利用CRISPR/Cas9技术高效创制长粒香型水稻

doi:10.16819/j.1001-7216.2020.0104

中国水稻科学 ›› 2020, Vol. 34 ›› Issue (5): 406-412.DOI: 10.16819/j.1001-7216.2020.0104

利用CRISPR/Cas9技术高效创制长粒香型水稻

徐善斌¹, 郑洪亮¹, 刘利锋³, 卜庆云², 李秀峰^2,^*(), 邹德堂^1,^*()

¹东北农业大学寒地粮食作物种质创新与生理生态教育部重点实验室, 哈尔滨 150030
²中国科学院东北地理与农业生态研究所, 哈尔滨 150081
³黑龙江省种业技术服务中心, 哈尔滨 150008

收稿日期:2020-01-10 修回日期:2020-04-16 出版日期:2020-09-10 发布日期:2020-09-10
通讯作者: 李秀峰,邹德堂
基金资助:
转基因生物新品种培育专项（2018ZX0800102B）;国家重点研发专项（2017YFD0100501）;黑龙江省自然科学基金（面上项目）（C2018065）;国家自然科学基金青年基因资助项目(31801327);黑龙江科技厅重点研发项目(GA18B101)

Improvement of Grain Shape and Fragrance by Using CRISPR/Cas9 System

Shanbin XU¹, Hongliang ZHENG¹, Lifeng LIU³, Qingyun BU², Xiufeng Li^2,^*(), Detang ZOU^1,^*()

¹Key Laboratory of Germplasm Enhancement, Physiology and Ecology of Food Crops in Cold Region, Ministry of Education, Northeast Agricultural University, Harbin 150030, China
²Northeast Institute of Geography and Agroecology, Key Laboratory of Soybean Molecular Design Breeding, Chinese Academy of Sciences, Harbin 150081, China
³Heilongjiang Seed Industry Technical Service Center, Harbin 150008

Received:2020-01-10 Revised:2020-04-16 Online:2020-09-10 Published:2020-09-10
Contact: Xiufeng Li, Detang ZOU

摘要/Abstract

摘要：

【目的】CRISPR/Cas9基因编辑技术已成为水稻分子育种的重要手段。为了促进水稻育种的发展,本研究以非香型粳稻品种龙粳11为试验材料,对GS3、GS9和Badh2基因进行编辑,以期获得能稳定遗传的长粒香水稻材料。【方法】利用CRISPR/Cas9技术,以GS3、GS9和Badh2为靶基因,构建敲除载体pYLCRISPR/Cas9-GS3/ GS9/Badh2-gRNA,通过农杆菌介导法,在龙粳11的GS3、GS9和Badh2基因中引入了特定的突变。【结果】T₂代无转基因的gs3/gs9/badh2纯合突变体与野生型龙粳11相比,粒长增加26.43%~27.01%,单株产量增加10.82%~12.11%,千粒重增加18.34%~41.36%,稻米变香,高效地将圆粒水稻变成长粒香型水稻。【结论】利用CRISPR/Cas9技术获得能够稳定遗传并具有长粒香品质的纯合突变株系,为组合多个品质性状提供了一种方便有效的方法,从育种角度加快了新品系创制过程。

关键词: 水稻, 粒型, 品质育种, 香米, CRISPR/Cas9

Abstract:

【Objective】 CRISPR/Cas9-mediated genome editing has become an important way for molecular breeding in rice. To promote rice breeding, the non-fragrant japonica rice variety Longjing11 was used as the test material. This study aims to edit GS3, GS9 and Badh2 genes for obtaining valuable and stable long-grain fragrant rice materials. 【Method】 The target genes GS3, GS9 and BADH2 were selected to construct the vector pYLCRISPR/ Cas9-GS3/ GS9/Badh2-gRNA by using CRISPR/Cas9 gene editing technology. The Agrobacterium-mediated transformation was used to transform Longjing11. Specific mutations were introduced into the GS3, GS9 and Badh2 genes in Longjing 11. 【Result】 The grain length of the transgenic free homozygote gs3/gs9/badh2 increased by 26.43% to 27.01%, yield per plant by 10.82% to 12.11%, 1000-grain weight by 18.34% to 41.36%, rice became fragrant as compared with wild type of Longjing 11. This study efficiently transformed round-grain rice into long-grain fragrant rice. 【Conclusion】The homozygous mutant lines featured by stable inheritance and long-grain fragrant quality were obtained by using CRISPR/Cas9 system. This study provides a convenient and effective way of combining multiple quality traits together, which could significantly accelerate breeding process from a breeding perspective.

