利用CRISPR/Cas9技术编辑OsOFP30基因创制水稻粒型突变体

doi:10.16819/j.1001-7216.2024.231002

中国水稻科学 ›› 2024, Vol. 38 ›› Issue (5): 507-515.DOI: 10.16819/j.1001-7216.2024.231002

利用CRISPR/Cas9技术编辑OsOFP30基因创制水稻粒型突变体

何勇¹^,^#, 刘耀威¹^,^#, 熊翔¹, 祝丹晨¹, 王爱群¹, 马拉娜¹, 王廷宝¹, 张健², 李建雄³, 田志宏¹^,^*()

¹长江大学生命科学学院/湿地生态与农业利用教育部工程研究中心, 湖北荆州 434025
²中国水稻研究所水稻生物育种全国重点实验室, 杭州 311401
³广西大学农学院/亚热带农业生物资源保护与利用国家重点实验室, 南宁 530004

收稿日期:2023-10-09 修回日期:2024-08-10 出版日期:2024-09-10 发布日期:2024-09-10
通讯作者: *email: zhtian@yangtzeu.edu.cn
作者简介:^#共同第一作者
基金资助:
水稻生物学国家重点实验室开放课题(20200106);亚热带农业生物资源保护与利用国家重点实验室开放课题(SKLCUSA-b202305);长江大学大学生创新创业训练计划项目(Yz2022223)

Creation of Rice Grain Size Mutants by Editing OsOFP30 via CRISPR/Cas9 System

HE Yong¹^,^#, LIU Yaowei¹^,^#, XIONG Xiang¹, ZHU Danchen¹, WANG Aiqun¹, MA Lana¹, WANG Tingbao¹, ZHANG Jian², LI Jianxiong³, TIAN Zhihong¹^,^*()

¹College of Life Science / Engineering Research Center of Ecology and Agricultural Use of Wetland, Ministry of Education, Yangtze University, Jingzhou 434025, China
²State Key Laboratory of Rice Biology and Breeding, China National Rice Research Institute, Hangzhou 311401, China
³College of Agriculture / State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi University, Nanning 530004, China

Received:2023-10-09 Revised:2024-08-10 Online:2024-09-10 Published:2024-09-10
Contact: *email: zhtian@yangtzeu.edu.cn
About author:^#These authors contributed equally to this work

摘要/Abstract

摘要：

【目的】研究水稻OFP家族转录因子OsOFP30对粒型的影响，创制新的粒型突变材料，为水稻粒型改良提供参考。【方法】以粳稻品种中花11为受体材料，利用CRISPR/Cas9技术对OsOFP30基因进行定向编辑；通过T₀、T₁和T₂代连续筛选，获得3类无外源片段插入的纯合突变体（长片段删除突变OsOFP30⁻⁸⁹、单碱基插入突变OsOFP30^+1G和OsOFP30^+1A）；分析粒型变异，进行生物信息学和测序分析，初步确定粒型变异原因。【结果】与野生型相比，3类突变体的粒宽和千粒重都显著降低，OsOFP30⁻⁸⁹和OsOFP30^+1G仅粒长和粒厚显著降低，穗型指标与野生型无明显差异，OsOFP30^+1A的粒长和粒厚与野生型无明显差异，仅一次枝梗数显著低于野生型；生物信息学分析表明，这3类突变体均由移码突变导致的翻译提前终止所产生，OsOFP30⁻⁸⁹突变蛋白长度为252个氨基酸，OsOFP30^+1G和OsOFP30^+1A突变蛋白长度均为282个氨基酸，两者仅第323位的核酸序列存在G和A的单碱基差异，导致突变蛋白第108位产生丝氨酸和天冬酰胺的差别。【结论】初步判断OsOFP30基因可调控水稻粒型变异。本研究创制了新的粒型突变材料，可为水稻粒型改良育种提供参考。

关键词: 水稻, OsOFP30, CRISPR/Cas9, 基因编辑, 粒型

Abstract:

