Screening Of The Functional Genes Interacting With Southern Tomato Virus By Yeast Two-hybrid Technique

Southern tomato virus(STV)as a new ds RNA virus was reported in 2009,but so far no virus particles have been found.This virus was detected from processing tomato Lige 87-5 in Xinjiang in 2012,and the whole genome sequence of the virus was obtained by PCR amplification.The virus can only spread through the seeds and pollen,can not be transmitted by mechanically,grafting,etc.The STV has been reported in different countries leaded to the different symptoms of tomatoes,and the diseased plants were infected by CMV,PVY,To MV and other viruses in the field at the same time,so it is not yet possible to determine the symptoms of processing tomatoes in Xinjiang after STV infection.In our laboratory,the preliminary study found that CMV,PVY are the most important virus in Xinjiang tomato,which often complex infection with STV,and the harm is very common.Objective: In order to understand the biological characteristics,pathogenesis and symptom p Henotype of STV,In this study,used the host factors as a breakthrough which interact with functional proteins of STV,CMV and PVY,and used the yeast two-hybrid technique to screen the host factors,to lay the foundation for the later study of the pathogenesis of virus and cultivate new varieties of processing tomatoes which resistant to virus.At the same time,this study preliminary explored the symptoms of processing tomatoes in Xinjiang after STV infection.Methods: 1.The STV CP and RdRp gene fragments were amplified by PCR,constructed the yeast two-hybrid bait expression vectors of p Sos-STV CP and p Sos-STV RdRp,and then transferred into cdc25 H yeast strains to verify the self-activation and toxic effects of bait protein.2.The host proteins which interact with the bait protein STV RdRp and CMV 2b were screened from the c DNA library of processing tomato of Xinjiang by Cyto Trap system,and sequenced.3.The host factors n were amplified by PCR,constructed the target gene expression vectors of p GADT7-n,and transferred to AH109 yeast competent cells with the corresponding bait gene expression vectors(p GBKT7-bait),the interaction between bait proteins(STV RdRp,STV CP,CMV 2b,PVY HC-Pro)and target proteins were verified by GAL4 yeast two-hybrid system.4.The biological information of PT2 was analyzed by biology online tool,and the PT2 subcellular localization vector was constructed,the transgenic tobacco obtained by Agrobacterium tumefaciens was used to observe the subcellular localization of PT2.5.Detected the virus of seeds from market-purchased and laboratory-preserved by one step RT-PCR,observed the symptoms of processing tomato infected with STV combined with hybrid breeding experiment.Results: 1.The expression vectors of p Sos-STV CP and p Sos-STV RdRp were successfully constructed,the self-activation results showed that STV CP had self-activating effect and STV RdRp had no self-activating effect and no toxicity.2.8 candidate host factors interacting with STV RdRp and 5 candidate host factors interacting with CMV 2b were screened by Cyto Trap system,included ribosomal protein,receptor-like protein kinase,heat shock cognate protein,D-sorbitol-6-p Hosp Hate dehydrogenase,potassium transporter,RNA binding protein,bromodomain-containing protein,zinc finger protein and unknown protein,etc.3.The corresponding target gene expression vectors(p GADT7-n)and the bait gene expression vectors(p GBKT7-bait)were successfully constructed,the results of rotary validation showed that CMV 2b might interact with Cab-1A in GAL4 system.4.The bioinformatics analysis of PT2 showed that the molecular weight of PT2 protein was about 87.89 k Da,which belonged to the stable protein,had no signal peptide,had 13 transmembrane regions,and the subcellular was localized in vacuole;the 35S:GFP-PT2 subcellular localization vector were successfully constructed,and four transgenic tobacco lines were obtained,fluorescence observation showed that the PT2 may be located in the chloroplast.4.The results of one-step RT-PCR showed that the detection rate of STV and CMV were 90.6% and 96.9% in the seeds from the market-purchased,much higher than the seeds from laboratory-preserved;compared with healthy tomato,there was no significantly changes in leaves and plants,but the shape of fruit of No.4 was significantly different(the fruit of STV was ovoid and healthy fruit was sp Herical),and the other was no obvious difference.Conclusions: 1.STV RdRp and CMV 2b can be applied to Cyto Trap yeast two-hybrid systems,STV CP is not suitable for Cyto Trap systems due to its self-activating effect,PVY HC-Pro also is not suitable for Cyto Trap systems because it or its bait fusion protein is toxic to cdc25H;while STV CP,STV RdRp,CMV 2b,PVY HC-Pro can be applied to GAL4 yeast two-hybrid systems.2.CMV 2b might interacts with Cab-1A in yeast strain AH109,which laid the foundation for the follow-up study;3.Four transgenic tobacco lines of 35S: GFP-PT2 were successfully obtained,fluorescence observation showed that the PT2 may be located in the chloroplast.4.According to the test results of STV,it is more serious,it can not be determined the symptoms of processing tomatoes infected with STV at present,and the differences in fruit shape may be related to breeds,and require the further study.