Enhancing Salt Tolerance of Rice by Knocking out OsEIL1 and OsEIL2 Via CRISPR/Cas9 System

doi:10.7668/hbnxb.20190798

Abstract

Abstract: In order to breeding salt tolerant rice varieties, a target was designed to simultaneously knockout OsEIL1 and OsEIL2 genes in rice variety Dongnong 427 by using CRISPR/Cas9 technology. Five mutant plants were obtained in T₀ via PCR and sequencing method, one of them was double genes mutant plant. In the T₁ mutant lines of this plant, four double gene homozygous mutant plants without T-DNA element were obtained. Salt tolerance experiment in seedling stage and agronomic trait analysis at maturity were carried out in T₂. The results indicated that after 200 mmol/L NaCl solution treatment for 5 days, the fresh weight and the dry weight of mutant plants were significantly higher than wild type plants, the Na⁺ content in shoot and root were significantly lower than wild type plants, however, the K⁺ content was no difference with wild type plants. After recovered for 7 days, the seedling survival rate of mutant plants(75.0%) was significantly higher than wild type plants(8.3%). The main agronomic traits didn't change significantly at maturity. In summary, this study improved the salt tolerance of rice variety Dongnong 427 by using CRISPR/Cas9 technology, and provided a theoretical and material basis for the cultivation of salt tolerant rice variety.

Key words: Rice, Salt tolerance, OsEIL1, OsEIL2, CRISPR/Cas9

CLC Number:

Q78
S511.03

MO Tianyu, XU Shanbin, ZOU Detang, WANG Jingguo, LIU Hualong, SUN Jian, JIA Yan, ZHAO Hongwei, ZHENG Hongliang. Enhancing Salt Tolerance of Rice by Knocking out OsEIL1 and OsEIL2 Via CRISPR/Cas9 System[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2021, 36(1): 71-80. doi: 10.7668/hbnxb.20190798.

