Improving Rice Quality By Using Gene Editing Technology To Target SSS?b Gene

Rice quality indicators mainly include appearance quality,taste quality,nutritional components and processing characteristics.The evaluation of rice taste quality is mostly based on the physical and chemical properties of starch,such as amylose content(AC),gel consistency(GC),gelatinization temperature(GT),which are closely related to the viscosity,softness and elasticity of rice.Starch is the main storage component of rice endosperm,including amylose and amylopectin.Its composition and structure have a decisive influence on the taste and quality of rice.As an important evaluation index of rice quality,the content of amylose has the greatest impact on rice quality.The structure of amylopectin and the distribution of its branch chain length also have an important effect on rice.Soluble starch synthase(SSS)is a key enzyme that catalyzes starch synthesis.It is mainly responsible for amylopectin synthesis and participates in the regulation of starch physicochemical indexes.At present,through the reasonable selection of starch synthesis related genes,different allele combinations can achieve the purpose of quality improvement,which is helpful to cultivate high quality rice germplasm.In this study,CRISPR/Cas9 gene editing technology was used to down regulate the expression of the rice cultivar Ningjing 4 starch synthase gene(SSS?b),editing the three target sites before,during and after the gene to obtain three different target site knockdown lines.The pedigrees were named SSS2-Q,SSS2-Z,and SSS2-H.The transgenic lines were identified for many years by molecular identification of the target genes and the determination of main agronomic traits,physiological and biochemical indicators,and the SSS2-Q lines that met the goal of cultivating high quality food quality were obtained.The main findings are as follows:1.Through gene amplification experiments,the molecular identification of SSS?b genes in different lines was performed.In Ningjing 4,the SSSIIb gene was edited by multiple targets,and three knockdown lines of SSS2-Q,SSS2-Z and SSS2-H were obtained.The molecular identification results showed that the target gene was edited at different positions,the SSS?b gene sequence in the knockdown lines had the different base changes with that of Ningjing 4,and plants excluding the transgene construction were obtained.2.The amylose content of the seeds of the knockdown lines were determined.Compared with Ningjing 4,the amylose content of the T1-3 generation of SSS2-Q lines were significantly lower than that of Ningjing 4,and the amylose content of the T1-3 generation of SSS2-Z lines had no significant change with Ningjing 4,while the amylose content of the T2-3 generation of SSS2-H lines were significantly higher than Ningjing 4.Comparing with the differences in the representative types of the transgenes at different knockdown sites,the SSS2-Q lines met the expected breeding goal,and other quality indicators of the lines were determined.3.The results of measuring the chain length distribution of amylopectin of rice flour showed that the SSS2-Q lines had a significantly lower intermediate chain length of degree of polymerization 10-18 compared to Ningjing 4,and the number of short chains DP 6-10 increased.The long chains of DP 18-25 increased slightly,and the long chains of DP greater than 25 did not change much.4.The determination of gel consistency showed that the gel consistency of the SSS2-Q lines was significantly higher than that of Ningjing 4.The gel consistency of the SSS2-Z lines was not significantly different from that of Ningjing 4,and the gel consistency of the SSS2-H lines was slightly decreased compared to Ningjing 4.The rapid viscosity analyzer(RVA spectrum)measurement results found that the SSS2-Q lines had characteristics of RVA spectrum such as hot paste viscosity,cool paste viscosity,peak time,and pasting temperature lower than Ningjing 4,and the breakdown value was higher than Ningjing 4.The SSS2-Z and SSS2-H lines had lower hot paste viscosity and cool paste viscosity than those of the Ningjing 4,but higher than those of the SSS2-Q lines.The breakdown value was higher than Ningjing 4 but lower than the SSS2-Q lines.5.In the endosperm of SSS2-Q lines 12 days after flowering,the expression level of starch synthesis related genes was detected and analyzed.Compared with Ningjing 4,the expression of SSS?b gene in SSS2-Q lines decreased to a significant level,while the expression levels of other starch synthase genes also changed to varying degrees,such as the significant increase in SSS?a and SBE?a gene expression.While the expression levels of SS?,SSS?a and SBE?b decreased.These genes played an important role in the formation of amylopectin.Therefore,the knockdown of the SSSIIb gene may change the amylopectin structure by affecting the amylopectin synthesis,thereby changing the physicochemical properties of starch.6.The appearance of mature seeds of SSS2-Q lines were transparent,and there was no powder phenotype of low-amylose rice appearance.Field investigation found that the number of grains per panicle of SSS2-Q lines was not different from that of Ningjing 4.The number of grains per panicle decreased significantly and the seed setting rate decreased.The grain length increased than SSS2-Q lines.There was no significant differences of other agronomic traits between SSS2-Q lines and Ningjing4.In order to create more functional rice varieties,this study also used CRISPR/Cas9 gene editing technology to knockdown the starch branching enzyme encoding gene SBE?b and starch synthase encoding gene SSS?a in rice variety Ningjing 7 to improve rice resistance starch content.To generation knockdown lines had been identified,and the target fragments had been edited for positive plants,providing a material basis for subsequent selection of resistant starch varieties.