• Identification results of mutants after editing OsGluB1 and OsGlb genes using CRISPR/Cas9 gene editing technology

Fig.1 Identification results of gene mutations in OsGluB1 and OsGlb after CRISPR/Cas9 gene editing

The OsGluB1 target gene mutates at this site by a deletion of 1 bp base A or C, while the OsGlb target gene mutates at this site by an addition of 1 bp base A. The tetraploid homozygous mutation types include OsGluB-1 deletion of 1 bp A and OsGlb insertion of 1 bp T (osGluB-1/osgb-4x-1), OsGluB-1 deletion of 1 bp C and OsGlb insertion of 1 bp T (osGluB-1/osgb-4x-2).

• Cytological changes in rice seeds after polyploidization and gene editing

→ 1. Thickness variation of paste layer

Fig.2 Scanning electron microscopy observation of changes in the aleurone layer structure of rice seeds after polyploidization and gene editing

→ 2. Changes in the number of cell layers in the aleurone layer

Fig.3 Observation of changes in endosperm structure of rice seeds with different ploidy levels under an optical microscope

→ 3. Structural changes of PSV (protein transport vesicles) and PB-II (protein body II)

Fig.4 Transmission electron microscopy observation of the thickness of rice seed endosperm layer and the number of protein bodies with different ploidy levels

We observed the structure of the aleurone layer cells under an optical microscope (Figure 3) and found that compared with diploid rice seeds, the number of aleurone layer cells in tetraploid rice seeds did not change significantly, but the thickness of the aleurone layer cells showed a significant thickening phenomenon. This result was verified by scanning electron microscopy and transmission electron microscopy observations of the seeds (Figures 2 and 4). The cell structure of the lateral and abdominal parts of HWC-2x and HWC-4x seeds showed that the number of layers of aleurone layer cells remained between 1-2, and the thickness of individual aleurone cells increased by 32.92% in HWC-4x compared to HWC-2x (Figure 4).

• Changes in Total Protein, Component Proteins, and Lysine Content in Polyploidization and Gene Editing Rice Seeds

→ 1. SDS-PAGE results

SDS-PAGE distinguishes between glutelin, prolamine, and globulin

Note: The protein content is determined using the Kjeldahl method, where there is a loss of component proteins. The difference between the sum of component protein content and the total protein content does not indicate that the remaining protein is albumin. And the loss of each component protein is consistent, and the ratio and trend of component protein content are consistent with the physiological state of rice.

→ 2. Changes in protein and lysine content of each component

Fig. 5 Changes of total protein, glutelin, gliadin and globulin contents in rice seeds after polyploidy and gene editing

Note: The protein content is determined using the Kjeldahl method, where there is a loss of component proteins. The difference between the sum of component protein content and the total protein content does not indicate that the remaining protein is albumin. And the loss of each component protein is consistent, and the ratio and trend of component protein content are consistent with the physiological state of rice.

Fig.6 Changes in Lysine Content of Rice Seeds after Polyploidization and Gene Editing

Figures 5 and 6 show that after polyploidization, the content of total rice endosperm storage protein glutelin, gliadin, globulin and lysine significantly increased; When OsGluB1 gene is mutated simply, the content of total protein and gliadin slightly increases, the content of glutelin and globulin slightly decreases, and the content of lysine significantly decreases; When OsGluB1 and OsGlb genes are mutated at the same time, the content of total protein, gliadin and lysine is significantly increased, and the content of glutelin and globulin is significantly reduced (the content of glutelin basically returns to the level of diploid wild type, and the content of globulin is reduced to almost negligible)

• Evaluation of experimental result

The total protein content of osglub1/osglb double gene mutant has been significantly higher than that of all rice varieties found in our HP activity, the glutelin content has returned to slightly higher than that of the wild type diploid, the relative ratio of gliadin and glutelin is significantly closer to 1:1 than that of the wild type diploid, almost without globulin, and the lysine content has reached the level of lysine in the ideal amino acid model of rice. Therefore, the result is very consistent with our expectations.

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