Key words: rice, grain shape, quality breeding, fragrant rice, CRISPR/Cas9

中图分类号:

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.

Shanbin XU, Hongliang ZHENG, Lifeng LIU, Qingyun BU, Xiufeng Li, Detang ZOU. Improvement of Grain Shape and Fragrance by Using CRISPR/Cas9 System[J]. Chinese Journal OF Rice Science, 2020, 34(5): 406-412.

图/表 7

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'）
B1-F	GCCGAGTGACATGGCAATGGCGG
B1-R	AAACCCGCCATTGCCATGTCACT
B2-F	GCCGCGATTGCTTCCTGCTCGGTT
B2-R	AAACAACCGAGCAGGAAGCAATCG
B3-F	GCCGCAAGTACCTCCGCGCAATCG
B3-R	AAACCGATTGCGCGGAGGTACTTG
U-F	CTCCGTTTTACCTGTGGAATCG
gRNA-R	CGGAGGAAAATTCCATCCAC
B1'	TTCAGAGGTCTCTCTCGCACTGGAATCGGCAGCAAAGG
B2	AGCGTGGGTCTCGTCAGGGTCCATCCACTCCAAGCTC
B2'	TTCAGAGGTCTCTCTGACACTGGAATCGGCAGCAAAGG
B3	AGCGTGGGTCTCGTCTTGGTCCATCCACTCCAAGCTC
B3'	TTCAGAGGTCTCTAAGACACTGGAATCGGCAGCAAAGG
BL	AGCGTGGGTCTCGACCGGGTCCATCCACTCCAAGCTC
Seq1-F	TTCAAAGCAAAGCACCAAGC
Seq1-R	GCTGGGGAAAACTTACAATG
Seq2-F	TCCACTCGACTCCTCACTCA
Seq2-R	GATTAGCGTAGGCCCTCACG
Seq3-F	ATCCATCTCCGTATCTCT
Seq3-R	GGTAGTCACCACCCTA
Hyg-F	ACGGTGTCGTCCATCACAGTTTGCC
Hyg-R	TTCCGGAAGTGCTTGACATTGGGGA
NOFS	GCGGTGTCATCTATGTTACTAG
M13F-47	CGCCAGGGTTTTCCCAGTCACGAC

图1 GS3、GS9和Badh2基因结构和靶点位置黑色序列为靶点序列,红色下划线序列为PAM序列。

Fig. 1. Gene structure and target site of GS3, GS9 and Badh2. The black sequence is the target sequence and the red underlined sequence is the PAM sequence.

图2 pYLCRISPR/Cas9-GS3/GS9/Badh2-gRNA载体

Fig. 2. pYLCRISPR/Cas9-GS3/GS9/Badh2-gRNA vector.

图3 T0代植株转基因检测 M-DM2000 DNA标记; N-阴性对照; P-阳性对照; 1~16代表T0植株。

Fig. 3. Transgenic detection of T0 generation plants. M, DM2000 DNA marker; N, Negative control; P, Positive control; Lanes 1-16, T0 generation plants.

图4 三基因纯合突变植株测序结果红色小写字母表示插入1 bp的突变,红色连字符表示缺失的序列。

Fig. 4. Sequencing result of homozygous plants with triple gene mutation. Red lowercase letters represent 1 bp insertions and the deleted sequences are shown by red hyphens.

