关键词: 山核桃, 核桃, 内根-贝壳杉烯氧化酶, 启动子, 遗传转化, 基因表达

Abstract:

Objective: Ent-kaurene oxidase (KO) is the key enzyme of gibberellins(GAs) biosynthetic pathway and the target enzyme of the plant GAs biosynthesis inhibitor paclobutrazol. Blocking of KO gene in the GAs biosynthetic pathway will affect the normal growth of plants. In this research, we cloned and analyzed the expression of CcKO (Caca0733S0131)gene and its promoter in Carya cathayensis, which is beneficial to further explore the function of CcKO in plant growth and development, especially the biological function related to plant height regulation, so as to help the creation of superior new germplasm of C. cathayensis. Method: The somatic embryos of C. cathayensis were used to clone 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. The amino acid sequence of this gene was obtained by BLAST online tool, and bioinformatics analysis and homology analysis were performed as well. Furthermore, the overexpression vector and promoter expression vector were transformed into Juglans regia 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. Result: A C. cathayensis CcKO open reading frame (ORF) was obtained, which was 1 563 bp and encoding 520 amino acids and molecular weight was 59.076 kDa. 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% homologous with JrKO, while 71%, 79%, 77% and 76% homologous with the amino acid sequences of Pyrus bretschneideri, Malus domestica, Castanea mollissima and Quercus suber. Heterologous genetic transformation of J. regia somatic embryos: The expression vector of CcKOpro∷GUS was successfully transferred into walnut somatic embryos by GUS staining and PCR verification, and CcKO gene was mainly located in vascular bundles; The overexpression vector of 35S∷CcKO∷GFP was successfully transformed into walnut somatic embryos by fluorescence detection and PCR verification; The phenotypic analysis showed that the plant height of the positive regenerated plants was significantly higher than the control. Moreover, the higher the expression abundance of CcKO, the higher the plant height. Real-time quantitative PCR and related analysis showed that the expression of the gene has spatially differences, and the expression level of stem was significantly higher than other tissues, and KO overexpression could increase the expression of GA20ox and decrease the expression of GA2ox in the GAs biosynthetic pathway of the J. regia positive regenerated plants. Conclusion: The coding region of CcKO was 1 563 bp and encoded 520 amino acids. The amino acid sequence encoded by this gene has the highest homologous with J. regia JrKO amino acid sequence, with the homologous of 96%; The promoter expression vector showed that the gene was mainly located in the vascular bundle. Real-time quantitative PCR and related analysis showed that the relative expression of KO mRNA in J. regia positive regenerated plants was significantly increased, and the regenerated plants showed obvious elongation characteristics as well. Moreover, the overexpression of CcKO in regenerated plants could result in the up-regulation of GA20ox expression and the down-regulation of GA2ox expression. The results of this study provide a basis for further analysis of the role of this gene in the growth and development of C. cathayensis and J. regia.

Key words: Carya cathayensis, Juglans regia, ent-kaurene oxidase gene, promoter, genetic transformation, gene expression