Abstract:

In order to clarify the biological function of gibberellin receptor GID1 in Pyrus betulifolia,and provide a good foundation for future development of P.betulifolia dwarf rootstocks using CRISPR/Cas9 gene editing technology.Pyrus betulifolia was used as the test material,and the PbGID1s genes were obtained by homologous cloning method.Bioinformatics analysis software was used to construct the gene structure and design the target sites;construction of sgRNA expression cassettes with targets into CRISPR/Cas9 expression vectors,through the mediation of Agrobacterium,the CRISPR/Cas9 expression vector was transferred into the cotyledons of P.betulifolia.Results showed that four PbGID1s were successfully cloned from P.betulifolia plants and named as PbGID1b-1,PbGID1b-2,PbGID1c-1 and PbGID1c-2. They all consisted of two exons and one intron found by gene structure analysis.Amino acid sequence comparison showed that all PbGID1s had the HGG and GXSXG conserved domains.Five gRNAs that could potentially edit all 4 PbGID1s simultaneously were successfully constructed into a single CRISPR/Cas9 vector,pYLCRISPR/Cas9P_35S-N.The results of the genetic transformation test of P.betulifolia showed that a total of 595 cotyledons of P.betulifolia were infiltrated,176 resistant buds and 33 positive plantlets were obtained,and the transformation efficiency reached 5.55%.A CRISPR/Cas9 vector was successfully constructed that could simultaneously target the PbGID1s family genes of P.betulifolia.Through the mediation of Agrobacterium,the vector was successfully transformed into P.betulifolia cotyledons,and positive plants were obtained.

Key words: Pyrus betulifolia, GA receptor, GID1, CRISPR/Cas9, Genetic transformation