Research Of Orientation Mutation Of Glutelin Gene GluA3 In Rice(Oryza Sativa L.)by CRISPR/Cas9 System

Protein is the second-largest nutrient substance in rice after starch,with the highest content of glutelin.The content and composition of glutelin will affect the quality index and nutritional value of rice directly.In this study,the CRISPR/cas9 system was utilized to edit Glu A3 gene（LOC＿Os03g31360/Os03g0427300）in rice,committing to improve the quality of rice as well as cultivate functional rice.We successfully got edited-plants through construction of dual-target knockout vector,plasmid transfection,Agrobacterium-mediated transformation,and plant tissue culture techniques.Then T₃generation mutants were detected and analyzed by these experiments including gene detection,agronomic traits observation,and transcription and translation level analysis.Finally,there were obtaining the following research results.（1）In this study,we successfully edited the Glu A3 gene in rice through CRISPR/cas9 system and obtained 27-types homozygous mutation plants.Mutation types involved the deletion,insertion,and substitution of single base,as well as the deletion and insertion of large fragments.Because of the gene mutation,leaded to the early termination of protein translation,which caused the lack of protein domain and the simplification of the spatial structure.Subsequently,the 11-types homozygous mutant plants,named m1-m11（‘Jiahua No.1’mutant:m1-m6,‘Yishendao No.1’mutant:m7-m11）,were used to proceed with the detection and analysis.（2）With the analysis of transcription level on mutant m1-m11,compared with the wild type,the expression level changed as follows:the Glu A3 gene was down-regulated,the Glu B4 and Glu B5 were up-regulated,the Glu C gene of‘Jiahua No.1’mutants were up-regulated,while there were no significant changes on Glu A1,Glu A2,and Glu D.Besides,there was no significant change in the gene expression of Glu C in‘Yishendao No.1’mutants,while it was down-regulated in other glutelin genes.（3）With the investigation of agronomic traits on the mutants m1-m11,it was found that there were significant differences between mutants and the wild types such as plant height,tiller number,panicle length,seed setting rate,and thousand-grain weight.（4）The protein content of wild types and mutants were detected and analyzed by SDS-PAGE and Near-Infrared Spectroscopy,respectively.SDS-PAGE analysis results demonstrated that the glutelin contents of mutants m4-m6 were slightly increased compared with the‘Jiahua No.1’,while the content of glutelin in m9-m11 mutants were a bit lower than‘Yishendao No.1’.Moreover,Near-Infrared Spectroscopy detection results illustrated that the total protein contents decreased in mutants m1,m2,m7,and m9-m11,while the m3-m6,and m8 were increased contrast to the wild types.In this study,the rice with low glutelin and total protein content were obtained,which could provide materials for the cultivation of functional rice suitable for patients with chronic kidney disease.At the same time,the rice with high glutenin and total protein content were also acquired,which could provide materials for the improvement of rice quality.What’s more,this can provide research direction and theoretical basis for CRISPR/cas9 system on editing other glutelin genes in rice.