Construction Of Expression Vectors Of Knocking Out Grape Gene By CRISPR/Cas9 System And Genetic Transformation

CRISPR/ Cas9 system is a new artificial gene editing tool which is widely researched and applied.It has the advantages of simpler operation,lower cost and short period.So far,site-directed editing of genes in living things such as wheat,rice,arabidopsis,Nicotiana benthamiana,apple,banana and mouse have been successfully implemented.The CRISPR system has also been successfully applied to grapes in recent two years,but it is not mature.In this experiment,VvOEE2 gene and VvPDS1 gene of Vitis vinifera cv.Thompson Seedless were taken as the target sites which were expected to be knocked out by CRISPR/Cas9.The main results are as follows:1.The recombined vectors were successfully constructed by dicotyledonous knock-out vector pP1 C.4 and pYLCRISPR/Cas9 which named as: pP1 C.4-OEE2-2,pP1 C.4-PDS1-1,pP1 C.4-PDS1-2 and pYL-OEE2-15.These four recombined carriers were transformed to somatic embryos of V.vinifera cv.Thompson Seedless respectively with agrobacterium.After two years of resistance screening and culture,80,0,63 and 13 resistant plants were obtained.The target mutation was not detected by the target site sequencing analysis of the resistant plants.2.The recombined carriers pJim-OEE2-1,pJim-OEE2-2,pJim-PDS1-1,pJimPDS1-2,pCambia-OEE2-1,pCambia-OEE2-2,pCambia-PDS1-1,pCambia-PDS1-2 were successfully constructed by vector pJim19 and pCambia1300 then transformed to V.vinifera cv.Thompson Seedless protoplasts.The green fluorescence-emitting protopla sts were observed under the fluorescence microscope which proved that the carriers were successfully transformed.No mutation of target sites was found after sequencing.It is speculated that the few intact protoplasts resulted in the reduce of transformati on efficiency during the preparation of protoplasts.The recombined carriers pCambiaOEE2-1,pCambia-OEE2-2,pCambia-PDS1-1,pCambia-PDS1-2 were transient transfor med to V.vinifera cv.Thompson Seedless leaves.A single base mutation in the P.OE E2-1 target site was detected successfully.