【Objective】To study the role of OsOFP30, a transcription factor of the rice OFP family, in regulating grain size and to create new mutant materials of grain size, thereby providing a new reference for the improvement of rice grain size.【Method】The japonica rice line Zhonghua 11 was utilized as a transgenic recipient to generate OsOFP30 mutants using the CRISPR/Cas9 system. Through continuous screening of the T₀, T₁, and T₂ generations, three types of homozygous mutants without foreign fragment insertions were obtained: the long segment deletion mutation OsOFP30⁻⁸⁹, the single base insertion mutation OsOFP30^+1G, and OsOFP30^+1A. Grain size was measured at maturity stages, and the potential reasons for the different grain sizes were analyzed through bioinformatics and sequencing.【Results】 Compared with the wild-type, the grain width and 1000-grain weight of all three types of mutants were significantly reduced. For OsOFP30⁻⁸⁹ and OsOFP30^+1G, only the grain length and grain thickness are significantly reduced, with no obvious differences in panicle shape indicators from the wild type. The grain length and grain thickness of OsOFP30^+1A show no obvious differences from the wild type, and only the number of primary branches is significantly lower than that of the wild type. Bioinformatics analysis revealed that the three types of mutants introduced premature translation termination due to a frameshift mutation. The OsOFP30⁻⁸⁹ mutant protein consists of 252 amino acids, while the OsOFP30^+1G and OsOFP30^+1A mutant proteins consist of 282 amino acids. Sequence analyses showed that OsOFP30^+1G and OsOFP30^+1A differ by a single base, with G and A at the 323rd position, respectively. This variation resulted in a change between serine and asparagine at the 108th position of the mutant protein. 【Conclusion】The OsOFP30 gene plays a crucial role in regulating rice grain size. This study successfully created new mutant materials affecting grain size, which will provide valuable references for the improvement of rice grain size.

Key words: rice, OsOFP30, CRISPR/Cas9, gene editing, grain size

何勇, 刘耀威, 熊翔, 祝丹晨, 王爱群, 马拉娜, 王廷宝, 张健, 李建雄, 田志宏. 利用CRISPR/Cas9技术编辑OsOFP30基因创制水稻粒型突变体[J]. 中国水稻科学, 2024, 38(5): 507-515.

HE Yong, LIU Yaowei, XIONG Xiang, ZHU Danchen, WANG Aiqun, MA Lana, WANG Tingbao, ZHANG Jian, LI Jianxiong, TIAN Zhihong. Creation of Rice Grain Size Mutants by Editing OsOFP30 via CRISPR/Cas9 System[J]. Chinese Journal OF Rice Science, 2024, 38(5): 507-515.

图/表 5

表1 本研究用到的引物列表

Table 1. List of primers used in this study

引物名称 Primer name	引物序列（5’→3’） Sequence(5’→3’)
gRT-OFP30	CAGATTCTCCACGACGACGGGTTTTAGAGCTAGAAAT	靶点扩增Target amplification
OsU6aT-OFP30	CCGTCGTCGTGGAGAATCTGCGGCAGCCAAGCCAGCA
U-F	CTCCGTTTTACCTGTGGAATCG	1^stPCR通用引物General primers for the first round PCR
gR-R	CGGAGGAAAATTCCATCCAC	1^stPCR通用引物General primers for the first round PCR
Pps-GGL	TTCAGAggtctcTctcg$\boxed{\hbox{ACTAGT}}$ATGGAATCGGCAGCAAAGG	2^nd PCR通用引物General primers for the second round PCR
Pgs-GGR	AGCGTGggtctcGaccg$\boxed{\hbox{ACGCGT}}$ATCCATCCACTCCAAGCTC	2^nd PCR通用引物General primers for the second round PCR
CrisprCX-F	CGGTGTCATCTATGTTACTA	菌检及测序
CrisprCX-R	GCAATAACTTCGTATAGGCT	Detection of positive clones and sequencing
Hgy-F	GGACTTCGGGGCAGTCCT	潮霉素抗性基因检测Detection of hygromycin resistance gene
Hgy-R	CGATGTAGGAGGGCGTGG	潮霉素抗性基因检测Detection of hygromycin resistance gene
XOFP30F	TTCCGCGGGTTCTACTAC	靶点所在区域基因序列扩增Amplification of the gene sequence in the region where the target site is located
XOFP30R	TCTTTCTCCTCCTCCGCTGG
XOFP30L	GGGAGGTTGAGGAAGGGT	阳性植株测序Sequencing of positive plants
OsOFP30-8gOFF-1F	CGTCGCAGCCACCTTACAAT	潜在高概率脱靶位点检测Detection of potential high-probability off-target sites
OsOFP30-8gOFF-1R	GCATTGTCGTCGTTGGGTGA
OsOFP30-8gOFF-1Seq	GCCGCCAACACCAACCAA
OsOFP30-8gOFF-2F	CATTGCCGGGCTGAACTCTT
OsOFP30-8gOFF-2R	TGGAGCTTGACGATGTTGCG
OsOFP30-8gOFF-2Seq	ACGGCCACTCCGGTAAGA
OsOFP30-10gOFF-F	TTCGCGACACAGCTCAAGTC
OsOFP30-10gOFF-R	TGCGACCTCCCCTTCTTCTT
OsOFP30-10gOFF-Seq	AAGTGCCGAACGAGCCGT

