Improving Soybean Resistance To Phytophthora Root Rot By Transforming Soybean With NPR1 Gene

Soybean(Glycine Max)is a rich source of nutrients that can be used to extract oil and make soy products,making it an indispensable delicacy on the dinner table.At the same time,as a high-quality feed protein,it plays an irreplaceable role in ensuring the supply of meat,eggs and milk.However,soybean yield is greatly affected by diseases,among which Phytophthora root rot has a great impact on soybean yield during the growth process.Phytophthora root rot may occur in every growth stage of soybean,and it is still a problem troubling the world up to now.Traditional pesticide control has little effect and will pollute the environment.Transgenic breeding provides a new and effective way to control soybean Phytophthora root rot.NPR1(Non-Expression of Pathogenesis Related 1)gene is a mutant gene isolated from Arabidopsis thaliana by locational cloning.It was found that the mutant plant-pathogenesisrelated protein gene was delayed in Arabidopsis thaliana with deletion of NPR1 gene and increased resistance to pathogenic microorganism.Based on this principle,in this experiment,soybean variety Jilin 30(JL30)and soybean strain JL30+ Gm CHR3 transgenic with disease resistance gene CHR3 were used as receptors.Molecular biological analysis and disease resistance identification were used to determine whether the target gene was integrated into the recipient plant.The results are as follows:1.The pollen tube channel method was used to harvest 553 seeds of JL30+ Gm CHR3 +NPR1 T0 generation,19 plants of JL30+ Gm CHR3 +NPR1T1 generation and 11 plants of T2 generation.408 seeds of JL30+NPR1T0 generation were harvested.7 plants of JL30+NPR1T1 generation and 5 plants of T2 generation were harvested.2.Three strains of JL30+ Gm CHR3 +NPR1 of T0 generation were obtained by Agrobacterium-mediated method;Fifty-eight seeds of T0 generation were harvested.Thirty-two plants of T1 generation and 34 plants of T2 generation were obtained from JL30+ Gm CHR3 +NPR1.Five plants of JL30+NPR1T0 generation were obtained,and 76 seeds of T0 generation were harvested.Thirty-five plants of JL30+NPR1T1 generation and 38 plants of T2 generation were harvested.3.Through Southern hybridization and PCR detection on transformed lines JL30+ Gm CHR3-1、JL30+Gm CHR3-5、JL30+Gm CHR3-8、JL30+ Gm CHR3-10、JL30-NPR1 and receptor JL30-CK,the results proved that the target gene NPR1 was successfully integrated into soybean genome in the form of single copy,and could be stable inherited in the offspring transformed plants.4.By fluorescence quantitative PCR detection transformation of plant stem,root,leaf,NPR1 gene expression,data show that NPR1 gene in the stem,root,leaf,seed were expressed,and expressed in root and leaf amount higher than the stems,and seeds,results show that NPR1 gene transcription level has been successfully expressed in soybean plant.5.Through the hypocotyl infection method turn strains infection of phytophthora root rot disease has the soybean plant,and death by soybean wilting condition and standard reference for disease resistance,the results show that transgenic strains JL30 + Gm CHR3 + + Gm CHR3 and disease resistance of NPR1 was obviously higher than that of control group JL30 + NPR1 strain,resistant to high grade,and JL30 + Gm CHR3 level for disease resistance,JL30 + NPR1 level for the fight,not transformation receptor JL30 for disease resistance level.It was confirmed that soybean resistance to Phytophthora root rot could be obtained through the polymerization of resistance genes.