水稻DMP1、DMP2、DMP3基因突变体的创制及其单倍体诱导能力鉴定

doi:10.16819/j.1001-7216.2025.231112

摘要/Abstract

摘要：

【目的】探究水稻DMP家族基因的单倍体诱导能力，为单倍体育种提供新的基因资源。【方法】筛选与ZmDMP基因高同源且在花粉中高表达的水稻DMP家族基因作为候选基因；利用CRISPR/Cas9多基因编辑技术，在籼粳杂交稻“春优84”中对筛选的DMP家族基因和单倍体诱导基因OsMTL创制单基因敲除以及多基因组合敲除突变体；对突变体进行形态学观察和花粉育性鉴定；调查统计转基因T₀材料的结实率和单倍体诱导率。【结果】在水稻RAP-DB数据库中共检索到13个玉米单倍体诱导基因ZmDMP的同源基因。OsDMP1和OsDMP2与ZmDMP同源性最高，相似度分别为41.24% 和37.32%；OsDMP1和OsDMP3在花药中表达量最高。因此，选择OsDMP1、OsDMP2、OsDMP3基因为候选基因。通过CRISPR/Cas9基因编辑技术创制了OsDMP1、OsDMP2、OsDMP3的单基因敲除和组合敲除突变体(osdmp1、osdmp2、osdmp3、osdmp1-osdmp2、osdmp1-osdmp3、osdmp1-osdmp2-osdmp3)，OsMTL单基因敲除突变体(osmtl)，以及OsDMP1、OsDMP2、OsDMP3与OsMTL基因的组合敲除突变体(osmtl-osdmp1、osmtl-osdmp2、osmtl-osdmp3、osmtl-osdmp1-osdmp2、osmtl-osdmp1-osdmp3、osmtl- osdmp1-osdmp2-osdmp3)。表型考察发现，相比较野生型，所有突变体的植株形态和花粉育性均未发生显著变化，而仅在包含osmtl的单基因敲除和多基因组合敲除突变体中结实率发生显著下降。单倍体鉴定结果显示，OsDMP1、OsDMP2、OsDMP3与OsMTL组合突变的单倍体诱导效率分别为0.4% ± 0.6%、2.6% ± 2.8%、1.4% ± 0.6%、1.5% ±1.3%、2.1% ± 2.4%、2.2% ± 0.6%，与osmtl突变体(1.5% ± 0.5%)无显著差异。然而，当OsDMP家族基因单个突变或多个组合突变时均无单倍体产生。【结论】本研究利用CRISPR/Cas9基因编辑技术成功创制了水稻OsDMP1、OsDMP2、OsDMP3、OsMTL单基因和多基因组合敲除突变体，发现了OsDMP1、OsDMP2和OsDMP3均无独立单倍体诱导能力，也不能提升OsMTL基因的单倍体诱导效率。本研究促进了对水稻中OsDMP同源基因的了解，为后续单倍体诱导基因研究提供参考。

关键词: 水稻, 单倍体诱导, DMP, OsMTL

Abstract:

