Application of CRISPR/Cas9 system to knock out GluB gene for developing low glutelin rice mutant

Abstract

The nutritional quality improvement is among the most integral objective for any rice molecular breeding programs. The seed storage proteins (SSPs) have greater role to determine the nutritional quality of any cereal grains. Rice contains relatively balanced amino acid composition and the SSPs are fractioned into albumins (ALB), globulins (GLO), prolamins (PRO) and glutelins (GLU) according to differences in solubility. GLUs are further divided into subfamilies: GluA, GluB, GluC, and GluD depending on resemblance in amino acid. The GLU protein accounts for 60-80% of total protein contents, encoded by 15 genes located on different chromosomes of rice genome. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system was employed to knockout Glu-B (LOC-Os02g15070) gene in non-basmati rice PK386 cultivar. The mutant displayed two base pair and three base pair mutation in the targeted regions. The homozygous mutant plant displayed reduction for both in total protein contents and GLU contents whereas, elevation in GLO, ALB and PRO. Moreover, the mutant plant also displayed reduction in physio-chemical properties e.g., total starch, amylose and gel consistency. The agronomic characteristics of both mutant and wild type displayed non-significant differences along with increase in higher percentage of chalkiness in mutant plants. The results obtained from scanning electron microscopy showed the loosely packed starch granules compared to wild type. The gene expression analysis displayed the lower expression of gene at 5 days after flowering (DAF), 10 DAF, 15 DAF and 20 DAF compared to wild type. GUS sub-cellular localization showed the staining in seed which further validated the results obtained from gene expression. Based on these findings it can be concluded Glu-B gene have significant role in controlling GLU contents and can be utilized in breeding programs to enhance the nutritional quality of rice, and may serve as healthy diet for patient allergic with high GLU contents.

Keywords: Amino acid composition; Irregularly-shaped protein bodies II; Low glutelin rice; Mutation breeding; Nutritional quality.

Fig. 1

CRISPR/Cas9 mediated editing of Glu-B gene. (A). The structure of the T-DNA region of the Cas9/guide RNA (gRNA) vector. Marker gene Hygromycin (Hyg) was driven by the CaMV35S (35 S) promoter whereas the gRNA was driven by the rice U6 promoter and the mpCas9 was driven by the Ubiquitin (Ubi) promoter. LB, Left border; RB, Right border. (B) The structure of Glu-B gene, two target sites were selected and sequenced in the mutant genotypes. (C) The Glu-A target site aligned with the Glu-B-1 (a 2 nucleotides deletion) and the Glu-B-2 (3 nucleotide deletion) mutant sequences in the same plant

Fig. 2

The performance of cv. PK386 (WT) and glu-A mutant. (A) Protein content (%), (B) Glutelin content (%), (C) Globulin content (%), (D) Albumin content (%), (E) Prolamin content (%), (F) Total starch content (%), (G) Amylose content (%), (H) Gel consistency (mm), (I) Gelatinization temperature (^oC), (J) Total Sugar content (%), (K) Paste viscosity; red curve shows PK386, Green curve shows glu-A mutant. Data are given as means ± SD from three replicates. Statistical comparisons were performed using Student’s t-test; all data were compared with WT (*P < 0.05, **P < 0.01)

Fig. 3

The agronomic and appearance quality related traits for WT and glu-A. Data are given as means ± SD from three replicates. Statistical comparisons were performed using Student’s t-test; all data were compared with WT (*P < 0.05, **P < 0.01)

Fig. 4

Scanning electron microscopy images for WT and glu-b (T₂ generation). (A) Mature endosperm of WT. (B) Central region of mature endosperm in WT (C) Peripheral region in mature endosperm in WT. (D) Mature endosperm of glu-b mutant. (E) Central region of mature endosperm in glu-b mutant. (F) Peripheral region in mature endosperm in glu-b mutant

Fig. 5

glu-b expression in cv. PK386 and T₂ generation of glu-b mutant. Transcript abundance as estimated by qRT-PCR in the R (roots), S (stem), L (leaves), LS (leaf sheath) at 5, 10, 15 and 20 DAF (days after flowering). Data are given as means ± SD from three replicates

Fig. 6

GUS expression in A, leaf B, leaf sheath C, stem D, root E, panicle F, brown rice and G, cut brown rice. Bars: 2 mm