• Login
    View Item 
    •   Home
    • Massey Documents by Type
    • Theses and Dissertations
    • View Item
    •   Home
    • Massey Documents by Type
    • Theses and Dissertations
    • View Item
    JavaScript is disabled for your browser. Some features of this site may not work without it.

    Molecular analysis of anthocyanin biosynthetic pathway genes in Cymbidium orchids : a thesis presentation in partial fulfilment of the requirements for the degree of Master of Science in Plant Molecular Biology at Massey University, Palmerston North, New Zealand

    Icon
    View/Open Full Text
    01_front.pdf (10.31Mb)
    02_whole.pdf (53.57Mb)
    Export to EndNote
    Abstract
    Anthocyanin biosynthesis was examined in cymbidium orchid (Cymbidium Swartz). Cymbidium orchids lack true red and purple/blue flowers, because they accumulate cyanidin and peonidin (pink) anthocyanins, but not pelargonidin (red) or delphinidin (blue). Transient gene expression studies showed that the use of a heterologous flavonoid biosynthetic gene, dihydroflavonol reductase (DFR) from Anthurium, enhanced production of pelargonidin in cymbidium floral tissues. Similarly, delphinidin was produced when a pansy flavonoid 3', 5'-hydroxylase (F3'5'H) was introduced. The maize Lc/C1 transcription factors in combination with DFR or F3'5'H was necessary in order to observe upregulation of the anthocyanin pathway and changes in anthocyanin pigment accumulation. The presence of the new anthocyanins was confirmed by TLC and HPLC. cDNA clones of the flavonoid biosynthetic genes chalcone synthase (CHS), DFR and flavonoid 3' hydroxylase (F3'H) were isolated using PCR. The full length cymbidium CHS (1173 bp) was ~85% identical at DNA level with CHS from Oncidium 'Gower Ramsey', a Phalaenopsis hybrid cultivar, as well as bibenzyl synthase from Phalaenopsis sp. The 792 bp partial-length F3'H sequence was ~66% identical with F3'H from Pelargonidiumxhortorum, Verbena hybrida and Sorghum bicolor, while the DFR sequence was highly homologous with the published cymbidium DFR. The deduced protein sequences contained domains or conserved residues typical of CHS and F3'H. Southern analysis showed both cymbidium CHS and F3'H are represented by small gene families, with CHS consisting of at least three members and F3'H up to three genes. By contrast, DFR is likely to be presented as a single gene. Using different coloured cymbidium cultivars, it was shown that DFR expression correlated with cyanidin production in the flower. The CHS clone was most highly expressed in leaf tissues and in late developmental stages in floral tissues of Vanguard Mas Beauty (a green cultivar). This expression pattern did not correlate with pigment production, and hence this gene is unlikely to be involved in anthocyanin production in flowers. F3'H expression was not detected in leaf and floral tissues at any developmental stages examined. DFR and CHS promoters were isolated by genome walking, in an attempt to identify organ specific promoters suitable for use in cymbidium. A 1544 bp DFR promoter and a 1561 bp CHS promoter were cloned upstream of a GFP reporter gene and transient gene expression studies showed that CHS promoter had the ability to drive GFP production in white and pink petal tissues and in leaf tissues. However, the DFR promoter activated GFP expression only in the white petal tissues. These transient gene expression studies also demonstrated that maize Lc/C1 transcription factors greatly enhanced the activities of both CHS and DFR promoters. The success of this transient expression system indicates that MYB and bHLH transcription factor are likely to be involved in anthocyanin production in cymbidium. Together, the results of this study confirm that a suite of molecular strategies to modify flower colour in cymbidium are feasible, as well as providing essential information on flavonoid and anthocyanin genes that expand our knowledge and understanding of this complex flowering plant.
    Date
    2008
    Author
    Wang, Lei
    Rights
    The Author
    Publisher
    Massey University
    URI
    http://hdl.handle.net/10179/12935
    Collections
    • Theses and Dissertations
    Metadata
    Show full item record

    Copyright © Massey University
    | Contact Us | Feedback | Copyright Take Down Request | Massey University Privacy Statement
    DSpace software copyright © Duraspace
    v5.7-2020.1-beta1
     

     

    Tweets by @Massey_Research
    Information PagesContent PolicyDepositing content to MROCopyright and Access InformationDeposit LicenseDeposit License SummaryTheses FAQFile FormatsDoctoral Thesis Deposit

    Browse

    All of MROCommunities & CollectionsBy Issue DateAuthorsTitlesSubjectsThis CollectionBy Issue DateAuthorsTitlesSubjects

    My Account

    LoginRegister

    Statistics

    View Usage Statistics

    Copyright © Massey University
    | Contact Us | Feedback | Copyright Take Down Request | Massey University Privacy Statement
    DSpace software copyright © Duraspace
    v5.7-2020.1-beta1