图5 T2代植株农艺性状考查数值用平均数±标准差表示,*,**分别表示在0.05和0.01水平上显著差异(t检验)。

Fig. 5. Agronomic traits of T2 generation plants. Values are shown as mean ± SD．*,**Significantly different at 0.05 and 0.01 levels by t-test, respectively．

图6 龙粳11及突变体中香味物质2-AP的含量

Fig. 6. 2-acetyl-1-pyrroline(2-AP) content of Longjing 11 and mutant plants.

参考文献 36

[1]	郭韬, 余泓, 邱杰, 李家洋, 韩斌, 林鸿宣. 中国水稻遗传学研究进展与分子设计育种[J]. 中国科学: 生命科学, 2019, 49(10): 1185-1212.
	Guo T, Yu H, Qiu J, Li J Y, Han B, Lin H X.Advances in rice genetics and breeding by molecular design in China[J]. Science in China: Life Sciences, 2019, 49(10): 1185-1212. (in Chinese with English abstract)
[2]	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.
[3]	Cai Y P, Chen L, Liu X J, Guo C, Sun S, Wu C X, Jiang B J, Han T F, Hou W S.CRISPR/Cas9-mediated targeted mutagenesis of GmFT2a delays flowering time in soya bean[J]. Plant Biotechnology Journal, 2018, 16(1): 176-185.
[4]	Qi W W, Zhu T, Tian Z R, Li C B, Zhang W, Song R T.High-efficiency CRISPR/Cas9 multiplex gene editing using the glycine tRNA-processing system-based strategy in maize[J/OL].BMC Biotechnology, 2016, 16(1): 58.
[5]	Zhang S J, Zhang R Z, Song G Q, Gao J, Li W, Han X D, Chen M L, Li Y L, Li G Y.Targeted mutagenesis using the Agrobacterium tumefaciens-mediated CRISPR-Cas9 system in common wheat[J/OL].BMC Plant Biology, 2018, 18(1): 302.
[6]	Wang P C, Zhang J, Sun L, Ma Y Z, Xu J, Liang S J, Deng J W, Tan J F, Zhang Q H, Tu L L, Daniell H, Jin S X, Zhang X L.High efficient multisites genome editing in allotetraploid cotton (Gossypium hirsutum) using CRISPR/Cas9 system[J]. Plant Biotechnology Journal, 2018, 16(1): 137-150.
[7]	Liu G Q, Li J Q, Godwin I D.Genome editing by CRISPR/Cas9 in sorghum through biolistic bombardment[J]. Methods in Molecular Biology, 2019, 1931: 169-183.
[8]	Samanta M K, Dey A, Gayen S.CRISPR/Cas9: An advanced tool for editing plant genomes[J]. Transgenic Research, 2016, 25(5): 561-573.
[9]	Ji X, Zhang H W, Zhang Y, Wang Y P, Gao C X.Establishing a CRISPR-Cas-like immune system conferring DNA virus resistance in plants[J/OL].Nature Plants, 2015, 1: 15144.
[10]	Hsu P D, Lander E S, Zhang F.Development and applications of CRISPR-Cas9 for genome engineering[J]. Cell, 2014, 157(6): 1262-1278.
[11]	Smih F, Rouet P, Romanienko P J, Jasin M.Double strand breaks at the target locus stimulate gene targeting in embryonic stem cells[J]. Nucleic Acids Research, 1995, 23(24): 5012-5019.
[12]	Song X J, Huang W, Shi M, Zhu M Z, Lin H X.A QTL for rice grain width and weight encodes a previously unknown RING-type E3 ubiquitin ligase[J]. Nature Genetics, 2007, 39(5): 623-630.
[13]	Fan C C, Xing Y Z, Mao H L, Lu T T, Han B, Xu C G, Li X H, Zhang Q F.GS3, a major QTL for grain length and weight and minor QTL for grain width and thickness in rice, encodes a putative transmembrane protein[J]. Theoretical and Applied Genetics, 2006, 112(6): 1164-1171.
