利用CRISPR/Cas9技术创制高效抗除草剂水稻
收稿日期: 2021-10-13
修回日期: 2021-12-07
网络出版日期: 2022-09-09
基金资助
黑龙江省杰出青年基金资助项目(JQ2020C003)
Improvement of Herbicide Resistance in Rice by Using CRISPR/Cas9 System
Received date: 2021-10-13
Revised date: 2021-12-07
Online published: 2022-09-09
【目的】培育抗除草剂品种在水稻育种中具有重要意义。利用CRISPR/Cas9基因编辑技术，以黑龙江优质粳稻品种为材料，编辑乙酰乳酸合酶ALS基因，创制具有抗除草剂特性的水稻材料。【方法】利用CRISPR/Cas9技术，以乙酰乳酸合酶ALS为靶基因，构建单碱基突变载体pH-nCas9-PBE-ALS，以松粳22、龙粳46和绥粳18为转化材料，利用农杆菌介导转化获得转基因植株，通过对转基因植株的突变位点进行测序结合除草剂喷施试验，鉴定基因型及表型。【结果】经分子水平检测验证，获得ALSS627N突变植株10株，ALSS627N且1884G-A但第628位氨基酸未改变突变植株1株，ALSS627N/G628E突变植株1株。相较于野生型，以上三类突变植株均具有较强抗除草剂特性。【结论】利用CRISPR/Cas9基因编辑技术获得具有抗除草剂特性，能够稳定遗传，不含转基因标记的纯合株系，可为抗除草剂水稻育种提供基础材料。
尹丽颖, 张元野, 李荣田, 何明良, 王芳权, 许扬, 刘欣欣, 潘婷婷, 田晓杰, 卜庆云, 李秀峰 . 利用CRISPR/Cas9技术创制高效抗除草剂水稻[J]. 中国水稻科学, 2022 , 36(5) : 459 -466 . DOI: 10.16819/j.1001-7216.2022.211004
【Objective】 It is of great significance to create herbicide-resistant rice varieties. The acetyllactic acid synthase (ALS) gene was edited to create rice varieties with excellent herbicide resistance using non-herbicide-resistant japonica rice varieties with good quality as materials. 【Methods】Using CRISPR/Cas9 technology, the single-base mutant vector pH-NCas9-PBA-ALS was constructed with acyllactic acid synthase (ALS) as the target gene. Songjing 22, Longjing 46 and Suijing 18 were used as transformation materials to obtain transgenic plants through Agrobacterium-mediated genetic transformation. Genotypes and phenotypes were identified by sequencing the mutation sites of transgenic plants and herbicide spraying test. 【Results】 Ten ALSS627N mutants, one ALSS627N and 1884G-A(the 628th amino acid unchanged mutant), and one ALSS627N/G628E mutant were obtained by molecular level detection. Compared with the wild type, the three types of mutants had stronger herbicide resistance. 【Conclusion】 Using CRISPR/Cas9 gene editing technology, we obtained homozygous mutant lines which are genetically stable and herbicide-resistant, providing basic materials for herbicide-resistant rice breeding.
Key words： rice; ALS; herbicide resistance; imazethapyr; CRISPR/Cas9; gene editing
| [1] | 徐振伟. 世界粮食危机与中国粮食安全[J]. 东北亚论坛, 2012(3): 30-37. |
| [1] | Xu Z W. World food crisis and food security of China[J]. Northeast Asia Forum, 2012(3): 30-37. (in Chinese with English abstract) |
| [2] | 张为农. 我国水稻除草剂的发展趋势[J]. 营销界(农资与市场), 2014(6): 84-86. |
| [2] | Zhang W N. Development trend of rice herbicide in China[J]. Marketing Industry(Agricultural Materials and Markets), 2014(6): 84-86. (in Chinese with English abstract) |
| [3] | Norsworthy J K, Scott R C, Bangarwa S K, Griffith G M, Wilson M J, Mccelland M. Weed management in a furrow-irrigated imidazolinone-resistant hybrid rice production system[J]. Weed Technology, 2011, 25(1): 25-29. |
| [4] | Kumar V, Bellinder R R, Gupta R K, Malik R K, Brainard D C. Role of herbicide-resistant rice in promoting resource conservation technologies in rice-wheat cropping systems of India: A review[J]. Crop Protection, 2008, 27(3-5): 290-301. |
| [5] | 李燕敏, 祁显涛, 刘昌林, 刘方, 谢传晓. 除草剂抗性农作物育种研究进展[J]. 作物杂志, 2017(2): 1-6. |
| [5] | Li Y M, Qi X T, Liu C L, Liu F, Xie C X. Progress of crop breeding on resistance to herbicides[J]. Crops, 2017(2): 1-6. (in Chinese with English abstract) |
| [6] | 崔莹. 新型转基因抗除草剂水稻培育[D]. 武汉: 华中农业大学, 2017. |
