(AtALS) Gene Editing And Analysis Of Transgenic Overexpression Herbicide Resistance In Arabidopsis Thaliana

Acetolactate Synthase(acetolactate-synthase,ALS),also known as Acetyl carboxylate Synthase(acetohydroxy acid synthase,AHAS),is an enzyme that exists only in microorganisms and plants.It has been reported that mutations at related sites of ALS gene can produce herbicide resistant plants.By subsequentially transform the sgRNA and donor template to the plant harbored the expressing of germ cell-specific expression promoter ProDD45 driven Cas9,our purples is to mutate the acetolactate synthetase(ALS)gene and substitute the endogenous AtALS in Arabidopsis thaliana.and provided grope for the analysis of resistance intensity and combination of different mutation sites to sulfonylureas and imidazoline herbicides.At the same time,mulitiple sites mutated AtALS was transgenic overexpressed in Arabidopsis and the effects on herbicide resistance was studied.The main results of our study are as follows:1.In vitro mutation of AtALS gene.Four loci of AtALS gene(P197S,R199 A,W574L and S653F)were mutated by overlap extension PCR method.According to the CRISPR/Cas9 mediated HDR repair,appropriate sgRNAs were designed and combined with mutated At ALS genome fragments to construct a single or multi-site mutated replacement vectors.The vectors contained the corresponding sgRNA driven by U6 promoter,the fragments containing site-specific substitutions(P197S and/or R199 A and/or W574 L and/or S653F)and the homologous arms adjacent to both ends of the fragment.2.Screening herbicide resistant plants of Arabidopsis thaliana.On the basis of ProDD45-Cas9 expression plants,we transformed our site-directed homologous replacement plasmid,screened resistant marker seedlings,and further screened herbicide-resistant seedlings in T2 generation.No herbicide resistant plants were obtained.3.The over-expression of GFP-fusion multiple sites mutated AtALS in plants.The overexpression vector of 4mAtALS-GFP mutated at four loci(P197S,R199 A,W574L and S653F)was constructed and fusion expressed with GFP.The plants were transformed by Agrobacterium-mediated floral dip method,and the positive plants were obtained.4.The green fluorescence signal of transgenic plants was observed by fluorescence microscope.The fusion expression of 4mAtALS-GFP could be observed by fluorescence microscope.It was found that green fluorescence signals were uniformly distributed in the leaves,stems and roots of Arabidopsis thaliana.Moreover,the green fluorescence signal showed phenotypic segregation in T2 generation plant population.By this method,the transgenic plants with high expression of4mAtALS-GFP fusion protein can be easily screened out.5.The expression of 4m AtALS-GFP protein was detected by Western Blot.6.The resistance test of sulfonylurea herbicides showed that 4mAtALS-GFP overexpressed plants were resistant to sulfonylurea herbicides.Compared with the control plants,transgenic Arabidopsis had better growth in the plants containing 5 ppm tribenuron-methyl herbicides.There is also some resistance to imazamox.Among them,in the experiment of spraying herbicide(Trme 25ppm+Imazamox 25 ppm),compared with the control,the transgenic line # 48 of m4AtALS-GFP showed obvious tolerance to tribenuron-methyl herbicide and imidazoline herbicide.In summary,our research explored the application of CRIPSR in ALS gene editing and systematic analysis of herbicides-resistant ALS mutation sites.The results of our mutation site combination provide a useful basis for modifying the structure of ALS gene and improving the herbicide tolerance of transgenic plants.