Functional Study Of The Gibberellin Oxidase Gene SdGA20ox1 In Nitraria Tangutoru

Sophora davidii（Franch.）Skeels is a typical deciduous shrub or small tree in the karst mountains of southwest China.The stems and leaves of S.davidii are tender and tasty,rich in protein,vitamins,mineral elements and amino acids,and are a high-quality fodder.The normal feeding of shorter livestock such as goats is seriously affected.By studying the functions of the genes involved in regulating the height of S.davidii,gene editing technology can be used to transform exogenous plant height genes into S.davidii for strain improvement to accelerate the selection and breeding of new semi-dwarf or dwarf S.davidii varieties for the development of local ecological livestock industry.gibberellin 2-oxidases（GA20oxs）The GA20ox gene has been investigated in several plants,however,no studies on the function of GA20ox gene in S.davidii have been reported.In this study,a GA20ox gene and its upstream promoter sequence were cloned from a high mutant of S.davidii and its function was systematically investigated by bioinformatics analysis,transgenic assay,yeast two-hybrid and bimolecular fluorescence complementation,etc.The main findings are as follows:1.SdGA20ox1 gene was cloned from S.davidii using subcloning method,its open reading frame was 1143 bp,encoding 380 amino acids,the gene sequence was submitted to NCBI,and the registration number was obtained as ON229923,the subcellular localization showed that SdGA20ox1 was localized in the nucleus and cytoplasm.Analysis of the conserved structural domains of the protein sequence showed that SdGA20ox1 belonged to the GA20ox family,and the results of SdGA20ox1 expression characterization showed that SdGA20ox1 expression was highest in stems and lowest in roots;SdGA20ox1 expression was induced by exogenous GA3,uniconazole,IAA,BR and ETH.The 1572 bp sequence upstream of ATG of SdGA20ox1 gene was obtained based on chromosome stepping technique,and Plant CARE online prediction analysis revealed that the promoter sequence not only contained basic cis-acting elements such as TATA-box and CAAT-box,but also two methyl jasmonate response elements,three light response elements,and one cis-acting element related to meristem expression.cis-acting element related to meristem expression.The SdGA20ox1_pro::GUS vector was successfully constructed to transform Arabidopsis.The GUS staining results of the transgenic plants showed that the GUS activity was higher in the leaf tip,stem tip meristem tissue and leaf margin projection of the seedlings of the transgenic plants.3,Overexpression of SdGA20ox1 in Arabidopsis resulted in higher seed germination rate and germination rate;overexpression of SdGA20ox1 also significantly promoted hypocotyl,root elongation in Arabidopsis seedlings.overexpression of SdGA20ox1 in Arabidopsis,transgenic plants had earlier flowering,lighter leaf color and lower chlorophyll content;the phenotype of mature plants remained significantly higher than wild type,in addition,transgenic In addition,the active GA1 and GA3 contents of the transgenic plants were significantly higher.4.By constructing a high-quality homogenized full-length c DNA library of S.davidii,the primary library volume was about 5.84×107 cfu/ml,the average insert length was 1000 bp,and the recombination rate was 100%.In vitro yeast two-hybrid screening yielded two proteins,rbc S and EIN4,that could interact with SdGA20ox1.The bimolecular fluorescence complementation verified that they could also interact in plants,and it was hypothesized that SdGA20ox1 might synergistically regulate the gibberellin metabolism and ethylene metabolism pathways with the candidate proteins to regulate the growth and development of S.davidii,although these inferences still need further experimental verification.The results of this study suggest that SdGA20ox1 is involved in the regulation of the plant shape of S.davidii,which provides an important theoretical basis for the breeding of new low and compact varieties of S.davidii.