CRISPR/Cas9-Mediated Editing Of The Starch Synthesis Gene Pullulan

Rice is one of main cereal crops around the world,of which endosperm occupys 90 percent of mature seeds.Fine endosperm development is essential for high grain yield and quality in rice.Endosperm chalkiness directly affects not only the appearance quality,but also relatives to processing and cooking and eating qualities.Thus,it is a great significance of the study in endosperm development and starch synthesis for high quality rice breeding.Using CRISPR/Cas9 technique debranching enzyme gene regulation rice PUL edit point,to obtain a set of important breeding value pul mutants.First,two guide RNA（gRNA）target site,crispr-PUL-T₁ and crispr-PUL-T2 were designed to edit the exon region of the PUL gene.The two targets were then constructed on the intermediate vector SK-GRNA;Finally,one step was used to connect the two intermediate assays to the linearized final vector pC1300-Cas9.1 Agrobacterium transformation and the mutation validation of transgenic T0 plantsDouble targets pC1300 Cas9::PUL1+2 vector was transformed into Agrobacterium EHA105 by heat shock,and then transformed it into pro-embryogenic callus of rice variety ZH11.The genomic DNA of the leaves of T0 plants was extracted by CTAB method,and then the double targets are used to design the primers of the target sequence which were processed by PCR-RE and Sanger sequencing.The results showed that Cas9 protein and hygromycin were totally determined positive by PCR;The mutation frequency of pul was as high as 90%,of which the InDel of single base and besides are themajor mutation,and the homozygous deletion rate of single base was about 51%.2 Determined of Agronomic TraitsThe results showed that the plant height,panicle length and sword length of Cas9-PUL were the same as the tiller number,grain number per spike,grain weight,grain length and grain width of T₁homozygous mutant.There was no significant difference in leaf width,and the number of tiller and seed setting rate decreased significantly.The grain length and 1000-grain weight increased significantly,and the length of homozygous mutant of Cas9-PUL was significantly increased significantly by more than5%.3 A Study on Cooking and Eating Quality of RiceThe T₀ of transplant lines were used for rice nutrition quality indicators.The total starch content,direct starch content,protein content,gel consistency and gelatinization temperature of T₀-1,T₀-6,T₀-11 and T₀-18 were detected.compared to the control（ZH11）T₀-6 decreased by 17%,reached a significant level,T₀-18 decreased by 20%,and reached a very significant level.The protein content of T₀-1 and T₀-18 increased,reaching a significant level.Gel consistency and gelatinization temperature.4 Quantification of the genes related expression of grain weight and starch synthesis by RT-PCRWhen knockouted mutant Cas-PUL,PUL gene was significantly reduced in RNA expression.The expression of T₀ grain weight related genes increased,and however,the expressions of related genes in the amylopectin synthesis decreased while their expressions in amylose starch synthesis（GBSSI）increased.The RNAs of T₀-1,T₀-6,T₀-11 and T₀-18 were extracted and compared with ZH11.The expression levels of GS2,GS3,GS5,GW7,GW8 and GL7 were detected,and the expression of GS in T0-6 and T0-pul-18 was increased compared with that of wild type.The expression levels of agarose synthesis genes GBSSI,SS,BE,ISA,AGPL1,DPE1were increased compared with those of ZH11,T₀-6and T₀-18,respectively.The starch synthase,starch branching enzyme,and starch debranching enzyme were significantly decreased during amylopectin synthesis.CRISPR/Cas9 technology enables to be successfully applied into editing the pullulanase gene,which not only provides fundamental material basis for rice nutrition quality and yield,but also speed s up the process of quality breeding.