Moderate Regulation Of Rice Grain Protein Content By Simultaneous Editing Of Glutelin Synthesis Genes Using The CRISPR/Cas9 System

Rice is one of the important food crops in Chhina.With the continuous improvement of living standards,the requirements for rice quality are also getting higher and higher.Rice grain protein content（GPC）directly affects rice quality.The improvement of rice quality through traditional breeding and is time-consuming and blind.With the development of molecular biology,gene editing technology has been widely used in cultivation.In this study,the CRISPR/Cas9 system was used to perform editing of the glutelin synthesis gene to study the effect of glutelin gene mutation on glutelin content to regulate the GPC.The results are as follows:1.We first investigated the total GPC of milled rice and the content of four fractions of 80 japonica cultivars.Correlation analysis showed that glutelin content was significantly positive correlated with the GPC and the expression level of glutelin synthesis gene family expect OsGluB4/5.2.CRISPR/Cas9 construct containing four sgRNAs which simultaneously targeting 8 genes（OsGluA1,OsGluA2,OsGluA3,OsGluB2,OsGluB6,OsGluB7,OsGluC,OsGluD）was developed and transformed an japonica variety NG9018.In T1 generation,only T-DNA free homozygous plants were chosen,resulting in seven different mutation types.3.SDS-PAGE and protein fraction analysis of the mutants showed that the glutelin of all mutants were significantly reduced by respectively 6.9%,19.2%,23.8%,27.1%,35.1%,35.4%and 45.6%.In the component proteins,albumin and glutelin decreased,prolamin and globulin had compensatory effect on the decrease.However,the total protein content of rice still gradually decreased with the increase of the number of knockout genes,and the range was 5.63 mg/g～20.15mg/g.4.The analysis of other storage substances in rice found that after the protein content decreased,the apparent amylose content increased.Statistical analysis showed that there was no significant difference in sucrose,fructose and total soluble sugar contents.Moderate regulation of glutelin content did not have much effect on the rest of the rice storage material.5.Compared with the wild type,most of the mutants showed a higher RVA profile,increased viscosity,except for NG9108-Ⅶ which had a sharp drop in glutelin content.The rice taste value and appearance of NG9108-Ⅶ with lower glutelin and total protein content were significantly decreased,and the rice hardness did not change significantly;the other mutants with moderately down-regulated rice protein content all showed higher taste value and appearance,the hardness of rice was significantly decreased.In conclusion,the direct mutagenesis of glutelin synthesis gene by CRISPR/Cas9 system can quickly reduce glutelin content and have positive effects on rice taste value,which provides a new strategy for rice quality improvement.