| [6] | Cui Y. Developing novel transgenic herbicide-tolerant rice[D]. Wuhan: Huazhong Agricultural University, 2017. (in Chinese with English abstract) |
| [7] | Wang M G, Mao Y F, Lu Y M, Wang Z D, Tao X P, Zhu J K. Multiplex gene editing in rice with simplified CRISPR-Cpf1 and CRISPR-Cas9 systems[J]. Journal of Integrative Plant Biology, 2018, 60(8): 626-631. |
| [8] | Li X F, Zhou W J, Ren Y K, Tian X J, Lü T X, Wang Z Y, Fang J, Chu C C, Yang J, Bu Q Y. High-efficiency breeding of early-maturing rice cultivars via CRISPR/Cas9-mediated genome editing[J]. Journal of Genetics and Genomics, 2017, 44(3): 175-178. |
| [9] | 周文甲, 田晓杰, 任月坤, 魏祥进, 高扬, 谢黎虹, 刘华招, 卜庆云, 李秀峰. 利用CRISPR/Cas9创造早熟香味水稻[J]. 土壤与作物, 2017, 6(2): 146-152. |
| [9] | Zhou W J, Tian X J, Ren Y K, Wei X J, Gao Y, Xie L H, Liu H Z, Bu Q Y, Li X F. Breeding of early-maturatity and fragrant rice via CRISPR/Cas9 mediated genome editing[J]. Soils and Crops, 2017, 6(2): 146-152. (in Chinese with English abstract) |
| [10] | Xu Y, Lin Q, Li X, Wang F, Chen Z, Wang J, Li W, Fan F, Tao Y, Jiang Y. Fine-tuning the amylose content of rice by precise base editing of the Wx gene[J]. Plant Biotechnology Journal, 2021, 19(1): 11-13. |
| [11] | 徐善斌, 郑洪亮, 刘利锋, 卜庆云, 李秀峰, 邹德堂. 利用CRISPR/Cas9技术高效创制长粒香型水稻[J]. 中国水稻科学, 2020, 34(5): 406-412. |
| [11] | Xu S B, Zheng H L, Liu L F, Bu Q Y, Li X F, Zou D T. Improvement of grain shape and fragrance by using CRISPR/Cas9 system[J]. Chinese Journal of Rice Science, 2020, 34(5): 406-412. (in Chinese with English abstract) |
| [12] | Antralina M, Istina I N, YuyunYuwariah, Simarmata T. Effect of difference weed control methods to yield of lowland rice in the SOBARI[J]. Procedia Food Science, 2015, 3: 323-329. |
| [13] | 苏少泉. 抗咪唑啉酮类除草剂作物的发展与未来[J]. 现代农药, 2006(1): 1-4. |
| [13] | Su S Q. The development and future of imidazolinone herbicide-resistant crops[J]. Modern Agrochemicals, 2006(1): 1-4. (in Chinese with English abstract) |
| [14] | Kiyoshi K, Koichiro K, Norihiko I, Tsutomu S, Atsunori F, Yoshiyuki T. A novel mutant acetolactate synthase gene from rice cells, which confers resistance to ALS-inhibiting herbicides[J]. Journal of Pesticide Science, 2007, 32(2): 89-98. |
| [15] | 任洪雷. 乙酰乳酸合成酶及ALS基因研究概述[J]. 中国农学通报, 2016, 32(26): 37-42. |
| [15] | Ren H L. Acetolactate synthase and ALS gene research[J]. Chinese Agricultural Science Bulletin, 2016, 32(26): 37-42. (in Chinese with English abstract) |
| [16] | Endo M, Osakabe K, Ono K, Handa H, Toki S. Molecular breeding of a novel herbicide-tolerant rice by gene targeting[J]. The Plant Journal, 2007, 52(1): 157-166. |
| [17] | Webster E P, Masson J A. Acetolactate synthase- inhibiting herbicides on imidazolinone-tolerant rice[J]. Weed Science, 2001, 49(5): 652-657. |
| [18] | 范方军, 王芳权, 李文奇, 王军, 朱金燕, 许杨, 仲维功, 杨杰. 抗咪草烟水稻资源的筛选[J]. 中国稻米, 2018, 24(6): 108-109. |
| [18] | Fan F J, Wang F Q, Li W Q, Wang J, Zhu J Y, Xu Y, Zhong W G, Yang J. Screening of imazethapyr-resistant rice resources[J]. China Rice, 2018, 24(6): 108-109. (in Chinese with English abstract) |
| [19] | Xiao G Y, Chen F, Meng Q C, Zhou H, Deng L H, Weng L S. Ecological risk and management of herbicide-resistant transgenic rice(Oryza sativa) in China[J]. Journal of Agricultural Biotechnology, 2015, 23(1): 1-11. |
| [20] | Boutsalis P, Karotam J, Powles S B. Molecular basis of resistance to acetolactate synthase-inhibiting herbicides in Sisymbrium orientale and Brassica tournefortii[J]. Pesticide Science, 1999, 55(5): 507-516. |
