利用CRISPR/Cas9技术创制Rc基因恢复红稻

doi:10.16819/j.1001-7216.2022.211205

中国水稻科学 ›› 2022, Vol. 36 ›› Issue (6): 572-578.DOI: 10.16819/j.1001-7216.2022.211205

利用CRISPR/Cas9技术创制Rc基因恢复红稻

张元野¹, 尹丽颖¹, 李荣田¹^,^*(), 何明良², 刘欣欣³, 潘婷婷⁴, 田晓杰², 卜庆云², 李秀峰²^,^*()

1.黑龙江大学生命科学学院, 哈尔滨 150080
2.中国科学院东北地理与农业生态研究所, 哈尔滨 150081
3.东北林业大学生命科学学院，哈尔滨 150040
4.黑龙江八一农垦大学农学院, 黑龙江大庆 163319

收稿日期:2021-12-07 修回日期:2022-03-10 出版日期:2022-11-10 发布日期:2022-11-10
通讯作者: 李荣田,李秀峰
基金资助:
黑龙江省杰出青年基金资助项目(JQ2020C003);中国科学院战略性先导科技专项(A类)(XDA28100000)

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

ZHANG Yuanye¹, YIN Liying¹, LI Rongtian¹^,^*(), HE Mingliang², LIU Xinxin³, PAN Tingting⁴, TIAN Xiaojie², BU Qingyun², LI Xiufeng²^,^*()

1. College of Life Science, Heilongjiang University, Harbin 150080, China
2. Northeast Institute of Geography and Agroecology, Key Laboratory of Soybean Molecular Design Breeding, Chinese Academy of Sciences, Harbin 150081, China
3. College of Life Science, Northeast Forestry University, Harbin 150040, China
4. College of Agriculture, Heilongjiang Bayi Agricultural University, Daqing 163319, China

Received:2021-12-07 Revised:2022-03-10 Online:2022-11-10 Published:2022-11-10
Contact: LI Rongtian, LI Xiufeng

摘要/Abstract

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

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

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.

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.

图/表 6

图1 Rc基因结构和靶点位置黑色序列为靶点序列，灰色下划线序列为PAM序列。

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.

表1 本研究中所用的引物

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

图2 T0代植株转基因检测 M―DM2000 DNA 标记; 1~14―T0 代植株; 15―阳性对照; 16―阴性对照。

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

图3 T1代转基因植株表型及测序结果鉴定 A―红米性状恢复株系与野生型表型对比（标尺=1 cm）；B―红米性状恢复株系与野生型碱基序列比对；C―红米性状恢复株系与野生型氨基酸序列比对; D―红米性状恢复株系与野生型bHLH结构域比对（红框内为bHLH结构域）。

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).

图4 T2代转基因植株耐盐碱能力鉴定 A~D分别为0、30、100、200 mmol/L NaCl溶液处理下各组的芽长；E~F分别为20和10 mmol/L Na2CO3下各组的芽长；G―200 mmol/L NaCl处理的芽长（标尺=2 cm）；H―20 mmol/L Na2CO3处理的幼苗生长情况（标尺=2 cm）。

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).

图5 T2代转基因植株耐盐碱相关基因相对表达量鉴定 A~C―200 mmol/L NaCl处理下耐盐标记基因表达量；D~F―20 mmol/L Na2CO3处理下耐碱标记基因表达量；G~I―200 mmol/L NaCl、20 mmol/L Na2CO3及空白对照Rc表达量。ns表示无显著差异、**、***、****表示分别在0.005、0.001、0.0001水平上显著差异（t检验）。KY180―空育180；SY453―上育453；KY-1―空育180的基因编辑株系；SY-1―上育453的基因编辑株系。

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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利用CRISPR/Cas9技术创制Rc基因恢复红稻

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

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