Cloning And Functional Analysis Of Gibberellin Related Gene CcKO In Hickory

Ent-kaurene oxidase（KO）is the key enzyme of gibberellins（GAs）biosynthesis pathway and the target enzyme of the plant GAs inhibitor Paclobutrazol.Blocking of KO gene in the GAs biosynthetic pathway will affect the normal growth of plants.In this research,the somatic embryos of Carya cathayensis were used as the experimental materials to cloned the sequence of KO gene and its promoter by homologous recombination and PCR amplification.35S::CcKO::GFP overexpression vectors and CcKOpro::GUS expression vectors were further constructed.And then analyzed the amino acid sequence homolgy and bioinformatics analysis by BLAST online tool.Furthermore,the overexpression vector and promoter expression vector were transformed into walnut somatic embryos which was mediated by Agrobacterium,and the plant regeneration experiments were carried out to obtain the positive regenerated plants to further analyze the biological functions of CcKO,aims to regulate the growth and development of hickory from the genes level by using biological technology and provide a theoretical basis for molecular assisted breeding of hickory dwarf/half dwarf new species.The main results of this study are as follows:1.Cloning of CcKO gene of hickory:A hickory CcKO open reading frame（ORF）was obtained,which was 1563 bp and encoding 520 amino acids and molecular weight was 59.076 kD.Amino acids homology analysis exhibited that CcKO contained a core functional domain of cytochrome P450 FXXGXRXCXG and transmembrane region near the N-terminus.BLAST analysis indicated that the amino acid sequence of CcKO was 96%homology with JrKO,while 74%,71%,72%and 77% homology with the amino acid sequence of Populus alba,Pyrus pyrifolia,Malus domestica and Castanea mollissima.2.Construction of the genetic transformation system of KO gene and promotor in hickory:（1）By treating mature somatic embryos of walnut with different dehydration time,it was found that with the extension of dehydration time,the germination rate of somatic embryos presented a trend of first increasing and then decreasing,and the germination rate of somatic embryos was the highest, reaching 78.33%after 8 days of dehydration.qPCR results showed that the expression of Jr DHN increased gradually with the extension of dehydration time,and decreased rapidly after rehydration. The results of Agrobacterium-mediated genetic transformation induced by different antibiotic types and concentrations showed that 300 mg/L Carbenicillin could effectively inhibit the growth of agrobacterium and promote somatic embryos proliferation.（2）Genetic transformation of KO gene and promotor in hickory:After GUS staining and PCR verification,CcKOpro::GUS promoter expression vector was successfully transferred into somatic embryos of walnut,and GUS positive rate of somatic embryos of E₁ generation was 30%,E₂ generation was 50%,and E₃ generation was 70%.The positive somatic embryos were cultured into plants,and the positive rate of regenerated plants was 75%Fluorescence detection and PCR verification showed that 35S::CcKO::GFP overexpression vector was successfully transferred into walnut somatic embryos,and the development of somatic embryos underwent spherical embryos,heart-shaped embryos,torpedo embryos and cotyledon embryos under stereoscopic fluorescence microscope,and the GFP positive rate of E₁ generation was 46%.The GFP positive rate of E₂ generation was 65%.The GFP positive rate of E₃generation was 83%.The positive somatic embryos were cultured into plants,and the positive rate of regenerated plants was 62.5%.3.Expression and biological function analysis of CcKO gene in hickory:qPCR results showed that CcKO gene expression is tissue specific,and the expression in stem is significantly higher than other tissues.Phenotype and correlation analysis results showed that the plant height of regenerated plants with CcKO gene overexpression was significantly higher than that of control,indicating that the expression level of this gene is positively correlated with plant height.CcKO gene overexpression could increase the expression of GA20ox and decrease the expression of GA2ox downstream of GAs synthesis pathway in walnut positive regenerated plants.Promoter GUS localization and correlation analysis showed that this gene is mainly located in vascular bundles,and regulates tissue metabolism and cell growth by combining with MYB transcription factor,thereby regulating plant growth and development.The results of this study provided theoretical basis for further analysis of the role of KO gene in the growth and development of walnut plants,and also provided technical reference for the study of other key enzymes synthesized by GAs in walnut plants.