Construction of CRISPR/Cas9 vector for editing IaVQ9 gene in Ipomoea aquatica

Huynh Thi Thu Hue, Nguyen Thi Bich Ngoc, Le Tat Thanh , Nguyen Thi Hoa, Le Minh Tri , Le Thi Bich Thuy
Author affiliations

Authors

  • Huynh Thi Thu Hue \(^1\) Institute of Genome Research, Vietnam Academy of Science and Technology, Hanoi, Vietnam
    \(^2\) Graduate University of Science and Technology, Vietnam Academy of Science and Technology, Hanoi, Vietnam
    https://orcid.org/0000-0003-2051-9638
  • Nguyen Thi Bich Ngoc \(^1\) Institute of Genome Research, Vietnam Academy of Science and Technology, Hanoi, Vietnam
  • Le Tat Thanh \(^1\) Institute of Genome Research, Vietnam Academy of Science and Technology, Hanoi, Vietnam
    \(^2\) Graduate University of Science and Technology, Vietnam Academy of Science and Technology, Hanoi, Vietnam
  • Nguyen Thi Hoa \(^3\) Academy of Military Science and Technology, Hanoi, Vietnam https://orcid.org/0009-0008-2843-056X
  • Le Minh Tri \(^3\) Academy of Military Science and Technology, Hanoi, Vietnam https://orcid.org/0000-0003-3494-5195
  • Le Thi Bich Thuy \(^4\) Institute of Biotechnology, Vietnam Academy of Science and Technology, Hanoi, Vietnam

DOI:

https://doi.org/10.15625/vjbt-19559

Keywords:

Ipomoea aquatica, gRNA, CRISPR/Cas9, VQ9, salt tolerance

Abstract

The group of proteins containing the VQ motif (named VQ proteins) is a family of plant-specific proteins with a FxxhVQxhTG conservative VQ-motif region. VQ proteins regulate many developmental processes, including responses to biotic and abiotic stresses, and seed development. The VQ9 protein has an interaction with the WRKY8 factor, when this interaction occurs, it causes a decrease in the DNA binding ability of WRKY8 to DNA, which plays a role in the regulation function of the plant to stress. Some mutations in the VQ9 gene increase salt tolerance in plants, suggesting that VQ9 acts antagonistically to regulate responses to salt conditions. This antagonism is consistent with an increase or decrease in the Na+/K+ ratio. Ipomoea aquatica is commonly grown and used as a vegetable in Southeast Asia. The research involved RNA extraction from I. aquatica leaves, followed by PCR sequencing to confirm the presence of the IaVQ9 gene. Subsequently, a specific guide RNA (gRNA) was designed using CRISPR-P ver.2.0 and inserted into the pRGEB31 vector, optimized for CRISPR/Cas9 applications. The gRNA-inserted vector was successfully transformed into E. coli DH10B and then into Agrobacterium tumefaciens EHA105, verified through colony PCR and restriction enzyme analysis. This process created a delivery system capable of editing the VQ9 gene in I. aquatica. This research represents a significant step towards improving crop resilience to salinity, addressing a critical challenge for agriculture in salt-affected regions. Future studies will focus on transferring the construct back into I. aquatica plants to assess its impact on enhancing salt tolerance, potentially contributing to sustainable crop production in adverse environmental conditions.

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References

Anthon C, Corsi GI, and Gorodkin J (2022) CRISPR on/off: CRISPR/Cas9 on- and off-target gRNA design. Bioinformatics 38: 5437-5439. http://doi.org/10.1093/bioinformatics/btac697

Buscaill P, Rivas S (2014) Transcriptional control of plant defence responses. Curr Opin Plant Biol 20: 35-46. http://doi.org/10.1016/j.pbi.2014.04.004.

Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini L A, Zhang F (2013) Multiplex Genome Engineering Using CRISPR/Cas Systems. Science 339: e819. http://doi.org/10.1126/science.1231143.

