《植物生理学报》 2021, 57(3): 643-654
芦笋性别决定基因Ao
通信作者：杨焕文；E-mail: mryanghuanwen@126.com；毛自朝；E-mail: mao2010zichao@126.com
摘 要：
为探究芦笋(Asparagus offcinalis)性别分化基因AoTDF1在花发育中的表达模式与分子功能, 本研究以四倍体‘紫色激情’两性株自交后代中雌、雄和两性株为材料, RT-PCR分别扩增AoTDF1的全长CDS(AoTDF1-P)序列及与拟南芥绒毡层发育调控AtAMS同源基因的全长CDS (AoAMS-P)序列, 构建基于黄色荧光蛋白(venus)的双分子荧光互补(bimolecular fluorescence complementation, BiFC)原核表达载体, 进行蛋白间互作检测; 同时用RNA原位杂交法检测AoTDF1-P在花发育早期的时、空特异性表达。结果显示:AoTDF1-P在雄花和两性花的无性别分化早期(T1)的雌蕊、雄蕊和花冠中均有较高表达, 在具性别分化早期(T2)降低表达; 而在性别分化后期(T3)两性花的花药绒毡层中专一地表达, 雄花无表达专一性, 但表达
量进一步降低; 而在T1、 T2、 T3期的雌花中AoTDF1-P均无表达。共聚焦显微观测到AoTDF1-P与AoAMS-P间产生较强的黄色荧光, 且AoTDF1-P和AoAMS-P自身均产生较弱的黄色荧光, 预示AoTDF1-P、AoAMS-P自身可形成同型聚体, 相互间能形成异型聚体调控复合物, 专一性地在芦笋雄花或两性花中通过调控下游基因的表达而促进雄性发育。研究为阐明AoTDF1分子机制奠定了基础。
关键词：芦笋; 性别决定; AoTDF1-P; 基因表达; 蛋白互作
收稿：2020-09-30 修定：2021-02-07
资助：国家自然科学基金(31360085)和云南省应用基础研究计划(2015FB143)
Corresponding author: YANG Huanwen; E-mail: mryanghuanwen@126.com; MAO Zichao; E-mail: mao2010zichao@126.com
Abstract:
In order to study the expression pattern and molecular function of AoTDF1, which is a sex determination gene, in flower development in asparagus, in this study, the female, male and romonocious asparagus which are selfed progeny of the andromonocious tetraploid asparagus cultivar ‘Purple Passion’, were used as plant materials. The full-length CDS sequence of named AoTDF1-P and the full-length CDS sequence of asparagus counterpart of ABORTED MICROSPORES (AMS), which regulates tapetum development in Arabidopsis thaliana named AoAMS-P were cloned by RT-PCR amplifcation respectively. And both the AoTDF1-P, and AoAMS-P were used to construct a pair of prokaryotic expression vector to detect protein integration with assay of bimolecular fluorescence complementation (BiFC) based on yellow fluorescence (venus); simultaneously, RNA in situ hybridization assay was used to detect the specifc expression of AoTDF1-P in the early stage of flower development. The results showed that AoTDF1-P is highly expressed in the pistils, stamens and corollas of both male and andromonociou flowers in the early stage of asexual differentiation (T1), and the expression level of gene is decreased in the early stage of gender differentiation (T2); while in the late sex differentiation stage (T3), the AoTDF1-P expression in andromonocious flowers are more specifc in tapetum of anther, however there is no specifc expression but continue reducing the expression level in the male flower; while the corresponding female flowers have no expression of AoTDF1-P in T1, T2, and T3 stage. Confocal microscopy observed that AoTDF1-P and AoAMS-P produced strong yellow fluorescence, and AoTDF1-P and AoAMS-P itself produced weak yellow fluorescence respectively, indicating that AoTDF1-P could specifc express and form homotypic or/and heterotypic regulate dimer/plymer complex in developing male and andromonocious flowers to promote male development by regulating the expression of downstream target genes in asparagus. The study provides evidence for elucidation of the molecular function of AoTDF1.
Key words: Asparagus ofcinalis; sex differentiation; AoTDF1-P; gene expression; protein interaction
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