Transformation Of Coat Protein Gene Of Thladiantha Dubia Mosaic Virus And Preliminary Analysis Of Its Disease Resistance

Plant viral diseases,as the second major disease that hinders the growth and development of plants,seriously affect the economic growth and development of agriculture in China.In recent years,with the development of genetic engineering technology,the control effect of plant viral diseases is remarkable.Among them,coat protein(CP)mediated plant viral resistance is relatively mature and effective.However,due to the variety of plant viruses,the range and effect of CP gene mediated resistance of different viruses are also different.Iin this study,the CPs of Thladiantha dubia mosaic virus(ThDMV)first discovered and reported in 2017 were selected as the research objects in order to add new members to the research of plant virus disease resistance and lay a foundation for further understanding the CPs function of ThDMV.The main contents are as follows:extract the total RNA from wild red leaf as the material,design the specific primers according to the existing ThDMV virus sequence,use RT-PCR technology to detect the ThDMV CP gene target size about 800bp by agarose gel electrochemistry,purify and recycle the target gene,connect the cloned pMD18-T vertor into E.coli DH5a expression,select the positive clone bacterium liquid by the preliminary examination of the bacterium liquid.Sequencing was completed to determine the correct ThDMV CP gene plasmid as the next experimental material.According to the obtained ThDMV CP sequence information and pCAMBIA1301,pCAMBIA1300,pGreen-35s polyclonal site information,primers were designed to add restriction sites to ThDMV CP.The recombinant plant expression vectors pCAMBIA1301-CP,pCAMB!A1300-CP and pGreen-35sCP were constructed by double restriction ligation of BamH Ⅰ and Hind Ⅲ.The constructed recombinant expression vectors were first transformed into E.coli DH5a for cloning and expression.Three recombinant plant binary expression vectors,pCAMBIA1301-CP,pCAMBIA1300-CP and pGreen-35sCP,were identified by bacterial liquid PCR and recombinant plasmid digestion.The three recombinant vectors were transformed into Agrobacterium tumefaciens LBA404 by electroshock transformation.Under the mediation of Agrobacterium tumefaciens LBA404,the recombinant expression vector was introduced into potato leaves.After preliminary RT-PCR detection,the successful potato was selected as plant material.The infection site of ThDMV CP gene in potato leaves was preliminarily analyzed by fluorescence in situ hybridization(FISH)and whole-tissue hybridization.Random distribution in leaves.Further,the crude enzyme solution was extracted from the leaves of plants infected with recombinant plant expression vector pCAMBIA1301-CP.The activities of plant defensive enzymes(superoxide dismutase SOD,peroxidase POD,catalase CAr)were detected within 5 days.The results showed that SOD,POD and CAT increased in varying degrees within 5 days of observation.ThDMV CP had a certain effect on potato resistance.Furthermore,the plants infected with recombinant plant expression vector pCAMBIA1301-CP were inoculated with PVY virus by friction inoculation.The resistance of potatoes to PVY mediated by ThDMV CP was detected by real-time fluorescence quantitative PCR(QPCR).