Cloning and functional analysis of CmPI, a class B flower development gene in Chinese chestnut

Abstract: 【Objective】A complete flower consists of four floral organs, including 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 ring flower organ structure from the classic one. Such as chestnut, poplar, walnut, jatropha, birch are catkins, their flowers are unisexual, and do not have petals and sepals. In these plants, the petals and sepals are replaced by the 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 the 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 regulatedby 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 the Class B flower organ development gene CmPI of C. mollissima were used to lay a foundation for the molecular regulation of bracteal dimorphism of C. mollissima.【Methods】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 the CmPI was performed using online tools. The temporal and spatial expression patterns of the 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. The overexpression vector 35S::CmPI was transferred into wild Arabidopsis thaliana to obtain overexpression plants for detecting the function of the CmPI in flower development. The expressions level of flower development genes of A. thaliana were analyzed.【Results】The coding sequence of the CmPI was 630 bp long, encoded 209 amino acids. The CmPI protein was a hydrophilic protein with a theoretical molecular weight of 82 015.9 Da and an isoelectric point of 5.1. The MEGAX was used to construct the phylogenetic tree of the PI homologous proteins of C. mollissima, Glycine max, Cucumis sativus, Vitis vinifera, Betaula platyphylla, Helianthus annuus, Liriodendron chinense, Salix purpurea, Nymphaea tetragona, Chenopodium quinoa and A. thaliana. The results showed that the CmPI had the closest relationship with that of B. platyphylla, followed by G. max, C. sativus and V. vinifera. The CmPI had conserved MADS domain (MADs-box) and K domain (K-box), and belonged to the type Ⅱ subfamily of MADS transcription factors. The CmPI was mainly expressed in the male flowers of Chinese chestnut. The expression of the CmPI in C. 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. The fluorescence signals of empty carriers were distributed on the cell membrane, cytoplasm and nucleus, while the fluorescence signals of the 35S::CmPIGFP were only distributed on the nucleus, indicating that the CmPI gene was localized in the nucleus. In early flower development of A. thaliana plants with overexpression of the CmPI, the sepals become narrow and there were gaps between the sepals, which could not wrap the internal flower organ structure. After flowering of the A. thaliana plants with overexpression of the CmPI, the sepals and petalization were scattered. The measurement results showed that the sepal width of the CmPI transgenic plants was 0.59 mm, which was significantly lower than that of the wild A. thaliana. The length of the sepal was 2.49 mm, which was significantly higher than that of the wild Arabidopsis. There was no significant difference in petal length between the CmPI transgenic plants and the wild individuals. The gene expression analysis of A, B, C and E genes related to the regulation of the sepals and petals in the transgenic lines and the wild type individuals showed that the class C gene AtAG was significantly higher in the CmPI-OE lines than the wild type Arabidopsis. And the expression levels of the class A gene AtAP1 and the class B gene AtAP3/AtPI, E class genes AtSEP1/AtSEP2/AtSEP3/AtSEP4 were lower that those of the wild type Arabidopsis.【Conclusion】The MADS- box gene CmPI is a B class gene in Chinese chestnut, which can lead to the petalization of the sepals and may be a key gene in inhibiting the further development of the bracts of the female flower.