关键词: 盐肤木, 可变剪接, RcAG, Rcag, 功能分析

Abstract:

Objective: AGAMOUS (AG) plays an important role in controlling the development of pistil and stamen. The cloning and functional analysis of AG homologous gene is helpful to clarify the function of AG gene in the flower organ development of Rhus chinensis, and lays a foundation for further exploration of the gender differentiation mechanism of R. chinensis flowers. Method: Based on existing transcriptome data in this laboratory, and combined with RACE technology, an AG homologous gene was cloned from R. chinensis flowers, and the structure and phylogenetic evolution of its encoded protein were analyzed. The real-time quantitative PCR was used to analyze the expression characteristics of AG homologous gene of R. chinensis in female, male and bisexual flowers at different flowering stages. Furthermore, the overexpression vector of AG homologous gene of R. chinensis was constructed, the transgenic plants was obtained by inflorescence staining and the transgenic plant phenotype was observed. Result: Two alternative splicing transcripts of AG homologous gene were cloned from R. chinensis, namely RcAG and Rcag with the deletion of the sixth exon. RcAG contains 729 bp open reading frame and can encode 242 amino acids. Rcag contains 687 bp open reading frame, encoding 228 amino acids. Phylogenetic tree analysis showed that RcAG was closely related to AG homologous proteins of Mangifera indica and Pistacia vera. qRT-PCR analysis showed that the abundance of RcAG and Rcag in female, male and bisexual flowers of R. chinensis showed a continuously increasing trend at different developmental stages, and the abundance of RcAG and Rcag in male flowers was significantly higher than that in female and bisexual flowers. Phenotypic observation of Arabidopsis thaliana showed that overexpression of RcAG in A. thaliana resulted in loss of petals and stamens, stigmatization of the tip of the sepal, and ovular structure at the edge of the base of the sepal. However, the number of petals increased and the number of stamens decreased in Rcag transgenic Arabidopsis. Conclusion: Based on the above results, Rcag loses the normal function of AG gene probably due to the absence of 42 bp base in K-region, and competes with RcAG to jointly regulate the development of female and stamen of R. chinensis. These results lay a foundation for the further exploration of RcAG and Rcag in the sex differentiation of R. chinensis.

Key words: Rhus chinensis, alternative splicing, RcAG, Rcag, functional analysis