Char SN, Neelakandan AK, Nahampun H, Frame B, Main M, Spalding MH, Becraft PW, Meyers BC, Walbot V, Wang K and Yang B. (2016) An Agrobacterium-delivered CRISPR/Cas9 system for high-frequency targeted mutagenesis in maize. Plant Biotechnol J 15:257-268. http://doi.org/ 10.1111/pbi.12611.

Chen P, Wei F, Cheng S, Ma L, Wang H, Zhang M, Mao G, Lu J, Hao P, Ahmad A, Gu L, Ma Q, Wu A, We H, Yu S (2020) A comprehensive analysis of cotton VQ gene superfamily reveals their potential and extensive roles in regulating cotton abiotic stress. BMC Genomics 21: e795. http://doi.org/10.1186/s12864-020-07171-z.

Dong JX, Chen CH, Chen Z X (2003) Expression profiles of the Arabidopsis WRKY gene superfamily during plant defense response. Plant Mol Biol 51: 21-37. http://doi.org/10.1023/a:1020780022549.

Doudna JA, Charpentier E (2014) Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science 346: e1258096. http://doi.org/10.1126/science.1258096

Fan D, Liu T, Li C, Jiao B, Li S, Hou Y, Luo K (2015) Efficient CRISPR/Cas9-mediated Targeted Mutagenesis in Populus in the First Generation. Scientific reports 5: 12217-12217. http://doi.org/10.1038/srep12217.

Guo R, Wang X, Han X, Chen X, Wang-Prusk G (2020) Physiological and transcriptomic responses of water spinach (Ipomoea aquatica) to prolonged heat stress. BMC Genomics 21:533 https://doi.org/10.1186/s12864-020-06953-9.

Hu Y, Chen L, Wang H, Zhang L, Wang F, Yu D (2013) Arabidopsis transcription factor WRKY8 functions antagonistically with its interacting partner VQ9 to modulate salinity stress tolerance. The Plant Journal 74: 730-745. http://doi.org/10.1111/tpj.12159.

Hu YR, Dong QY, Yu DQ (2012) Arabidopsis WRKY46 coordinates with WRKY70 and WRKY53 in basal resistance against pathogen Pseudomonas syringae. Plant Sci 185: 288-297. http://doiorg/10.1016/j.plantsci.2011.12.003.

Jiang W, Zhou H, Bi H, Fromm M, Yang B, Weeks DP (2013) Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice. Nucleic acids research 41: e188. http://doi.org/10.1093/nar/gkt780.

Kadkhodaei S, Memari HR, Abbasiliasi S, Rezaei MA, Movahedi A, Shun TJ, Ariff AB (2016) Multiple overlap extension PCR (MOE-PCR): an effective technical shortcut to high throughput synthetic biology. RSC Advances 6: 66682-66694. http://doi.org/10.1039/C6RA13172G.

Ma X, Zhang Q, Zhu Q, Liu W, Chen Y, Qiu R, Wang B, Yang Z, Li H, Lin Y, Xie Y, Shen R, Chen S, Wang Z, Chen Y, Guo J, Chen L, Zhao X, Dong Z, Liu YG (2015) A Robust CRISPR/Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants. Molecular Plant 8: 1274-1284. http://doi.org/10.1016/j.molp.2015.04.007.

Nguyen NT, Vu HT, Nguyen TT, Nguyen L-AT, Nguyen M-CD, Hoang KL, Nguyen KT, Quach TN (2019) Co-expression of Arabidopsis AtAVP1 and AtNHX1 to Improve Salt Tolerance in Soybean. Crop Sci 59:1133-1143. http://doi.org/10.2135/cropsci2018.10.0640.

Pan C, Ye L, Qin L, Liu X, He Y, Wang J, Chen L, Lu G (2016) CRISPR/Cas9-mediated efficient and heritable targeted mutagenesis in tomato plants in the first and later generations. Sci Rep 6: e24765. http://doi.org/10.1111/pbi.12832.