[14]	Zhao D S, Li Q F, Zhang C Q, Zhang C, Yang Q Q, Pan L X, Ren X Y, Lu J, Gu M H, Liu Q Q. GS9 acts as a transcriptional activator to regulate rice grain shape and appearance quality[J/OL]. Nature Communications, 2018, 9(1): 1240.
[15]	Liu J F, Chen J, Zheng X M, Wu F Q, Lin Q B, Heng Y Q, Tian P, Cheng Z J, Yu X W, Zhou K N, Zhang X, Guo X P, Wang J L, Wang H Y, Wan J M.GW5 acts in the brassinosteroid signalling pathway to regulate grain width and weight in rice[J/OL].Nature Plants, 2017, 3: 17043.
[16]	Ishimaru K, Hirotsu N, Madoka Y, Murakami N, Hara N, Onodera H, Kashiwagi T, Ujiie K, Shimizu B I, Onishi A, Miyagawa H, Katoh E.Loss of function of the IAA-glucose hydrolase gene TGW6 enhances rice grain weight and increases yield[J]. Nature Genetics, 2013, 45(6): 707-711.
[17]	Che R H, Tong H N, Shi B H, Liu Y Q, Fang S R, Liu D P, Xiao Y H, Hu B, Liu L C, Wang H R, Zhao M F, Chu C C. Control of grain size and rice yield by GL2-mediated brassinosteroid responses[J/OL].Nature Plants, 2015, 2: 15195.
[18]	Qi P, Lin Y S, Song X J, Shen J B, Huang W, Shan J X, Zhu M Z, Jiang L, Gao J P, Lin H X.The novel quantitative trait locus GL3.1 controls rice grain size and yield by regulating Cyclin-T1;3[J]. Cell Research, 2012, 22(12): 1666-1680.
[19]	Li Y B, Fan C C, Xing Y Z, Jiang Y H, Luo L J, Sun L, Shao D, Xu C J, Li X H, Xiao J H, He Y Q, Zhang Q F.Natural variation in GS5 plays an important role in regulating grain size and yield in rice[J]. Nature Genetics, 2011, 43(12): 1266-1269.
[20]	Wang Y X, Xiong G S, Hu J, Jiang L, Yu H, Xu J, Fang Y X, Zeng L J, Xu E B, Xu J, Ye W J, Meng X B, Liu R F, Chen H Q, Jing Y H, Wang Y H, Zhu X D, Li J Y, Qian Q.Copy number variation at the GL7 locus contributes to grain size diversity in rice[J]. Nature Genetics, 2015, 47(8): 944-948.
[21]	Si L Z, Chen J Y, Huang X H, Gong H, Luo J H, Hou Q Q, Zhou T Y, Lu T T, Zhu J J, Shang G Y, Chen E W, Gong C X, Zhao Q, Jing Y F, Zhao Y, Li Y, Cui L L, Fan D L, Lu Y Q, Weng Q J, Wang Y C, Zhan Q L, Liu K Y, Wei X H, An K, An G, Han B.OsSPL13 controls grain size in cultivated rice[J]. Nature Genetics, 2016, 48(4): 447-456.
[22]	Wang S K, Wu K, Yuan Q B, Liu X Y, Liu Z B, Lin X Y, Zeng R Z, Zhu H T, Dong G J, Qian Q, Zhang G Q, Fu X D.Control of grain size, shape and quality by OsSPL16 in rice[J]. Nature Genetics, 2012, 44(8): 950-954.
[23]	Li M R, Li X X, Zhou Z J, Wu P Z, Fang M C, Pan X P, Lin Q P, Luo W B, Wu G J, Li H Q.Reassessment of the four yield-related genes Gn1a, DEP1, GS3, and IPA1 in rice using a CRISPR/Cas9 system[J/OL].Frontiers in Plant Science, 2016, 7: 377.
[24]	Sakthivel K, Sundaram R M, Rani N S, Balachandran S M, Neeraja C N.Genetic and molecular basis of fragrance in rice[J]. Biotechnology Advances, 2009, 27(4): 468-473.
