• Polyploidization increases protein content

Figure 1. analysis of nutritional quality of diploid and tetraploid rice seeds

• Gene regulation and metabolic regulation increase protein content

In order to understand how the protein content of rice increases after polyploidization, we consulted our second PI. During her doctoral study, she explored the temporal expression patterns of various glutelin subunits in different rice lines through quantitative proteomic analysis of seeds based on tandem mass tags, immunoblot analysis, and the results showed that polyploidization increased the content of glutelin by affecting the biosynthesis, transport, and deposition of glutelin, while the changes in glutelin accumulation between tetraploid and diploid rice were mainly manifested in the initial time, duration, and relative level of various glutelin gene expression.She took six pairs of materials such as 9311-2x, 9311-4x, a3-2x and a3-4x as the research objects, extracted the total protein of brown rice for SDS-PAGE analysis. The seed storage proteins of different varieties have similar SDS-PAGE protein profiles, and the bands of glutelin precursor, glutelin acidic subunit, basic subunit, globulin and prolamine can be clearly observed. She carried out image scanning analysis on the SDS-PAGE band map, and determined that glutelin was mainly composed of three band regions: glutelin precursor or multimer with the size of about 57kda, glutelin acidic subunit with the size of about 37kDa and glutelin basic subunit with the size of about 19kDa. The total glutelin content was counted; In addition, scan and analyze globulin with the size of about 26kDa and prolamine with the size of 10-16kda. The total protein content is obtained by the sum of all bands, and then divide into four groups to draw a histogram (Fig. 2). Comparing the protein content of the three components and the total protein content, it can be seen that compared with diploid, the increase rate of 9311 glutelin was 51.7%, prolamine was 28.87%, globulin was 33%, and total protein was 44.34%; A3 glutelin increased by 23.94%, prolamine by 50.51%, globulin by 9.23%, and total protein by 29.57%; The increase of glutelin content of the two pairs of materials was higher than that of prolamine and globulin, which was consistent with the test results of the kit.

Figure 2. difference of protein content of each component in two pairs of diploid and tetraploid rice seeds

• Mutant GluB1 and Glb genes increase lysine content and almost remove globulin

For the experimental design of gene editing, through modeling prediction and literature review, we found that mutation of GluB1 and Glb genes can increase the content of lysine and reduce the content of globulin to almost No. Our study of the second PI found several interesting relationships through the mutation of glub1 and GLB genes in diploid and tetraploid backgrounds. The content of glutelin and globulin did not change in single mutant (osglub-2x, osglub-4x), but significantly decreased in double mutant (osglub/osglb-2x, osglub/osglb-4x). This shows that globulin is a protein controlled by single gene family, and the mutation of globulin gene directly leads to the complete inability of globulin synthesis; The synthesis of glutelin is significantly different from that of glutelin due to its change trend and synthesis mechanism.

Figure 3. analysis of protein content of single mutant and double mutant components in rice with different ploidy

• Gene dosage compensation effect

Through consulting the literature, we found that GluB1 and Glb genes have a negative transcriptome regulation relationship with other subtype proteins of glutelin family and other rice storage proteins, and our second PI study clarified the direction of compensatory expression between GluB1 and Glb and lysine rich storage proteins.

Figure 4. Effect of glub1 and GLB gene mutations on expression levels of other storage proteins