| Maize(Zea mays L.)starch is an essential source of energy and nutrition for human survival.Maize starch,especially high amylose corn starch,is also an important raw material in many fields.In addition,Resistant Starch(RS)generally comes from high amylose,which can reduce the incidence of intestinal diseases and blood glucose and insulin response,and is beneficial to maintain health.In common maize,it usually contains only 25% amylose or even less,so its resistant starch content is also low,usually less than 1%.Therefore,increasing amylose content in maize to increase resistant starch content has become one of the important breeding methods.Previous studies through RNAi or mutagens have found that starch branching enzyme SBE,especially SBEⅡb,has a significant effect on the fine structure and properties of starch,and its mutated starch contains very high amylose and RS,while SBE1 has little effect on the composition and properties of starch,but its mutated starch digestibility is slightly reduced.At present,modern biotechnology represented by transgenic technology,especially CRISPR/Cas9 gene editing technology,has been widely used in crop breeding improvement,providing a new feasible technology for crop breeding.In this study,CRISPR/ Cas9 gene editing technology was used for site-directed gene editing of ZmSBEⅠ and ZmSBEⅡb genes related to amylopectin synthesis in the starch synthesis pathway to obtain new maize germplasm with high amylose.The main research results are as follows:(1)According to the sequence analysis of target genes ZmSBEⅠ and ZmSBEⅡb,appropriate sgRNA was designed and amplified for verification,and the CRISPR/Cas9 editing vector was constructed.The sequencing sequence analysis showed that the vector was successfully constructed and transformed.(2)PCR amplification and sequencing analysis were performed on the editing sites of T1 genes,and single gene mutations of sbe1,sbe2 b and double gene mutations of sbe1sbe2 b were screened out to determine their mutation types.The lines without transgene were selected by bar gene detection for propagation and experimental analysis.(3)Comparison of agronomic traits between the wild type and the three mutants showed that the 100-grain weight of the three mutants decreased significantly,the wild type was 32.67 g,and sbe1sbe2 b decreased the most by 14 g.(4)Scanning electron microscopy of different mutants showed that the shape of starch grains in sbe1 mutants had little change,while irregular and long starch grains were found in sbe2 b and sbe1sbe2 b mutants,and large starch grains decreased.Grain size analysis showed that the starch grain size of the three mutants decreased,and sbe2 b and sbe1sbe2 b decreased by 2.5μm and 2μm,respectively,which was consistent with the scanning electron microscope observation.(5)The amylose content(AC)of sbe1 mutant was 30.95%,which showed no significant change compared with the wild type,while the AC of the other two mutants was significantly increased,among which the AC of sbe2 b mutant was53.43%,and the AC of sbe1 sbeb mutant was 48.21%.In addition,the resistant starch content of sbe1sbe2 b mutant was 7% higher than that of sbe2 b,reaching 34.04%.The chain length distribution analysis of different mutants showed that the short chain(DP< 20)of sbe2 b and sbe1sbe2 b mutants decreased while the long chain(DP > 20)increased,and the gelatinization temperature of sbe1 and sbe1sbe2 b mutants also increased,but the gelatinization enthalpy decreased,which may be related to the formation of resistant starch.In conclusion,using CRISPR/Cas9 gene editing technology to simultaneously target the key enzymes of maize starch synthesis ZmSBE1 and ZmSBEⅡb,new maize germplasm containing high amylose and resistant starch can be created. |
