Journal of Food, Agriculture and Environment

Functional expression analysis of an acyl-ACP thioesterase FatB1 from Arachis hypogaea L. seeds in Escherichia coli

Author(s):

Gao Chen ¹, Qingfang He ^{1, 2}, Ning Xuan ¹, Zhenying Peng ¹, Lei Shan ¹, Guiying Tang¹, Yuping Bi ^{1, *}

Recieved Date: 2012-07-04, Accepted Date: 2012-09-30

Abstract:

Acyl-acyl carrier protein thioesterases are intraplastidial enzymes that determine the types and amounts of fatty acids found in plants and then release them into the cytosol to be incorporated into glycerolipids. To better understand the role of the FatB class of acyl-acyl carrier protein thioesterase, a 1.2 kb cDNA fragment of AhFatB1 was amplified using RT-PCR method from the developing seeds of Arachis hypogaea L. Sequence analysis shows that it encodes 413 amino acids with molecular mass of 45.47 kDa, a 2996 bp DNA fragment was isolated and the genomic DNA sequence has six exons and five introns. Real-time quantitative PCR and semi-quantitative RT-PCR analyses showed that AhFatB1 was expressed in all tissues of A. hypogaea L., especially in stems, and was also highly expressed in immature seeds harvested at 70 days after pegging. To further identify the functions of AhFatB1, the prokaryotic expression vectors of GST-AhFatB1 were constructed and expressed in E. coli BL21 (DE3) and the fusion protein was obtained. The transformants cultured at 37°C and 25°C were collected, dried and  fatty acids measured by gas chromatography (GC). The results showed that AhFatB1 can increase the contents of myristic acid (14:0), palmitoleic acid (16:1) and oleic acid (18:1) in bacteria effectively. These results indicates that Acyl-ACP Thioesterases of peanut were at least partially responsible for improving the synthesis of upstream substrate of PUFAs. Together, the results demonstrate that AhFatB1 from developing seeds of Arachis hypogaea L. can be used for transgenic development of Arachis hypogaea L. and Glycine max to alter the fatty acid profiles.

Keywords:

Arachis hypogaea L., acyl-acyl carrier protein thioesterase, fatty acid, quantitative real-time PCR, semi-quantitative RT-PCR, prokaryotic expression

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Journal: Journal of Food, Agriculture and Environment
Year: 2012
Volume: 10
Issue: 3&4
Category: Agriculture
Pages: 332-336

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