Resolution And Functional Reconstruction Of Glyphosate-resistant Gene AroA_A1501

Since the commercialization of GM crops,herbicide tolerance has been the main character of GM crops.In 2019,the planting area of herbicide tolerance GM crops accounted for 46%of the global planting area of GM crops.The large-scale planting of GM herbicide-tolerant crops has brought enormous economic and social benefits,but the emergence of glyphosate-resistant weeds has also caused widespread concern about their environmental risks.The transgenic crops bred by gene splicing technology can avoid the influence of target traits escaping into the environment,and it is an effective measure to prevent the gene drift and the emergence of glyphosate-tolerant weeds.In order to develop rice with glyphosate tolerance of aroA_A15011501 gene by gene splicing technique,the splicing sites of aroA_A15011501 Gene were screened by structural analysis,and the splicing sites were obtained by functional complementation test,the glyphosate tolerance and enzyme kinetic parameters of the protein reassembled by Intein were compared by prokaryotic system analysis,and the suitable splicing sites were obtained,the results provide technical support for breeding of glyphosate-tolerant transgenic rice by gene resolution technology,and provide technical reserve for controlling gene drift and preventing the emergence of glyphosate-tolerant weeds by gene resolution technology.The specific findings are as follows:1)Bioinformatics analysis predicted the primary,secondary,and tertiary structures of the EPSPS protein encoded by the aroA_A15011501 gene,from which 13 possible splicing sites were predicted.aroA_A1501was divided into N-terminal?An?and C-terminal?Ac?by PCR,and the fusion genes An-In and Ic-Ac were formed by fusion PCR with the N-terminal In and C-terminal Ic of Ssp DnaE intein,respectively.2)The prokaryotic expression vectors of An-In or Ic-Ac,as well as An-In and Ic-Ac were constructed based on pETDuet-1 vector,and the aroA deficient strain ER2799 was introduced for complementary function test.It was found that only ER2799 recombinant strains containing 140N-In and Ic-141C,224N-In and Ic-225C,230N-In and Ic-231C,and complete aroA_A15011501 could grow in restricted culture with 20 mm glyphosate MOPS.RT-PCR results showed that the complete aroA_A15011501 gene could not be amplified in the recombinant strain ER2799 containing 140N-In and Ic-141C,224N-In and Ic-225C,230N-In and Ic-231C.The results indicated that the recombinant ER2799 strain could be resistant to glyphosate by intein-mediated reassembly of aroA_A15011501 at 140/141,224/225 and 230/231 sites.3)The recombinant strain ER2799 containing different plasmids was inoculated into MOPS medium with different concentration of glyphosate,and the tolerance of glyphosate was analyzed.The results showed that the glyphosate tolerance of the functional proteins reassembled from aroA_A15011501 was different,and the glyphosate tolerance of 140/141 was 20 mm,the tolerance of glyphosate to glyphosate was lower than that of intact protein,and the tolerance of glyphosate to glyphosate after 224/225 site splitting was similar to that of intact protein,however,the tolerance of glyphosate to 120 mM glyphosate after 230/231 was twice as high as that to glyphosate after 230/231.4)The expression of exogenous genes in different ER2799 recombinant strains was analyzed by Q-PCR.It was found that the expression of the fusion genes 140N-In,224N-In and 230N-In was higher than that of the complete aroA_A15011501 gene,while the expression of the fusion genes Ic-141C,Ic-225C and Ic-231C were lower than that of aroA_A1501,and the expression of Ic-231C was the lowest,the results showed that the tolerance of ER2799 recombinant strains to glyphosate was not caused by the difference of gene expression.5)The results of ELASA enzyme activity assay showed that the activity of the functional protein reassembled from the protein was almost the same as that of the intact protein after being separated at140/141 and 224/225,the enzyme activity of the reassembled functional protein was higher than that of the intact protein after being separated at 230/231,reaching 0.05 difference.6)The kinetic parameters of EPSPS synthase from different ER2799 recombinant strains were analyzed.It was found that the activity of EPSPS was 17.861±0.267,IC50 was about 10880,Km value was 56.8,Ki value was 29.4 after 230/231 split.The activity of EPSPS was 5.33±0.533,IC50 was about 2700,Km was about 82.4,Ki was about 48.6 after 140/141 split.,and the activity of EPSPS was about 9.27±0.133,IC50 was about 4770,Km was about 125.7,Ki was about 79 after 224/225 split.The enzyme activity of complete aroA_A15011501 was 9.29±0.133,IC50 was about 4600,Km value was122.28,Ki value was 85.The results showed that the activity of EPSPS was the highest after 230/231and the binding ability of EPSPS to substrate and glyphosate was the strongest.7)Plant expression vectors containing 140N-In and Ic-141C,224N-In and Ic-225C,230N-In and Ic-231C were constructed using DT-2300 as basic vector.The transgenic plants with complete aroA_A1501gene,140N-In and Ic-141C,224N-In and Ic-225C,230N-In and Ic-231C were obtained 26,24,21,20strains repectively.8)Glyphosate tolerance of transgenic rice seedlings was analyzed and 2000 ppm glyphosate tolerance was found in transgenic rice seedlings with gene aroA_A1501,230N-In and Ic-231C...