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References

[1] Ma C Q, Wang Y G, Gu D, Nan J D, Chen S X, Li H Y. Overexpression of S-Adenosyl-l-Methionine synthetase 2 from sugar beet M14 increased Arabidopsis tolerance to salt and oxidative stress[J].International Journal of Molecular Science, 2017,18(4):847. doi:10.3390/ijms18040847.
[2] Ouhibi C, Attia H, Rebah F, Msilini N, Chebbi M, Aarrouf J, Urban L,Lachaal M.Salt stress mitigation by seed priming with UV-C in lettuce plants:growth, antioxidant activity and phenolic compounds[J].Plant Physiology and Biochemistry, 2014,83:126-133.doi:10.1016/j.plaphy.2014.07.019.
[3] Munns R, Tester M.Mechanisms of salinity tolerance[J].Annual Review of Plant Biology, 2008,59:651-681. doi:10.1146/annurev.arplant.59.032607.092911.
[4] 杨劲松.中国盐渍土研究的发展历程与展望[J]. 土壤学报,2008,45(5):837-845. doi:10.3321/j.issn:0564-3929.2008.05.010.
Yang J S. Development and prospect of the research on salt affected soils in China[J]. Acta Pedologica Sinica,2008,45(5):837-845.
[5] Khush G S. What it will take to feed 5.0 billion rice consumers in 2030[J].Plant Molecular Biology, 2005,59(1):1-6. doi:10.1007/s11103-005-2159-5.
[6] Li Y, Xiao J H, Chen L L, Huang X H, Cheng Z K, Han B, Zhang Q F, Wu C Y. Rice functional genomics research:past decade and future[J].Molecular Plant, 2018,11(3):359-380. doi:10.1016/j.molp.2018.01.007.
[7] Lutts S, Kinet J M, Bouharmont J. Changes in plant response to NaCl during development of rice(Oryza sativa L.) varzieties differing in salinity resistance[J]. Journal of Experimental Botany, 1995,46(293):1843-1852. doi:10.1093/jxb/46.12.1843.
[8] Huang X Y, Chao D Y, Gao J P, Zhu M Z, Shi M, Lin H X. A previously unknown zinc finger protein, DST, regulates drought and salt tolerance in rice via stomatal aperture control[J].Genes Development, 2009,23(15):1805-1817. doi:10.1101/gad.1812409.
[9] El Mahi H, Pérez-Hormaeche J, De Luca A, Villalta I, Espartero J, Gámez-Arjona F, Fernández J L, Bundó M, Mendoza I, Mieulet D, Lalanne E, Lee S Y, Yun D J, Guiderdoni E, Aguilar M, Leidi E O, Pardo J M, Quintero F J. 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. doi:10.1104/pp.19.00324.
[10] Suzuki K, Costa A, Nakayama H, Katsuhara M, Shinmyo A, Horie T. OsHKT2;2/1-mediated Na⁺ influx over K⁺ uptake in roots potentially increases toxic Na⁺ accumulation in a salt-tolerant landrace of rice Nona Bokra upon salinity stress[J].Journal of Plant Research, 2016,129(1):67-77. doi:10.1007/s10265-015-0764-1.
[11] Qiu D Y, Xiao J, Xie W B, Liu H B, Li X H, Xiong L Z,Wang S P. Rice gene network inferred from expression profiling of plants overexpressing OsWRKY13, a positive regulator of disease resistance[J].Molecular Plant, 2008,1(3):538-551. doi:10.1093/mplant/ssn012.
[12] Hou X, Xie K B, Yao J L, Qi Z Y, Xiong L Z.A homolog of human ski-interacting protein in rice positively regulates cell viability and stress tolerance[J].Proceedings of the National Academy of Sciences of the United States of America, 2009,106(15):6410-6415. doi:10.1073/pnas.0901940106.
[13] Zhu J K. Salt and drought stress signal transduction in plants[J].Annual Review of Plant Biology,2002,53:247-273. doi:10.1146/annurev.arplant.53.091401.143329.
[14] Erpen L, Devi H S, Grosser J W, Dutt M. Potential use of the DREB/ERF, MYB, NAC and WRKY transcription factors to improve abiotic and biotic stress in transgenic plants[J].Plant Cell, Tissue and Organ Culture, 2017,132(1):1-25. doi:10.1007/s11240-017-1320-6.
[15] Mao C Z, Wang S M, Jia Q J, Wu P. OsEIL1, a rice homolog of the Arabidopsis EIN3 regulates the ethylene response as a positive component[J].Plant Molecular Biology,2006,61(1-2):141-152.doi:10.1007/s11103-005-6184-1.
[16] 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. doi:10.1104/pp.15.00353.
[17] Cermak T, Doyle E L, Christian M, Wang L, Zhang Y, Schmidt C, Baller J A, Somia N V, Bogdanove A J, Voytas D F.Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting[J]. Nucleic Acids Research, 2011,39(17):7879-7879. doi:10.1093/nar/gkr739.
[18] Lloyd A, Plaisier C L, Carroll D, Drews G N.Targeted mutagenesis using zinc-finger nucleases in Arabidopsis[J].Proceedings of the National Academy of Sciences of the United States of Americ a, 2005,102(6):2232-2237. doi:10.1073/pnas.0409339102.
[19] Cong L, Ran F A, Cox D, Lin S L, Barretto R, Habib N, Hsu P D, Wu X B, Jiang W Y, Marraffini L A, Zhang F.Multiplex genome engineering using CRISPR/Cas systems[J].Science, 2013,339(6121):819-823.doi:10.1126/science.1231143.
[20] Barrangou R,Fremaux C,Deveau H,Richards M,Boyaval P,Moineau S, Romero D A, Horvath P.CRISPR provides acquired resistance against viruses in prokaryotes[J].Science, 2007,315(5819):1709-1712.doi:10.1126/science.1138140.