| [21] | 陈竹锋, 王承旭, 柳威, 唐晓艳, 邓兴旺. 水稻抗除草剂蛋白及其在植物育种中的应用:CN102586215A[P]. 2012-2-18. |
| [21] | Chen Z F, Wang C X, Liu W, Tang X Y, Deng X W. Rice herbicide-resistant protein and its application in plant breeding: CN102586215A[P]. 2012-2-18. (in Chinese) |
| [22] | 赵炳然, 袁定阳, 韶也, 毛毕刚, 袁智成, 胡远艺, 彭彦, 罗武中. 一种水稻抗除草剂蛋白与基因及其应用:CN106867977A[P]. 2017-2-20. |
| [22] | Zhao B R, Yuan D Y, Shao Y, Mao B G, Yuan Z C, Hu Y Y, Peng Y, Luo W Z. Application of novel herbicide resistance proteins and genes in rice: CN106867977A[P]. 2017-2-20. (in Chinese with) |
| [23] | 王芳权, 杨杰, 范方军, 李文奇, 王军, 许扬, 朱金燕, 费云燕, 仲维功. 水稻抗咪唑啉酮类除草剂基因ALS功能标记的开发与应用[J]. 作物学报, 2018, 44(3): 324-331. |
| [23] | Wang F Q, Yang J, Fan F J, Li W Q, Wang J, Xu Y, Zhu J Y, Fei Y Y, Zhong W G. Development and application of the functional marker for imidazolinone herbicides resistant ALS gene in rice[J]. Acta Agronomica Sinica, 2018, 44(3): 324-331. (in Chinese with English abstract) |
| [24] | 张保龙, 王金彦, 陈天子, 凌溪铁. 粳稻的ALS突变型基因及其蛋白和应用: CN106868028A[P]. 2017-2-20. |
| [24] | Zhang B L, Wang J Y, Chen T Z, Ling X T. Application of ALS mutant gene and protein in japonica rice: CN106868028A[P]. 2017-2-20. (in Chinese) |
| [25] | 张喜娟, 来永才, 王俊河, 孟英, 唐傲, 董文军, 冷春旭. 黑龙江省直播稻的发展现状与对策[J]. 黑龙江农业科学, 2015(8): 142-145. |
| [25] | Zhang X J, Lai Y C, Wang J H, Meng Y, Tang A, Dong W J, Leng C X. Development situation and countermeasures of rice direct seeding cultivation in Heilongjiang Province[J]. Heilongjiang Agricultural Sciences, 2015(8): 142-145. (in Chinese) |
| [26] | Li H, Li X F, Xu Y, Liu H L, He M L, Tian X J, Wang Z Y, Wu X J, Bu Q Y, Yang J. High-efficiency reduction of rice amylose content via CRISPR/Cas9-mediated base editing[J]. Rice Science, 2020, 27(6): 445-448. |
| [27] | 宋贵生, 冯德江, 魏晓丽, 唐家斌, 朱祯. 水稻乙酰乳酸合成酶基因的克隆和功能分析[J]. 中国农业科技导报, 2007(3): 66-72. |
| [27] | Song G S, Feng D J, Wei X L, Tang J B, Zhu Z. Isolation and functional analysis of rice acetolactate-synthase (ALS)[J]. Journal of Agricultural Science and Technology, 2007(3): 66-72. (in Chinese with English abstract) |
| [28] | 毕俊国, 谭金松, 刘毅, 张安宁, 王飞名, 刘国兰, 余新桥, 罗利军. 抗咪唑啉酮类除草剂水稻种质的筛选鉴定[J]. 植物遗传资源学报, 2020, 21(4): 804-808. |
| [28] | Bi J G, Tan J S, Liu Y, Zhang A N, Wang F M, Liu G L, Yu X Q, Luo L J. Screening and identification of rice germplasm resistant to imidazolinone herbicide[J]. Journal of Plant Genetic Resources, 2020, 21(4): 804-808. (in Chinese with English abstract) |
| [29] | 陈天子, 余月, 凌溪铁, 张保龙. EMS诱变水稻创制抗咪唑啉酮除草剂种质[J]. 核农学报, 2021, 35(2): 253-261. |
| [29] | Chen T Z, Yu Y, Ling X T, Zhang B L. Screening of imidazolinone-resistant rice from EMS-mutated populations[J]. Journal of Nuclear Agricultural Sciences, 2021, 35(2): 253-261. (in Chinese with English abstract) |
| [30] | Wang F Q, Xu Y, Li W Q, Chen Z H, Wang J, Fan F J, Tao Y J, Jiang Y J, Zhu Q H, Yang J. Creating a novel herbicide-tolerance OsALS allele using CRISPR/Cas9- mediated gene editing[J]. The Crop Journal, 2021, 9(2): 305-312. |
| [31] | 张保龙, 王金彦, 凌溪铁, 陈天子, 邓惠清, 吴魁. 使植物具有除草剂抗性的水稻ALS突变型蛋白, 基因及其应用: CN107090447A[P]. 2017. |
| [31] | Zhang B L, Wang J Y, Ling X T, Chen T Z, Deng H Q, Wu K. Rice plants having a mutant ALS herbicide resistance protein, genes and their application: CN107090447A[P]. 2017. (in Chinese with English abstract) |
/
| 〈 |
|
〉 |