| Rice,as the most important food crop in the world,was the main food source for more than 65% of our population.Early research on rice focused on increasing yield,however,it has gradually moved closer to improving quality in recent years.Starch was the main component of rice.Its amylose content was an important reference index for evaluating the cooking and eating quality of rice.Generally speaking,rice with low amylose content showed better elasticity and soft glutinous properties.This work aimed to use CRISPR/Cas9 gene editing technology to reduce the amylose content of indica rice R287 and then in order to improve the cooking and eating quality of rice.Therefore,a new type of indica rice standard breeding material for glutinous rice was expected to be developed.GBSSI,which is encoded by wax gene Wx,can control the synthesis of amylose.The effects of Wx gene mutation on starch biosynthesis related genes and some agronomic traits in rice were studied by studying the expression levels of starch biosynthesis related genes in transformed plants.In this work,we constructed a target vector of Wx gene and used Agrobacterium tumefacien-mediated transformation to infect callus of indica rice R287,which was made of amylose material,thus reducing the synthesis of amylose.By studying the expression level of starch synthesis genes in transformed plants after transcription,and measured the starch content of wild-type R287 plants in control group and mature seeds of transformed plants,the relationship between the genes involved in starch synthesis can be understood more intuitively.The results are as follows:1.Searched the Wx gene sequence according to NCBI database,then designed target sites and constructed the vector CRISPR/Cas9-Wx(A-D)on the 3rd exon,6th intron,5’UTR region and 7th exon,respectively.2.The Agrobacterium containing the above-mentioned vector infects the recipient material into callus,and the transformed plant is obtained by screening and differentiation.The positive identification of the hygromycin resistance gene of the transformed plants was performed,and then the positive plants were sequenced and analyzed.Three strains of CRISPR/Cas9-Wx A homozygous mutant materials and seven strains of double heterozygous mutant materials were obtained,all of which had a base at the 9th base of the third exon of the Wx gene(1760 base of the Wx gene)In the follow-up experiments,three CRISPR/Cas9-Wx A strains(a1-a3)were obtained through cultivation and selection of the insertion mutation of the base;in the same way,four CRISPR/Cas9-Wx B strains(b1-b4)were obtained.Mutations such as insertions and deletions occurred at the 70 th base of the 6th intron of the Wx gene(the2457th base of the Wx gene)respectively;the four CRISPR/Cas9-Wx C strains(c1-c4)were found in the Wx Insertion,deletion and other mutations occurred around the 369 th base of the gene(5’ UTR region);six CRISPR/Cas9-Wx D strains(d1-d6)were located at the 77 th base of the 7th exon of the Wx gene.3.We collected mature seeds,dried them and peeled them to determine the starch content.The amylose content of seeds in the mutants a1,a2,and a3 was between 4.71% and5.27%,which was about 17% different from the 22.85% of the wild-type R287 plant seed in the control group,while the b and c series the amylose content of mutant seeds was not significantly changed compared with that of wild-type R287 seeds;The content of amylopectin in the seeds of mutants a1,a2,and a3 is between 94.43% and 95.49%,which is about 17.28%-18.34% different from the seed amylopectin content of wild-type R287 plants in the control group.There was no significant difference between the amylopectin content and the wild-type R287 plant seed amylopectin content.4.Seed endosperm was taken during the filling stage for fluorescence quantitative analysis of genes Wx,AGPlar,SSI,SSII-3,SSIII-2,PUL and other starch synthesis-related genes.It was found that in mutants a1 and a2,Wx gene expression was reduced by 4 times,while AGPlar and SSI gene expression was increased by 2 times,PUL and SSII-3 gene expression was increased by 3 times,and SSIII-2 was increased by approximately 7 times the amount of expression.This is consistent with the results of the decrease in amylose content and the increase in amylopectin content in the above mutants a1 and a2.There was no significant difference between the gene expression levels of the b and c series mutants and the wild-type R287 plants of the control group. |