[25]	Chen S H, Yang Y, Shi W W, Ji Q, He F, Zhang Z D, Cheng Z K, Liu X N, Xu M L.Badh2, encoding betaine aldehyde dehydrogenase, inhibits the biosynthesis of 2-acetyl-1-pyrroline, a major component in rice fragrance[J]. Plant Cell, 2008, 20(7): 1850-1861.
[26]	Shan Q W, Zhang Y, Chen K L, Zhang K, Gao C X.Creation of fragrant rice by targeted knockout of the OsBadh2 gene using TALEN technology[J]. Plant Biotechnology Journal, 2015, 13(6): 791-800.
[27]	孙慧宇, 宋佳, 王敬国, 刘化龙, 孙健, 莫天宇, 徐善斌, 郑洪亮, 邹德堂. 利用CRISPR/Cas9技术编辑Badh2基因改良粳稻香味[J]. 华北农学报, 2019, 34(4): 1-8.
	Sun H Y, Song J, Wang J G, Liu H L, Sun J, Mo T Y, Xu S B, Zheng H L, Zou D T.Editing Badh2 gene to improve the fragrance of japonica rice by CRISPR/Cas9 technology[J]. Acta Agricultuae Boreali-Sinica, 2019, 34(4): 1-8. (in Chinese with English abstract)
[28]	曾栋昌, 马兴亮, 谢先荣, 祝钦泷, 刘耀光. 植物CRISPR/Cas9多基因编辑载体构建和突变分析的操作方法[J]. 中国科学: 生命科学, 2018, 48(7): 783-794.
	Zeng D C, Ma X L, Xie X R, Zhu Q L, Liu Y G.A protocol for CRISPR/Cas9-based multi-gene editing and sequence decoding of mutant sites in plants[J]. Science in China: Life Sciences, 2018, 48(7): 783-794. (in Chinese with English abstract)
[29]	Xie X R, Ma X, Zhu Q L, Zeng D C, Li G S, Liu Y G, CRISPR-GE: A convenient software toolkit for CRISPR- based genome editing[J]. Molecular Plant, 2017, 10(9): 1246-1249.
[30]	Ma X L, Zhang Q Y, Zhu Q L, Liu W, Chen Y, Qiu R, Wang B, Yang Z F, Li H Y, Lin Y R, Xie Y Y, Shen R X, Chen S F, Wang Z, Chen Y L, Guo J X, Chen L T, Zhao X C, Dong Z C, Liu Y G.A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants[J]. Molecular Plant, 2015, 8(8): 1274-1284.
[31]	Liu W Z, Xie X R, Ma X L, Li J, Chen J H, Liu Y G.DSDecode: A web-based tool for decoding of sequencing chromatograms for genotyping of targeted mutations[J]. Molecular Plant, 2015, 8(9): 1431-1433.
[32]	Zhang Y C, Li S M, Xue S J, Yang S H, Huang J, Wang L.Phylogenetic and CRISPR/Cas9 studies in deciphering the evolutionary trajectory and phenotypic impacts of rice ERECTA genes[J/OL]. Frontiers in Plant Science, 2018, 9: 473.
[33]	Wang M, 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.
[34]	Zhang J S, Zhang H, Botella J R, Zhu J K.Generation of new glutinous rice by CRISPR/Cas9-targeted mutagenesis of the Waxy gene in elite rice varieties[J]. Journal of Integrative Plant Biology, 2018, 60(5): 369-375.
[35]	Wang Y, Geng L Z, Yuan M L, Wei J, Jin C, Li M, Yu K, Zhang Y, Jin H B, Wang E, Chai Z J, Fu X D, Li X G.Deletion of a target gene in indica rice via CRISPR/Cas9[J]. Plant Cell Reports, 2017, 36(8): 1333-1343.
[36]	Nan J Z, Feng X M, Wang C, Zhang X H, Wang R S, Liu J X, Yuan Q B, Jiang G Q, Lin S Y.Improving rice grain length through updating the GS3 locus of an elite variety Kongyu 131[J]. Rice, 2018, 11(1): 21.

