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植物学报 >

2024 , Vol. 59 >Issue 4: 533 - 543

DOI: https://doi.org/10.11983/CBB24015

研究论文

盐肤木APETALA3/DEFICIENS同源基因的克隆与功能分析

  • 顾磊 ,
  • 张棋 ,
  • 张霞 ,
  • 杨冰冰 ,
  • 王芳岚 ,
  • 刘文 ,
  • 陈发菊
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  • 三峡大学生物技术研究中心, 三峡区域植物遗传与种质创新重点实验室(三峡大学), 宜昌 443002
*E-mail: chenfj616@163.com

收稿日期: 2024-01-30

  录用日期: 2024-03-30

  网络出版日期: 2024-04-17

基金资助

国家自然科学基金(32370393);湖北省中央引导地方科技发展专项(2022BGE265)

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Cloning and Functional Analysis of APETALA3/DEFICIENS Homologous Gene from Rhus chinensis

  • Lei Gu ,
  • Qi Zhang ,
  • Xia Zhang ,
  • Bingbing Yang ,
  • Fanglan Wang ,
  • Wen Liu ,
  • Faju Chen
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  • Key Laboratory of Three Gorges Regional Plant Genetics and Germplasm Enhancement (CTGU), Biotechnology Research Center, China Three Gorges University, Yichang 443002, China

Received date: 2024-01-30

  Accepted date: 2024-03-30

  Online published: 2024-04-17

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摘要

AP3/DEF (APETALA3/DEFICIENS)基因为MADS-box基因家族的B类基因, 在花发育过程中主要参与调控花瓣和雄蕊发育。对盐肤木(Rhus chinensis) AP3/DEF同源基因进行克隆及功能分析, 有助于探究其在盐肤木雄蕊发育过程中的作用。采用RT-PCR技术获得盐肤木AP3/DEF同源基因CDS; 利用NCBI CD Search对其序列和结构域进行比较分析; 利用酵母双杂交系统, 对AP3/DEF同源蛋白与盐肤木中其它MADS-box转录因子进行蛋白互作验证; 通过实时荧光定量PCR分析盐肤木AP3/DEF同源基因的时空表达模式; 用过表达拟南芥(Arabidopsis thaliana)验证盐肤木AP3/DEF同源基因在花器官发育中的功能。结果表明, 克隆得到2个盐肤木AP3/DEF同源基因分别命名为RcAP3 (GenBank: OR962160)和RcTM6 (GenBank: OR962159), 根据其氨基酸保守结构域比对及系统进化分析, 发现这2个蛋白序列与漆树科的芒果(Mangifera indica)和阿月浑子(Pistacia vera) AP3/DEF同源蛋白亲缘关系最近。酵母双杂交结果表明, RcAP3和RcTM6与盐肤木B类蛋白RcPI、C类蛋白RcAG和Rcag存在互作关系, 但与A类和E类蛋白不存在互作关系。实时荧光定量PCR分析结果显示, RcAP3和RcTM6基因在不同性别盐肤木花芽快速发育期高表达, 在花芽发育早期和开花后表达水平较低; RcAP3在雌花、雄花和两性花的花芽分化过程中均维持较高的表达水平, 而RcTM6在两性花中显著表达, 在雄花和雌花中表达量很低。两性花快速生长期, RcAP3在花瓣和雄蕊中高表达且差异很小, 而RcTM6在雄蕊中的表达量显著高于其它花器官。RcAP3基因能恢复拟南芥ap3-3突变体花瓣和雄蕊的缺陷表型, RcTM6过表达则导致拟南芥花瓣、雄蕊和子房缩短, 花药败育, 表明盐肤木中同属AP3/DEF亚家族的同源基因RcAP3和RcTM6存在功能分化。RcAP3促进花瓣和雄蕊发育, 而RcTM6抑制雄蕊发育。研究结果为进一步研究盐肤木性别分化的分子机制奠定了基础。

关键词： 盐肤木; RcAP3; RcTM6; 表达模式; 功能分析

本文引用格式

顾磊 , 张棋 , 张霞 , 杨冰冰 , 王芳岚 , 刘文 , 陈发菊 . 盐肤木APETALA3/DEFICIENS同源基因的克隆与功能分析[J]. 植物学报, 2024 , 59(4) : 533 -543 . DOI: 10.11983/CBB24015

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 exploring 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 R. 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 R. 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 R. chinensis, but not with the A-class and E-class proteins. Real-time fluorescence quantification results demonstrated that RcAP3 and RcTM6 were highly expressed during the rapid growth stage of flower buds in different sexes of R. chinensis, with lower expression levels during the early bud development and after flowering. RcAP3 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 R. chinensis.

Key words： Rhus chinensis; RcAP3; RcTM6; expression pattern; functional analysis

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