Screening Of Host Factors That Interact With Wheat Yellow Mosaic Virus CP By Yeast Two-hybrid System And The Resistance Evaluation Of Wheat Cutivars

Wheat yellow Mosaic virus was one member of Bymovirus,transmitted by Polymyxa graminis which perennial lived in the soil.The coat protein was the unparalleled structure protein of virus,was very important pathogenic factor,and played multiple roles in the process of virus infection.It carried identification information with vector and the host,and the main functions were packaging the virus particles,participating in the spread by vector,identification with vector and the host,regulating RNA replication,virus' cell movement and system infection,especially for producing symptoms of host.Due to the hard vector,planting resistant cultivars was the only effective method to control the disease.We used Wheat yellow Mosaic virus coat protein as a bait to screen the host factors in Wheat yeast two hybrid c DNA library in this paper,and more than 200 cultivars were identified in the field.The results provided a scientific basis for the Wheat yellow Mosaic virus pathogenic mechanism research and more materials for field.1.In order to screen the wheat proteins which interact with the coat protein of wheat yellow Mosaic virus,wheat c DNA library and bait vector p GBK-CP were constructed.Total RNA extracted from the wheat leaf and stem.After the separation of m RNA,the wheat c DNA fragments were directional transferred to the purpose vector using the GATE-WAY technology.After homologous recombination reaction,the wheat yeast two hybrid c DNA library was constructed.According to the wheat yellow mosaic virus coat protein open reading frame information in NCBI,build the bait vector p GBK-CP,the titer of yeast two hybrid c DNA library was 5.86 x 106 CFU/ml,the capacity is 2.34 x 107 CFU,restructuring at a rate of95.8%,only one clone was smaller than 1 kb.Successfully build wheat c DNA library which can be used in the yeast two-hybrid system.The bait could express a fusion protein which had a60 k Da molecular weight,it had no autoactivation and toxicity for the yeast.2.Screening wheat protein genes which interact with wheat yellow mosaic virus coat protein by yeast two-hybrid,and using co-transformation to testify it.After the examination,several positive clones were obtained via the yeast two-hybrid screening,four of this clones were phosphoenolpyruvate carboxykinase gene segment,one was phenylalanine ammonia-lyase gene segment,two were ATP-dependent zinc metalloprotease FTSH 2 gene segment,three were isolate Ta ENO-b enolase gene segment.The interaction relationships of wheat yellow mosaic virus coat protein and PEPCK,PAL proteins were confirmed through co-transformation test.3.This study also focus on the identification of WYMV resistance of wheat cultivars with grading evaluation method.In the last three years,field experiment results showed that immune cultivars take the more proportion of 276 wheat cultivars,63.77% in 2013,74.64% in2014 and 66.67% in 2015.Resistance cultivars take the least part,3.62% in 2013,2.54% in2014 and 4.71% in 2015.Moderate resistance culivars were 18.12% in 2013,18.12% in 2014 and 25.72% in 2015.Susceptible culivars were 14.49% in 2013,4.71% in 2014 and 2.90% in2015.There had 100 wheat cultivars displayed immune to virus in three years,e.g.Fumai 936,Huaimai 18,Lianmai 2,et al.The results provide scientific basis for the disease control and disease-resistant breeding works.