水稻DMP1、DMP2、DMP3基因突变体的创制及其单倍体诱导能力鉴定
收稿日期: 2023-11-15
修回日期: 2023-12-12
网络出版日期: 2025-01-14
基金资助
国家水稻产业技术体系(CARS-01)
Generation of Rice DMP1, DMP2 and DMP3 Mutants and Identification of Their Haploid Induction Ability
Received date: 2023-11-15
Revised date: 2023-12-12
Online published: 2025-01-14
【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。
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 . DOI: 10.16819/j.1001-7216.2025.231112
【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
| [1] | 陈海强, 刘会云, 王轲, 张双喜, 叶兴国. 植物单倍体诱导技术发展与创新[J]. 遗传, 2020, 42(5): 466-482. |
| Chen H Q, Liu H Y, Wang K, Zhang S X, Ye X G. Development and innovation of haploid induction technologies in plants[J]. Hereditas (Beijing), 2020, 42(5): 466-482. (in Chinese with English abstract) | |
| [2] | Dunwell J M. Haploids in flowering plants: origins and exploitation[J]. Plant Biotechnology Journal, 2010, 8(4): 377-424. |
| [3] | 相志国, 海燕, 康明辉, 赵永英. 单倍体的产生途径及其在作物遗传育种中的应用[J]. 河南农业科学, 2011, 40(11): 17-21. |
| Xiang Z G, Hai Y, Kang M H, Zhao Y Y. Generation ways of haploid and its application in crop genetics and breeding[J]. Journal of Henan Agricultural Sciences, 2011, 40(11): 17-21. | |
| [4] | Coe E H. A line of maize with high haploid frequency[J]. American Naturalist, 1959, 93(873): 381-382. |
| [5] | Prigge V, Xu X W, Li L, Babu R, Chen S J, Atlin G N, Melchinger A E. New insights into the genetics of in vivo induction of maternal haploids, the backbone of doubled haploid technology in maize[J]. Genetics, 2012, 190(2): 781-793. |
| [6] | Kelliher T, Starr D, Richbourg L, Chintamanani S, Delzer B, Nuccio M L, Green J, Chen Z, McCuiston J, Wang W, Liebler T, Bullock P, Martin B. MATRILINEAL, a sperm-specific phospholipase, triggers maize haploid induction[J]. Nature, 2017, 542(7639): 105-109. |
| [7] | Gilles L M, Khaled A, Laffaire J B, Chaignon S, Gendrot G, Laplaige J, Bergès H, Beydon G, Bayle V, Barret P, Comadran J, Martinant J P, Rogowsky P M, Widiez T. Loss of pollen-specific phospholipase NOT LIKE DAD triggers gynogenesis in maize[J]. The EMBO Journal, 2017, 36(6): 707-717. |
| [8] | Liu C X, Li X, Meng D X, Zhong Y, Chen C, Dong X, Xu X W, Chen B J, Li W, Li L, Tian X L, Zhao H M, Song W B, Luo H S, Zhang Q H, Lai J S, Jin W W, Yan J B, Chen S J. A 4-bp insertion at ZmPLA1 encoding a putative phospholipase a generates haploid induction in maize[J]. Molecular Plant, 2017, 10(3): 520-522. |
| [9] | Yao L, Zhang Y, Liu C, Liu Y, Wang Y, Liang D, Liu J, Sahoo G, Kelliher T. OsMATL mutation induces haploid seed formation in indica rice[J]. Nature Plants, 2018, 4(8): 530-533. |
| [10] | Liu C X, Zhong Y, Oi X L, Chen M, Liu Z K, Chen C, Tian X L, Li I L, Jiao Y Y, Wang D, Wang Y W, Li M R, Xin M M, Liu W X, Jin W W, Chen S J. Extension of the in vivo haploid induction system from diploid maize to hexaploid wheat[J]. Plant Biotechnology Journal, 2020, 18(2): 316-318. |
| [11] | Cheng Z X, Sun Y, Yang S H, Zhi H, Yin T, Ma X J, Zhang H S, Diao X M, Guo Y, Li X H, Wu C Y, Sui Y. Establishing in planta haploid inducer line by edited SiMTL in foxtail millet (Setaria italica)[J]. Plant Biotechnology Journal, 2021, 19(6): 1089-1091. |
