Objective To investigate the expression characteristics and regulatory relationship of miR396b and its target gene JmSVP during heterodichogamous flower development in Juglans mandshurica, and to clarify the sequence features and spatiotemporal expression pattern of JmSVP, so as to provide a basis for elucidating the molecular regulatory mechanism underlying heterodichogamous flower development and sex differentiation in J. mandshurica.

Method Female-first and male-first J. mandshurica plants were used as experimental materials. Female and male flower buds, as well as young leaves, stems, and fruits, were collected during the physiological differentiation stage (April 3 to April 30) and morphological differentiation stage (May 4 to May 26). Total RNA was extracted and reverse-transcribed into cDNA. The full-length CDS of JmSVP was cloned, followed by sequence homology, conserved domain, and phylogenetic analyses. The targeted cleavage relationship between miR396b and JmSVP was verified using RLM 5′RACE, and the expression patterns of miR396b and JmSVP in different organs and in female and male flower buds at different developmental stages were analyzed by qRT-PCR.

Result The full-length CDS of JmSVP was 687 bp, encoding a protein of 228 amino acids containing conserved MADS-box and K-box domains. Phylogenetic analysis showed that JmSVP clustered with SVP proteins from closely related species such as walnut, indicating high sequence homology. RLM 5′RACE confirmed that JmSVP is a direct downstream target of miR396b during flower development in J. mandshurica, and the cleavage site was located between the 10th and 11th nucleotides of the mature miR396b sequence. Moreover, the expression trends of miR396b and JmSVP were opposite during the critical transition from physiological to morphological differentiation. qRT-PCR analysis showed that JmSVP was expressed in stems, leaves, female flower buds, male flower buds, and fruits, but was significantly more highly expressed in flower buds, with the highest expression detected in female flower buds. During the development of female-first and male-first flower buds in Juglans mandshurica, JmSVP showed significant differences in expression among different developmental stages, reproductive types, and flower-bud sexes, exhibiting clear spatiotemporal specificity overall. Its high-expression dominant type shifts with developmental progression from female flowers in the early stage to male flowers in the later stage.

Conclusion The miR396b-JmSVP regulatory module may participate in the differentiation and development of heterodichogamous flower buds in J. mandshurica, and the expression of both is likely co-regulated by the dual factors of “reproductive type + flower bud sex”. These findings provide theoretical support for further elucidating the molecular regulatory mechanism of flower development in J. mandshurica.