Cloning and functional analysis of CcGA3ox gene from hickory (Carya cathayensis)

Abstract:【Objective】Gibberellin (GAs) is an essential natural plant hormone in plants and plays an indispensable role in seed germination, stem elongation, floral organ induction and development , fruit formation, chlorophyll expression. Therefore, the process of anabolism of GAs is very important for the growth of plants. Gibberellin 3-oxidases (GA3ox) are key enzymes in the biosynthetic pathway of GAs which directly catalyze inactive GAs (GA9/GA20) to bioactive GAs (GA1/GA4) to regulate the 、dynam-ic balance of GAs in plants and regulate plant growth and development process, especially in regulating plant height. Carya cathayensis is an important economic tree species in China and has extremely high value in nutrition, industry and medicine. In recent years, the demand for hickory has gradually in-creased, but most hickory trees are high and grow on hillside ditches, resulting in difficulty of harvest and management. Therefore, the creation of dwarf or semi-dwarf new germplasms is very important fo  the sustainable development of the hickory industry. At present, there are on a few studies on the GA3ox gene in fruit trees. In this study, through the cloning and bioinformatics analysis of the CcGA3ox gene and the construction of genetic vector in hickory, we further explored the role of the CcGA3ox gene in regulating the growth and development of fruit trees, especially in regulating plant height, so as to help dig and utilize more high-quality genes in hickory.【Methods】Firstly, according to the full-length CDS sequence of the GA3ox gene of hickory and the Clon Express Ⅱ One Step Cloning Kit of Vazyme, the full-length cloning primers were designed using the software Primer 5.0 based on In-Fusion cloning technology, and then the target gene was amplified by PCR amplification system. Based on the cloning results, the bioinformatics analysis of the CcGA3ox was carried out, the gene structure characteristics were analyzed by ORF Finder open reading, the conserved regions of genes were analyzed by NCBI, the physicochemical properties of amino acids were analyzed by ExPASy, and the subcellular localiza- tion was predicted by PSORT online analysis software, TMHMM ServerV.2.0 software was used for pro-tein sequence transmembrane region analysis, MEGA7.0 software was used for multi-sequence align-ment analysis of amino acid sequences, and a multi-species phylogenetic tree was established. At the same time, pC1300 plasmids were double-digested by BamHⅠ and SalⅠ, then the PCR products and double-digested products were recovered respectively. The 35S::CcGA3ox::GFP fusion expression vec-tor was used to transform the competent cells of Escherichia coli DH5α by using One step cloning kit of Vazyme .The positive clones were screened by primer PCR at full length of the amplified gene, and the positive clones were selected for sequencing validation. The constructed vector 35S::CcGA3ox::GFP was transfected into Agrobacterium GV3101 competent cells. The correct strains were identified by PCR and enriched after extended culture with rifampicin and kanamycin of LB liquid medium, and then suspended in liquid DKW medium containing acetylsyringone. The well-grown walnut somatic embryos were selected, and were immersed in suspension for 10-15 min, then were cultured in DKW solid medi-um containing acetylsyringone for 3 days, and then were placed in DKW solid medium containing 80mg·L-1 hygromycin and 300 mg·L-1carboxybenzylpenicillin to screen positive somatic embryos. The ef-fect of 35S::CcGA3ox::GFP expression on the fluorescence excitation of somatic embryos was ob-served under white light and blue excitation light (OD=488 nm) by stereofluorescence microscopy, and the somatic embryos and regenerated plants were positively identified by PCR. Phenotypic changes were observed on days 0 and 15, and plant height and internode length were determined. Fluorescence quantitative PCR (qRT-PCR) was used to determine the changes of gene expression of the CcGA3ox in regenerated plants. The chlorophyll content of regenerated plants was determined by ultraviolet spectro-photometer using ethanol to extract chlorophyll.【Results】The open reading frame of the CcGA3ox was 1 116 bp in length and encoded 371 amino acids with a molecular weight of 40.69 kDa. Bioinformatics analysis showed that the CcGA3ox was an acidic hydrophilic protein with conserved domains typical of 2OG-FeⅡ-Oxy. Subcellular localization prediction indicated that it was located in 56.5% of the cyto-plasm, 21.7% in the nucleus and 13% in the mitochondria. Analysis of the transmembrane region of the protein showed that the transmembrane region of the protein was zero. The results of amino acid phylo- genetic tree comparison showed that the gene had the closest genetic relationship with pecan 98.6% ho-mology, and 97.84%, 84.64%, 83.56% homology with walnut, Chinese chestnut and broad leaved oak re-spectively. The results of somatic fluorescence microscopy showed that 35S::CcGA3ox::GFP overex-pression vector was successfully introduced into walnut somatic embryos, and underwent four complete growth and development life cycles: spherical embryo, heart embryo, torpedo embryo and cotyledon em-bryo. Phenotypic observation showed that the height and internode length of the transformed plants were significantly increased compared with those of the control plants. The results of qRT-PCR showed that the height of the positive regenerated plants was significantly different from that of the control plants, and the expression level was higher, indicating that its expression abundance was positively correlated with the plant height. At the same time, the CcGA3ox gene affected the expression of chlorophyll, result-ing in the reduction of the content of chlorophyll in the regenerated plants of 35S::CcGA3ox::GFP. Com- pared with the control plants, the average content of chlorophyll a decreased by 41.5%, the average con- tent of chlorophyll b decreased by 29.3%, and the total chlorophyll decreased by 34.4%.【Conclusion】The amino acid sequence of the CcGA3ox gene had the highest homology with that of the CcGA3ox of pecan (98.6%). The expression of the CcGA3ox CDNA in transformed walnut significantly increased,and the regenerated plants showed obvious elongation characteristics, and the chlorophyll content obvi-ously declined. The GA3ox gene would play a key role in the growth of plant height in walnut.