| [12] | Zhong Y, Liu C X, Qi X L, Jiao Y Y, Wang D, Wang Y W, Liu Z K, Chen C, Chen B J, Tian X L, Li J L, Chen M, Dong X, Xu X W, Li L, Li W, Liu W X, Jin W W, Lai J S, Chen S J. Mutation of ZmDMP enhances haploid induction in maize[J]. Nature Plants, 2019, 5(6): 575-580. |
| [13] | Zhong Y, Chen B J, Li M R, Wang D, Jiao Y Y, Qi X L, Wang M, Liu Z K, Chen C, Wang Y W, Chen M, Li J L, Xiao Z J, Cheng D H, Liu W X, Boutilier K, Liu C X, Chen S J. A DMP-triggered in vivo maternal haploid induction system in the dicotyledonous Arabidopsis[J]. Nature Plants, 2020, 6(5): 466-472. |
| [14] | Wang N, Xia X Z, Jiang T, Li L L, Zhang P C, Niu L F, Cheng H M, Wang K J, Lin H. In planta haploid induction by genome editing of DMP in the model legume Medicago truncatula[J]. Plant Biotechnology Journal, 2022, 20(1): 22-24. |
| [15] | Li Y F, Li D, Xiao Q, Wang H D, Wen J, Tu J X, Shen J X, Fu T D, Yi B. An in planta haploid induction system in Brassica napus[J]. Journal of Integrative Plant Biology, 2022, 64(6): 1140-1144. |
| [16] | Zhong Y, Wang Y W, Chen B J, Liu J C, Wang D, Li M R, Qi X L, Liu C X, Boutilier K, Chen S J. Establishment of a dmp based maternal haploid induction system for polyploid Brassica napus and Nicotiana tabacum[J]. Journal of Integrative Plant Biology, 2022, 64(6): 1281-1294. |
| [17] | Wang C, Shen L, Fu Y P, Yan C J, Wang K J. A simple CRISPR/Cas9 system for multiplex genome editing in rice[J]. Journal of Genetics and Genomics, 2015, 42(12): 703-706. |
| [18] | Wang C, Liu Q, Shen Y, Hua Y F, Wang J J, Lin J R, Wu M G, Sun T T, Cheng Z K, Mercier R, Wang K J. Clonal seeds from hybrid rice by simultaneous genome engineering of meiosis and fertilization genes[J]. Nature Biotechnology, 2019, 37(3): 283-286. |
| [19] | Liu Q, Wang C, Jiao X Z, Zhang H W, Song L L, Li Y X, Gao C X, Wang K J. Hi-TOM: A platform for high-throughput tracking of mutations induced by CRISPR/Cas systems[J]. Science China: Life Sciences, 2019, 62(1): 1-7. |
| [20] | Ma X L, Chen L T, Zhu Q L, Chen Y L, Liu Y G. Rapid decoding of sequence-specific nuclease-induced heterozygous and biallelic mutations by direct sequencing of PCR products[J]. Molecular Plant, 2015, 8(8): 1285-1287. |
| [21] | 曹跃炫, 严绘景, 王克剑, 刘朝雷. 苗期快速分选水稻人工无融合生殖克隆种子[J]. 中国水稻科学, 2022, 36(6): 656-662. |
| Cao Y X, Yan H J, Wang K J, Liu C L. Rapid identification of rice clonal seeds generated by synthetic apomixis at seedling stage[J]. Chinese Journal of Rice Science, 2022, 36(6): 656-662. (in Chinese with English abstract) | |
| [22] | Liu C L, He Z X, Zhang Y, Hu F Y, Li M Q, Liu Q, Huang Y, Wang J, Zhang W, Wang C, Wang K J. Synthetic apomixis enables stable transgenerational transmission of heterotic phenotypes in hybrid rice[J]. Plant Communications, 2023, 4(2): 100470. |
| [23] | Liu C L, Wang J, Lu H W, Huang Y, Yan H J, Liang H, Wang C, Wang K J. Engineering synthetic apomixis in different hybrid rice varieties using the Fix strategy[J]. New Crops, 2024(1): 100003. |
