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| Cloning and Identification of LEAFY Gene in Chinese Milk Vetch (Astragalus sinicus) |
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Abstract Abstract Astragalus sinicus is an important nectariferous plant and green manure crop. It plays an important role in promoting crop yield. Basing on the local farming system, selecting flowering varieties is an important breeding target of Astragalus sinicus. LEAFY (LFY) is a plant-specific flower meristem specific-gene. However, the study on LEAFY gene of Astragalus sinicus is not reported yet. In this study, in order to explore the function of LEAFY gene in Astragalus sinicus, the sequence of LEAFY in Astragalus sinicus was cloned by Illumina Hiseq method and rapid-amplification of cDNA ends (RACE) technology, and the bioinformatics analysis of LEAFY gene was performed by bioinformatics analysis software; The expression characteristics of the LEAFY gene in different tissues were analyzed by qRT-PCR; The function of LEAFY gene of Astragalus sinicus was identified after expression vector construction, infection and statistical analysis of phenotypic characteristics of T2 transgenic homozygous lines were performed. The results showed that the full-length cDNA of the LEAFY gene (GenBank No. MH352146) in Astragalus sinicus was 1 400 bp in length and contained the open reading frame of 1 191 bp; The encoded protein contains 396 amino acids, including 22 α-helices and 5 β-sheets, which has a molecular weight of 44.72 kD and a theoretical isoelectric point of 6.41; The similarity of the encoded amino acid sequences to the LEAFY proteins of Medicago truncatula, Medicago sativa, Cicer arietinum, and other species were all above 80%, which was highly homologous. The qRT-PCR analysis of LEAFY gene showed that the LEAFY gene was expressed in various organs, and the order from high to low of the expression levels were flower buds, flowers, leaves, leaf buds, roots, stems and pods. The results of the experiments with Arabidopsis thaliana over expressing LEAFY showed that the number of bolting days of transgenic Arabidopsis thaliana was 3 days earlier than that of wild type, and the time of transgenic A. thaliana flowering was 2 days earlier than that of wild type. The average number of flowers was 1.79 more than that of wild type. In summary, this study showed that the LEAFY gene had the high homology with the leguminous species, and was the highest expressed in the flower bud, and verified that the LEAFY gene might regulate the flowering mechanism. These results provide a scientific basis for the further development and utilization of A. thaliana by molecular technology, and are of great significance to agricultural production.
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Received: 22 January 2018
Published: 20 July 2018
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