high-throughput screen for genes from castor that boost hydroxy fatty acid accumulation in seed oils of transgenic Arabidopsis

• Published in: The Plant journal : for cell and molecular biology Vol. 45; no. 5; pp. 847 - 856
• Main Authors: Lu, Chaofu, Fulda, Martin, Wallis, James G, Browse, John
• Format: Journal Article
• Language: English
• Published: Oxford, UK Oxford, UK : Blackwell Science Ltd 01.03.2006; Blackwell Science Ltd; Blackwell Science
• Subjects: Arabidopsis; Arabidopsis - physiology; Arabidopsis thaliana; Biolistics - methods; Biological and medical sciences; biosynthesis; biotechnology; castor; cDNA libraries; Chromatography, Gas - methods; Cloning, Molecular - methods; complementary DNA; endosperm; engineering; enzymes; Fatty Acids - biosynthesis; full‐length cDNA library; Fundamental and applied biological sciences. Psychology; gas chromatography; Gene Library; genes; Genes, Plant; genetic engineering; Genetic Vectors; high‐throughput; hydroxy fatty acids; industry; Metabolism; Metabolism. Physicochemical requirements; Molecular Probe Techniques; oilseed crops; oilseeds; Plant physiology and development; Plants, Genetically Modified; polymerase chain reaction; ricinoleate; Ricinus communis - genetics; seed oils; seeds; Seeds - metabolism; transcriptome; Transformation, Genetic; transgenic plants; transgenic plants; Seeds; Arabidopsis; Genomics; Biosynthesis; Fatty acids; Arabidopsis thaliana; Complementary DNA; Gene; Cruciferae; Dicotyledones; Angiospermae; high-throughput; full-length cDNA library; ricinoleate; Spermatophyta; Genetic engineering; Transgenic plant; castor
• ISSN: 0960-7412; 1365-313X
• DOI: 10.1111/j.1365-313X.2005.02636.x

It is desirable to produce high homogeneity of novel fatty acids in oilseeds through genetic engineering to meet the increasing demands of the oleo-chemical industry. However, expression of key enzymes for biosynthesis of industrial fatty acids usually results in low levels of desired fatty acids in transgenic oilseeds. The abundance of derivatized fatty acids in their natural species suggests that additional genes are needed for high production in transgenic plants. We used the model oilseed plant Arabidopsis thaliana expressing a castor fatty acid hydroxylase (FAH12) to identify genes that can boost hydroxy fatty acid accumulation in transgenic seeds. Here we describe a high-throughput approach that, in principle, can allow testing of the entire transcriptome of developing castor seed endosperm by shotgun transforming a full-length cDNA library into an FAH12-expressing Arabidopsis line. The resulting transgenic seeds were screened by high-throughput gas chromatography. We obtained several lines transformed with castor cDNAs that contained increased amounts of hydroxy fatty acids in transgenic Arabidopsis. These cDNAs were then isolated by PCR and retransformed into the FAH12-expressing line, thus confirming their beneficial contributions to hydroxy fatty acid accumulation in transgenic Arabidopsis seeds. Although we describe an approach that is targeted to oilseed engineering, the methods we developed can be applied in many areas of plant biotechnology and functional genomic research.