Using CRISPR Technology To Construct Strawberry ABA Signaling Pathway Candidate Gene Mutant Library

Forest strawberry Fragaria vesca（F.vesca）has a small genome（2n=14,240 Mb genome）,a short reproductive cycle,and is easy to survive.It can rely on the propagation of seeds and stolons,and the advantages of easy transformation,This makes it one of the model plants for studying the functional genomics of Rosaceae and the development and maturity of non-respiratory changing fruits.Abscisic acid（ABA）has been shown to play a very important role in the development and maturation of strawberry fruit.In recent years,although breakthroughs have been made in the influence of ABA signaling pathway key factors on strawberry fruit development and maturation in model plant forest strawberry,most of them adopt spraying,injection and soaking and other transient transformation methods.The effects of key factors on the ABA signaling pathway on strawberry fruit development and maturity were explored by inhibiting or enhancing gene expression,without using stable transformation methods to study.Therefore,we hope to introduce CRISPR/cas9 plant expression vectors into strawberry explants with the help of plant transformation and CRISPR/Cas9technology.Through plant transformation technology,a mutant library of strawberry ABA signal-related genes was established to further study the effects of various components on the ABA signaling pathway on strawberry fruit development and maturity.It will provide some references and basis for future research on plant hormone signaling pathway and strawberry fruit development.The main research contents and results are as follows:1.Select key factors in the ABA signaling pathway:Fve PP2C、Fve PYR/PYL/RCAR、Fve Sn RK2、Fve ABI4、Fve ABI5、Fve NCED5、Fve BG3and Fve UGT,a suitable sg RNA was selected at the 5’end of the gene sequence and a protein functional domain,respectively,and the plasmid p CBC-DT1T2 and p HSE401 were used as materials to construct a CRISPR/cas9 vector.The results showed that the recombinant plasmid was successfully constructed,the sg RNA fragment was correct and no base mutation was generated.2.Because stable plant tissue culture often takes a long time,it is necessary to test the CRISPR vector and the target we designed before plant tissue culture.The constructed CRISPR/cas9 vector was transiently injected into tobacco by Agrobacterium-mediated method.Because the GFP fragment was inserted into the CRISPR/cas9 vector,the results showed that all the fluorescence was expressed in tobacco.The experimental results indirectly indicate that the CRISPR/cas9 vector was successfully constructed.Then select the Fve ABI5 and Fve NCED5 genes and continue to transiently inject the constructed CRISPR/cas9 vectors with diploid strawberry fruits during RS1 period（22-24 days after pollination）through Agrobacterium-mediated method.After 7 days,the fluorescence of the fruit was observed under a stereo microscope,and it was found that 80%of the fruits in the RS1 period injected contained fluorescence.Continue to extract DNA from strawberry fruits with GFP fluorescence after injection,and PCR-amplify the fragments containing sg RNA,connect the product to the T vector,transform into E.coli competent and randomly select a certain number of monoclonals for sequencing.Editing was found to occur at the sg RNA.As a result,it was found that editing occurred at the sg RNA.The types of editing included base replacement,base insertion,single base deletion,and large fragment deletion.The above experimental results indicate that the CRISPR/cas9 vector can be injected transiently during the diploid strawberry RS1 period to verify the success of the constructed vector and whether the selected target sg RNA plays an editing role.3.Taking the leaves of forest strawberry’Yellow Wonder’as explants,the genetic transformation system for strawberry CRISPR/cas9 technology application is:leaf pre-cultivation for 7 to 10 days;Agrobacterium GV3101suspension with a bacterial concentration of OD₆₀₀=0.6 was used to infect the leaf disc tissue for 30 minutes;washed three times with sterile water and placed on a co-culture medium containing sterile filter paper for 3 days in the dark;The leaf disc tissue was washed again three times with sterile water,and transferred to a delayed medium containing 250 mg/L carbenzyl and 250mg/L termeltine for 1 to 2 weeks in the dark;afterwards,transfer to 250mg/L of carbenzyl,250 mg/L of temetin and 2 mg/L of hygromycin Continue selective culture on the medium.