Impovement For Amylose Content By CRISPR/Cas9 Editing In Maize

Maize（Zea mays L.）is an important food crop,it is not only used for food and feeding,but also the main raw materials of starch industry.According to the molecular structure,starch can be divided into amylose and amylopectin.The glutinous maize,which is all made of amylopectin,tastes better.While high amylose varieties with high amylose content have higher processing value in starch industry.Although the traditional maize breeding technology has made remarkable achievements,however,it is still difficult to meet the needs of agricultural production and starch industry due to the lack of progress in improving the quality of high amylose.Modern biotechnology,represented by transgenetic technology,provides a new technological approach for maize quality improvement.Especially CRISPR/Cas9gene editing technology,can knock out the key genes of amylopectin synthesis directionally,to create the needed new germplasm which used for breeding high amylose varieties.In this study,seven CRISPR/Cas9 vectors targeting maize endogenous starch branching enzyme（SBE）gene Zm SBEI、Zm SBEIIb and soluble starch synthetase（SSS）gene Zm SSSIII were constructed and used to transform maize embryonic calli by Agrobacterium mediation.Through herbicide resistance screening,PCR,southern hybridization identification and target sequence determination and so on,the positive transgenic lines with target mutation and without selection marker gene were selected.Then identifying the phenotype of agronomic characters through field experiments,determination of amylose content,the morphology and structure of starch granules in endosperm were observed under scanning electron microscope.The main results are as follows:1.The target sequences of maize endogenous genes Zm SBEI、Zm SBEIIb and Zm SSSIII of recipient inbred line C01 were amplified and analyzed,designed the single-stranded guide RNA,construction of CRISPR/Cas9 editing vector,double enzyme digestion identification and sequence analysis showed that the construction was successful.2.Embryogenic callus were induced by immature embryos of recipient inbred lines C01,Agrobacterium-mediated transformation method of maize embryonic callus,through herbicide resistance screening and PCR identification,a total of 17 T₀generation plants were obtained.Through self-crossing breeding,Southern hybridization,PCR identification and sequence analysis,we selected Zm SBEI、Zm SBEIIb and Zm SSSIII gene single mutation,Zm SBEI and Zm SBEIIb gene double mutation,Zm SBEIIb and Zm SSSIII gene double mutation and Zm SBEI、Zm SBEIIb、Zm SSSIII gene triple mutation without Bar selective marker gene one for each T₁generation.3.Except for the Zm SBEI gene single mutant line,the content of amylose in the other five T₁lines was significantly higher than that of the untransformed receptor inbred line C01.The plant height and ear height of most lines were significantly higher than those of the untransformed receptor line,the 100-grain weight was significantly less than that of the untransformed receptor line,but the number of rows per ear and the number of grains per row did not change.4.Scanning electron microscopy observation showed that except for the Zm SBEI gene single mutant line,the starch granules in endosperm of the other five T₁lines were smaller,arranged closely,and there are some irregular rod-shaped particles,it is similar to the high amylose mutant line with Zm SBEIIb gene mutation.5.Sequence analysis shows that,the high amylose mutant line GEMS0067 with Zm SBEIIb gene mutation,the 9th exon is completely deletion,amylose content up to70%,starch granules in endosperm are smaller,arranged closely,and there are some irregular rod-shaped particles also,starch has high gelatinization temperature,stronger thermal stability,the proportion of starch chain length was significantly higher than that of wild-type control.6.The mutation efficiencies of the three target points of the Zm SBEI,Zm SBEIIb and Zm SSSIII genes was as high as 49.48%,63.85%and 68.75%,respectively,in the17 transgenic events transformed by the CRISPR/Cas9 vector harboring the comfirmed sg RNA sequences.This result can provide reference for related research.In conclusion,CRISPR/Cas9 gene editing technology was used to knock out endogenous starch branching enzyme genes Zm SBEI,Zm SBEIIb and soluble starch synthase gene Zm SSSIII simultaneously,high amylose maize germplasm can be created,the phenotype was similar to the high amylose mutant line with the Zm SBEIIb gene mutation,it is expected to be used in high amylose quality breeding after further identification.