表2 潜在脱靶位点信息

Table 2. Information of potential off-target sites

编号Number	脱靶位点的位置 Position of off-target site	脱靶位点的核苷酸序列 Sequences of off-target site	错配碱基数 Number of mismatch	脱靶概率 Off-score
1	Chr08: −27321337	gAGcTcCTCCgCGACGACGG CGG	4	0.190
2	Chr08: −11564691	CtGcTgCTCCACGACGACGG CGG	3	0.147
3	Chr10: +17270887	CgccTTCTCtACGACGACGG CGG	4	0.102

图1 OsOFP30 sgRNA靶点、重叠PCR结果及阳性克隆筛选 A: OsOFP30靶点; B: 重叠PCR (M: DL2000 DNA标记; CK: 空白对照; 1st: 重叠PCR第一轮PCR产物; 2nd：重叠PCR第二轮PCR产物)；C: PCR筛选阳性克隆(M: DL2000 DNA标记; CK: 空白对照; 1~11: 待检测菌株克隆)。

Fig. 1. Schematic diagram of OsOFP30 target site, the result of overlapping PCR and screening for positive clones A, Schematic diagram of OsOFP30 target site; B, Result of overlapping PCR(M, DL2000 DNA Marker; CK, Blank control; 1st, Overlapping PCR products of the first round; 2nd, Overlapping PCR products of the second round); C, PCR screening for positive clones by PCR(M, DL2000 DNA Marker; CK, Blank control; 1-11, Clones of strain to be tested).

图2 OsOFP30阳性植株筛选与无外源片段插入纯合突变体类型分析 A: T0部分转基因阳性植株HPT抗性基因PCR检测（M: DL2000 DNA标记）; B: T0部分转基因阳性植株Cas9蛋白编码基因PCR检测（M: Trans2K® Plus II DNA Marker; C: 无外源片段插入纯合突变体类型; D: 无外源片段插入纯合突变体编码蛋白结构示意。CK: 空白对照; ―: 阴性对照; +: 阳性对照; 1-14: 检测样品; OsOFP30代表野生型; OsOFP30右上角数字或字母代表不同的突变体类型; 黑色框代表保守结构域; 示意图上方数字代表从该基因ATG编码蛋白开始的顺序。

Fig. 2. Screening of OsOFP30 positive plants and analysis of homozygous mutants without foreign fragment insertion A, PCR detection of HPT resistance gene in some positive transgenic plants of T0 generation (M, DL2000 DNA Marker); B, PCR detection of Cas9 protein-coding gene in some positive transgenic plants of T0 generation (M, Trans2K® Plus II DNA Marker); C, The homozygous mutant type without foreign fragment insertion; D, The structure of the protein encoded by the homozygous mutant without foreign fragment insertion. CK, Blank control; -, Negative control; +, Positive control; 1-14, Test sample. OsOFP30 represents wild-type, the number or letter in the upper right corner of OsOFP30 represents different mutant types, the black box represents the structural domain, and the number at the top of the schematic diagram represents the sequence starting from the ATG encoded protein of the gene.

图3 OsOFP30突变体穗型和粒型观察 A: 穗长(bar=10 cm); B: 穗型(bar=10 cm); C: 穗长; D: 一次枝梗数; E: 单穗重; F:粒长(bar=1 cm); G:粒宽(bar=1 cm); H: 粒长; I: 粒宽; J: 粒厚; K: 千粒重。图中OsOFP30代表野生型，OsOFP30右上角带数字或字母代表不同的突变体类型。**和*分别代表突变体与野生型间的差异达0.01和0.05显著水平。

Fig. 3. Panicle phenotypes and grain shape of OsOFP30 mutant A, Panicle length (bar=10 cm); B, Panicle phenotypes; C, Panicle length (bar=10 cm); D, Number of primary branches; E, Grain weight per panicle; F, Grain length (bar=1 cm); G, Grain width (bar=1 cm); H, Grain length; I, Grain width; J, Grain thickness; K, 1000-grain weight. OsOFP30 represents the wild type, and the number or letter at the upper right corner of OsOFP30 represents different mutant types. ** and * represent that the difference between the mutant and the wild type reaches the significant levels of 0.01 and 0.05, respectively.

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利用CRISPR/Cas9技术编辑OsOFP30基因创制水稻粒型突变体

Creation of Rice Grain Size Mutants by Editing OsOFP30 via CRISPR/Cas9 System

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