Lehrstuhl für Pflanzenzüchtung

Identification and characterization of mutations in the phytic acid biosynthesis pathway genes in oilseed rape

Phytate

Background

Oilseed rape (rapeseed, Brassica napus L., genome AACC, 2n=38) is the most important oilseed crop in Europe. It stands in second position for oil production in the world after soybean. Dry mature seeds from rapeseed accumulate oil (45-50%) and proteins (20-25%), while the meal remaining after oil extraction contains about 40% protein. The availability of rapeseed meal as a protein supplement in animal feed is limited due to the toxicity and digestibility problems caused by anti-nutritional components like sinapine, glucosinolates and phytate.

Phytic acid (m-inositol hexakisphosphate) is the principal storage form of phosphorus in B.napus seeds with levels ranging from 2-4% of the seed weight. Phytate provides 65 to 85% of the total phosphorus in seeds. Humans and non-ruminant animals lack phytase, the digestive enzyme to hydrolyze phytic acid, which reduces the amount of bio-available phosphorus in their diet. The negatively charged phytic acid molecule also chelates minerals such as calcium, potassium, magnesium, iron and zinc and lowers their bioavailability. In addition to the nutritional effects of phytate in animal diets, there are also environmental consequences as undigested phytic acid increases phosphate in animal manure and can pollute water systems.

Phytic acid has also other important functions in plants (DNA repair, signaling pathway, cell membrane formation, plant defense reactions etc.) and is synthesized via two pathways (lipid dependent and lipid independent). Many synthesis and transporter genes were identified previously in various crop species by mutant and knock-out analysis. This project aims at developing an efficient and high throughput method to knock out key genes involved in the phytic acid pathway in B.napus via Next Generation Sequencing and CRISPR/Cas9 technology to provide a deeper understanding of the biological function of various genes involved in the pathway.

Objectives

  • Identification and characterization of phytic acid synthesis and transporter genes in B. napus in silico and in planta
  • Detection of point mutations within B. napus phytic acid synthesis genes using TILLING by Illumina Next Generation Sequencing
  • Molecular and phenotypic characterization of single and double mutants
  • Knockout of phytic acid synthesis genes by CRISPR/Cas9 genome editing
  • Expression profiling, enzyme and transporter measurements of selected target genes

Results

Eight biosynthetic genes and one transporter gene were identified in the phytic acid pathway for the production of phytic acid in Arabidopsis. The corresponding paralogs were identified in Brassica napus as target genes for the knockout analysis. After expression analysis in developing seeds of control cultivar Express 67, we selected 20 paralogs of the 5 key synthesis and transporter genes BnITPK, BnMIK, BnIPK, BnOslpa and BnMRP5 for knock-out by CRISPR/Cas9 and 12 highly expressed paralogs of BnMIPS, BnIPK2b, BnMIK, BnIPK, BnOslpa and BnMRP5 for mutant screening by TILLING. The screening of our Express 617 EMS TILLING population and first CRIPSR Cas9 experiments are currently on the way. 

Project team

M.Sc. Niharika Sashidhar
Dr. Hans-Joachim Harloff
Prof. Christian Jung

Scientific Partners

Institute of Clinical Molecular Biology (IKMB), University of Kiel
Botanical Institute, University of Kiel

Commercial Partners

Norddeutsche Pflanzenzucht Hans-Georg Lembke KG

Publications

H.-J. Harloff, S. Lemcke, J. Mittasch, A. Frolov, J. Wu, F. Dreyer, G. Leckband, C. Jung (2012): A mutation screening platform for rapeseed (Brassica napus L.) and the detection of sinapine biosynthesis mutants. Theoretical and Applied Genetics 124, 957-969

Financial Support

Funding has been provided by the DFG grant No. JU 205/26-1

 

Updated: 22-May-2017            Responsible for this webpage:  Niharika Sashidhar