cDNA Libraries Methods and Applications
The numerous vital applications of complementary DNA (cDNA) technology have changed dramatically as the technology has advanced over recent years. In cDNA Libraries: Methods and Protocols, expert researchers provide current techniques that reflect t
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. Introduction Plant oils in most oilseed crops are comprised primarily of only a few fatty acids, such as palmitic, stearic, oleic, linoleic, and linolenic acids. However, certain plant species produce fatty acids with special chemical structures such as alterations in fatty acyl chain length, double-bond positions, and oxygenated functional groups (1). Physical or chemical properties make some of these unusual fatty acids valuable for their applications in numerous industrial products. In recent years, considerable efforts have been made to genetically engineer oilseed crops for production of such unusual Chaofu Lu et al. (eds.), cDNA Libraries: Methods and Applications, Methods in Molecular Biology, vol. 729, DOI 10.1007/978-1-61779-065-2_3, © Springer Science+Business Media, LLC 2011
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fatty acids, and thus provide a low-cost feedstock resource for the oleo-chemical industry (2–5). However, the results of these experiments have typically been disappointing, producing, in most cases, plant lines with very low yields of the desired fatty acids (2–4, 6). For example, ricinoleic acid (12-hydroxyoctadeccis-9-enoic acid; 18:1-OH) biosynthesis in castor (Ricinus communis L.) is catalyzed by the oleate D12-hydroxylase (FAH12) (7). Heterologous expression of FAH12 in the model oilseed plant Arabidopsis produced only up to 17% hydroxy fatty acids in seed oils. This level is much lower than that found in castor oil, where ricinoleate constitutes ~90% of the total fatty acids. Other efforts to produce unusual fatty acids, such as acetylenic, monoenoic, eleostearic, and parinaric acids, have likewise found that accumulation of unusual fatty acids in transgenic seeds is far below the proportion found in their respective natural sources (3, 4, 6). These results show that expressing the single catalytic enzymes required for unusual fatty acid biosynthesis is insufficient to create transgenic plants producing large amounts of these fatty acids in seed storage oil. Since the unusual fatty acids occur in abundance in their natural sources, we believe that additional necessary components for increased accumulation of unusual fatty acids in transgenic plants can be obtained from the source species. Although we describe our approach for a specific application, the methods we have developed are applicable to many questions on biotechnology and functional genomics research. We described a high-throughput approach to screen genes from castor that may boost hydroxy fatty acids accumulation in seed oils of transgenic Arabidopsis (8). The procedure is comprised of five major steps: (a) construction of a full-length cDNA library in a high-throughput l phage vector from developing endosperm of castor seeds, and introduction of the cDNA library into a high-throughput plant expression binary vector, (b) transformation of the full-length cDNA library into the FAH12 transgenic line, and production of seeds from individual transgenic plants, (c) highthroughput screening of fatty acid composition of transgenic
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