Objective Upstream regulatory factors of boron toxicity-related gene MIR397 in citrus were identified to understand the underlying mechanism for possible genetic improvement on the plants.

Method A homogenized yeast one-hybrid (Y1H) three-frame cDNA library on Xuegan (Citrus sinensis) and sour pumelo (C. grandis) was constructed by SMART RACE technology. Upstream regulators of MIR397 were screened based on the cis-elements, including ABRE, GCN4, Box II-like, and ERE, as baits.

Results The capacity of the 3 frame cDNA libraries were 1.5×10⁶, 1.5×10⁶, and 1.6×10⁶ CFU, respectively. The amplification sizes of the inserted fragments in the cDNA library ranged from 500 to 4 000bp with a recombination rate of 100% and zero redundancy. With a titer of 4×10⁹ CFU·mL⁻¹, the requirements for constructing Y1H cDNA library was met. Twenty-three expression sequence tags (ESTs) were obtained from the Y1H screening. Bioinformatics analysis revealed that 60.87% of the ESTs were related to plant growth and development, stress response, signal transduction, and transcriptional regulation, 17.39% to protein translation, and the remainders annotated as proteases. HAP3, BBX, and EIN3 might participate in the citrus response to boron toxicity by interacting with ERE cis elements. Point-to-point Y1H verification showed that HAP3, BBX, and EIN3 interacted with ERE elements.

Conclusion Three transcription factors that interacted with the ERE element of the MIR397 promoter were identified through Y1H library screening. The information obtained paved the way for further studies on the molecular networks involved in the citrus responses to boron toxicity.