Scientia Agricultura Sinica ›› 2024, Vol. 57 ›› Issue (2): 227-235.doi: 10.3864/j.issn.0578-1752.2024.02.001

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

CRISPR/Cas9-Mediated Editing of MODD Enhances Rice Dormancy

GUO NaiHui1,2(), ZHANG WenZhong1(), SHENG ZhongHua2(), HU PeiSong1,2()   

  1. 1 Rice Research Institute, Shenyang Agricultural University, Shenyang 110866
    2 China National Rice Research Institute/China National Rice Improvement Centre, Hangzhou 310006
  • Received:2023-06-13 Accepted:2023-07-24 Online:2024-01-16 Published:2024-01-19

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Abstract:

【Objective】 Dormancy is an important agronomic trait of rice. Proper dormancy can inhibit the preharvest sprouting of rice and is a key factor to ensure yield and quality. However, the genes and regulatory networks of rice dormancy regulation still need further study. The MODD encoded a protein with unknown function, and it negatively regulate rice abscisic acid signaling and drought resistance, but its function in regulating rice dormancy is unknown. Studying the function of MODD in regulating rice dormancy will help to improve the rice dormancy regulatory network, and at the same time provide a new theoretical basis and germplasm resources for genetic breeding of preharvest sprouting resistance.【Method】 Based on the gene sequences published in the RGAP database, a CRISPR-Cas9 knockout vector for MODD was constructed, and the calli of Zhonghua 11 was transformed through agrobacterium mediated genetic transformation to obtain transgenic rice plants. The MODD knockout homozygous lines were screened and identified using PCR amplification, sequencing technology, and qRT-PCR technology. The amino acid sequences of the two mutant lines (KO-1 and KO-2) were obtained according to the CDS of the two mutant lines, and then the protein sequences of ZH11 and the two mutant lines (KO-1 and KO-2) were compared by DNAMAN. The homologous genes of MODD in rice were screened using Linux system. Take the seeds 35 days after heading and investigated the germination rate of ZH11 and knockout lines. The yeast hybridization and LUC experiments were used to verify the upstream gene of MODD. 【Result】 Six MODD homologous genes were found in rice, which were LOC_Os07g41160, LOC_Os03g30570, LOC_Os03g53630, LOC_Os04g35430, LOC_Os03g17050, LOC_Os06g01170. The knockout vector was successfully constructed and transferred it into ZH11, two homozygous mutant lines (KO-1 and KO-2) were obtained. The qRT-PCR results showed that the expression level of MODD in the two mutant line (KO-1 and KO-2) was significantly reduced. Protein sequence analysis showed that the frameshift mutations of KO-1 and KO-2 caused the early termination of protein translation. The germination rate of the two mutant lines (KO-1 and KO-2) was significantly lower than that of ZH11 by 15% and 15% respectively on the third day after water absorption; After that, the difference gradually expanded and reached the maximum on the 6th day, which was significantly lower than that of ZH11 by 35% and 35% respectively. The preharvest sprouting of two mutant lines (KO-1 and KO-2) was significantly lower than that of ZH11. The results of Y1H experiment showed that ABI5 could bind to the promoter region of MODD in yeast, and the binding range was further reduced to less than 300bp. LUC results showed that the fluorescence value of ABI5 was 2.6 times that of none alone, indicating that ABI5 could activate the expression of MODD.【Conclusion】 Knocking out MODD could increase seed dormancy, and MODD may regulate seed dormancy through ABA signaling pathway.

Key words: rice, dormancy, ABI5, MODD

Table 1

The primers used in this study"

引物名称Primer name 引物序列Primer sequence (5′-3′) 用途Usage
MODD-CRISPR-F TGTGTGGGATGAATGGAGGAAGCGAA CRISPR/Cas9载体构建
CRISPR/Cas9 vector construction
MODD-CRISPR-R AAACTTCGCTTCCTCCATTCATCCCA
CX-F TCCCAGTCACGTTGTAA 筛选阳性克隆Screening for positive clones
pLACZ-MODD-F ATCTGTCGACCTCGAGACTCAAACGGACGAAATATGTGG 酵母单杂Y1H
pLACZ-MODD-R GAGCACATGCCTCGAGTCCGGCAAGCTAGCTAGCCA
PB42AD-ABI5-F TGCCTCTCCCGAATTCATGGCATCGGAGATGAGCAA
PB42AD-ABI5-R CGAGTCGGCCGAATTCTCACCACATGCAGCTGCCGC
pLACZ-MODD-1-F ATCTGTCGACCTCGAGACTCAAACGGACGAAATATGTGG
pLACZ-MODD-1-R GAGCACATGCCTCGAGGGCCGCTGCGACGAG
pLACZ-MODD-2-F ATCTGTCGACCTCGAGGTACGGCCCGGCCC
pLACZ-MODD-2-R GAGCACATGCCTCGAGTTCTCACCCGATCCACACC
pLACZ-MODD-3-F ATCTGTCGACCTCGAGACGTGTCGGTGTGGAGGG
pLACZ-MODD-3-R GAGCACATGCCTCGAGGAGCCGAAGCCGACCAG
pLACZ-MODD-4-F ATCTGTCGACCTCGAGCTACGGCGATGGTCGGCTTGA
pLACZ-MODD-4-R GAGCACATGCCTCGAGTCCGGCAAGCTAGCTAGCCA
p190Luc-MODD-F GGCCAGTGCCAAGCTTACTCAAACGGACGAAATATGTGG LUC活性检测 LUC activity test
p190Luc-MODD-R AGGGTCTTGCAGATCTTCCGGCAAGCTAGCTAGCCA
NONE-ABI5-F TAGAACTAGTGGATCCATGGCATCGGAGATGAGCA
NONE-ABI5-R GCTTGATATCGAATTCTCACCACATGCAGCTGCC
OsActin-qF GTCCTCTTCCAGCCTTCCTT 实时荧光定量分析 qRT-PCR
OsActin-qR CTCATCCTGTCAGCAATGCC
MODD-qF GCAAGAGGGTTGAAGGCTTC
MODD-qR GGGATAGGGTTGACGACGAT

Fig. 1

Homology analysis of MODD gene The red line represented the gene registration number of MODD"

Fig. 2

Schematic diagram of MODD target site and BGK03 vector A: Schematic diagram of MODD target site, the red triangle represented the target position; B: Schematic diagram of BGK03 vector, the red arrow represented the insertion position of gRNA"

Fig. 3

Identification of modd mutant line A: Base types of the two mutant lines, the protospacer adjacent motif (PAM) site is depicted as underline and blue, deleted nucleotides are depicted as dashes, and inserted nucleotides are shown in red; B: Sequencing results of two mutant lines; C: MODD gene expression of the two mutant lines, **: P<0.01, the same as below; D: Amino acid types of two mutant lines, the dark background represented the same amino acids as the wild-type and mutant lines. KO-1 and KO-2: MODD mutant lines"

Fig. 4

Germination phenotype of modd mutant A: Pre-harvest sprouting phenotype of modd mutant and ZH11 on the sixth day of germination; B: Germination rate of modd mutant and ZH11; C: Germination rate of modd mutant and ZH11 after breaking dormancy"

Fig. 5

The expression of MODD was activated by ABI5 A: ABI5 combined with the promoter of MODD was verified by Y1H; B: Reduce the promoter region of MODD where ABI5 binding, the red font represented the region where ABI5 binds to the MODD promoter; C: LUC verification of ABI5 activating the expression of MODD"

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