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Construction of a full-length cDNA library from castor endosperm for high-throughput functional screening.
Methods Mol Biol. 2011; 729:37-52.MM

Abstract

It is desirable to produce high homogeneity of novel fatty acids in oilseeds through genetic engineering to meet increasing demands by 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 unusual 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. We described previously 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 a FAH12-expressing Arabidopsis line. The resulting transgenic seeds can be screened by high-throughput gas chromatography. The most critical step of the approach is the construction of a full-length cDNA library. In this chapter, we describe in detail the construction of the cloning vectors and a full-length cDNA library from developing castor seed endosperms. The approach we describe has broad applicability in many areas of biology.

Authors+Show Affiliations

Department of Plant Sciences and Plant Pathology, Montana State University, Bozeman, MT, USA. clu@montana.eduNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article

Language

eng

PubMed ID

21365482

Citation

Lu, Chaofu, et al. "Construction of a Full-length cDNA Library From Castor Endosperm for High-throughput Functional Screening." Methods in Molecular Biology (Clifton, N.J.), vol. 729, 2011, pp. 37-52.
Lu C, Wallis JG, Browse J. Construction of a full-length cDNA library from castor endosperm for high-throughput functional screening. Methods Mol Biol. 2011;729:37-52.
Lu, C., Wallis, J. G., & Browse, J. (2011). Construction of a full-length cDNA library from castor endosperm for high-throughput functional screening. Methods in Molecular Biology (Clifton, N.J.), 729, 37-52. https://doi.org/10.1007/978-1-61779-065-2_3
Lu C, Wallis JG, Browse J. Construction of a Full-length cDNA Library From Castor Endosperm for High-throughput Functional Screening. Methods Mol Biol. 2011;729:37-52. PubMed PMID: 21365482.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Construction of a full-length cDNA library from castor endosperm for high-throughput functional screening. AU - Lu,Chaofu, AU - Wallis,James G, AU - Browse,John, PY - 2011/3/3/entrez PY - 2011/3/3/pubmed PY - 2011/6/15/medline SP - 37 EP - 52 JF - Methods in molecular biology (Clifton, N.J.) JO - Methods Mol Biol VL - 729 N2 - It is desirable to produce high homogeneity of novel fatty acids in oilseeds through genetic engineering to meet increasing demands by 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 unusual 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. We described previously 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 a FAH12-expressing Arabidopsis line. The resulting transgenic seeds can be screened by high-throughput gas chromatography. The most critical step of the approach is the construction of a full-length cDNA library. In this chapter, we describe in detail the construction of the cloning vectors and a full-length cDNA library from developing castor seed endosperms. The approach we describe has broad applicability in many areas of biology. SN - 1940-6029 UR - https://www.unboundmedicine.com/medline/citation/21365482/Construction_of_a_full_length_cDNA_library_from_castor_endosperm_for_high_throughput_functional_screening_ L2 - https://dx.doi.org/10.1007/978-1-61779-065-2_3 DB - PRIME DP - Unbound Medicine ER -