【Objective】 A complete flower consists of four floral organs, which are sepals, petals, stamens, and carpels from the outside to the inside. In the ‘ABC(D)E’ flower development regulation model, each round of flower organ development is specifically regulated by corresponding genes. The variation of plant flowers in the process of evolution has produced a different four-wheeled flower organ structure from the classic one. Such as chestnut, poplar, walnut, jatropha, birch are catkins, their flowers are unisexual, and no petals and sepals. In these plants, the petals and sepals are replaced by bracts. The development of bracts may be co-regulated by A, B and E genes. As a typical representative of Fagaceae plants, the female and male flowers of chestnut differ greatly in morphology. The male bracts of chestnut are small and thin, and the stamens are easy to extend out during flowering. The female flowers of chestnut are usually 3 to 5 grouped together in clusters at the base of mixed inflorescences. The female flower cluster is surrounded by numerous bracts. After the female flower is fertilized, the bracts develop into epicarp.The sexual dimorphism of chestnut bracts may also be regulated by MADS-box gene. PI gene is one of class B genes, which is involved in the development regulation of petals and stamens, and is one of the ideal genes for studying the development regulation of chestnut bracts. In this study, the Castanea mollissima variety 'Yanshan Hongli' was used as experimental material. Homology cloning, evolutionary analysis, spatio-temporal expression analysis and functional verification of Class B flower organ development gene CmPI of Castanea mollissima were used to lay a foundation for the molecular regulation of bracteal dimorphism of Castanea mollissima.【Method】The PI homologous gene CmPI was retrieved from the genome data of Chinese chestnut. The coding region sequence was cloned by RT-PCR. Bioinformatics analysis of CmPI was performed using online tools. The temporal and spatial expression patterns of CmPI in different tissues and flower development stages of Chinese chestnut were analyzed by fluorescence quantitative PCR (qRT-PCR). The 35S::CmPI-GFP fusion vector was constructed to transform tobacco leaves instantaneously for subcellular localization analysis. Overexpression vector 35S::CmPI was transferred into wild Arabidopsis thaliana to obtain overexpression plants for detecting the function of CmPI in flower development. The expressions level of flower development genes of Arabidopsis thaliana were analyzed.【Results】 The coding sequence of CmPI was 630bp long, encodes 209 amino acids. CmPI protein is a hydrophilic protein with a theoretical molecular weight of 82015.9 Da and an isoelectric point of 5.1. MEGAX was used to construct the phylogenetic tree of PI homologous proteins of Castanea mollissima, Glycine max, Cucumis sativus, Vitis vinifera, Betaula platyphylla, Helianthus annuus, Liriodendron chinense, Salix purpurea, Nymphaea tetragona, Chenopodium quinoa and Arabidopsis thaliana. The results showed that CmPI has the closest relationship with Betaula platyphylla, followed by Glycine max, Cucumis sativus and Vitis vinifera. CmPI has conserved MADS domain (MADs-box) and K domain (K-box), and belongs to the type II subfamily of MADS transcription factors. CmPI is mainly expressed in male flowers of Chinese chestnut. The expression of CmPI in Castanea mollissima male flower kept increasing until 31th May. The constructed 35S::CmPI-GFP vector and 35S::GFP empty vector were infected by Agrobacterium-mediated method to carry out instantaneous expression. Fluorescence signals of empty carriers were distributed on the cell membrane, cytoplasm and nucleus, while fluorescence signals of 35S::CmPI-GFP were only distributed on the nucleus, indicating that CmPI gene was localized in the nucleus. In early flower development of Arabidopsis thaliana plants with overexpression of CmPI, the sepals become narrow and there are gaps between the sepals, which can not wrap the internal flower organ structure. After flowering of Arabidopsis thaliana plants with overexpression of CmPI, the sepals and petalization were scattered. The measurement results showed that the sepal width of CmPI transgenic plants was 0.59 mm, which was significantly lower than that of wild Arabidopsis thaliana. The length of the sepals was 2.49 mm, which was significantly higher than that of wild Arabidopsis. There was no significant difference in petal length between CmPI transgenic plants and wild individuals. The gene expression analysis of A, B, C and E genes related to the regulation of sepals and petals in transgenic lines and wild type individuals showed that class C gene AtAG was significantly higher in CmPI-OE lines than wild type Arabidopsis. And the expression levels of class A gene AtAP1 and class B gene AtAP3/AtPI, E class genes AtSEP1/AtSEP2/AtSEP3/AtSEP4 were lower that wild type Arabidopsis.【Conclusion】 The MADS-box gene CmPI is a B class gene in Chinese chestnut, which can lead to the petalization of sepals and may be a key gene in inhibiting the further development of female flower bracts.
PDF （）