Cloning and functional analysis of gibberellin 2-oxidase1 gene of pecan (Carya illinoensis)

Abstract:【Objective】Changing plant type can effectively improve crop yield, quality and resistance to stresses The research in this field mainly focuses on the creation and application of semi- dwarfing and dwarfing mutants. Different from the breeding objectives of crops, the innovative cultivation of germplasm resources of grafted rootstocks and scions is usually carried out separately for the needs of variegated cultivation of fruit crops. The creation of new rootstock germplasm mainly focuses on important traits such as plant stature, stress resistance and adaptability. Gibberellins (GAs) is one of the five class of hormones that control all aspects of plant growth and development. Semi-dwarfing and dwarfing mutants may shed light on the mode of action of gibberellin. The GA2ox is one of the key genes that reduce endogenous bioactive GA content and lead to semi- dwarfing and dwarfing plant. So far, few studies have been reported on the cloning of the genes related to gibberellin synthesis in Carya illinoensis (pecan). To construct the dwarf pecan plant line for the selection of the rootstock, CiGA2ox1 was isolated and cloned from pecan, and the effects on the growth and development were studied by overexpression of the CiGA2ox1, so as to provide theoretical reference for functional analysis of the gene and breeding.【Methods】The full length of CiGA2ox1 gene was isolated from pecan somatic embryos by RT-PCR. The nucleotide sequence and the corresponding amino acid sequence verified by sequencing were analyzed by BLASTn and BLASTp in the database of National Biotechnology Information Center (NCBI). The sequence of the CiGA2ox1 was analyzed by bioinformatics MEGA 7.0 software to construct the phylogenetic tree. The transient expression was observed by subcellular localization in tobacco. The constructed plant fusion expression vector 35S::CiGA2ox1::GFP, empty vector pC1300- GFP and Marker were respectively transferred into Agrobacterium tumefaciens, injected into tobacco epidermis and the tobacco plants were cultured under low light for 2 days. The labeled tobacco leaves were made into a pack and observed with a Zeiss LSM710 confocal laser microscope and photographed. Then, 35S::CiGA2ox1::GFP overexpression vector was transformed into pecan somatic embryos by agrobacterium to obtain CiGA2ox1 overexpression plant lines. The modified pC1300 plasmid was doubly digested by BamHI and SalI, and the doubly digested products were recovered. The target band and plasmid vector were connected by ClonExpress Ultra One Step Cloning Kit. The somatic embryos of the pecan overexpressing CiGA2ox1 gene were placed under Carl Zeiss stereo d13covery V12 (Axio cam MRC system) and stimulated by blue light (488 nm). The transformed somatic embryos with green fluorescence excitation were identified by PCR. qRT-PCR was used to analyze the expression levels of the related genes in the transformed pecan lines. At the same time, the terminal buds of the wild-type and positive regenerated plant lines with the same growth conditions were cut, and cultured for 2 weeks for phenotype observation. Chlorophyll was extracted by acetone ethanol (1∶1) from leaves of the transformed and wild-type lines. IBM SPSS Statistics 25 software was used for one-way anova, and Graphpad7.0 software was used to plot the results of the above physiological indicators.【Results】The length of the CiGA2ox1 gene was 1053 bp and encoded 350 amino acids. It contained a conserved 2OG-FeⅡ- Oxy domain and three Fe2 + binding sites. The relative molecular weight was 39.22 ku, the isoelectric point (pI) was 6.55, and the molecular formula is C1761H2750N464O516S17. The transmembrane region value of CiGA2ox1 protein had no transmembrane region. The sequence phylogenetic analysis showed that the CiGA2ox1 was the most closely related to the JrGA2ox1 in walnut. The subcellular mapping of the tobacco leaves showed that the CiGA2ox1 gene was localized in the nucleus and cell membrane. The result suggested that the CiGA2ox1 might have catalytic function on plasma membrane. After gene trans formation, fluorescence detection and PCR verification showed that the 35S::CiGA2ox1::GFP overexpression vector was successfully transformed into the pecan somatic embryos and the plants were regenerated. The relative expression of the CiGA2ox1 gene significantly increased in transformed plants compared with the WT. Compared with the wild-type plants, the internode of the transformed plants was shortened and dwarfed. The plant height was about 0.64 times as high as that of wild-type, indicating that the CiGA2ox1 gene of pecan had a negative effect on plant height. The transformed plant had reduced the leaf length, darkened the green color and increased the chlorophyll content. It showed that GA was an important signal to regulate chlorophyll biosynthesis. Reducing the concentration of endogenous bioactivity GA by overexpression of the GA2ox1 would increase the content of chlorophyll in plants.【Conclusion】The CiGA2ox1 gene mainly played a negative role in regulating plant height and a positive role in regulating chlorophyll content in pecan.