Cloning and Functional Analysis of APETALA3/DEFICIENS homologous gene from Rhus chinensis

doi:10.11983/CBB24015

Abstract

Abstract: The APETALA3/DEFICIENS (AP3/DEF) gene is a B-class gene of the MADS-box gene family, which is mainly involved in the regulation of petal and stamen development during flower development. Cloning and analysis of the AP3/DEF homologous genes from Rhus chinensis can help explore their role in the development of stamen. The CDS of AP3/DEF homologous gene was obtained using RT-PCR; its sequence and structural domains were compared and analyzed by NCBI CD Search; interactions between AP3/DEF homologous proteins and other MADS-box transcription factors in Rhus chinensis were verified by using yeast two-hybrid system; Real-time fluorescence PCR was used to analyze the spatial and temporal expression patterns of the AP3/DEF homologous genes; Overexpression in Arabidopsis was conducted to verify the function of the AP3/DEF homologous genes in flower organ development. Two coding sequences of AP3/DEF homologous genes in Rhus chinensis were cloned and named RcAP3 (GenBank: OR962160) and RcTM6 (GenBank: OR962159). Based on the alignment of their conserved amino acid domains and phylogenetic analysis, these two protein sequences showed the closest genetic relationship to the AP3/DEF homologous gene-encoded proteins in the Anacardiaceae family, including Mangifera indica and Pistacia vera. The yeast two-hybrid results showed that RcAP3 and RcTM6 interacted with the B-class protein RcPI, the C-class proteins RcAG and Rcag in Rhus chinensis, but not with the A-class and E-class proteins. Real-time fluorescence quantification results demonstrated that RcAP3 and RcTM6 genes were highly expressed during the rapid growth stage of flower buds in different sexes of Rhus chinensis, with lower expression levels during the early bud development and after flowering. RcAP3 gene exhibited high expression levels during buds differentiation in female, male, and hermaphrodite flowers, while RcTM6 showed significant expression in hermaphrodite flowers and very low expression in male and female flowers. During the rapid growth stage of hermaphrodite flowers, RcAP3 was highly expressed in both petals and stamens with minimal differences, whereas RcTM6 showed significantly higher expression in stamens compared to other floral organs. RcAP3 could restore the petal and stamen phenotypes in the ap3-3 mutant of Arabidopsis, while overexpression of RcTM6 resulted in shortened petals, stamens, and aborted ovaries in Arabidopsis. Functional differentiation exists between the AP3/DEF subfamily homologous genes, RcAP3 and RcTM6. RcAP3 promotes petal and stamen development, while RcTM6 inhibits stamen development. These findings provide a foundation for further research on the molecular mechanisms underlying sex differentiation in Rhus chinensis.

Key words: Rhus chinesnsis, RcAP3, RcTM6, expression pattern, functional analysis

Gu Lei, Zhang Qi, Zhang Xia, Yang Bingbing, Wang Fanglan, Liu Wen, Chen Faju. Cloning and Functional Analysis of APETALA3/DEFICIENS homologous gene from Rhus chinensis[J]. Chinese Bulletin of Botany, DOI: 10.11983/CBB24015.

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB24015

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