Characterization and Evaluation of OsLCT1 and OsNramp5 Mutants Generated Through CRISPR/Cas9-Mediated Mutagenesis for Breeding Low Cd Rice

doi:10.1016/j.rsci.2019.01.002

Abstract

Abstract:

To explore how rice (Oryza sativa L.) can be safely produced in Cd-polluted soil, OsLCT1 and OsNramp5 mutant lines were generated by CRISPR/Cas9-mediated mutagenesis. One of OsLCT1 mutant (lct1×1) and two of OsNramp5 mutants (nramp5×7 and nramp5×9) were evaluated for grain Cd accumulation and agronomic performances. In paddy field soil containing approximately 0.9 mg/kg Cd, lct1×1 grains contained approximately 40% (0.17 mg/kg) of the Cd concentration of the wild type parental line, less than the China National Food Safety Standard (0.20 mg/kg). Both OsNramp5 mutants showed low grain Cd accumulation (< 0.06 mg/kg) in the paddy (approximately 0.9 mg/kg Cd) or in pots in soil spiked with 2 mg/kg Cd. However, only nramp5×7 showed normal growth and yield, whereas the growth of nramp5×9 was severely impaired. The study showed that lct1×1 could be used to produce rice grains safe for human consumption in lightly contaminated paddy soils and nramp5×7 used in soils contaminated by much higher levels of Cd.

Key words: cadmium, rice, OsNramp5, OsLCT1, genome-editing, heavy metal contamination, CRISPR, Cas9

Songmei Liu, Jie Jiang, Yang Liu, Jun Meng, Shouling Xu, Yuanyuan Tan, Youfa Li, Qingyao Shu, Jianzhong Huang. Characterization and Evaluation of OsLCT1 and OsNramp5 Mutants Generated Through CRISPR/Cas9-Mediated Mutagenesis for Breeding Low Cd Rice[J]. Rice Science, 2019, 26(2): 88-97.

Figures/Tables 5

Fig. 1. Schematic diagram of the genes and sgRNA target sites for CRISPR/Cas9-mediated mutagenesis of OsLCT1 (A) and OsNramp5 (B), together with a hydrophobic plot of the wild type (WT) and mutant proteins.Exons, introns, and untranslated regions are indicated by solid boxes, lines, and hollow boxes, respectively. The protospacer adjacent motif sequences (Meharg et al, 2013) prior to the target sequences are boxed with dot lines. The primers Pe1-F/-R, Pe7-F/-R and Pe9-F/-R used for genotyping the mutations and their positions are indicated by arrows. Mutations within or around the target sequences of the mutant lines lct1×1, nramp5×7, and nramp5×9 are shown in lowercase letters (for insertion) or ‘-’ (for deletion). The hydrophobicity (red curves) and the estimated transmembrane domains (filled boxes) of the predicted proteins were drawn using the SOSUI program (http://harrier.nagahama-i-bio.ac.jp/sosui/). For nramp5×9, the position of the deletion is shown in a circle in the WT hydrophobic plot and the deleted amino acid sequence is shown by uppercase letters.

Table 1. Development and analysis of T0 rice plants for targeted mutagenesis of OsLCT1 and OsNramp5.

Plasmid	No. of transgenic plants	No. of plants tested for mutation	No. of mutated plants
Plasmid	No. of transgenic plants	No. of plants tested for mutation	Total	Monoallelic heterozygous	Biallelic heterozygous	Biallelic homozygous
pH-lct1×1	69	69	15	10	5	0
pH-nramp5×7	6	6	1	0	1	0
pH-nramp5×9	49	4 ^a	4	0	3	1
^a Due to technical reasons, we failed to amplify the fragment encompassing the target site in exon 9 (Fig. 1-B) for the other plants.

Fig. 2. Mutations identified within and around the target sites of OsLCT1 and OsNramp5 generated through CRISPR/Cas9-mediated genome editing in rice in T1 generation.The protospacer adjacent motif sequences are boxed. The 20-nt target sequences are underlined. Dashes indicate deletions and lowercase letters indicate inserted nucleotides.

Fig. 3. Cadmium content in brown rice grains of the mutant lines nramp5×7, nramp5×9, lct1×1 and wild type Xidao 1.A, Pot experiment in 2016. B, Pot experiment in 2017. C, Field experiment in 2017. Plants were grown in soil spiked with 2 or 10 mg/kg Cd. Different letters above columns indicate significant difference at the 0.05 level. The data presented are Mean ± SE (n = 3).

Fig. 4. Plant morphology (A) and yield-related traits (B) of Xidao 1 and its OsLCT1 and OsNramp5 mutants grown in a paddy field at Hangzhou, China, in 2017.G1, Xidao 1; G2, nramp5×7; G3, nramp5×9; G4, lct1×1. Data are presented as Mean ± SE (n = 6). Different letters above columns indicate significant differences at the 0.05 level.

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