Yeast one-hybrid library construction and screening of upstream regulators of <i>MIR397</i> in boron-treated <i>Citrus</i> leaves

HUANG Jing-Hao; LIN Xiongjie; CHEN Mulan; RAO Wenhua; HE Jingyi; GAO Fangluan; FAN Guocheng

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Fujian Journal of Agricultural Sciences > 2025 > Accepted Manuscript

HUANG J - H，LIN X J，CHEN M L，et al. Yeast one-hybrid library construction and screening of upstream regulators of MIR397 in boron-treated Citrus leaves[J]. Fujian Journal of Agricultural Sciences，2025，40（X）：1−10.

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Yeast one-hybrid library construction and screening of upstream regulators of MIR397 in boron-treated Citrus leaves

1.
Pomological Institute, Fujian Academy of Agricultural Sciences/Fujian Key Laboratory of Plant Nutrition and Fertilizer, Fuzhou, Fujian　350013, China
2.
Sanming Agriculture and Rural Bureau, Sanming, Fujian　365000, China
3.
Institute of Plant Protection, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian 350013, China
4.
College of Plant Protection, Fujian Agriculture and Forestry University, Fuzhou, Fujian　350002, China

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Received Date: October 09, 2024
Revised Date: November 07, 2024
Available Online: April 10, 2025

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Abstract

Abstract

Objective
To gain a deeper understanding of the mechanism underlying boron toxicity in citrus and provide scientific basis for genetic improvement of citrus resistance to boron toxicity, upstream regulatory factors to MIR397, one of key boron toxicity-responded gene in citrus, were screened and identified in this study.
Method
A homogenized yeast one-hybrid three-frame cDNA Library was constructed by SMART RACE technology using ‘Xuegan’ (Citrus sinensis) and sour pumelo (C. grandis) as materials, and upstream regulators of boron toxicity responsive MIR397 were screened using cis-elements, including ABRE, GCN4, Box II-like and ERE, as baits.
Results
The results show that the capacity of the three frame cDNA libraries were 1.5×10⁶,1.5×10⁶ and 1.6×10⁶ CFU, respectively. The amplification sizes of inserted fragments in the cDNA library ranged from 500 to 4000 bp, with a recombination rate of 100% and a redundancy rate of 0. The titer of the cDNA library is 4×10⁹ CFU·mL⁻¹, which met the requirements for constructing yeast one-hybrid cDNA library. Twenty three expression sequence tags (ESTs) were obtained from yeast one-hybrid (Y1H) screening of the library. Bioinformatics analysis revealed that 60.87% of the ESTs are related to plant growth and development, stress response, signal transduction and transcriptional regulation, and 17.39% are related to protein translation, while the rest of ESTs are annotated as proteases. HAP3, BBX, and EIN3 may participate in citrus response to boron toxicity by interacting with ERE cis-elements. Point-to-point Y1H verification showed that HAP3, BBX, and EIN3 interact with ERE elements.
Conclusion
Three transcription factors that interact with the ERE element of the MIR397 promoter were identified through yeast one hybrid library screening, laying the foundation for further molecular network research on citrus responses to boron toxicity.
- Citrus,
- boron toxicity,
- three-frame cDNA library,
- promoter,
- regulator

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Yeast one-hybrid library construction and screening of upstream regulators of MIR397 in boron-treated Citrus leaves

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