Study On Apple GA20ox Gene By Gene Editing And Transcriptome Sequencing

Apple(Malus domesica Borkh.)belongs to the deciduous tree of the Rosaceae family and is one of the fruit tree species with the most extensive cultivation area and high economic and nutritional value in the world.Gibberellin(GA)is an important plant endogenous hormone that plays an important regulatory role in the entire life cycle of higher plant growth and development.GA20-oxidase is a key enzyme in the third stage of gibberellin synthesis pathway.It is encoded by a multi-gene family composed of seven family members.GA20-oxidase has the function of promoting the synthesis of active gibberellin and belongs to positive regulation.Therefore,the expression level of GA20 ox gene in plants will directly affect the synthesis of biologically active GA in plants,and ultimately affect the growth and development of plants.In this study,transcriptome sequencing was used to analyze the gene expression differences between GA20 ox RNAi silenced dwarf plants and wild-type plants in Hanfu,in order to explore the gene expression in apple dwarfing process and the mechanism of GA20 ox affecting plant dwarfing;and try to apply it in apples CRISPR/Cas9 gene editing system,the CRISPR/Cas9 system that edits the GA20 ox gene is introduced into apples through Agrobacterium-mediated leaf disc transformation to knock out the expression of the GA20 ox gene and block the biosynthetic pathway of GA to obtain transgenic dwarfs.Rootstocks were used to analyze the feasibility of the gene editing system in apples and to explore the gene editing effect of GA20 ox.The main research contents are as follows:1.The MdGA20ox1 gene in the synthetic pathway of apple gibberellin was cloned.The CDS sequenced was 1179 bp in length,encoding 392 amino acids.The secondary structure prediction of the protein encoded by the gene shows that,the α-helix accounted for 33.42%,the extended main chain accounted for 19.90%,the β-turn accounted for 8.67%,and the random coil accounted for 38.01%,which was a stable protein.The phylogenetic tree analysis showed that MdGA20ox1 belonged to the first subfamily of the GAox family.The amino acid sequence alignment showed that MdGA20ox1 and MdGA20ox5 were highly homologous,and the homology was as high as 94.94%.Subcellular localization showed that the gene was located in the nucleus.The promoter sequence of 2032 bp upstream of MdGA20ox1 gene was cloned for cis-acting element analysis,and it was found that the promoter region of this gene contains many light-responsive elements and hormone-responsive elements.MdGA20ox1 was tested for transcriptional activity,and the results showed that MdGA20ox1 had no transcriptional activation activity.2.The MdGA20ox2 gene in the synthetic pathway of apple gibberellin was cloned.The CDS sequenced was 1131 bp in full length,encoding 376 amino acids.The secondary structure prediction of the protein encoded by the gene shows that,the α-helix accounted for36.73%,the extended main chain accounted for 19.03%,the β-turn accounted for 8.04%,and the random coil accounted for 36.19%,which was a stable protein.Phylogenetic tree analysis showed that MdGA20ox2 belonged to the first subfamily of the GAox family.Subcellular localization showed that the gene was located in the nucleus.The 1844 bp upstream promoter sequence of MdGA20ox2 gene was cloned for cis-acting element analysis,and it was found that the promoter region of this gene contains many light-responsive elements and hormone-responsive elements,as well as meristem expression-related and seed-regulation-related cis-acting elements.3.The MdGA20ox5 gene in the synthetic pathway of apple gibberellin was cloned.The sequenced CDS is 1179 bp in length,encoding 392 amino acids.The secondary structure prediction of the protein encoded by the gene shows that,the α-helix accounts for 32.49%,the extended main chain accounts for 19.90%,the β-turn accounts for 8.93%,and the random coil accounts for 38.78%,which is a stable protein.Phylogenetic tree analysis showed that MdGA20ox5 belonged to the first subfamily of GAox family.Subcellular localization showed that the gene was located in the nucleus.The promoter sequence of 1947 bp upstream of MdGA20ox5 gene was cloned and analyzed for cis-acting elements.It was found that the promoter region of the gene contained many light-responsive elements and hormone-responsive elements,as well as low temperature-responsive cis-acting elements.4.The gene editing vectors of MdGA20ox2 and MdGA20ox5 were constructed.Through Agrobacterium-mediated apple genetic transformation,the editing vectors were transferred into the leaves of apple ’GL-3’ to obtain transgenic lines,to explore the gene editing situation and analyze the effect of gene editing.5.Transcriptome data of Hanfu apple MdGA20 ox gene RNAi silenced transgenic dwarf plants and Hanfu control at the shoot stage were obtained by transcriptome sequencing,and some genes with significant differences in expression were verified by semi-quantitative methods.The validation results were consistent with the transcriptome data,indicating the reliability of the transcriptome data.Through the analysis of KEGG pathway,it was found that the main pathways related to the formation of plant dwarf traits caused by the synergistic effect of GA20 ox genes are phenylpropanoid biosynthesis pathway,plant hormone signal transduction pathway and diterpenoid biosynthesis pathway.Some differentially expressed genes that may play a role in the formation of plant dwarf traits were screened.