Lehrstuhl für Pflanzenzüchtung

Cloning of genes from the tocopherol pathway in oilseed rape

Tocopherol

 

Background

Tocopherols are a class of molecules characterized by a fully saturated tail derived from phytyl diphosphate. They can be divided into α-, β-, γ- and δ-tocopherol which differ in their number and position of methyl substituents on the aromatic ring. Tocopherols, mainly α-tocopherol, are an essential component of human nutrition known as vitamin E.

The rapeseed (Brassica napus L.) oil contains α-, γ- and small amounts of δ-tocopherol. The tocopherol content of modern canola varieties ranges between 400 and 1000 ppm in oil. Due to the high nutritional importance of vitamin E higher contents are desired. For tocopherol synthesis, six enzymes are required. All of them have been cloned from A. thaliana during the last years.

Objectives

  • Cloning of the B. napus genes encoding for PDS1, VTE1, VTE2, VTE3, VTE4 and VTE5
  • Characterization and functional analysis of these genes
  • Genetic mapping of the genes
  • Candidate Gene association mapping with Brassica napus tocopherol genes in a worldwide B. napus diversity set
  • Development of functional marker
     

Results

Using PCR-based methods, we were able to clone full-length sequences from all the B. napus genes All sequences have showed high similarity (78 - 82 %) to the corresponding A. thaliana sequences.

The genomic sequence of the BnPDS1 gene has a length of about 1500 bp and consists of two exons separated by a 76 bp intron. Functional analyses were done by cloning BnaX.PDS1 sequences into an E.coli expression strain. Since the gene PDS1 encodes for the enzyme p-hydroxyphenylpyruvat dioxygenase, expression in E. coli results in a brown color of the medium due to the oxidation and polymerization of the product homogentisate.

For BnaX.VTE4, we could detect 4 transcribed copies. The coding sequences range between 1044 and 1047 bp. The analysis of partial genomic sequences revealed the existence of 5 introns. We transformed one of the coding sequences under the control of the 35 S promoter into A. thaliana and analysed the seeds of transgenic plants. Overexpression of the BnaX.VTE4 sequence led to an altered tocopherol composition with a large increase of the α-tocopherol fraction.

The BnaX.VTE3, which was first isolated by our project partner (Prof. Meng), has a length of 1200 bp. The gene consists of three exons separated by two small introns. Using polymorphisms within this gene, we were able to develop a CAPS marker for the "Mansholt" x "Samourai" mapping population. The BnaX.VTE3 locus could be mapped to chromosome N07. We have transformed the gene under the control of the 35S promoter into A. thaliana and analysed the seeds of transgenic plants. Overexpression resulted in an increase of α-tocopherol and the seeds showed reduced δ-tocopherol contents.

For an association study, a field trial was made with 94 winter type rapeseed accessions at two different locations in Germany. The B. napus collection was phenotyped for the tocopherol forms α-, γ-, and δ- by HPLC analysis. The tocopherol contents and compositions were determined and haplotypes from different candidate genes are being analysed. The aim of this study is to use the candidate-gene association approach to identify polymorphisms linked to genes for tocopherol content and composition in B. napus. The discovered SNPs will be converted to high-throughput markers and tested for marker assisted selection.

Project team

Dr. Steffi Fritsche
Prof. Dr. Christian Jung

This project ended in 2013

Scientific Partners

Prof. Dr. Dr. h.c. Wolfgang Friedt, University of Gießen
Prof. Jingling Meng, National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan, China

Commercial Partners

Dr. Martin Frauen, Norddeutsche Pflanzenzucht Hans-Georg Lembke KG, Hohenlieth, Germany

Publications

Fritsche, S., Wang, X., Nichelmann, L., Suppanz, I., Hadenfeldt, S., Endrigkeit, J., Meng, J., Jung, C. (2014) Genetic and functional analysis of tocopherol biosynthesis pathway genes in rapeseed (Brassica napus L.) Plant Breeding, 133: 470-479

Bagheri,H., El-Soda,M., Kim,H.K., Fritsche, S., Jung,C., Aarts,M.G.M. (2013)  Genetic analysis of health- related secondary metabolites in the new Brassica rapa recombinant inbred line population. International Journal of Molecular Sciences 14 (15561 - 15577)

