Development of Aromatic Salt-tolerant Rice Based on CRISPR/Cas9 Technology

doi:10.16819/j.1001-7216.2023.220907

Abstract

Abstract:

【Objective】 It is of importance to promote salt-tolerant and fragrant rice breeding. We obtained transgene free homozygous rice materials with salt-tolerance and fragrance by CRISPR/Cas9-mediated editing of Badh2 and OsRR22 in japonica variety Lianjing 11. 【Method】 Target sites were designed according to knockout efficiency of editing sites in Badh2 and OsRR22 gene sequence. The pH-Ubi-Badh2-OsRR22 knockout vector was constructed and transferred into the recipient variety Lianjing 11 by the Agrobacterium-mediated technology. PCR detection for hygromycin, Cas9 marker and target gene sequencing were performed in the transgenic progenies to obtain badh2-osrr22 homozygous lines without exogenous gene insertion. The seed traits and salt tolerance at seedling stage were analyzed. 【Result】 Compared with the background material Lianjing 11, the 2-AP content of T₂ significantly increased. After treated with 128 mmol/L NaCl solution for 14 days, the seedling height, the fresh and the dry weight of T₂ line 21-30 increased by 15.2%, 45.2% and 13.2%, respectively. 【Conclusion】 The successful editing of Badh2 and OsRR22 by CRISPR/Cas9 technology developed aromatic salt-tolerant homozygous lines without exogenous gene insertion, speeding up the breeding process of pyramiding multiple traits in rice.

Key words: rice, CRISPR/Cas9, salt-tolerant, fragrance, Badh2, OsRR22

摘要：

【目的】 为了促进耐盐香稻育种，利用CRISPR/Cas9系统对粳稻品种连粳11的Badh2和OsRR22基因进行编辑，以期快速获得一批不含有转基因成分且具有耐盐性和香味的纯合水稻材料。【方法】 根据Badh2和OsRR22基因序列中编辑位点的敲除效率设计靶位点，构建pH-Ubi-Cas9-Badh2-OsRR22敲除载体，利用农杆菌介导法转入受体品种连粳11中。对转基因后代进行潮霉素和Cas9标记PCR检测以及靶基因测序，获得无外源基因插入的badh2-osrr22纯合株系，并对后代种子性状和苗期耐盐性进行分析。【结果】 T₂代成熟种子中2-AP含量较背景材料连粳11显著增加，千粒重、粒长、粒宽无明显变化；128 mmol/L氯化钠处理14 d后株系21-30较连粳11苗高增加15.2%，苗鲜质量增加45.2%，苗干质量增加13.2%。【结论】 利用CRISPR/Cas9技术对Badh2和OsRR22基因编辑获得能稳定遗传且无外源基因插入的耐盐香稻材料，加快了水稻多性状聚合的选育进程。

关键词: 水稻, CRISPR/Cas9, 耐盐, 香味, Badh2, OsRR22

LI Jingfang, WEN Shuyue, ZHAO Lijun, CHEN Tingmu, ZHOU Zhenling, SUN Zhiguang, LIU Yan, CHEN Haiyuan, ZHANG Yunhui, CHI Ming, XING Yungao, XU Bo, XU Dayong, WANG Baoxiang. Development of Aromatic Salt-tolerant Rice Based on CRISPR/Cas9 Technology[J]. Chinese Journal OF Rice Science, 2023, 37(5): 478-485.

李景芳, 温舒越, 赵利君, 陈庭木, 周振玲, 孙志广, 刘艳, 陈海元, 张云辉, 迟铭, 邢运高, 徐波, 徐大勇, 王宝祥. 基于CRISPR/Cas9技术创制耐盐香稻[J]. 中国水稻科学, 2023, 37(5): 478-485.

