Breeding of Rc Function Restoration Red Rice via CRISPR/Cas9 Mediated Genome Editing

doi:10.16819/j.1001-7216.2022.211205

Abstract

Abstract:

【Objective】 It is of great value to restore cultivated rice varieties to red rice with good rice quality and strong stress resistance. CRISPR/Cas9 gene editing technology was used to edit the Rc gene, restoring the red seed coat, and laying a data foundation for the improvement of rice quality and resistance.【Methods】 Using CRISPR/Cas9 technology, the target gene Rc mutant vector pYLCRISPR/ Cas9-Rc-gRNA was constructed and transformed into transgenic plants with Kongyu 180 and Shangyu 453 as materials. The results were verified by sequencing and phenotypic observation. 【Results】 Two Rc mutants were obtained. For KY-1, with 4 bases deleted from 1414 bp to 1417 bp, the terminator was replaced by phenylalanine. SY-1 also lost a base at 1411 bp, which resulted in the transformation from the terminator to aspartic acid. The two kinds of editing materials restored red rice phenotype and had certain saline-alkali tolerance.【Conclusion】 Using CRISPR/Cas9 technique, we obtained homozygous lines with red seed coat, providing basic materials for the improvement of red rice.

Key words: rice, Rc, red episperm, salinity tolerance, CRISPR/Cas9

摘要：

【目的】将栽培稻品种恢复为米质优、抗逆性强的红稻具有较大的研究价值。利用CRISPR/Cas9基因编辑技术，编辑原花青素转录调节因子Rc基因，恢复红种皮特性，以改良水稻米质，提升抗逆性。【方法】利用CRISPR/Cas9技术，以Rc为靶基因，构建突变载体pYLCRISPR/Cas9-Rc-gRNA，以空育180、上育453为材料，转化获得转基因植株，通过测序手段和表型观察验证成果。【结果】分子水平检测获得Rc突变材料2种，其中KY-1在1414―1417 bp缺失4个碱基，终止子突变为苯丙氨酸；SY-1在1411 bp处缺失1个碱基，终止子突变为天冬氨酸。2种编辑材料均恢复为红米表型，且具有一定耐盐碱能力。【结论】利用CRISPR/Cas9基因编辑技术成功获得恢复红种皮表型的纯合株系，为红米改良提供基础材料。

关键词: 水稻, Rc, 红种皮, 耐盐碱, CRISPR/Cas9

ZHANG Yuanye, YIN Liying, LI Rongtian, HE Mingliang, LIU Xinxin, PAN Tingting, TIAN Xiaojie, BU Qingyun, LI Xiufeng. Breeding of Rc Function Restoration Red Rice via CRISPR/Cas9 Mediated Genome Editing[J]. Chinese Journal OF Rice Science, 2022, 36(6): 572-578.

张元野, 尹丽颖, 李荣田, 何明良, 刘欣欣, 潘婷婷, 田晓杰, 卜庆云, 李秀峰. 利用CRISPR/Cas9技术创制Rc基因恢复红稻[J]. 中国水稻科学, 2022, 36(6): 572-578.

Figures/Tables 6

Fig. 1. Gene structure and target site of Rc. The black sequence is the target sequence and the gray underlined sequence is the PAM sequence.

Table 1. Primers used in this research.

引物名称 Primer name	引物序列（5'-3'） Primer sequence（5'-3'）
CAS-U6a-Rc1-LP	GCCGCGCAAGTGGATGCCATCCA
CAS-U6a-Rc1-RP	AAACTGGATGGCATCCACTTGCG
CAS-U6b-Rc2-LP	GTTGGCACTGAAATCACCTTGGA
CAS-U6b-Rc2-RP	AAACTCCAAGGTGATTTCAGTGC
M13-F	GTAAAACGACGGCCAGT
Rc-F	CACAGAGAATGCTCAAGA
Rc-R	CGGCTTTATAGAAATAGAGG
HPT-F	TGCGCCCAAGCTGCATCAT
HPT-R	TGAACTCACCGCGACGTCTGT
qRT-bZIP71-F	AGGGATGATGAGGAACTTGG
qRT-bZIP71-R	GGTATGCATGCGATCATTTC
qRT-NHX1-F	ACATTGGAACGCTGGATGTA
qRT-NHX1-R	TCACAACACCTTCACCGAAT
qRT-CHX11-F	AGATCCTGGGTGGCATCTT
qRT-CHX11-R	AGGAAGAGGAAGAGCAGCAG
qRT-IRO2-F	TCGCCGTGGCTGGACCTAGAC
qRT-IRO2-R	CCCCAACACTCCTGGTTTGCAG
qRT-IDEF1-F	GAGGCTAGTCTTCCACCTTTG
qRT-IDEF1-R	TGGCCAGTACCTGTACTTAAAC
qRT-IRT1-F	TCACCGCATCTGGTCATACT
qRT-IRT1-R	GAGATTGAGGAGAGGCTTGG
qRT-Rc-F	ACACTAACAACACTGACACT
qRT-Rc-R	CTCTTACCACTTCTGACATCT

Fig. 2. Transgenic detection of T0 generation plants. M, DM2000 DNA marker; 1-14, T0 generation plants; 15, Positive control; 16, Negative control.

Fig. 3. Phenotype and sequencing identification of T1 transgenic plants. A, Phenotype comparison of red rice and wild type(bar=1 cm); B, Sequence comparison between red rice and wild type; C, Comparison of amino acid sequences between red rice and wild type; D, Comparison of bHLH domains between red rice and wild type(the bHLH domain is in the red box).

Fig. 4. Identification of salt and alkali tolerance of T2 generation transgenic plants. A, Bud length in each group under different concentrations of salt treatment; E-F, Bud length in each group under different concentrations of alkali treatment; G, Growth of seedlings treated with 200 mmol/L NaCl (scale=2 cm); H, Growth of seedlings treated with 20 mmol/L Na2CO3 (scale =2 cm).

Fig. 5. Identification of saline-alkali tolerance related gene expression levels in T2 transgenic plants. A, Salt-tolerant gene expression level under 200 mmol/L NaCl treatment; D-F, Identification of alkali-tolerant gene expression level under 20 mmol/L Na2CO3 treatment; G-I, Rc expression level of 200 mmol/L NaCl, 20 mmol/L Na2CO3 and blank control. ns means no significant difference; **,*** and **** mean significant difference at 0.005, 0.001 and 0.0001 levels, respectively (t test). KY180, Kongyu 180；SY453, Shangyu 453；KY-1, Gene editing line of Kongyu 180; SY-1, Gene editing line of Shangyu 453.

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