【Objective】Exploring the haploid induction ability of rice DMP family genes to provide new gene resources for haploid breeding.【Methods】Selecting OsDMP family genes with high homology to the ZmDMP gene and high expression level in pollen as candidate genes. Using CRISPR/Cas9 multi-gene editing technology to create single-gene knockout and multi-gene combination knockout mutants of the selected OsDMP family genes and haploid induction gene OsMTL in the indica and japonica hybrid rice Chunyou 84. Conducting morphological observations and pollen fertility identification on the mutants. Investigating and statistically analyzing the seed setting rate and haploid induction rate of the T₀ materials. 【Results】A total of 13 homologous genes of maize haploid induction gene ZmDMP were retrieved from the rice RAP-DB database. OsDMP1 and OsDMP2 showed the highest homology with ZmDMP, with similarity percentages of 41.24% and 37.32%, respectively; Moreover, OsDMP1 and OsDMP3 were the most highly expressed OsDMP genes in anthers. Therefore, OsDMP1, OsDMP2 and OsDMP3 were selected as candidate genes. Using CRISPR/Cas9 gene editing technology, single-gene knockout and combined knockout mutants of OsDMP1, OsDMP2, and OsDMP3 were created (osdmp1, osdmp2, osdmp3, osdmp1-osdmp2, osdmp1-osdmp3, osdmp1-osdmp2-osdmp3). Additionally, single-gene knockout mutants of OsMTL were generated (osmtl), as well as combined knockout mutants between OsDMP1, OsDMP2, OsDMP3, and OsMTL (osmtl-osdmp1, osmtl-osdmp2, osmtl-osdmp3, osmtl-osdmp1-osdmp2, osmtl-osdmp1-osdmp3, osmtl-osdmp1-osdmp2-osdmp3). Phenotypic investigation showed that compared with the wild type, plant morphology and pollen fertility of all mutants did not visibly change, but the seed setting rate decreased significantly in single gene knockout and multi-gene combination knockout mutants containing osmtl. Haploid detection results indicated that the haploid induction efficiency of the combined mutants between OsDMP1, OsDMP2, OsDMP3 and OsMTL was 0.4%±0.6%, 2.6%±2.8%, 1.4%±0.6%, 1.5%±1.3%, 2.1%±2.4%, 2.2%± 0.6%, respectively, which was not significantly different from that of osmtl mutant (1.5%±0.5%). However, no haploid was produced when OsDMP family genes were mutated individually or in combination. 【Conclusion】CRISPR/Cas9 gene editing technology was used to create single and multi-gene knockout mutants between OsDMP1, OsDMP2, OsDMP3, and OsMTL in rice. It was found that OsDMP1, OsDMP2 and OsDMP3 had no independent haploid induction ability, nor could it improve the haploid induction efficiency of OsMTL genes. These results promoted the understanding of OsDMP homologous genes in rice, and provided reference for the subsequent studies on the haploid induction gene.

Key words: rice, haploid induction, DMP, OsMTL

胡风越, 王健, 王春, 王克剑, 刘朝雷. 水稻DMP1、DMP2、DMP3基因突变体的创制及其单倍体诱导能力鉴定[J]. 中国水稻科学, 2025, 39(1): 55-66.

HU Fengyue, WANG Jian, WANG Chun, WANG Kejian, LIU Chaolei. Generation of Rice DMP1, DMP2 and DMP3 Mutants and Identification of Their Haploid Induction Ability[J]. Chinese Journal OF Rice Science, 2025, 39(1): 55-66.

图/表 9

表1 水稻DMP同源家族基因

Table 1. The DMP homologous genes in rice

重命名Rename	基因登录号Accession number	描述 Description	长度Length/aa	相似性Identity	E值 E value
OsDMP1	LOC_Os05g48840	Protein of unknown function DUF679 family protein	239	137	3e⁻³³
OsDMP2	LOC_Os08g01530	Protein of unknown function DUF679 family protein	225	132	1e⁻³¹
OsDMP3	LOC_Os01g29240	Protein of unknown function DUF679 family protein	226	120	4e⁻²⁸
OsDMP4	LOC_Os07g22510	Protein of unknown function DUF679 family protein	263	119	9e⁻²⁸
OsDMP5	LOC_Os03g25440	Protein of unknown function DUF679 family protein	194	112	2e⁻²⁵
OsDMP6	LOC_Os06g24490	Protein of unknown function DUF679 family protein	254	110	5e⁻²⁵
OsDMP7	LOC_Os01g65992	Protein of unknown function DUF679 domain containing protein	240	107	4e⁻²⁴
OsDMP8	LOC_Os01g27100	Protein of unknown function DUF679 domain containing protein	319	107	7e⁻²⁴
OsDMP9	LOC_Os01g29330	Protein of unknown function DUF679 family protein	241	100	8e⁻²²
OsDMP10	LOC_Os01g29280	Protein of unknown function DUF679 family protein	265	96.3	1e⁻²⁰
OsDMP11	LOC_Os07g45080	Protein of unknown function DUF679 family protein	189	90.9	5e⁻¹⁹
OsDMP12	LOC_Os01g27120	Protein of unknown function DUF679 domain containing protein	226	83.2	9e⁻¹⁷
OsDMP13	LOC_Os12g23310	Protein of unknown function DUF679 domain containing protein	111	53.1	1e⁻⁷

