Reseach Of Orientation Mutation Of SSIII? And PPDK Related To Starch Synthesis In Rice (Oryza Sativa L.) By CRISPR/Cas9 System

Rice is one of main cereal crops around the world,and is also one of the model plants for genomic research.Starch is the main storage material in endosperm of mature rice seeds.After eating rice,starch will enter the human body,the blood glucose will increase rapidly at the same time.Which will cause a serious physical burden for diabetic patients.Therefore,the research on changing the starch content in rice seeds has become an important goal of researchers.In this study,we use CRISPR/Cas9 technology to edit starch and related genes of rice,and we conclude as the following:1.Using CRISPR/Cas9 technology,we successfully edited two genes related to starch synthesis SS?a(LOC_Os08g09230)and PPDK(LOC_Os05g33570).2.In this study,we got the following results: SS?a gets 3 types of homozygous mutants by CRISPR/Cas9 technology:(1)Two bases(AG)are missing;(2)One base A is inserted;(2)Missing 347 bp near the target and inserting 9bp(TGAGCTAAG).PPDK gets 3 types of homozygous mutants:(1)One base T is inserted;(2)Missing 3 bases(GGG);(3)Missing one base A.3.The expression at the transcriptional level of mutants was analyzed.We found that the expression level of the mutant edited genes was lower than that of the wild plants,while the expression level of other genes related to starch synthesis showed different degrees of change.There is a 5-10 fold down-regulation of the gene expression level of the ppdk mutant for starch synthesis.The expression levels of SSI and SSII genes in ssIIIa mutants were up-regulated,while the expression levels of other related genes were decreased or not significantly changed.4.The cross section of mature mutant seeds was observed by cold field emission scanning electron microscope,we found that significant changes were seen in the form and size of starch granules in the ppdk mutant and the ssIIIa dual-target mutant,and the 1000-grain weight has also been significantly reduced while no significant changes were observed in the single-target mutant of ssIIIa.5.Staining I-KI of the mutant revealed that the ppdk mutants have certain shallowed in color compared to the wild type,while the ssIIIa mutant did not change significantly.6.Detection of resistant starch content in mutant seeds showed that the resistant starch content of PPDK mutant was not obvious,while the content of ssIIIa mutant was increased significantly.This study used the CRISPR/Cas9 technology to successfully edit the genes related to rice starch synthesis,which provids an experimental basis for the study of functional rice using CRISPR/Cas9 technology.