Construction Of Novel Rice Germplasm With High Level Of Resistant Starch By Mutating SBE3 And SS? Through CRISPR/Cas9 System

Rice is an important cereal crops that feed more than half of the population in the world.The composition and content of rice nutrients are closely related to human health.Starch is the major dietary source of carbohydrates and consists of two types of molecules,amylose(Am)and amylopectin(Ap),in which the fraction of ingested starch that escapes into the human large intestine is called resistant starch.Amylose content is related to the resistant starch content of grain starch.In cooked foods,the amylose molecules rapidly recombine upon cooling to form a precipitate or gel,forming a complex that resists digestion,while the amylopectin molecules are slow to recombine,thus forming a complex that is easier to digest.Therefore,high amylose(AC)and high-resistant starch(RS)rice varieties help to reduce glycemic index in diabetic patients.In addition,diabetic patients often have kidney disease,which causes protein metabolism disorders.Reducing the protein content in rice can help relieve kidney metabolic pressure in kidney patients.Therefore,eating rice with high resistance to starch and low protein is beneficial to the health of diabetic and kidney patients.In recent years,the incidence of diabetes in China has increased year by year,and it is urgent to cultivate rice germplasm with high resistance to low starch.Studies have shown that the starch branching enzyme gene SBE3 and the starch synthase gene SS? play a major role in determining the fine structure and physical properties of starch.Among the chemically mutagenized mutants or RNA interference plants of these two genes,the content of amylose and resistant starch was higher than that of the wild type.In this study,low-gluten rice was used as a receptor material,and the CRISPR/Cas9 system was used to construct functional deletion mutants of SBE3 and SS?genes respectively,in order to create a new rice variety with high resistance to starch low gluten.In this study,the amylose content and the resistant starch content of the above homozygous mutant lines were determined by Megazyme/K-AMY method and Megazyme/K-RSTAR method.The results showed that the average content of amylose in the three homozygous mutant lines of SBE3 was 16.75%,and the average content of resistant starch was 5.6%,which was significantly higher than 6.16%and 5.5%of the recipient varieties.The average content of amylose in the three homozygous mutant lines of SSIII gene was 17.35%,which was significantly higher than that of the recipient variety,while the level of resistant starch increased less.Scanning electron microscopy(SEM)analysis showed that the starch granules of sbe3 and ss? mutants were significantly different in arrangement,size,spacing,transparency and shape compared with wild type.It is consistent with the level of change of amylose and resistant starch.The phenotypic analysis showed that the phenotypes such as plant height and grain type of sbe3 and ss?mutants were not significantly different from those of the recipient varieties.The soluble sugar content of the mutant and recipient varieties was determined by ion chromatography.The results showed that the average content of sucrose in the sbe3 mutant lines reached 26.6?g/mg,which was significantly higher than that of the recipient variety of 22.3 ?g/mg.The contents of glucose,trehalose,fructose and maltose were not significantly different from the wild type.The average content of sucrose in the ss? mutant lines was 12 ?g/mg,which was slightly higher than that of the recipient variety.The other soluble sugar content was not significantly different from the recipient variety.In summary,the high-amylose plants can be obtained by site-directed mutagenesis of the SBE3 and SS? genes by the CRISPR/Cas9 system.Site-directed mutagenesis of SBE3 can also increase the content of resistant starch.This study provides an important genetic germplasm for cultivating rice varieties with a high-resistance starch low-gluten background.