Cloning And Functional Analysis Of GmITPK3 In Soybean

Inositol 1,3,4-triphosphate 5/6-kinase(ITPK)is a conserved multifunctional enzyme existing in animals,plants and nematodes.Phosphoinositol metabolism plays an important role in extracellular stimulation and signal transduction.There are few reports on the study of inositol phosphate biosynthesis pathway and characterization of related enzymes.Four genes of soybean GmITPK family were analyzed by real-time fluorescence quantitative analysis to detect the relative expression of soybean GmITPK family genes under various tissues and abiotic stresses.GmITPK3 gene was selected for research.In order to analyze the function of soybean GmITPK3 gene,its expression vector was cloned and constructed,and its bioinformatics analysis was carried out;The GmITPK3 protein was induced and purified in prokaryotic system and its enzyme activity was determined;The subcellular localization of GmITPK3 protein was observed in tobacco;Using yeast system and Arabidopsis stable genetic transformation system to verify gene function;Soybean plants with GmITPK3 gene were obtained through stable genetic transformation system of soybean.The main research results are as follows:1.Fluorescent quantitative PCR was used to analyze the expression of soybean GmITPK family genes in various tissues and under different abiotic stress conditions,with no tissue specific expression.It was found that the expression of GmITPK3 gene was significantly up-regulated in leaves under saline alkali stress.2.The GmITPK3 gene was cloned,and the complete sequence and coding protein of soybean GmITPK gene family were identified through bioinformatics analysis.The evolution and classification analysis were carried out through sequence alignment.The phylogenetic tree analysis showed that GmITPK could be divided into three subfamilies.3.The gene was constructed into p ET-28a(+)prokaryotic expression vector,and the GmITPK3 protein was induced and purified.The protein was well expressed after IPTG induction by Western blot detection.The enzyme activity detection showed that it had inositol phosphokinase activity,which was 0.1983U/m L according to the measured enzyme activity.4.The p GDG-GmITPK3 subcellular localization vector constructed in this study was transiently expressed in Bens tobacco.Through laser confocal microscope observation,it was found that GmITPK3 gene was located in cytoplasm.Using the defective Saccharomyces cerevisiae system,the growth of yeast cells under abiotic stress was analyzed.The results showed that overexpression of GmITPK3 gene enhanced the resistance of yeast to ABA and saline alkali stress.5.Seven transgenic Arabidopsis lines were obtained by catkin impregnation method.The germination rate of transgenic Arabidopsis with GmITPK3 gene was detected.The results showed that there was almost no difference in the germination rate in the medium containing saline alkali stress,but the transgenic Arabidopsis with GmITPK3 gene in phenotype grew better than the wild type,while the mutant grew worst.At the same time,phenotypic identification in the soil showed that the transgenic lines grew better than the wild type lines,and the mutant was the most yellow and dry,indicating that the GmITPK3 gene had the function of salt tolerance.6.In order to further study the functional mechanism of transgenic soybeans,this paper conducted a study on the stable inheritance of GmITPK3 gene in soybeans.At present,a T1 generation transgenic soybean plant has been obtained.