Construction of yeast library during walnut adventitious root formation and screening of upstream regulatory factors of JrAGD3 gene

Upstream regulatory factors of the JrAGD3 gene were screened by constructing a yeast library and using yeast one-hybrid technology, aiming to provide a theoretical basis for elucidating the molecular mechanism by which the JrAGD3 gene mediates rejuvenation and promotes adventitious root formation.

Tissues from rejuvenated walnut cuttings undergoing adventitious root formation were used as materials. A yeast cDNA library was constructed using SMART (Switching mechanism At 5′ end of RNA template) technology. The promoter sequence of the JrAGD3 gene was used as a bait to screen for upstream regulatory factors via the yeast one-hybrid (Y1H) system.

The promoter region of JrAGD3 contains multiple types of cis-acting elements, which are associated with light response regulation, hormone response regulation, meristem expression, and circadian rhythm regulation. The constructed cDNA library had a capacity of 3×10⁶ CFU/mL, with a total clone count of 1.20×10⁷ CFU, an average insert fragment length of approximately 1 000 bp, and a recombination rate of 100%. Through yeast one-hybrid screening, 19 potential upstream regulatory factors of JrAGD3 were identified, including two transcription factors: WOX4 and WRKY11.

This study successfully constructed a high-quality yeast library for the rejuvenation-induced adventitious root formation process in walnut cuttings, providing a valuable resource for screening interacting proteins using the yeast one-hybrid system. Using the JrAGD3 gene promoter sequence as bait, WOX4 and WRKY11 transcription factors were identified, preliminarily revealing the potential transcriptional regulatory network of JrAGD3. These findings not only enriched the molecular understanding of adventitious root formation in walnut but also provided new insights for optimizing cutting propagation systems and improving the clonal propagation efficiency of walnut and other important economic forest tree species.