Identification Of The SKP2A Gene And The Mechanism Of IAA Growth Hormone Regulation In Amorpha Fruticosa

Auxin is a key factor in the growth and development of plants.It regulates the extension and division of plant cells and affects the development of root length and lateral roots.Amorpha fruticosa has a wide range of growth adaptability and strong vitality.Explore the auxin regulation mechanism of Amorpha fruticosa is of great significance for promoting plant growth and development and improving resistance.In this study,the AfSKP2A（S-phase kinase-associated protein 2A）gene of Amorpha fruticosa was cloned by genetic engineering technology,and its nucleotide structure and the regulatory mechanism by the auxin IAA were studied.The results were as follows:1.The full-length nucleotide of AfSKP2A gene is 1514bp,of which the 3’non-coding region is 392bp.The software prediction results show that the ORF region of the AfSKP2A gene encodes 374 amino acids,and its protein molecular weight is 40.78k Da,which is a hydrophobic stable protein.The primary structure of AfSKP2A protein includes the n-terminal Fbox domain（47-87）and the C-terminal LRRs domain（105-329）.According to the phylogenetic tree analysis,Amorpha fruticosa and Lupinus albiflora belong to the same branch and are closest to chickpeas.According to the prediction of online software,the SKP2A protein of Amorpha fruticosa is in the cytoplasm.2.By analyzing the expression characteristics of the AfSKP2A gene space,it was found that the AfSKP2A gene was expressed in stems,roots,skins,flowers,and ears,and the expression level in roots was the highest.The expression levels in roots and leaves were different when treated with indoleacetic acid（IAA）,abscisic acid（ABA）,and salicylic acid（SA）,and the induced expression levels in leaves treated with IAA and salicylic acid（SA）reached 31.1 times and 535.5 times of the control,respectively.By analyzing the temporal expression characteristics of the AfSKP2A gene,it was found that under different abiotic stress treatments such as NaCl,NaHCO₃,15%PEG6000,Fe Cl₃,and 1%H₂O₂,although the expression levels in roots and leaves were different,the differences were not significant.3.By constructing the recombinant expression vector of PGWB5-AfSKP2A-GFP,the fluorescence signal of AfSKP2A-GFP fusion protein was mainly detected in the cytoplasm by Agrobacterium tumefaciens injection method,so it was determined that the subcellular localization of AfSKP2A protein was in the cytoplasm.4.The unknown protein BPM（ran-binding protein M homolog protein）interacting with AfSKP2A protein was screened by yeast two-hybrid technology,and the yeast two-hybrid related vector p DEST22（AD）-BPM was constructed.The interaction between AfBPM protein and AfSKP2A protein was further verified by co-transformation.5.By constructing the recombinant plasmid p YES2-AfSKP2A expressed by yeast,the response and stress resistance of auxin（SA,IAA）were tested in yeast strain IV.It was found that auxin（SA,IAA）can accelerate the growth of yeast and improve the resistance of yeast to the stress of salt（NaCl）and metal ions（Fe Cl₃,Cu SO₄）.It may be that the AfSKP2A gene regulates the expression of stress-related genes such as auxin（SA,IAA）,salt（NaCl）,and metal ions（Fe Cl₃,Cu SO₄）,which makes it sensitive to hormones,salts,and metal ions.6.To study its nucleotide structure,specific primers were designed to amplify its Fbox and LRRs genes,and the recombinant plasmids p CAMBIA1300-LRRs-CLuc and pCAMBIA1300-Fbox-NLuc were constructed by seamless cloning.Through qualitative observation of firefly enzyme complementary technology,it was verified that there was an interaction between Fbox and LRRs protein.Through quantitative analysis,it was found that IAA could promote the interaction between Fbox protein and LRRs protein,and it was confirmed that there was an interaction between Fbox protein and LRRs protein,which was promoted by IAA.7.Tobacco plants with AfSKP2A and segmented genes initiated by Ca MV35S were transformed by the Agrobacterium-mediated method,and tobacco plants with AfSKP2A,Fbox,and LRRs were successfully obtained.After IAA treatment,the differences between the three transformed gene lines were compared.It was found that the root length of tobacco increased,the number of lateral roots increased,and some strains became more branched,indicating that the AfSKP2A gene was regulated by IAA.8.The recombinant plasmid was transformed into Populus davidiana by the Agrobacterium-mediated leaf disc method,and Populus davidiana was successfully obtained by PCR molecular detection.During the growth of Populus davidiana,it was found that the colored leaves of Populus davidiana over-expressed AfSKP2A turned red,and its chlorophyll content decreased after detection.Through the detection of related gene expression,it was found that the expression levels of the oxidative stress gene and enolase gene increased,which once again proved that the AfSKP2A gene was regulated by auxin IAA.