图1 不同物种DMP蛋白系统进化分析 A. thaliana: 拟南芥；B. distachyon: 二穗短柄草；C. melo: 甜瓜；M. acuminata: 小果野芭蕉；M. truncatula: 苜蓿；P. trichocarpa: 三角叶杨；S. lycopersicum: 番茄； V. vinifera: 葡萄；Z. mays: 玉米。采用MEGA7软件中的最大似然法构建，Bootstrap为1000。ZmDMP、AtDMP8、AtDMP以及水稻中DMP同源蛋白加粗显示。

Fig. 1. Phylogenetic analysis of DMP proteins in various species A. thaliana, Arabidopsis thaliana; B. distachyon, Brachypodium distachyon; C. melo, Cucumis melo; M. acuminate, Musa acuminata; M. truncatula, Medicago truncatul; P. trichocarpa, Populus trichocarpa; S. lycopersicum, Solanum lycopersicum; V. vinifera, Vitis vinifera; Z. mays, Zea mays. The maximum likelihood method in MEGA7 software was adopted. Bootstrap = 1000. ZmDMP, AtDMP8, AtDMP and rice DMP homologous proteins are shown in bold.

图2 同源比对与表达谱分析筛选水稻DMP家族候选基因 A: OsDMP家族蛋白与ZmDMP、AtDMP8、AtDMP9蛋白序列相似性比较；B: OsDMP家族基因的表达谱预测。

Fig. 2. Candidate genes of rice DMP family by homologous comparison and expression profile analysis A, Similarity analysis of OsDMP family proteins with ZmDMP, AtDMP8 and AtDMP9 proteins; B, Online expression analysis of OsDMP family genes.

图3 OsDMP1、OsDMP2、OsDMP3和OsMTL的CRISPR/Cas9多基因敲除载体结构示意图靶基因gRNA的启动子为U3，Cas9蛋白的启动子为Actin。

Fig. 3. Structure schematic diagram of CRISPR/Cas9 multi-gene knockout vectors for OsDMP1, OsDMP2, OsDMP3 and OsMTL genes The promoter of the target gene gRNA is U3, and the promoter of Cas9 protein is Actin.

表2 转基因T0植株突变比例

Table 2. Proportion of mutations in T0 transgenic plants

编号 Code	转化株数 No. of transgenic lines	突变株数 No. of mutant lines	突变株占比（突变株数／转化株数） Proportion of mutant lines (No. of mutant lines / No. of transgenic lines) (%)	双等位突变株数 No. of biallelic mutant lines	双等位突变株占比（双等位突变株数／转化株数） Proportion of biallelic mutant plants (No. of biallelic mutant lines/No. of transgenic lines) (%)
D1	24	21	87.5	18	75.0
D2	21	17	81.0	14	66.7
D3	39	38	97.4	36	92.3
D1-2	24	24	100.0	19	79.2
D1-3	24	23	95.8	11	45.8
D1-2-3	37	35	94.6	24	64.9
osmtl	22	15	68.2	12	54.5
MD1	23	21	91.3	15	65.2
MD2	21	13	61.9	12	57.1
MD3	24	19	79.2	12	50.0
MD1-2	24	15	62.5	9	37.5
MD1-3	24	19	79.2	14	58.3
MD1-2-3	40	33	82.5	14	35.0

图4 转基因T0植株突变类型蓝色字母表示靶位点序列；蓝色下划线表示原间隔相邻基序(Protospacer adjacent motif, PAM)；红色字母表示“插入”突变的碱基；红色中划线表示“缺失”突变。

Fig. 4. Mutations of transgenic T0 plants Blue letters indicate the target site sequences; blue underscores indicate the protospacer adjacent motif (PAM); red letters indicate ‘insertion’ mutant bases; and red hyphens indicate ‘deletion’ mutations.

