Creating High-resistant Starch Rice By Simultaneous Editing Of SS3a And SS3b

The prevalence of chronic diseases like obesity,overweight,and type Ⅱ diabetes is increasing as people’s living standards are rising and their lifestyles are changing.Consumption of resistant starch(RS)helps to keep postprandial blood glucose levels stable and fends off disorders that are associated to them.Rice,the main source of carbohydrates for most people,generally contains less than 1%RS,which is insufficient to have a beneficial effect on the human body.As a result,enhancing RS content in rice is now considered to be "mainstream".In this study,SSⅢa/SS3a and SSⅢb/SS3b encoding soluble starch synthetase Ⅲ genes were co-knocked out by CRISPR/Cas9 genome editing technology in Nipponbare.Single and double homozygous mutant lines with various mutation types were produced.Through agronomic traits,digestive traits,physicochemical quality assessment,starch fine structure measurement and starch grain morphology observation,It revealed that the synergistic mechanism of SS3a and SS3b in the synthesis of rice leaf and endosperm starch and the creation of RS,and created new nutritionally healthy rice germplasm with high RS content of 9.54%-9.73%(wild type 0.58%)and significantly lower digestion rate,which provides a new strategy for breeding new rice varieties with high RS content and helps to further elucidate function of starch synthesisrelated genes in cereals.The exact outcomes are listed below:1.Bioinformatics Analysis of SS3a and SS3b revealed that both were highly conserved in terms of gene structures,protein domains and amino acid sequences.The analysis of expression patterns revealed that SS3a and SS3b were both significantly expressed in endosperm and nutritional organs like leaves,respectively.Several homozygous mutant lines,including SS3a single mutants(ss3a),SS3b single mutants(ss3b)and their double mutant lines(ss3a-ss3b),were obtained after multiple generations of genotype and phenotypic screening with SS3a and SS3b co-knocked out by CRISPR/Cas9 genome editing technology in Nipponbare.The agronomic traits of the mutant lines were examined,and it was found that the development of the mutant lines did not change significantly compared to the wild type at the seedling and filling stages and that the grain length and width of the different mutants were similar to those of the wild type,but the grain thickness and 1000grain weight of the ss3a and ss3a-ss3b mutants were significantly lower.2.In terms of digestive characteristics,the ss3a mutants showed a significant increase in RS content and a great decrease in total digestible starch content as well as digestion rate,but the ss3a-ss3b mutants had an increased effect based on ss3a.While there was no significant change between the ss3b mutants and the wild type.Despite this,the SS3b mutation had no significant effect on the physicochemical quality of endosperm starch.In other words,total amylose content,AAC,triglyceride content,pasting temperature and viscosity of mutants have the same level with the wild type.Meanwhile,the SS3a-SS3b mutation resulted in a highly significant enhance in AAC and triglyceride content and a significant decrease in total amylose content,pasting temperature and RVA profile.These suggest that SS3a and SS3b have synergistic effects in rice endosperm starch synthesis and RS formation.3.In terms of the fine structure of starch,the endosperm of the ss3b mutant showed no significant change in starch molecular weight distribution,whereas the endosperm containing the SS3a mutation(ss3a and ss3a-ss3b mutants)showed a significant increase in the proportion of straight chain starch and a significant decrease in the proportion of branched starch long chains(Ap2),with both changes being greater in the ss3a-ss3b mutant.XRD results showed that the relative crystallinity of endosperm starch was significantly reduced,and the proportion of amylose-lipid complexes was significantly increased in the ss3a and ss3a-ss3b mutants,whereas no significant changes were observed in the endosperm starch of the ss3b mutant.The significant increase in contents of amylose and amylose-lipid complexes was the main reason for the significant enhance in RS content in the relevant mutants.4.There was no significant change in grain morphology and appearance of the ss3b mutants.But the chalky grain percentage and chalkiness degree were significantly increased result from SS3a mutation.In the endosperm of the ss3a and ss3a-ss3b mutants,the morphology of the starch granules and compound starch granule complex was aberrant and loosely arranged,and the abnormality was more severe in the ss3a-ss3b mutants whereas ss3b showed no appreciable change.This result explained the chalky phenotype of the related mutations.5.In the leaves,the SS3a mutation had no significant effect on the instantaneous starch composition and fine structure,while the ss3b mutants showed a significant decrease in total starch content and the proportion of branched starch B-chains(DP>12)and a slight increase in AAC.The total starch content and the proportion of branched starch B-chains in leaves of ss3a-ss3b mutants were further reduced based on the SS3b mutation,and there was no significant change in AAC.In the purified leaf transient starch,the morphology of ss3a starch granules did not change significantly compared with the wild type,the ss3b starch granules were irregular and had a rough surface,and the morphology of starch granules in the mutants was more abnormal.These indicate that SS3b plays a major role in the synthesis of leaf transient starch,and there is also a significant synergistic effect between SS3a and SS3b.