[21] Puchta H. The repair of double-strand breaks in plants:mechanisms and consequences for genome evolution[J].Journal of Experimental Botany, 2005,56(409):1-14. doi:10.1093/jxb/eri025.
[22] Huang L Y, Zhang R, Huang G F, Li Y X, Melaku G, Zhang S L, Chen H T, Zhao Y J, Zhang J, Zhang Y S, Hu F Y. Developing superior alleles of yield genes in rice by artificial mutagenesis using the CRISPR/Cas9 system[J].The Crop Journal, 2018,6(5):475-481. doi:10.1016/j.cj.2018.05.005.
[23] Miao C B, Xiao L H, Hua K, Zou C S, Zhao Y, Bressan R A, Zhu J K. Mutations in a subfamily of abscisic acid receptor genes promote rice growth and productivity[J].Proceedings of the National Academy of Sciences of the United States of America, 2018,115(23):6058-6063. doi:10.1073/pnas.1804774115.
[24] Shan Q W, Wang Y P, Li J, Zhang Y, Chen K L, Liang Z, Zhang K, Liu J X, Xi J J, Qiu J L, Gao C X. Targeted genome modification of crop plants using a CRISPR-Cas system[J].Nature Biotechnology, 2013,31(8):686-688. doi:10.1038/nbt.2650.
[25] Wang Y P, Cheng X, Shan Q W, Zhang Y, Liu J X, Gao C X, Qiu J L. Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew[J].Nature Biotechnology, 2014,32(9):947-951. doi:10.1038/nbt.2969.
[26] Do P T, Nguyen C X, Bui H T, Tran L T N, Stacey G, Gillman J D, Zhang Z J, Stacey M G. Demonstration of highly efficient dual gRNA CRISPR/Cas9 editing of the homeologous GmFAD2-1A and GmFAD2-1B genes to yield a high oleic, low linoleic and alpha-linolenic acid phenotype in soybean[J].BMC Plant Biology, 2019,19(1):311. doi:10.1186/s12870-019-1906-8.
[27] Jacobs T B, LaFayette P R, Schmitz R J, Parrott W A. Targeted genome modifications in soybean with CRISPR/Cas9[J]. BMC Biotechnology, 2015,15:16. doi:10.1186/s12896-015-0131-2.
[28] Liang Z, Zhang K, Chen K L, Gao C X. Targeted mutagenesis in Zea mays using TALENs and the CRISPR/Cas system[J].Journal of Genetics and Genomics, 2014,41(2):63-68. doi:10.1016/j.jgg.2013.12.001.
[29] Svitashev S, Young J K, Schwartz C, Gao H R, Falco S C, Cigan A M. Targeted mutagenesis, precise gene editing, and site-specific gene insertion in maize using Cas9 and guide RNA[J].Plant Physiol, 2015,169(2):931-945. doi:10.1104/pp.15.00793.
[30] Li A X,Jia S G,Yobi A,Ge Z X,Sato S J,Zhang C,Angelovici R,Clemente T E,Holding D R.Editing of an alpha-kafirin gene family increases, digestibility and protein quality in sorghum[J].Plant Physiology, 2018, 177(4):1425-1438. doi:10.1104/pp.18.00200.
[31] 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].Nat Biotechnol, 2019,37(3):283-286. doi:10.1038/s41587-018-0003-0.
[32] Tang L, Mao B G, Li Y K, Lü Q M, Zhang L P, Chen C Y, He H J, Wang W P, Zeng X F, Shao Y, Pan Y L, Hu Y Y, Peng Y, Fu X Q, Li H Q, Xia S T, Zhao B R.Knockout of OsNramp5 using the CRISPR/Cas9 system produces low Cd-accumulating indica rice without compromising yield[J]. Scientific Reports, 2017,7(1):14438. doi:10.1038/s41598-017-14832-9.
[33] 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(3):47. doi:10.1007/s11032-019-0954-y.
[34] Xie X R, Ma X L, 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. doi:10.1016/j.molp.2017.06.004.
[35] 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. doi:10.1016/j.molp.2015.04.007.
[36] Rowland L J, Nguyen B. Use of polyethylene glycol for purification of DNA from leaf tissue of woody plants[J].Biotechniques, 1993,14(5):734-736. doi:10.1002/bip.360330513.
[37] 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.doi:10.1016/j.molp.2015.05.009.
[38] 方玉洁,覃永华,熊立仲.水稻叶片Na⁺,K⁺含量测定(火焰光度法)[J]. Bio-Protocol, 2018:Bio-101 e1010149. doi:10.21769/BioProtoc.1010149.
Fang Y J, Qin Y H, Xiong L Z.Determination of Na⁺, K⁺ content in rice leaf(flame photometric method)[J]. Bio-Protocol, 2018:Bio-101e1010149.
[39] 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 L, Li H Q. Reassessment of the four yield-related genes Gn1a, DEP1, GS3, and IPA1 in rice using a CRISPR/Cas9 system[J].Frontiers in Plant Science, 2016,7:377. doi:10.3389/fpls.2016.00377.
[40] Tang X, Liu G Q, Zhou J P, Ren Q R, You Q, Tian L, Xin X H, Zhong Z H, Liu B L, Zheng X L, Zhang D W, Malzahn A, Gong Z Y, Qi Y P, Zhang T, Zhang Y. A large-scale whole-genome sequencing analysis reveals highly specific genome editing by both Cas9 and Cpf1(Cas12a) nucleases in rice[J].Genome Biology, 2018,19:84. doi:10.1186/s13059-018-1458-5.
[41] Tsai S Q, Joung J K. Defining and improving the genome-wide specificities of CRISPR Cas9 nucleases[J].Nature Reviews Genetics, 2016,17(5):300-312. doi:10.1038/nrg.2016.28.

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