Construction Of Piezo Knockout Vector And Anti-Tomv Virus Vector And Its Transformation Of Agrobacterium

Piezo gene exists in plants,and the gene is highly homologous to the animal.It is speculated that the function of the encoded protein should be related to stress,but there is no report on the function of the gene on plants.Therefore,the total RNA of tomato young fruit was extracted by TRIzol method,and the Piezo gene fragment of tomato was amplified by PCR.Using the online tool to find the target,the complete sgRNA expression cassette fragment was constructed by using the pYLsgRNA-AtU3b-LacZ plasmid as a template by Overlapping PCR.The sgRNA expression cassette was cloned into the PYL CRISPR/CAS9Pubi-N expression vector by the "Golden Gate" cloning method to construct the Piezo knockout vector,and the Agrobacterium was successfully transformed to explore the function of the Piezo gene on plants.Tomato mosaic virus(ToMV)is a worldwide distributed plant virus whose host is mainly tomato.After the virus infects tomatoes,the cultivation area is greatly reduced,causing major economic losses in agriculture.Recent studies have found that the CRISPR/Cas13 a system is a class 2 VI-A ribonuclease capable of targeting and cleavage of genomic single-stranded RNA(ssRNA)molecules,which can be exploited for research against plant RNA viruses.In this study,Cas13 a with high GC content was synthesized based on tomato codon preference,and the Pubi promoter and NOS terminator were amplified by PYLCRISPR/CAS9Pubi-N plasmid and the three were ligated to construct the Cas13 a expression system fragment.We designed and synthesized four different region-specific crRNA fragments targeting ToMV virus,and amplified the U3 promoter and its terminator with the pYLsgRNA-AtU3b-LacZ plasmid as a template to construct a crRNA expression fragment.The Cas13 a expression system fragment and the crRNA expression fragment were fused with the PYLCRISPR/CAS9Pubi-N backbone fragment into a plasmid,and the plasmid was successfully transformed into Agrobacterium,which was the basis for Cas13a-mediated tomato antiviral research.In summary,this study successfully constructed the Piezo knockout vector using the CRISPR/Cas9 system;The tomato anti-Tomv virus vector was successfully constructed using the CRISPR/Cas13 a system.These results provide valuable assistance in studying the functionof the Piezo gene in plants and reducing tomato ToMV virus.