Cloning and Expression Analysis of LpluF3′5'H in Lupinus polyphyllus 'Lupine'
HU Jun-Shu, ZHU Yun-Tao, ZHAO Yi-Ran, YANG Jie, SUN Ting-Ting, HE Heng-Bin*
Beijing Key Laboratory of Flower Germplasm Innovation & Molecular Breeding/National Engineering Research Center for Floriculture/Beijing Laboratory of Urban and Rural Ecological Environment/Key Laboratory of Genetics and Breeding in Forest Trees & Ornamental Plants Ministry of Education, College of Landscape Architecture, Beijing Forestry University, Beijing 100083, China
Abstract：The flavonoid-3', 5'-hydroxylase (F3′5′H) gene is crucial for the development of blue flowers in nature. To provide more resources and reference for blue flower breeding by cloning and expression analysis of F3′5′H in Lupinus polyphyllus, the petals of L. polyphyllus 'Lupine' were used as the material to clone LpluF3′5′H and the sequence was used for bioinformatics analysis by software such as MEGA 10.0 and online websites. The subcellular localization of LpluF3'5'H protein was determined by tobacco injection method. qPCR was used to analyze the expression of LpluF3′5′H in petals with different colors and developmental stages. The LpluF3′5′H cDNA sequence had the encoding region of 1 620 bp encoding 539 amino acids, and was named LpluF3′5′H (GenBank No. OQ657278). Bioinformatics predicted that LpluF3′5′H belongs to the CYP75A subfamily, cytochrome P450 superfamily. Phylogenetic analysis revealed that the protein encoded by LpluF3′5′H was the highest similarity to the F3'5'H protein of L. angustifolius, and the monocotyledonous plants F3'5'H protein clustered into another family. Subcellular localization showed that LpluF3'5'H was localized in chloroplasts. qPCR analysis indicated that the expression of LpluF3′5′H could be detected in different development stages and petals. Besides, LpluF3′5′H showed the stronger expression in 'Lupine Blue' and lower in 'Lupine White'. In the different developmental stages of 'Lupine Blue', the expression levels of LpluF3′5′H peaked in the second developmental stage and then decreased. Among the different petals of 'Lupine Blue', LpluF3′5′H was the most highly expressed in the wings, followed by the vexils, and lowest expressed in the keels. LpluF3′5′H might play an important role in the flower color formation of 'Lupine Blue', and the study provides a basis for further research into the flower color mechanism of blue-flowered L. polyphyllus formation.
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