利用CRISPR/Cas9技术高效创制长粒香型水稻

Improvement of Grain Shape and Fragrance by Using CRISPR/Cas9 System

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

图/表 7

参考文献 36

相关文章 15

编辑推荐

Metrics

[1]	郭展, 张运波. 水稻对干旱胁迫的生理生化响应及分子调控研究进展[J]. 中国水稻科学, 2024, 38(4): 335-349.
[2]	韦还和, 马唯一, 左博源, 汪璐璐, 朱旺, 耿孝宇, 张翔, 孟天瑶, 陈英龙, 高平磊, 许轲, 霍中洋, 戴其根. 盐、干旱及其复合胁迫对水稻产量和品质形成影响的研究进展[J]. 中国水稻科学, 2024, 38(4): 350-363.
[3]	许丹洁, 林巧霞, 李正康, 庄小倩, 凌宇, 赖美玲, 陈晓婷, 鲁国东. OsOPR10正调控水稻对稻瘟病和白叶枯病的抗性[J]. 中国水稻科学, 2024, 38(4): 364-374.
[4]	丁正权, 潘月云, 施扬, 黄海祥. 基于基因芯片的嘉禾系列长粒优质食味粳稻综合评价与比较[J]. 中国水稻科学, 2024, 38(4): 397-408.
[5]	候小琴, 王莹, 余贝, 符卫蒙, 奉保华, 沈煜潮, 谢杭军, 王焕然, 许用强, 武志海, 王建军, 陶龙兴, 符冠富. 黄腐酸钾提高水稻秧苗耐盐性的作用途径分析[J]. 中国水稻科学, 2024, 38(4): 409-421.
[6]	吕宙, 易秉怀, 陈平平, 周文新, 唐文帮, 易镇邪. 施氮量与移栽密度对小粒型杂交水稻产量形成的影响[J]. 中国水稻科学, 2024, 38(4): 422-436.
[7]	胡继杰, 胡志华, 张均华, 曹小闯, 金千瑜, 章志远, 朱练峰. 根际饱和溶解氧对水稻分蘖期光合及生长特性的影响[J]. 中国水稻科学, 2024, 38(4): 437-446.
[8]	刘福祥, 甄浩洋, 彭焕, 郑刘春, 彭德良, 文艳华. 广东省水稻孢囊线虫病调查与鉴定[J]. 中国水稻科学, 2024, 38(4): 456-461.
[9]	陈浩田, 秦缘, 钟笑涵, 林晨语, 秦竞航, 杨建昌, 张伟杨. 水稻根系和土壤性状与稻田甲烷排放关系的研究进展[J]. 中国水稻科学, 2024, 38(3): 233-245.
[10]	缪军, 冉金晖, 徐梦彬, 卜柳冰, 王平, 梁国华, 周勇. 过量表达异三聚体G蛋白γ亚基基因RGG2提高水稻抗旱性[J]. 中国水稻科学, 2024, 38(3): 246-255.
[11]	尹潇潇, 张芷菡, 颜绣莲, 廖蓉, 杨思葭, 郭岱铭, 樊晶, 赵志学, 王文明. 多个稻曲病菌效应因子的信号肽验证和表达分析[J]. 中国水稻科学, 2024, 38(3): 256-265.
[12]	朱裕敬, 桂金鑫, 龚成云, 罗新阳, 石居斌, 张海清, 贺记外. 全基因组关联分析定位水稻分蘖角度QTL[J]. 中国水稻科学, 2024, 38(3): 266-276.
[13]	魏倩倩, 汪玉磊, 孔海民, 徐青山, 颜玉莲, 潘林, 迟春欣, 孔亚丽, 田文昊, 朱练峰, 曹小闯, 张均华, 朱春权. 信号分子硫化氢参与硫肥缓解铝对水稻生长抑制作用的机制[J]. 中国水稻科学, 2024, 38(3): 290-302.
[14]	周甜, 吴少华, 康建宏, 吴宏亮, 杨生龙, 王星强, 李昱, 黄玉峰. 不同种植模式对水稻籽粒淀粉含量及淀粉关键酶活性的影响[J]. 中国水稻科学, 2024, 38(3): 303-315.
[15]	关雅琪, 鄂志国, 王磊, 申红芳. 影响中国水稻生产环节外包发展因素的实证研究：基于群体效应视角[J]. 中国水稻科学, 2024, 38(3): 324-334.