Figures/Tables 7

References 26

[1]	El Mahi H, Perez-Hormaeche J, de Luca A, Villalta I, Espartero J, Gamez-Arjona F, Fernandez J L, Bundo M, Mendoza I, Mieulet D. A critical role of sodium flux via the plasma membrane Na⁺/H⁺ exchanger SOS1 in the salt tolerance of rice[J]. Plant Physiology, 2019, 180(2): 1046-1065.
[2]	Yang A, Dai X Y, Zhang W H. A R2R3-type MYB gene, OsMYB2, is involved in salt, cold, and dehydration tolerance in rice[J]. Journal of Experimental Botany, 2012, 63(7): 2541-2556.
[3]	Wang R, Jing W, Xiao L G, Jin Y K, Shen L K, Zhang W H. The rice high-affinity potassium pransporter1;1 is involved in salt tolerance and regulated by an MYB-type transcription factor[J]. Plant Physiology, 2015, 168(3): 1076-1090.
[4]	Liu S P, Zheng L Q, Xue Y H, Zhang Q, Wang L, Shuo H X. Overexpression of OsVP1 and OsNHX1 increases tolerance to drought and salinity in rice[J]. Journal of Plant Biology, 2010, 53: 444-452.
[5]	张霞, 唐维, 刘嘉, 刘永胜. 过量表达水稻 OsP5CS1 和OsP5CS2 基因提高烟草脯氨酸的生物合成及其非生物胁迫抗性[J]. 应用与环境生物学报, 2014, 20(4): 717-722.
	Zhang X, Tang W, Liu J, Liu Y S. Co-expression of rice OsP5CS1 and OsP5CS2genes in transgenic tobacco resulted in elevated proline biosynthesis and enhanced abiotic stress tolerance[J]. Chinese Journal of Applied & Environmental Biology, 2014, 20(4): 717-722. (in Chinese with English abstract)
[6]	Li H W, Wang X P. Overexpression of the trehalose- 6-phosphate synthase gene OsTPS1 enhances abiotic stress tolerance in rice[J]. Planta, 2011, 234(5): 1007-1018.
[7]	Lan T, Zheng Y L, Su Z L, Yu S B, Song H B, Zheng X Y, Lin G G, Wu W R. OsSPL10, a SBP-box gene, plays a dual role in salt tolerance and trichome formation in rice (Oryza sativa L.)[J]. G3: Genes, Genomes, Genetics, 2019, 9(12): 4107-4114.
[8]	Yang C, Ma B, He S J, Xiong Q, Duan K X, Yin C C, Chen H, Lu X, Chen S Y, Zhang J S. MAOHUZI6/ETHYLENE INSENSITIVE3-LIKE1 and ETHYLENE INSENSITIVE3-LIKE2 regulate ethylene response of roots and coleoptiles and negatively affect salt tolerance in rice[J]. Plant Physiology, 2015, 169(1): 148-165.
[9]	Koh S, Lee S C, Kim M K, Koh J H, Lee S, An G, Choe S, Kim S R. T-DNA tagged knockout mutation of rice OsGSK1, an orthologue of Arabidopsis BIN2, with enhanced tolerance to various abiotic stresses[J]. Plant Molecular Biology, 2007, 65(4): 453-466.
[10]	Takagi H, Tamiru M, Abe A, Yoshida K, Uemura A, Yaegashi H, Obara T, Oikawa K, Utsushi H, Kanzaki E, Mitsuoka C, Natsume S, Kosugi S, Kanzaki H, Matsumura H, Urasaki N, Kamoun S, Terauchi R. MutMap accelerates breeding of a salt-tolerant rice cultivar[J]. Nature Biotechnology, 2015, 33(5): 445-449.
[11]	Zhang A N, Liu Y, Wang F M, Li T F, Chen Z H, Kong D Y, Bi J G, Zhang F Y, Luo X X, Wang J H, Tang J J, Yu X Q, Liu G L, Luo L J. Enhanced rice salinity tolerance via CRISPR/Cas9-targeted mutagenesis of the OsRR22 gene[J]. Molecular Breeding, 2019, 39: 47.
[12]	李晶岚, 陈鑫欣, 石翠翠, 刘方惠, 孙静, 葛荣朝. OsRPK1基因过表达和RNA干涉对水稻苗期耐盐性的影响[J]. 作物学报, 2020, 46(8): 1217-1224.
	Li J L, Chen X X, Shi C C, Liu F H, Sun J, Ge R C. Effects of OsRPK1 gene overexpression and RNAi on the salt-tolerance at seedling stage in rice[J]. Acta Agronomica Sinica, 2020, 46(8): 1217-1224. (in Chinese with English abstract)
[13]	魏晓东, 张亚东, 赵凌, 路凯, 宋雪梅, 王才林. 稻米香味物质2-乙酰-1-吡咯啉的形成及其影响因素[J]. 中国水稻科学, 2022, 36 (2): 131-138.
	Wei X D, Zhang Y D, Zhao L, Lu K, Song X M, Wang C L. Research progress in biosynthesis and influencing factors of 2-acetyl-1-pyrroline in fragrant rice[J]. Chinese Journal of Rice Science, 2022, 36(2): 131-138. (in Chinese with English abstract)
[14]	Jiang M, Liu Y H, Li R Q, Li S, Tan Y Y, Huang J Z, Shu Q Y. An inositol 1, 3, 4, 5, 6-pentakisphosphate 2-kinase 1 mutant with a 33-nt deletion showed enhanced tolerance to salt and drought stress in rice[J]. Plants, 2021, 10(1): 23.
[15]	Bradbury L M, Fitzgerald T L, Henry R J, Jin Q S, Waters D L. The gene for fragrance in rice[J]. Plant Biotechnology Journal, 2005, 3(3): 363-370.
[16]	Chen S H, Yang Y, Shi W W, Ji Q, He F, Zhang Z D, Heng Z K, Liu X N, Xu M L. Badh2, encoding betaine ldehyde dehydrogenase, inhibits the biosynthesis of -acetyl-1-pyrroline, a major component in rice fragrance[J]. Plant Cell, 2008, 20(7): 1850-1861.
[17]	Bradbury L M, Gillies S A, Brushett D J, Waters D L, Henry R J. Inactivation of an amino aldehyde dehydrogenase is responsible for fragrance in rice[J]. Plant Molecular Biology, 2008, 68(4-5): 439-449.
[18]	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.
[19]	Hui S Z, Li H J, Mawia A M, Zhou L, Cai J Y, Ahmad S, Lai C K, Wang J X, Jiao G A, Xie L H, Shao G N, Sheng Z H, Tang S Q, Wang J L, Wei X J, Hu S K, Hu P S. Production of aromatic three-line hybrid rice using novel alleles of BADH2[J]. Plant Biotechnology Journal, 2022, 20(1): 59-74.
[20]	Miao J, Guo D, Zhang J, Huang Q, Qin G, Zhang X, Wan J, Gu H, Qu L. Targeted mutagenesis in rice using CRISPR-Cas system[J]. Cell Research, 2013, 23(10): 1233-1236.
[21]	Yoshida S, Forno D A, Cook J H, Gomez K A. Laboratory Manual for Physiological Studies of Rice[M]. International Rice Research Institute, Philippines, 1976: 61-66.
[22]	Liu Y, Wang B X, Li J, Song Z Q, Lu B G, Chi M, Yang B, Liu J B, Lam Y W, Li J X, Xu D Y. Salt-response analysis in two rice cultivars at seedling stage[J]. Acta Physiologiae Plantarum, 2017, 39: 215.
[23]	Wang B X, Xu B, Liu Y, Li J F, Sun Z G, Chi M, Xing Y G, Yang B, Li J, Liu J B, Lu B G, Xu D Y, Bello B K. A novel mechanism of the signaling cascade associated with the SAPK10-bZIP20-NHX1 synergistic interaction to enhance tolerance of plant to abiotic stress in rice[J]. Plant Science, 2022, 323: 111393.
[24]	Xu Y, Lin Q P, Li X F, Wang F J, Chen Z H, Wang J, Li W Q, Fan F J, Tao Y J, Jiang Y J, Wei X D, Zhang R, Yang J, Gao C X. Fine-tuning the amylose content of rice by precise base editing of the Wx gene[J]. Plant Biotechnology Journal, 2021, 19(1): 11-13.
[25]	Liu X X, Liu H L, Zhang Y Y, He M L Li R T, Meng W, Wang Z Y, Li X F, Bu Q Y. Fine-tuning flowering time via genome editing of upstream open reading frames of heading date 2 in rice[J]. Rice, 2021, 14(1): 59.
[26]	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.
	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)

