Subjects: Biology >> Botany >> Applied botany submitted time 2021-12-19 Cooperative journals: 《广西植物》
Abstract： A number of Magnolia (Magnoliaceae) species bloom twice each year, instead of once in most other species in this family, which is a desirable ornamental trait. To investigate the molecular mechanism of the flower bud differentiation during the second bloom each year in these Magnolia species, quantitative real-time PCR (qRT-PCR) was frequently used as a sensitive gene expression technique that relies on the stability of reference genes for data normalization. Therefore, the identification of reference gene(s) suitable for molecular characterization during Magnolia flower bud differentiation in second bloom is of great interest. In this study, gene expression in the leaf and flower bud tissues of M. liliflora ‘Hongyuanbao’ at different stages of the flower bud differentiation during second bloom was analyzed. Based on transcriptomic sequencing data, eight constitutively expressed genes, including UBC (ubiquitinconjugating enzyme), ACT (actin), β-TUB (tubulin beta), β-TUB5 (tubulin beta), α-TUB3 (tubulin alpha), PEPC (phosphoenolpyruvate carboxylase), ACP2 (acyl carrier protein 2), ACP3 (acyl carrier protein 3), were selected as candidate reference genes for qRT-PCR. Comprehensively analysis was conducted using four softwares including geNorm, NormFinder, BestKeeper and RefFinder. Primer Premier 5 was used to design the primers. PCR products of all the eight candidate reference genes were analyzed by gel electrophoresis which showed sharp bands with the expected size. Each melting curve showed a single peak, which indicated the high specificity of PCR primers. The general assessment by the four different softwares ranked β-TUB, β-TUB5 and α-TUB3 as the most stable reference genes, whereas UBC and ACT were the lest stable. The reference genes were further evaluated by analyzing the relative expression of TFL1 gene with either single or in combination of β-TUB5, α-TUB3, β-TUB, which showed highly consistent results. In contrast, ACT and UBC did not effectively standardize the expression level of TFL1. In general, through this study, we have identified β-TUB5, α-TUB3 and β-TUB as the most suitable reference genes for qRT-PCR analysis of gene expression in flower bud differentiation during the annual second bloom of M. liliflora ‘Hongyuanbao’, which provides useful tools for investigating the regulatory mechanisms in Magnolia flowering.
Subjects: Biology >> Botany >> Applied botany submitted time 2021-08-09 Cooperative journals: 《广西植物》
Abstract： UDP-flavonoid 3-O-glucosyltransferase (3GT) is one of the important catalytic enzymes in the anthocyanin biosynthesis pathway. To study the function of 3GT in anthocyanin biosynthesis of Magnolia liliflora, M. liliflora ‘Hongyuanbao’ was employed as materials. Primers were designed based on the 3GT sequence obtained from the transcriptome database of M. liliflora ‘Hongyuanbao’, and the structural gene Ml3GT1 in anthocyanin biosynthesis pathway was cloned by RT-PCR (reverse transcription-PCR), and its bioinformatics and expression pattern were analyzed. The results were as follows: (1) The cDNA of Ml3GT1 was 1 836 bp, and the open reading frame was 1 374 bp, encoding 457 amino acid residues. The relative molecular weight of Ml3GT1 was 49.37 kDa, and its isoelectric point was 6.04. (2) The deduced amino acid sequence of Ml3GT1 contains a conserved plant secondary product Glycosyltransferase signature sequence (PSPG box). (3) Results of the phylogenetic analysis showed that Ml3GT1 was closely relative to 3GT proteins from Freesia hybrida, Petunia × hybrida, and Ipomoea batatas. (4) Results of fluorescence quantitative PCR revealed that Ml3GT1 has spatio-temporal specificity, with the highest expression level in flowers, the lower expression level in young leaves and old leaves, and trace expression level in roots and stems; With the development of flowers, the expression level of Ml3GT1 gene decreased first, then increased, and showed the highest expression level at the fully-opening stage. These results suggest that Ml3GT1 may be involved in flavonoid glycosylation. This study will lay a foundation for the flower and color breeding of Magnolia plants.
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