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Rational Improvement of Rice Yield and Cold Tolerance by Editing the Three Genes OsPIN5b, GS3, and OsMYB30 With the CRISPR-Cas9 System

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Rational Improvement of Rice Yield and Cold Tolerance by Editing the Three Genes OsPIN5b, GS3, and OsMYB30 With the CRISPR-Cas9 System

Yafei Zeng et al. Front Plant Sci.

Abstract

Significant increases in rice yield and stress resistance are constant demands for breeders. However, high yield and high stress resistance are often antagonistic to each other. Here, we report several new rice mutants with high yield and excellent cold tolerance that were generated by simultaneously editing three genes, OsPIN5b (a panicle length gene), GS3 (a grain size gene) and OsMYB30 (a cold tolerance gene) with the CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-associated protein 9) system. We edited two target sites of each gene with high efficiency: 53% for OsPIN5b-site1, 42% for OsPIN5b-site2, 66% for GS3-site1, 63% for GS3-site2, 63% for OsMYB30-site1, and 58% for OsMYB30-site2. Consequently, the ospin5b mutants, the gs3 mutants, and the osmyb30 mutants exhibited increased panicle length, enlarged grain size and increased cold tolerance, respectively. Then nine transgenic lines of the ospin5b/gs3, six lines of ospin5b/osmyb30 and six lines of gs3/osmyb30 were also acquired, and their yield related traits and cold tolerance corresponded to the genes being edited. Additionally, we obtained eight ospin5b/gs3/osmyb30 triple mutants by editing all three genes simultaneously. Aside from the ospin5b/gs3/osmyb30-4 and ospin5b/gs3/osmyb30-25 mutants, the remaining six mutants had off-target events at the putative off-target site of OsMYB30-site1. The results also showed that the T2 generations of these two mutants exhibited higher yield and better cold tolerance compared with the wild type. Together, these results demonstrated that new and excellent rice varieties with improved yield and abiotic stress resistance can be generated through gene editing techniques and may be applied to rice breeding. Furthermore, our study proved that the comprehensive agronomic traits of rice can be improved with the CRISPR-Cas9 system.

Keywords: CRISPR/Cas9; GS3; OsMYB30; OsPIN5b; cold tolerance; grain length; panicle length.

Figures

Figure 1
Figure 1
The ospin5b/gs3/osmyb30-4 and ospin5b/gs3/osmyb30-25 were precisely edited with CRISPR/Cas9 system. (A) Schematic diagram of the CRISPR/Cas9 vector. The pYLCRISPR/Cas9 binary vector was based on the pCAMBIA1300 backbone which contained the Kanamycin resistance gene. HPT encoded hygromycin B phosphotransferase, which could be driven by the cauliflower mosaic virus 35S promoter (P35S). The Cas9 was driven by the maize ubiquitin promoter (Pubi) and used to edit target sites. Tnos was the terminator of nopaline synthase gene which was chose to terminate the expression of Cas9 gene. U6a, small nuclear RNA promoters, was employed to facilitate the expression of multiple sgRNA cassettes. The six sgRNA cassettes also were inserted behind U6a. (B) Six target sequences alignment in ospin5b/gs3/osmyb30-4 and ospin5b/gs3/osmyb30-25. Blue represents the target sequences. Red represents the changing base.
Figure 2
Figure 2
CRISPR/Cas9-induced ospin5b mutants and the expression level of cell cycle-related genes. (A) Schematic representation of the OsPIN5b genomic region. The two target sites of OsPIN5b and PCR primers were marked in first exon. (B) The phenotype of panicles and data statistics of ospin5b-1, ospin5b-13 and WT, Bar = 5 cm. (C) The expression level of marker gene OsYUC1 in WT, ospin5b-1 and ospin5b-13. Data are means ± SD from three biological replicates. Student’s t test, **P < 0.001, *0.001 < P < 0.05.
Figure 3
Figure 3
CRISPR/Cas9-induced gs3 mutants and the expression level of cell cycle-related genes. (A) Schematic representation of the GS3 genomic region. The two target sites of GS3 and PCR primers were marked in first and fifth exons. (B) The grain phenotype of gs3–9, gs3–21 and WT, and statistics for the average grain length. Bar = 1 cm. (C) The expression level of marker gene OsH1 and OsCYCT1 in WT, gs3–9 and gs3–21. Data are means ± SD from three biological replicates. Student’s t test, **P < 0.001, *0.001 < P < 0.05.
Figure 4
Figure 4
The phenotype analysis of osmyb30 mutants and the expression level of downstream BMY10 gene. (A) Schematic representation of the OsMYB30 genomic region. The two target sites of OsMYB30 and PCR primers were marked in second exon. (B) The seedlings of osmyb30-7, osmyb30-11 and WT after being treated in 4°C chamber. (C) The survival rate of osmyb30 mutants after 4°C treatment. (D) The expression level of marker gene OsBMY10 in WT, osmyb30-7 and osmyb30-11. Data are means ± SD from three biological replicates. Student’s t test, **P < 0.001, *0.001 < P < 0.05.
Figure 5
Figure 5
The phenotypes of ospin5b/gs3-2, ospin5b/osmyb30-17 and gs3/osmyb30-15 mutants. (A) The panicles and data statistics of ospin5b/gs3-2, ospin5b/osmyb30-17 and gs3/osmyb30-15 mutants, Bar = 5 cm. (B) The grain phenotype of ospin5b/gs3-2, ospin5b/osmyb30-17 and gs3/osmyb30-15, and statistics for the average grain length. Bar = 1 cm. (C) The seedlings and survival rate of ospin5b/gs3-2, ospin5b/osmyb30-17 and gs3/osmyb30-15 after being treated in 4°C chamber. Data are means ± SD from three biological replicates. Student’s t test, **P < 0.001, *0.001 < P < 0.05.
Figure 6
Figure 6
The phenotypes of ospin5b/gs3/osmyb30-4, ospin5b/gs3/osmyb30-25 and the predicted protein structure of OsPIN5b and OsMYB30 in ospin5b/gs3/osmyb30-4, ospin5b/gs3/osmyb30-25. (A) Whole plant morphology, survival rate after cold treatment and grain size in WT, ospin5b/gs3/osmyb30-4, ospin5b/gs3/osmyb30-25. Bar = 20 cm. (B) The OsPIN5b and OsMYB30 protein structures of WT, ospin5b/gs3/osmyb30-4 and ospin5b/gs3/osmyb30-25 were predicted by SWISS-MODEL. The observed differential regions were highlighted in green.

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