Preliminary Study On The Function Of SlAFO4A Gene In Processing Tomato

Processing tomatoes as one of the important economic crops,has an important development and utilization value,the virus disease is the impact of processing tomato yield and quality of one of the main pathogens,resulting in reduced production of tomatoes or even productive.The main viruses in Xinjiang include: ToMV,CMV,PVY and STV,in which CMV and PVY have serious harm to tomato plants.RNA silencing is the main defense mechanism of plant autonomy against virus infection,which plays an important role in plant antiviral.RNA silencing complex(RISC)is the main component of RNA silencing,which can mediate the target gene mRNA Specific protein,so as to achieve RNA silencing effect,Argonaute(AGO)protein is composed of RNA silencing complex of the core protein,RNA silencing is necessary.AGO protein is found in plants for the first time and is a highly conserved alkaline protein.Its protein family members have four domains: N-terminal domain,PIWI domain,PAZ domain and MID domain.The PAZ domain mainly recognizes two bases that are prominent at the 3 ’end of the siRNA and are responsible for binding to RNA.The PIWI domain is structurally and functionally similar to RNase H,and RISC has the activity of endonuclease to cleave the target gene.AGO protein family members were divided into two subgroups,AGO subgroup and PIWI subgroup,respectively.The number of AGO protein family members in different species was different,and its main function was also different.At present,there are eight members of the known AGO protein family in tomato,in which SlAGO1 A and SlAGO1 B may affect plant height and flowering time.SlAGO7 is found to be related to leaf and flower organ formation.SlAGO4 A is found to be related to plant resistance.OBJECTIVE: To study the interaction between PVY HC-Pro and CMV 2b by yeast two-hybrid method,subcellular localization,overexpression and interference of transgenic plants.The results were as follows:(1)To further study its function in accordance with.Methods: The phraggae was constructed by comparing the SlAGO4 A gene with the amino acid sequence of the protein domain of the AGO protein family,and the protein domain analysis was carried out by SMART.The bait vector and the target vector were designed by using the bait vector and the target vector sequence in the GAL4 yeast two-hybrid system.The bait vector and the target vector were constructed by using the SlAGO4 A sequence in GenBank and the PVY HC-Pro and CMV2 b sequences in this laboratory.The yeast two-hybrid system The experimental method was carried out for yeast two-hybrid experiments.The GFP fusion vector was constructed and the genetic transformation was carried out by Agrobacterium tumefaciens-mediated method.The primers were designed according to the sequence of SlAGO4 A gene and subcellular localization vector.GFP fluorescence was observed by laser confocal microscopy.The recombinant expression vector and the vector were constructed by SlAGO4 A gene sequence and PIWI protein domain sequence.The genetic transformation of tomato was carried out by Agrobacterium tumefaciens infection method.The relative expression of target gene was analyzed by qRT-PCR.Results: Phylogenetic analysis showed that SlAGO4 A had similarity with Arabidopsis AtAGO4 amino acid and was attributed to AtAGO4 subgroup.SlAGO4 A has an AGO protein family member with a total of N-terminal domain,PAZ domain,MID domain and PIWI domain,and also has DUF1785 and ArgoL2 domains.BD-CMV 2b,BD-PVY HC-Pro and the target expression vector AD-SlAGO4 A were successfully constructed to determine the interaction between SlAGO4 A and PVY HC-Pro and CMV 2b by GAL4 yeast two-hybrid system.The GFP fusion vector 35S: GFP-SlAGO4 A was successfully constructed and transformed into tobacco plants.Five transgenic tobacco lines were obtained.The results of laser confocal microscopy showed that SlAGO4 A was located on the nucleus and the cell membrane.The recombinant expression vector 35S: SlAGO4 A and the interference vector 35S: RiPIWI were successfully constructed and the tomato plants were transformed into 6 transgenic tomato plants and 5 transgenic tomato lines.The expression of SlAGO4 A gene in all transgenic tomato plants Were significantly increased,compared with the normal plant growth rate of about 6-76 × between the fluctuations,proved successful access to overexpressing plants.CONCLUSION: SlAGO4 A gene may be similar to that of AtAGO4 by phylogenetic results.According to the yeast two-hybrid experiment,we determined that SlAGO4 A had no interaction with PVY HC-Pro and CMV2 b.Five GFP fusion transgenic lines were successfully obtained,and SlAGO4 A was located on the nucleus and guard cell membrane by fluorescence observation.Six overexpression and 5 transgenic tomato lines were obtained.To lay the foundation for the study of its function,and to provide a theoretical basis for further study of the gene in tomato.