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Tissue-Specific CRISPR/Cas9 System of Cotton Pollen with GhPLIMP2b and GhMYB24 Promoters

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Abstract

CRISPR/Cas9 technology is a powerful tool for improving crop genetic traits. However, CRISPR/Cas9 system for efficient expression of cotton germ cells has still not been established. In this study, we developed a tissue-specific vectors to drive Cas9 expression with GhPLIMP2b and GhMYB24 promoters and established the effective method to transform cotton pollen by Agrobacterium vacuum infiltration. GhPLIMP2b and GhMYB24 promoters of cotton pollen were cloned into Cas9 expression vectors. The sgRNAs targetting to CLA1, ERA1 and GGB (drought-resistant negative regulation genes) were designed and constructed into GhPLIMP2b and GhMYB24 promoter vectors. Cotton pollens were tranformed by Agrobacterium vacuum infiltration with GhPLIMP2b and GhMYB24 promoter vectors. The results of clone sequencing shown that mutation types of the sequence were mainly base substitutions wth the frequency from 3.29 to 6.45%. Eleven potential off-target sites were chosen and two sites were observed. Our results indicated that GhPLIM2bP::Cas9 and GhMYB24P::Cas9 editing vectors achieved targeted edition of endogenous genes in cotton pollen, but there were a few off-target effects. This study provides an effective gene-editing system for the rapid acquisition of cotton mutants using pollen as a transgenic receptor.

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Acknowledgements

This work was supported by the Natural Science Foundation of China (316604330); the Fundamental Research Funds for the Central Universities (KYYJ201701); Xinjiang Uygur Autonomous Region postgraduate research and innovation project (XJ2019G131); Xinjiang Agricultural University cotton team development fund (XNMH2019003)

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Authors and Affiliations

  1. College of Agriculture, Engineering Research Centre of Cotton of Ministry of Education, Xinjiang Agricultural University, 311 Nongda East Road, Urumqi, 830052, China

    Jianfeng Lei, Peihong Dai, Jiyang Li, Mi Yang, Xiuqin Li, Wanqi Zhang, Guantong Zhou & Xiaodong Liu

  2. State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing, Jiangsu, China

    WangzhenGuo

Authors
  1. Jianfeng Lei
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  2. Peihong Dai
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  3. Jiyang Li
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  4. Mi Yang
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  5. Xiuqin Li
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  6. Wanqi Zhang
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  7. Guantong Zhou
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  8. WangzhenGuo
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  9. Xiaodong Liu
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Contributions

XDL and JFL designed the experiment; JFL, PHD, JYL, MY, XQL, WQZ, GTZ and WZG performed the experiments and analyzed the data; JFL and XDL wrote the manuscript. All the authors agreed on the contents of the paper and post no conflicting interest.

Corresponding author

Correspondence to Xiaodong Liu.

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Lei, J., Dai, P., Li, J. et al. Tissue-Specific CRISPR/Cas9 System of Cotton Pollen with GhPLIMP2b and GhMYB24 Promoters. J. Plant Biol. 64, 13–21 (2021). https://doi.org/10.1007/s12374-020-09272-4

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  • DOI: https://doi.org/10.1007/s12374-020-09272-4

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