Cloning,Expression Analysis And Functional Identification Of Soybean Gmaro A Gene

The soybean occupies a very important position in our grain industry and is closely related to our life.However,the overgrowth of weeds in the field is one of the main reasons for the decrease of soybean crops.In order to effectively control weeds in the field,glyphosate occupies a dominant position in herbicide market because of its efficient herbicidal advantage.But while glyphosate takes advantage of its efficient weeding,it also indiscriminately harms crops.Therefore,the cultivition of glyphosate-resistant crops has become one of the research hotspots for scientists.Glyphosate resistance gene is the basis of the cultivation of the glyphosate resistant crops.In the current controversial situation of food safety of genetically modified crops,it is particularly important to excavate glyphosate resistance genes derived from plants with higher safety.In this study,the expression of GmaroA gene(GmaroA1 and GmaroA2)encoding EPSPS enzyme in Jiyu 72 soybean was analyzed.The GmaroA1 and GmaroA2 genes were cloned into plant expression vector,and the functions of GmaroA1 and GmaroA2 genes were identified by transient transformation system of soybean hair roots.The glyphosate resistance related functions of Arabidopsis thaliana were further identified by overexpression of glyphosate resistance and resupplement of GmaroA1 gene by mutants in Arabidopsis thaliana.Finally,the cloning and prokaryotic expression analysis of T(183)I and P(187)S double amino acid mutation GmaroA1(TIPS)gene encoding enzyme activity center of GmaroA1 gene were carried out.The specific results of the study are as follows: 1.The expression of GmaroA1/GmaroA2 gene in different tissues and glyphosate stress of soybean was analyzed by Real-time fluorescence quantitative PCR.The results showed that:The relative expression of GmaroA1/GmaroA2 gene in roots and leaves of soybean was higher than that of the other tissues.After glyphosate stress treatment for different time,it was found that the expression of GmaroA1 and GmaroA2 genes in root and leaf tissues of soybean seedlings increased adaptively at first,but after 2 or 3 days,the growth homeostasis of soybean seedlings was destroyed and the gene expression was significantly decreased.2.GmaroA1 and GmaroA2 genes were cloned from Jiyu 72 soybean by RT-PCR using RT-PCR technique.Sequence homology analysis showed that the sequence alignment of GmaroA1 gene was consistent with that of Williams82 soybean in GenBank,while the sequence of GmaroA2 gene was found to be 10 bases different from that of Williams82 soybean after multiple cloning.3.Mediated by Agrobacterium tumefaciens K599,GmaroA1 and GmaroA2 genes were transformed into induced soybean hair roots.The results showed that:Under different concentrations of glyphosate stress,the growth of soybean hairy root lines overexpressing GmaroA1/GmaroA2 gene was better than that of the control group,the accumulation of shikimic acid in leaves was significantly lower than that of the control group,and the chlorophyll content was higher than that of the control group.It is preliminarily proved that GmaroA1 and GmaroA2 genes may be involved in shikimic acid pathway and are related to glyphosate tolerance.4.The gene GmaroA1 was transferred into wild type Arabidopsis thaliana and mutant Arabidopsis thaliana by Agrobacterium tumefaciens EH105 mediated Arabidopsis thaliana.Glyphosate resistance phenotypes of transgenic Arabidopsis thaliana were analyzed.It was found that the overexpression and replenishment of transgenic Arabidopsis thaliana grew well,while the growth of Arabidopsis thaliana in the control group was inhibited by glyphosate.It was further proved that GmaroA1 gene could improve the tolerance of Arabidopsis glyphosate.5.The GmaroA1(PS)gene and GmaroA1(TIPS)gene of T(183)I and P(187)S amino acid mutation of GmaroA1 gene encoding enzyme activity center were successfully cloned by OverlapPCR.These genes were constructed into pET-22 b prokaryotic expression vector,and the protein of GmaroA1 and its editing form gene induced by IPTG was proved to be well expressed in E.coli by Western Blot detection.It can lay a foundation for further analysis of resistance function and enzyme activity of GmaroA1 and its edited genes in E.coli prokaryotic system.