Table 1

Primers for vector construction and genotype"

Fig. 1

Schematic diagram of target sites of glutelin-coding genes and T-DNA region of CRISPR vector A: GluA1 and GluA2 gene structure, yellow and orange boxes represent the exons of GluA1 and GluA2 genes respectively, short black lines between exons represent introns, blue and green lines indicate the locations of gene editing target sites, underlined red letters indicate the sequences of gene editing target sites, and underlined black letters indicate the protospacer adjacent motif (PAM) sequence; B: GluA3 gene structure, the light blue box represents the exon of GluA3 gene, short black lines between exons represent introns, short pink and pink lines indicate the location of gene editing target sites, underlined red letters indicate the sequences of gene editing target site, and underlined black letters indicate the PAM sequence; C: Diagram of gene editing vector T-DNA segment, ZmpUBI: promoter of maize Ubiquitin1 gene. SpCas9: Cas9 gene derived from Streptococcus pyogenes after codon optimization for rice; NOST: NOS terminator; 3: rice U3b promoter; 6: rice U6b promoter; GluA1/2-1: the first target site of GluA1 and GluA2 genes; GluA1/2-2: the second target site of GluA1 and GluA2 genes; GluA3-1: the first target site of GluA3 gene; GluA3-2: the second target site of GluA3 gene; 35S Promoter: Cauliflower mosaic virus 35S promoter; HygR: hygromycin resistance gene; 35ST: 35S terminator; LB: T-DNA left boundary; RB: T-DNA right boundary."

Fig. 2

Genotype identification and Sanger sequencing peak map A: Genotyping by agarose gel electrophoresis; B: Chromatograms of Sanger sequencing; C: Identification of transgenic elements. Marker: DNA molecular weight standards; WT: wild-type japonica rice cultivar SX867; LGC-1: recipient cultivar; +CK: positive control of CRISPR plasmid; -CK: PCR negative control; LGC1: wild-type LGC1 locus; Lgc1: mutant Lgc1 locus; GluA1: GluA1 locus; GluA2: GluA2 locus; GluA3: GluA3 locus; Cas9: Cas9 gene cassette; Hyg: hygromycin resistant gene cassette. Longer bands of GluA1, GluA2, and GluA3 in A represent wild-type genotypes, and shorter bands represent mutant genotypes. The “*” and “·” under the letters in B represent the bases flanking the deletion region of target genes; Left arrows in A and C indicate the bands of DNA molecular weight standards."

Fig. 3

SDS-PAGE and amino acid composition determination in rice A: SDS-PAGE of proteins from brown rice; B: SDS-PAGE of proteins from polished rice; C: results of amino acid composition determination in rice. M: protein marker; WT: wild-type japonica rice cultivar SX867; LGC-1: transgenic receptor cultivar; GluA-2-11 and GluA-3-6: low-glutelin lines generated in this research. The right vertical lines in A and B indicate different components of rice storage protein. Asp, Glu, Ser, etc. in C represent different amino acids. Different letters indicate significant differences at the P < 0.01."

Fig. 4

Detection results of main nutrients in rice A: total protein content of brown rice; B: total protein content of milled rice; C: total starch content of brown rice; D: total fatty acid content of brown rice. A-D: each sample was measured in triplicate, and different lowercase letters represent significant differences at the P < 0.01. E-G: the results of scanning electron microscopy (SEM) of mature seeds of LGC-1, GluA-2-11, and GluA-3-6. The scale bar: 10 μm. H-J: total ion chromatogram of fatty acid content determination."

Table 2

Statistical results of appearance quality"

Table 3

Statistical results of food quality"

Fig. 5

Rice plant type and rice appearance phenotype A: the morphology of LGC-1, GluA-2-11, and GluA-3-6 mature plants; B-D: seed appearance of LGC-1, GluA-2-11, and GluA-3-6; E-G: the appearance of LGC-1, GluA-2-11, and GluA-3-6 polished rice. In A, the scale bar: 20 cm; in B-G, the scale bar: 1 cm."

Table 4

Statistical results of agronomic trait"