引物名称 Primer name	引物序列 Primer sequence(5’-3’)
Badh2-S1-F	GGCATGGTGGAAAAAGTCCTATAG	构建Badh2敲除载体 Construction of Badh2 knockout vector
Badh2-S1-R	AAACCTATAGGACTTTTTCCACCA	构建Badh2敲除载体 Construction of Badh2 knockout vector
Badh2-F	CCTTTGTCATCACACCCTGG	Badh2敲除靶点测序鉴定
Badh2-R	TAACTGCCTTCCTTGCCACG	Sequencing for Badh2 knockout target
OsRR22-S1-F	GGCACTCAACTGACCATACAACTC	构建OsRR22敲除载体
OsRR22-S1-R	AAACGAGTTGTATGGTCAGTTGAG	Construction of OsRR22 knockout vector
OsRR22-F	TAGGAGGAAGTTCGGTAATCGT	OsRR22敲除靶点测序鉴定
OsRR22-R	ACATTTTCCCTGGTGAGTTTCT	Sequencing for OsRR22 knockout target
Cas9-F	CCTTCTGTGTGGTCTGGTTC	载体检测
Cas9-R	AGACAATCACCCCCTGGAAC	Vector assay
Hyg-F	CTATTTCTTTGCCCTCGGAC	转基因检测
Hyg-R	ATGCCTGAACTCACCGCGAC	Transgenic detection
qPCR SOS1-F	ATTGCAGCTGAGCATGTACG	实时荧光定量PCR
qPCR SOS1-R	AGAGCTTGCTTTCGTGTGAC	Quantitative real-time PCR
qPCR OsMYB2-F	GGGCTGAAACGCACAGGCAAGA	实时荧光定量PCR
qPCR OsMYB2-R	CTGCTTGGCGTGCTTCTGC	Quantitative real-time PCR
qPCR OsNHX1-F	GTGACAGACCTGGCAAATCC	实时荧光定量PCR
qPCR OsNHX1-R	TCGACACAGCTCCTCTCATC	Quantitative real-time PCR
qPCR OsHKT1;1-F	TGCCAGAAGTTGTTGAAGCC	实时荧光定量PCR
qPCR OsHKT1;1-R	CCCAGGAACATCACCAGGAT	Quantitative real-time PCR
qPCR OsP5CS1-F	GTCAGAGTGGACTGATGGCT	实时荧光定量PCR
qPCR OsP5CS1-R	GCCTTTCTAGTGCTGATGGC	Quantitative real-time PCR
UBIQUITIN-F	GCTCCGTGGCGGTATCAT	实时荧光定量PCR
UBIQUITIN-R	CGGCAGTTGACAGCCCTAG	Quantitative real-time PCR

