Gene Editing And Functional Analysis Of BpGA3ox And BpGA2ox Gene Family In Betula Platyphylla

Because of the biological characteristics of woody plants such as long growth cycle and complex genome ploidy,it is extremely difficult to improve plant type.Betula platyphylla,as a pioneer tree widely distributed in north China,has important ecological,economic and medicinal value.Because of its efficient and stable zygote embryo genetic transformation,Betula platyphylla can be another model plant for the genetic engineering research of trees besides poplar.The new varieties of Betula platyphylla created by this method can also provide the basis for the optimization of forest tree varieties.Gibberellin 3 oxidase（GA3ox）and Gibberellin 2oxidase（GA2ox）are important plant hormones involved in plant type regulation in oxidase biosynthesis.GAox plays an important role in regulating the homeostasis of gibberellin in plants.In this study,two gene family members of Betula platyphylla were identified and their expression pattern were analyzed.At the same time,CRISPR/Cas9 technology was used to carry out targeted knockout mutants of all members in functional domain and specific domain regions,and the regulatory functions of BpGA3ox2 and BpGA2ox3 genes on plant height of Betula platyphylla were preliminarily discussed.The main results are as follows:1.Bioinformatics analysis showed that there were 12 gibberellin oxidase genes（BpGAoxs）in Betula platyphylla,including 2 GA20ox genes,2 GA3ox genes and 8 GA2ox genes.Multiple sequence alignment,conserved domain analysis and protein structure analysis showed that the function of gibberellin oxidase was conserved.Phylogenetic tree and collinear analysis showed that the oxidase genes in woody plants were evolutionally-conserved and in accordance with the relationship and law of species evolution.The analysis of cis-acting elements in promoter regions showed that 12 genes had elements related to light response,plant hormones,stress and meristem.2.RT-PCR was used to analyze the expressions of BpGA3ox and BpGA2ox gene family members in different tissue parts of roots,stems,leaves and flowers.The results of gibberellin（GA₃）and its inhibitor（Uni）treatment showed that BpGA3oxs in leaves showed a relatively complex response trend,which may be involved in the feedback regulation as well as GA₃ synthesis.However,only a few genes in the BpGA2oxs showed obvious induction tendency,which indicated the existence of functional differentiation among the members of the gene family.3.A total of 22 target sites were designed in the conserved functional domain and specific region for BpGA3ox and BpGA2ox gene family members of Betula platyphylla,respectively,and the genetic transformation of its zygote embryos was carried out using CRISPR/Cas9 technology.Three targets of BpGA3ox2 gene and three targets of BpGA2ox3 gene infected 991 and 724explants,244 and 134 were resistant,and 15 and 4 were edited,respectively.The average conversion efficiency was 14.32%and 13.53%,and the average editing efficiency was 9.61%and 4.08%,respectively.4.bpga3ox2 and bpga2ox3 editing sites were sequenced and we found that editing methods included base mutation（substitution,insertion）and fragment deletion;The plant height and stem length of bpga3ox2 mutants were lower than those of wild type.The plant height and stem length of bpga2ox3 mutants were higher than those of the wild type at the same time,and the number of internodes of all the mutant materials had no significant change.