RISBZ1 And RPBF Promoter Editing Strategies Based On CRISPR/Cas9 Regulate Rice Quality

Rice is one of the important food crops in our country.With the continuous improvement of people’s living standards,the demand for rice quality is also becoming increasingly intense.Rice endosperm is mainly composed of starch,protein and lipids,and their content and composition will directly affect rice quality.Therefore,it is worth trying to improve rice quality by regulating the content and composition of stored substances in rice endosperm.At present,rice quality improvement by traditional breeding methods is not only time-consuming and laborious,but also blind.Studies have shown that rice RISBZ1 and RPBF genes are key transcription factors that regulate the expression of rice storage substance synthesis genes.The appearance of rice grain of the lofunction-type mutants deteriorates significantly and can hardly be used effectively in production practice.In this study,CRISPR/Cas9 gene editing system was used to fine-edit the promoter regions of RISBZ1 and RPBF in order to obtain excellent transformants with fine-tuned expression levels,improved rice quality and excellent agronomic traits.The main research results are as follows:1.By predicting the cis-acting elements of RISBZ1 and RPBF promoters,10 and 7 specific targets were selected at the key sites of RISBZ1 and RPBF gene promoters,respectively,and a two-site editing vector was constructed to transform the conventional japonica rice variety Nipponharu.A total of 12 homozygous mutations were screened for RISBZ1 promoter region by sequencing and additive implantation,which were ZM2,ZM12-1,ZM12-2,ZM3,ZM34,ZM56,ZM8,ZM78-1,ZM78-2,ZM10,ZM910-1 and ZM910-2.Seven homozygous mutation types were screened for the RPBF promoter region,namely FM12-1,FM12-2,FMM4-1,FMM4-2,FMM4-3,FM45 and FM67.2.The results of qRT-PCR analysis on 5,15 and 25 days after anthesis showed that the expression levels of RISBZ1 and RPBF increased gradually with grain filling,and fell back at 25 days after anthesis.The expression level of RPBF was higher than that of RISBZ1.In RISBZ1 mutation types,the expression levels of ZM2,ZM12-1,ZM12-2 and ZM56 were significantly down-regulated,while the expression levels of ZM8 and ZM78-1 were significantly up-regulated.Among the seven mutation types of RPBF,the expression levels of FMM4-1,FMM4-2,FMM4-3 and FM12-2 were significantly down-regulated,while the expression levels of FM45 and FM67 were significantly up-regulated.3.The expression levels of RISBZ1 and RPBF genes were significantly positively correlated with the content of gluten,with the correlation coefficients reaching 0.802 and 0.866.The down-regulation of RISBZ1 and RPBF will significantly reduce the grain gluten content,while the protein content of other components will be compensatively increased.On the contrary,the expression of RISBZ1 and RPBF is up-regulated,and the content of the four components of protein will be synergistically increased.In addition,in RISBZ1 and RPBF down-regulated mutants,amylose content in grains was down-regulated by about 2.01%-2.97%.4.The main agronomic traits of some mutants showed that although some agronomic traits of some mutants were different from those of the wild type,there was no fixed rule,which was suspected to be caused by environmental factors or tissue culture effects.5.The chalkiness of eight types of RISBZ1-related mutants showed that the chalkiness of ZM2,ZM12-1,ZM12-2,ZM56,ZM8 and ZM10 increased significantly compared with the wild type,while the chalkiness of ZM3 and ZM34 decreased significantly.The chalkiness of five mutant types of RPBF was changed,and the chalkiness was increased compared with that of the wild type.