Weeds are one of the important factors that harm crop yields. The glyphosate-tolerant EPSP synthase (EPSPS) gene aroA in microorganisms is widely used to cultivate herbicide resistance in crops. Since the world's first large-scale planting of genetically modified herbicide resistant crops in 1996, it has developed rapidly for more than a decade. However, at present, only a very small number of genes that are truly commercially used to cultivate herbicide resistant crops, such as the CP4-EPSP synthase gene derived from Agrobacterium tumefaciens cloned by international giant Monsanto. How to break the monopoly of multinational companies in the herbicide resistant crop industry has become an urgent mission.
The main contents of this project include: screening of glyphosate herbicide resistant microorganisms; establishing a large-scale isolation technology for the glyphosate target enzyme EPSP synthetase gene; completing the prokaryotic expression, purification and enzymology kinetics study of the screened aroA gene, calculating and comparing enzyme kinetics parameters such as Michaellian constant and glyphosate inhibition constant of EPSPS from different sources on the substrates phosphoenolpyruvate (PEP) and 3-phosphoshikimic acid (S3P), laying a foundation for selecting an aroA gene suitable for application; Carry out research on chemical synthesis, structure optimization and in vitro directed molecular evolution of aroA gene. Through innovative mutant gene library construction and in vitro directed molecular evolution methods, a batch of aroA mutant genes with improved enzyme specific activity and enhanced glyphosate tolerance were screened; Combined with the results of directed evolution of genes in vitro, the secondary and tertiary structures of proteins, and using multi-site mutation technology to explore new loci closely related to enzyme activity, substrate affinity and glyphosate resistance, providing new clues for the genetic modification research of aroA; Complete plant genetic transformation and functional research of the aroA gene, and verify the tolerance of the new aroA gene to glyphosate herbicide through the obtained transgenic plants. The disciplines involved in this project include molecular biology, biochemistry, genetic engineering, plant physiology, microbiology and enzyme engineering.
Comprehensive comparison with similar research at home and abroad, this project is original in the isolation, directed evolution and functional research of aroA gene, and the overall research level is internationally advanced. The key inventions and innovations of the project include: 1) The establishment of a simple and efficient technical system suitable for large-scale isolation of aroA genes; 2) For the first time, a new aroA gene encoding EPSPS was screened from extreme microorganisms using the relevant technical system. It has only 35% homology with CP4 EPSPS, and its substrate affinity and Kcat/Km are 13 and 29 times higher than CP4 EPSPS. It is the best EPSPS found so far; 3) For the first time, the type II aroA gene with high substrate affinity was cloned from Ochrobacter hominis, clarifying the key sites of EPSPS involved in substrate binding and glyphosate resistance; 4) For the first time, the type I aroA gene of EPSPS with low sensitivity to glyphosate was isolated from antagonistic plant pathogenic microorganisms, and multiple regions closely related to glyphosate sensitivity and substrate affinity were found. Using module replacement, differences in the glyphosate sensitivity region and substrate affinity region of EPSPS were found; 5) An efficient in vitro directional shuffling technology for aroA gene was established. This method has large library capacity, high mutation frequency, and reliable mutation sites. New aroA genes with Kcat/Km and Ki/Km increased by 6.3 and 1.7 times compared with CP4-EPSPS were obtained by gene shuffling in vitro.
The project has published a total of 7 SCI papers, of which 2 are top international academic journals on environmental microorganisms and 1 is an international core journal on applied microorganisms, which has attracted great attention from domestic and foreign peer experts; the project has obtained 4 authorized patents; The core technology of the project's invention patent reflects multiple advantages in the expression of some important antigen and antibody proteins, creating good economic benefits for the company using it. From 2011 to 2013, the added output value was 10.52 million yuan and the added profit was 4.2 million yuan. The large-scale aroA gene screening system established in this project has been widely used to mine new aroA genes. More than 100 million hm2 of glyphosate resistant crops transformed with CP4EPSP gene are promoted globally every year, which shows the potential commercial value of the new aroA gene obtained in the future. This project is of great significance in promoting the cultivation of new varieties of herbicide-resistant crops.