Screening And Identification Of Glyphosate-resistant Soybean Resources And Study On EPSPS Genes In Soybean

Glyphosate is a killing herbicide.Spraying glyphosate can effectively eliminate the harm of weeds in the field and reduce the production cost.Globally,glyphosate-resistant transgenic soybeans are widely planted abroad,and China is an important non-transgenic soybean planting base.How to create and identify glyphosate-resistant non-transgenic soybean germplasm resources and cultivate glyphosate-resistant non-transgenic soybean varieties are the key points to reduce costs and increase efficiency.Mutation has the characteristics of high m utation frequency and wide variation,and has become an important technical means to create excellent mutant germplasm resources.In the early stage of this study,a number of new germplasm resources with excellent characteristics were created by using various mutagenesis techniques,and the physicochemical indexes of resistant materials were analyzed to clarify the physicochemical mechanism of glyphosate resistance.The key gene EPSPS(Glyma.03G027400.1)of glyphosate resistance was cloned to analyze the genetic differences,which laid a foundation for the subsequent screening of glyphosate-resistant resources,so as to accelerate the breeding process of non-transgenic glyphosate-resistant soybeans.The main results are as follows:(1)Screening of resistant materials: In this study,635 materials selected from 200,000 M 2generation materials planted in the field to M5 generation materials were sprayed with glyphosate with a concentration of 1.31 kg a.i hm-2,and 17 resistant germplasm resources with different glyphosate resistance levels were screened out.Mutant 1456-3 was the mutant of Dongnong 60,which showed strong glyphosate resistance and could be used as the basic material for glyphosate-resistant resource breeding.(2)The shikimic acid content and chlorophyll content of resistant materials 1456-3 and Dongnong 60(wild type)after glyphosate treatment were analyzed.With the increase of treatment concentration and time,shikimic acid content and chlorophyll content in leaves of resistant material 1456-3 did not change significantly,while shikimic acid content in leaves of Dongnong 60(wild type)increased rapidly and chlorophyll content decreased significantly,so shikimic acid content and chlorophyll content in materials after glyphosate treatment we re the key to screening resistant materials.Superoxide dismutase(SOD)and peroxidase(POD)of resistant material 1456-3 and Dongnong 60(wild type)were determined.The results showed that SOD and POD in resistant material 1456-3 did not change significantly,indicating that there was no oxidative stress reaction,and the SOD and POD activities of Dongnong 60(wild type)gradually increased,and in glyphosate(1.31 kg a.i.hm-2)The enzyme activity of resistant material 1456-3 maintained normal activity,indicating that SOD and POD activities were the key biochemical index factors for glyphosate resistance.(3)According to the cloning and sequencing of EPSPS(Glyma.03G027400.1)gene,the base sequence was 1581 bp,and a total of 526 amino acids were translated.The results of sequence alignment showed that the base mutation between the resistant material 1456-3 and Dongnong 60 was located in the conserved domain,The mutation of 67 bp leads to the mutation of the 23 rd amino acid from proline to serine,that of 73 bp leads to the mutation of the 25 th amino acid from glutamic acid to lysine,that of 160 bp leads to the mutation of the 54 th amino acid from leucine to phenylalanine,that of 199 bp leads to the mutation of the 67 th amino acid from methionine to valine,and that of 503 bp leads to the mutation of the 168 th amino acid from isoleucine to serine.The mutation of 775 bp led to the mutation of the 259 th amino acid from isoleucine to valine,the mutation of 1079 bp led to the mutation of the 360 th amino acid from tryptophan to serine,and the mutation of 1288 bp led to the mutation of the 430 th amino acid from alanine to threonine.Through the above comparison,it was analyzed that the change of amino acids was the reason why the resistant material 1456-3 was resistant to glyphosate.