

Potato (Solanum tuberosum) is the fourth most important food crop in the world. Potato crop is mainly planted in winter in Guangxi. Since winter patato fills the gap in the market of fresh potato it has a great potential for development. Yet, viral diseases adversely impact the growing of the potato industry by causing heavy loss of the yield. The purpose of this study was to investigate the viral diseases on winter potato in Guangxi and to develop an anti-virus transgenic potato germplasm. Surveys were made in the main potato growing areas in Guangxi for viral disease incidence and for identification of the identities of the causal agents. Meanwhile, candidate genes of host factors required by virus replication were cloned and functionally characterized from potato. The main results are as follows.1. Incidence of viral diseases in winter potato in Guangxi and the identities of the causal viral agentsSurveys of potato viral diseases were conducted during2010to2012. Incidences of2～10%were observed. By using virus-specific RT-PCR method, potato virus Y (PVY) and potato virus S (PVS) were detected from samples collected in2010, and PVY, PVS and potato virus A (PVA) were identified from samples collected in2011. For samples collected in2012, a combination of methods including indirect ELISA, small RNA deep sequencing, and RT-PCR was used to idenitify and verify the viruses that infect winter patota crop. PVY, PVS, PVA, Potato virus M (PVM), Potato virus H (PVH), and Potato leafroll virus (PLRV), and Potato spindle tuber viroid (PSTVd) were uncovered. Isolates of PVS were sequenced and it was revealed that significant divergence exists among the isolates.2. Cloning and functional analysis of candidate host factor genes StTOM1-A, StTOMl-B, and StTOM3from potato.Homologs of AtTOMl and AtTOM3, genes encoding host factors required for the replication of viruses in Arabidopsis, StTOM1-A, StTOM1-B and StTOM3from patato, were cloned and characterized. The cDNA foll length of StTOMl-A is1227bp, encoding283amino acid residues, with a34bp5’UTR and a344bp3’UTR. The foll-length genomic sequence of the gene is6475bp, includes10exons and9introns. The sizes of exons and introns are in the range of44bp～415bp and76bp～1129bp, respectively. Similarly, the cDNA of StTOM1-B has a high degree of sequence identity with StTOMl-A whose cDNA is composed of1287bp with an open reading frame of867bp encoding a289aa protein, and the genomic DNA for the gene is7627bp, including11exons and10introns. The sizes of the exons for StTOM1-B are between44bp to466bp, and the sizes of introns are between75bp to2746bp. The length of the StTOM3cDNA is1254bp, with an ORF of891bp encoding a297aa protein, and the genomic fragment that contains this gene is6245bp in length, composed of11exons and10introns. The sizes of exons and introns are in the range of41bp--403bp and77bp-1241bp, respectively.Phylogenetic analysis showed that StTOM1-A, StTOM1-B and StTOM3shares high homology with host factor genes TOM1and TOM3of Arabidopsis, tobacco and tomato. Hydrophobicity analysis revealed that there are seven transmembrane domains in the proteins encoded by StTOMl-A, StTOM1-B, and StTOM3. There is only one copy for each of these genes in potato and all of them were expressed constitutively, according to the Southern blot and the temporal spatial expression analyses.3. Construction of new antiviral transgenic potato germplasm.Plant binary expression vectors carrying RNAi elements for StTOM1-B, StTOM3, and StTOM1-B, StTOM3were constructed and introduced into potato explants via Agrobacterium tumefaciens-mediated transformation. A total of17transgenic potato plants verified by PCR and Southern analysis were obtained, of which6plants were with StTOM1-B RNAi fragment,4plants with StTOM3RNAi fragment, and7plants with StTOM1-B IStTOM3RNAi fragment. Quantitative RT-PCR showed that the expression of StTOM1-B and StTOM3had been significant inhibited after the transformation of plant binary expression vector. The StTOM1-B mRNA expression accounted for only7.73%of negative control in StTOM1-B RNAi transgenic plants, and the StTOM3was only11.74%in StTOM3RNAi transgenic plants. In transgenic plants with StTOM1-B IStTOM3RNAi fragment, expression of StTOM1-B was 21.71%, and StTOM3was18.76%, respectively. Virus inoculation test showed that the transgenic plants can effectively inhibit the infection of tobacco mosaic virus, but they showed no obvious inhibition for PVY, PVS and PVM. It indicated that StTOM1-B and StTOM3are host factors which can support the replication of TMV in potato, but they are not host factors for PVY, PVS and PVM.