Xingxing Wang, Chunyu Zhang, Lingjuan Li, Steffi Fritsche, Jessica Endrigkeit, Wenying Zhang, Yan Long, Christian Jung, Jinling Meng (2012). Unravelling the genetic basis of seed tocopherol content and composition in rapeseed (Brassica napus L.). PLoS ONE, November 2012, Volume 7, Issue 11, e50038

Fritsche, S. Cloning and functional characterization of tocopherol biosynthesis pathway related genes of from oilseed rape (Brassica napus L.) Doctoral thesis, Christian-Albrechts University, (2012).

Fritsche S, Wang X, Li J, Stich B, Kopisch-Obuch F, Endrigkeit J, Leckband G, Dreyer F, Friedt W, Meng J, Jung C (2012) A candidate gene-based association study of tocopherol content and composition in rapeseed (Brassica napus). Frontiers in Plant Genetics and Genomics, June 2012, Volume 3, Article 129

Steffi Fritsche, Xingxing Wang, Nian Wang, Jessica Endrigkeit, Jinquan Li, Benjamin Stich, Martin Frauen, Wolfgang Friedt, Jinling Meng, Christian Jung (2012) A candidate gene association analysis for tocopherol content and composition in rapeseed (Brassica napus). Plant and Animal Genome XX, 14.-18. January 2012, San Diego, USA

Fritsche S., Wang X., Wang N., Endrigkeit J., Friedt W., Dreyer F., and Jung, C. (2011) A candidate-gene association study identifies Single Nucleotide Polymorphisms associated with Brassica napus tocopherol content and composition. International rapeseed congress Prague 2011

J. Endrigkeit, C Zhang, W. Ecke, D. Cai, J. Meng, and C. Jung (2009) Genetic mapping, cloning and functional characterization of the BnaX.VTE4 gene encoding a γ-tocopherol methyltransferase from oilseed rape. Theor.Appl.Genet, 119: 567-575.

Jessica Endrigkeit, Xingxing Wang, Chunyu Zhang, Jinling Meng and Christian Jung (2008) Development of Functional Markers for Tocopherol Content in Brassica napus._ Brassica2008, Lillehammer, Norway, 8 – 12th September 2008

L. Li, Chunyu Zhang, Xingxing Wang, Jessica Endrigkeit, Christian Jung and Jinling Meng (2008) Tocopherol variation and QTL detection in seed of Brassica napus. Brassica2008, Lillehammer, Norway, 8 – 12th September 2008

J. Endrigkeit and C. Jung (2007) Mapping and cloning of genes for tocopherol synthesis in Brassica napus L. First International Symposium on SECONDARY METABOLISM IN PLANT SEEDS:CURRENT STATUS AND FUTURE APPLICATIONS Potsdam, Germany February 21-24, 2007

Endrigkeit J. Identifikation und Charakterisierung von Genen der Tocopherol-Biosynthese aus Raps (Brassica napus L.) 2007. Doctoral thesis Kiel

J. Endrigkeit and C. Jung (2006) Mapping and cloning of genes for tocopherol synthesis in oilseed rape 15. Brassica Workshop Wageningen 30.9-4.10.2006

C. Jung (2006) Der Einsatz der Gentechnik zur züchterischen Verbesserung von Nutzpflanzen: Wie der Mensch sich die Natur nutzbar macht. Plant Genetics Conference Kiel, 20.-23.9.06

J. Endrigkeit and C. Jung (2006) Mapping and cloning of genes for tocopherol synthesis in oilseed rape. Plant Genetics Conference Kiel, 20.-23.9.06

J. Endrigkeit, C. Jung (2006) Isolierung und Charakterisierung von Genen der Tocopherolsynthese aus Raps (Brassica napus) Vortr. Pflanzenzüchtg. 69,  49-54

Financial Support (past three years)

Funding has been provided by the Stiftung Schleswig-Holsteinische Landschaft Kiel and by the Deutsche Forschungsgemeinschaft under grant No. DFG JU 205/14-1

 

Responsible for this webpage: C. Jung