Establishment Of Strawberry CRISPR/Cas9 Genome Editing Technology System And Analysis Of FaMAPK12 Gene Function

Strawberry is not only an important horticultural crop,but also has become a model material for the study of fruit development and maturation mechanism because of its short growth cycle,small genome,and stable genetic transformation.The CRISPR/Cas9 genome editing technology is an important technology in the field of life science research that has emerged in recent years.It is accurate,convenient,and highly efficient.Its application in strawberries will greatly promote strawberry genetic improvement and molecular biology research.Previous studies in our laboratory have showed that sucrose can be used as a signal to regulate strawberry fruit development and maturation.Therefore,this study first established the CRISPR/Cas9-mediated genome editing technique in strawberry,and on this basis,also studied the mechanism of sucrose signal transduction regulating strawberry fruit ripening.First,diploid(Fragaria x vesca)and octaploid(Fragaria x ananassa)strawberry fruits were used as material to study the feasibility of using CRISPR/Cas9 to perform genome editing in strawberry fruit transient transformation system.This study designed a dual-target genome editing vector containing two sgRNAs targeting FvMYB10 and FvCHS,respectively.After transient transformation of strawberry fruit and detection by PCR-Restriction Enzyme Assay(PCR-RE)and sequencing,it was found that both target sites were mutated.The above results indicate that genomic editing can be performed in strawberry fruit transient transformation system using CRISPR/Cas9.After the genome editing in fruit transient transformation system was realized,the feasibility of single target genome editing in the stable genetic transformation system of diploid strawberry plants was further studied.In this study,FvPDS was selected as the target gene,and in the TO plant,22 albino plants and 1 chimeric plant were obtained.After sequencing,the genome editing efficiency was 25%in the TO plants,and the proportions of the plants with double allele editing,homozygous editing,and heterozygous editing were 12.5%,8.3%,and 4.2%.The above results indicate that single-targeted genome editing can be performed in a stable genetic transformation system of diploid strawberry plants using CRISPR/Cas9.In order to further study whether multi-target genome editing can be performed in a stable genetic transformation system of diploid strawberry plants,four-target genome editing was performed in this study.After sequencing,the efficiency of genome editing in the TO generation plants was 14.5%.The proportions of single-,double-,and triple-target genome editing plants were 10.9%,1.8%,and 1.8%,respectively,but four-target editing plants were not detected.Compared with single target genome editing,the editing efficiency of multi-target genomes editing was significantly reduced and the proportion of heterozygous plants was increased.The qRT-PCR method was used to analyze the expression levels of Cas9 and sgRNA in the edited and non-edited plants.It was found that Cas9 expression was a key factor affecting the genome editing of strawberry.Based on the successful establishment of genome editing technology in strawberry,octoploid strawberry(Fragaria x ananassa)was used as the material to study the mechanism of sucrose signal transduction.In this study,the role of mitogen-activated protein kinases(MAPKs)in sucrose signal transduction was explored.Analysis of the response of FaMAPKs family members to sucrose signal revealed that FaMAPK12 may play an important role in sucrose signal transduction.Overexpression of FaMAPK12 promoted fruit anthocyanin accumulation in strawberry.On the contrary,silencing of FaMAPK12 inhibited fruit anthocyanin accumulation.Y2H and BiFC analysis showed that FaMAPK12 interacts with FaMYB10,an important transcription factor that controls the accumulation of anthocyanin in strawberry fruit,and FaMAPKK4,an upstream signal component of MAPK.The study of GUS activity assay in strawberry fruit showed that sucrose could significantly promote the expression regulation of FaCHS,a key gene of anthocyanin synthesis,by the signal module of'FaMAPK12-FaMYB 10',and proved that sucrose signal could regulate strawberry anthocyanin accumulation through the signal pathway of 'FaMAPK12-FaMYB10-FaCHS.In summary,this study successfully established CRISPR/Cas9-mediated single gene editing and multi-genomic editing techniques in strawberry,and further revealed the signal transduction mechanism of sucrose signal regulating strawberry fruit anthocyanin accumulation.These results laid the foundation for the systematic analysis of the molecular mechanisms underlying strawberry fruit development and maturation regulation.