引物名称 Primer name	引物序列 Primer sequence(5’-3’)
Badh2-S1-F	GGCATGGTGGAAAAAGTCCTATAG	构建Badh2敲除载体 Construction of Badh2 knockout vector
Badh2-S1-R	AAACCTATAGGACTTTTTCCACCA	构建Badh2敲除载体 Construction of Badh2 knockout vector
Badh2-F	CCTTTGTCATCACACCCTGG	Badh2敲除靶点测序鉴定
Badh2-R	TAACTGCCTTCCTTGCCACG	Sequencing for Badh2 knockout target
OsRR22-S1-F	GGCACTCAACTGACCATACAACTC	构建OsRR22敲除载体
OsRR22-S1-R	AAACGAGTTGTATGGTCAGTTGAG	Construction of OsRR22 knockout vector
OsRR22-F	TAGGAGGAAGTTCGGTAATCGT	OsRR22敲除靶点测序鉴定
OsRR22-R	ACATTTTCCCTGGTGAGTTTCT	Sequencing for OsRR22 knockout target
Cas9-F	CCTTCTGTGTGGTCTGGTTC	载体检测
Cas9-R	AGACAATCACCCCCTGGAAC	Vector assay
Hyg-F	CTATTTCTTTGCCCTCGGAC	转基因检测
Hyg-R	ATGCCTGAACTCACCGCGAC	Transgenic detection
qPCR SOS1-F	ATTGCAGCTGAGCATGTACG	实时荧光定量PCR
qPCR SOS1-R	AGAGCTTGCTTTCGTGTGAC	Quantitative real-time PCR
qPCR OsMYB2-F	GGGCTGAAACGCACAGGCAAGA	实时荧光定量PCR
qPCR OsMYB2-R	CTGCTTGGCGTGCTTCTGC	Quantitative real-time PCR
qPCR OsNHX1-F	GTGACAGACCTGGCAAATCC	实时荧光定量PCR
qPCR OsNHX1-R	TCGACACAGCTCCTCTCATC	Quantitative real-time PCR
qPCR OsHKT1;1-F	TGCCAGAAGTTGTTGAAGCC	实时荧光定量PCR
qPCR OsHKT1;1-R	CCCAGGAACATCACCAGGAT	Quantitative real-time PCR
qPCR OsP5CS1-F	GTCAGAGTGGACTGATGGCT	实时荧光定量PCR
qPCR OsP5CS1-R	GCCTTTCTAGTGCTGATGGC	Quantitative real-time PCR
UBIQUITIN-F	GCTCCGTGGCGGTATCAT	实时荧光定量PCR
UBIQUITIN-R	CGGCAGTTGACAGCCCTAG	Quantitative real-time PCR