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Identification of a cDNA that encodes a 1-acyl-sn-glycerol-3-phosphate acyltransferase from Limnanthes douglasii

Brown, Adrian P.; Brough, Clare L.; Kroon, Johan T.M.; Slabas, Antoni R.

Authors

Adrian P. Brown

Clare L. Brough

Antoni R. Slabas



Abstract

Two different techniques were used to isolate potential cDNAs for acyl-CoA: 1-acyl-sn-glycerol-3-phosphate acyltransferase (LPA-AT) enzymes from Limnanthes douglasii. Both heterologous screening with the maize pMAT1 clone and in vivo complementation of the Escherchia coli mutant JC201 which is deficient in LPA-AT activity, were carried out. Clones identified by these procedures were different. Homology searches demonstrated that the clone isolated by heterologous probing, pLAT1, encodes a protein which is most similar to the maize (open reading frame in pMAT1) and yeast SLC1 proteins, which are putative LPA-AT sequences. This L. douglasii sequence shows much lower homology to the E. coli LPA-AT protein PlsC, which is the only LPA-AT sequence confirmed by over-expression studies. The clone isolated by complementation, pLAT2, encodes a protein with homology to both SLC1 and PlsC. It was not possible to over-express the complementing protein encoded by pLAT2 but further experimentation on membranes from complemented JC201 demonstrated that they possess a substrate specificity distinctly different from PlsC and similar to Limnanthes sp. microsome specificity. This data strongly supports the contention that pLAT2 is an LPA-AT clone. Northern blot analysis revealed different expression patterns for the two genes in pLAT1 and pLAT2. Transcription of the gene encoding the insert of pLAT2 occurred almost exclusively in developing seed tissue, whilst the cDNA of pLAT1 hybridised to poly(A)+ mRNA from seed, stem and leaf, demonstrating more widespread expression throughout the plant. Southern blot analysis indicated that the cDNA of pLAT2 was transcribed from a single-copy gene while that for pLAT1 was a member of a small gene family.

Citation

Brown, A. P., Brough, C. L., Kroon, J. T., & Slabas, A. R. (1995). Identification of a cDNA that encodes a 1-acyl-sn-glycerol-3-phosphate acyltransferase from Limnanthes douglasii. Plant Molecular Biology, 29(2), 267-278. https://doi.org/10.1007/bf00043651

Journal Article Type Article
Acceptance Date Jul 12, 1995
Publication Date Oct 31, 1995
Deposit Date Sep 9, 2021
Journal Plant Molecular Biology
Print ISSN 0167-4412
Electronic ISSN 1573-5028
Publisher Springer
Volume 29
Issue 2
Pages 267-278
DOI https://doi.org/10.1007/bf00043651
Public URL https://durham-repository.worktribe.com/output/1241084