| [24] | Zhao X, Xu X W, Xie H X, Chen S J, Jin W W. Fertilization and uniparental chromosome elimination during crosses with maize haploid inducers[J]. Plant Physiology, 2013, 163(2): 721-731. |
| [25] | Qiu F Z, Liang Y L, Li Y, Liu Y Z, Wang L M, Zheng Y L. Morphological, cellular and molecular evidences of chromosome random elimination in vivo upon haploid induction in maize[J]. Current Plant Biology, 2014(1): 83-90. |
| [26] | Karimi-Ashtiyani R, Ishii T, Niessen M, Stein N, Heckmann S, Gurushidze M, Banaei-Moghaddam A M, Fuchs J, Schubert V, Koch K, Weiss O, Demidov D, Schmidt K, Kumlehn J, Houben A. Point mutation impairs centromeric CENH3 loading and induces haploid plants[J]. Proceedings of the National Academy of Sciences of the United States of America, 2015, 112(36): 11211-11216. |
| [27] | Kelliher T, Starr D, Wang W, Mccuiston J, Zhong H, Nuccio M L, Martin B. Maternal haploids are preferentially induced by CENH3-tailswap transgenic complementation in maize[J]. Frontiers in Plant Science, 2016, 7: 414. |
| [28] | Lü J, Yu K, Wei J, Gui H P, Liu C X, Liang D W, Wang Y L, Zhou H J, Carlin R, Rich R, Lu T C, Que Q D, Wang W C, Zhang X P, Kelliher T. Generation of paternal haploids in wheat by genome editing of the centromeric histone CENH3[J]. Nature Biotechnology, 2020, 38(12): 1397-1401. |
| [29] | Jiang C L, Sun J, Li R, Yan S J, Chen W, Guo L, Qin G C, Wang P C, Luo C, Huang W J, Zhang Q H, Fernie A R, Jackson D, Li X, Yan J B. A reactive oxygen species burst causes haploid induction in maize[J]. Molecular Plant, 2022, 15(6): 943-955. |
| [30] | Sarkar K R, Coe E H. A genetic analysis of the origin of maternal haploids in maize[J]. Genetics, 1966, 54(2): 453-464. |
| [31] | Khanday I, Skinner D, Yang B, Mercier R, Sundaresan V. A male-expressed rice embryogenic trigger redirected for asexual propagation through seeds[J]. Nature, 2019, 565(7737): 91-95. |
| [32] | Conner J A, Podio M, Ozias-Akins P. Haploid embryo production in rice and maize induced by PsASGR-BBML transgenes[J]. Plant Reproduction, 2017, 30(1): 41-52. |
| [33] | Wei X, Liu C L, Chen X, Lu H W, Wang J, Yang S L, Wang K J. Synthetic apomixis with normal hybrid rice seed production[J]. Molecular Plant, 2023, 16(3): 489-492. |
| [34] | Underwood C J, Vijverberg K, Rigola D, Okamoto S, Oplaat C, Camp R, Radoeva T, Schauer S E, Fierens J, Jansen K, Mansveld S, Busscher M, Xiong W, Datema E, Nijbroek K, Blom E J, Bicknell R, Catanach A, Erasmuson S, Winefield C, van Tunen A J, Prins M, Schranz M E, van Dijk P J. A PARTHENOGENESIS allele from apomictic dandelion can induce egg cell division without fertilization in lettuce[J]. Nature Genetics, 2022, 54(1): 84-93. |
| [35] | Zhang X C, Shi C, Li S L, Zhang B, Luo P, Peng X B, Zhao P, Dresselhaus T, Sun M X. A female in vivo haploid-induction system via mutagenesis of egg cell-specific peptidases[J]. Molecular Plant, 2023, 16(2): 471-480. |
| [36] | Cyprys P, Lindemeier M, Sprunck S. Gamete fusion is facilitated by two sperm cell-expressed DUF679 membrane proteins[J]. Nature Plants, 2019, 5(3): 253-257. |
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