Cloning And Functional Analysis Of Soybean GmBIN2 Gene

In the natural environment,plants often suffer from abiotic stress factors such as drought,freezing injury and high salinity,that can greatly restrict plant growth and development,even cause yield reduction.Among them,drought and salt damage are major factors that affect crop yields.At present,the available water resources are scarce,and soil salinization is very serious.The production safety of soybean and other crops has been seriously threatened.During the evolution of plant life,a complex regulatory system has formed to combat the impact of adverse environments.Protein kinase could be involved in plant stress signal transduction process through phosphorylation of substrates,thereby further regulating the damage caused by external stresses to plants.Therefore,plant protein kinase plays a very important role in the resistance pathway.In this study,we selected the soybean glycogen synthesis kinase GmBIN2,screened under different concentrations of 2,4-D treatment in‘Dongnong 50’to determine the differentially expressed genes in the previous work of our laboratory.Using‘Dongnong 50’as experimental material,the tissue-specific expression of GmBIN2 gene and the expression pattern of GmBIN2 gene under NaCl,PEG and ABA treatments were analyzed by real-time PCR.At the same time,genetic transformation of Arabidopsis and soybean was carried out,and the genetic phenotype,physiological indicators and stress related gene expression levels of transgenic plants were analyzed,in order to explore the potential function of plants overexpressed GmBIN2 in improving the resistance to stresses.The main results were as following:1.The full-length coding sequence of the GmBIN2 gene was 1125bp,encoding a 374 amino acid polypeptide with a 42.4kDa molecular weight and 8.84 isoelectric point.AtBIN2 had the highest homology with GmBIN2 at 88%identity,and they both belonged to Subgroup II.The predicted three-dimensional structure indicated that the GmBIN2 protein contained 18α-helices and 16β-sheets.The GmBIN2 structure contained a conserved Serine/Threonine protein kinase catalytic domain.2.GmBIN2 expression was increased by salt and drought stresses,but was not significantly affected by the ABA treatment.The expression of GmBIN2 is tissue specific,the highest in the seed,followed by the stem and root.3.The prokaryotic expression vector was constructed,and the expression of Western Blotting was analyzed.The results showed when the IPTG concentration was 0.1mM,the expression level of GmBIN2 protein was the highest,and the protein molecular weight was about 42kDa.4.Expression vector pCAMBIA3301-GmBIN2 was transformed into Arabidopsis plants,the obtained transgenic Arabidopsis overexpressed GmBIN2 gene had higher germination rate and root length than wild-type under salt and drought stresses,and GmBIN2 may activate a Ca²⁺-dependent signal system to enhance salt resistance of transgenic Arabidopsis.5.Transgenic Arabidopsis overexpressed GmBIN2 gene could upregulate the expression of RD29A and AtCBL1,which related to stress,indicating that GmBIN2 gene may enhance salt tolerance of transgenic Arabidopsis by activating related genes in salt signaling pathway.6.Soybean hairy root system was constructed,and overexpression of GmBIN2 in transgenic roots showed significantly higher relative root growth rate than the control when subjected to salt and mannitol treatments.Physiological indicators measurement results showed that transgenic soybean roots had lower relative conductivity,higher proline content and SOD activity under salt and drought stresses than the control.7.Soybean genetic transformation was carried out and four T₁ transgenic soybean plants were successfully obtained,and drought resistance was tested using T₃ generation plants.The results showed that after natural drought for 25d,the growth of transgenic soybean plants was significantly better than that of non transgenic plants.