图5 野生型和突变体形态学观察、花粉育性分析和结实率统计 A~N: 成熟期植株的株型、穗型，标尺为5 cm；a~n: 花粉碘染结果，标尺为100 μm；O: 野生型和突变体结实率比较和分析，采用邓肯新复极差法 (Duncan’s multiple-range test) 进行均值差异显著性分析，字母a、b表示在α = 0.05水平差异显著。

Fig. 5. Morphologic observation, pollen fertility analysis and seed setting rate of wild-type and mutants A-N, The morphology of mature plant and panicle, bar = 5 cm; a-n, Results of pollen iodine staining, bar = 100 μm; O, Comparison and analysis of seed setting rate in wild-type and mutants. Duncan's Multiple Range test was used to analyze the significance of mean difference, and letters a and b indicate significant difference at α = 0.05 level.

图6 分子标记与流式细胞术鉴定植株倍性 A: 水稻12条染色体上的InDel标记在不同材料中的凝胶电泳带型; chr: 染色体(chromosome)的简写；C84: 春优84的父本；16A: 春优84的母本春江16A；Haploid: 单倍体；RID: 重组自交二倍体。B: 二倍体和单倍体的流式细胞术分析；PI: 碘化丙啶(propidium iodide)。

Fig. 6. Ploidy identification with InDel markers and flow cytometry A, Gel electrophoresis band pattern of InDel markers on 12 chromosomes of rice in different materials; chr, Abbreviation of chromosome; C84, Male parent of CY84; 16A, Female parent of CY84; RID, Recombinant inbred diploid. B, Ploidy analysis of diploid and haploid by flow cytometry; PI, Propidium iodide.

表3 不同类型突变体单倍体诱导效率

Table 3. Haploid induction rate of different type mutants

编号 Number	基因型 Genotype	单倍体诱导效率%（单倍体数/检测数） Haploid induction rate % (Haploid/Tested number）			均值±标准差Mean±SD(%)
编号 Number	基因型 Genotype	株系1 Line 1	株系2 Line 2	株系3 Line 3	均值±标准差Mean±SD(%)
WT	WT	0.0%(0/92)	0.0%(0/93)	0.0%(0/93)	0.0
D1	osdmp1	0.0%(0/179)	0.0%(0/186)	0.0%(0/172)	0.0
D2	osdmp2	0.0%(0/182)	0.0%(0/179)	0.0%(0/186)	0.0
D3	osdmp3	0.0%(0/186)	0.0%(0/183)	0.0%(0/185)	0.0
D1-2	osdmp1-osdmp2	0.0%(0/978)	0.0%(0/165)	0.0%(0/172)	0.0
D1-3	osdmp1-osdmp3	0.0%(0/1662)	0.0%(0/176)	0.0%(0/175)	0.0
D1-2-3	osdmp1-osdmp2-osdmp3	0.0%(0/1393)	0.0%(0/181)	0.0%(0/169)	0.0
osmtl	osmtl	1.1%(1/93)	1.3%(1/78)	2.2%(2/93)	1.5±0.5
MD1	osmtl-osdmp1	0.0%(0/102)	0.0%(0/104)	1.2%(1/86)	0.4±0.6 ns
MD2	osmtl-osdmp2	1.3%(1/77)	6.5%(4/62)	0.0%(0/58)	2.6±2.8 ns
MD3	osmtl-osdmp3	2.2%(2/90)	0.9%(1/107)	1.2%(1/82)	1.4±0.6 ns
MD1-2	osmtl-osdmp1-osdmp2	3.2%(3/94)	0.0%(0/84)	1.4%(1/72)	1.5±1.3 ns
MD1-3	osmtl-osdmp1-osdmp3	5.4%(5/93)	0.9%(0/108)	0.0%(0/64)	2.1±2.4 ns
MD1-2-3	osmtl-osdmp1-osdmp2-osdmp3	1.6%(2/129)	1.9%(2/108)	3.1%(3/96)	2.2±0.6 ns

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