Redillas MCFR, Park SH, Lee JW, Kim YS, Jeong JS, Jung H, Bang SW, Hahn TR, Kim JK (2012) Accumulation of trehalose increases soluble sugar contents in rice plants conferring tolerance to drought and salt stress. Plant Biotechnol Rep 6:89-96. http://doi.org/10.1007/s11816-011-0210-3.

Shan Q, Wang Y, Li J, Zhang Y, Chen K, Liang Z, Zhang K, Liu J, Xi JJ, Qiu JL, Gao C (2013) Targeted genome modification of crop plants using a CRISPR-Cas system. Nature Biotechnology 31: e686. http://doi.org/10.1038/nbt.2650.

Shi J, Gao H, Wang H, Lafitte HR, Archibald RL, Yang M, Hakimi SM, Mo H, Habben JE (2017) ARGOS8 variants generated by CRISPR-Cas9 improve maize grain yield under field drought stress conditions. Plant Biotechnology Journal 15: 207-216. http://doi.org/10.1111/pbi.12603.

Song W, Zhao H, Zhang X, Lei L and Lai J (2016) Genome-Wide Identification of VQ Motif-Containing Proteins and their Expression Profile Under Abiotic Stresses in Maize. Front. Plant Sci 5: e1177. http://doi.org/10.3389/fpls.2015.01177.

Tian J, Zhang J, Francis F (2024) The role and pathway of VQ family in plant growth, immunity, and stress response. Planta 256: e16. http://doi.org/10.3389/fimmu.2020.612452.

Ülker B, Somssich IE (2004) WRKY transcription factors: from DNA binding towards biological function. Curr opin plant biol 7: 491-498. http://doi.org/10.1016/j.pbi.2004.07.012.

Wani SH, Kumar V, Khare T, Guddimalli R, Parveda M, Solymosi K, Suprasanna P, Kishor PK (2020) Engineering salinity tolerance in plants: progress and prospects. Planta 251:1-29. http://doi.org/10.1007/s00425-020-03366-6.

Wang L, Chen L, Li R, Zhao R, Yang M, Sheng J, Shen L (2017) Reduced Drought Tolerance by CRISPR/Cas9-Mediated SlMAPK3 Mutagenesis in Tomato Plant. J Agric Food Chem. 65: 8674-8682. http://doi.org/10.1021/acs.jafc.7b02745.

Xie K, Yang Y (2013) RNA-Guided Genome Editing in Plants Using a CRISPR-Cas System. Molecular Plant 6: 1975-1983. http://doi.org/10.1093/mp/sst119.

Yan H, Wang Y, Hu B, Qiu Z, Zeng B, Fan C (2019) Genome-Wide characterization, Evolution, and Expression Profiling of VQ Gene Family in Response to Phytohormone Treatments and Abiotic Stress in Eucalyptus grandis. International Journal of molecular sciences 20: e1765. http://doi.org/10.3390/ijms20071765.

Zhang A, Liu Y, Wang F, Li T, Chen Z, Kong D, Bi J, Zhang F, Luo X, Wang J, Tang J, Yu X, Liu G, Luo L (2019) Enhanced rice salinity tolerance via CRISPR/

Cas9-targeted mutagenesis of the OsRR22 gene. Mol Breeding 39: e47. http://doi.org/ 10.1007/s11032-019-0954-y.

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Published

30-09-2024

How to Cite

Hue, H. T. T., Ngoc, N. T. B., Thanh , L. T., Hoa, N. T., Tri , L. M., & Thuy, L. T. B. (2024). Construction of CRISPR/Cas9 vector for editing <i>IaVQ9</i> gene in <i>Ipomoea aquatica</i>. Vietnam Journal of Biotechnology, 22(3), 437–449. https://doi.org/10.15625/vjbt-19559

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