Yuan Yijun, Chen Yinhua, Yan Shan, Liang Yuanxue, Zheng Yusheng, Dongdong Li
Department of Biotechnology, Hainan University, Haikou, Hainan 570228, China.
Hainan Key Laboratory for Sustainable Utilisation of Tropical Bioresource, Hainan University, Haikou, Hainan 570228, China.
Funct Plant Biol. 2013 Feb;41(1):80-86. doi: 10.1071/FP13050.
Coconut (Cocos nucifera L.) contains large amounts of medium chain fatty acids, which mostly recognise acyl-acyl carrier protein (ACP) thioesterases that hydrolyse acyl-ACP into free fatty acids to terminate acyl chain elongation during fatty acid biosynthesis. A full-length cDNA of an acyl-ACP thioesterase, designated CocoFatB1, was isolated from cDNA libraries prepared from coconut endosperm during fruit development. The gene contained an open reading frame of 1254 bp, encoding a 417-amino acid protein. The amino acid sequence of the CocoFatB1 protein showed 100% and 95% sequence similarity to CnFatB1 and oil palm (Elaeis guineensis Jacq.) acyl-ACP thioesterases, respectively. Real-time fluorescent quantitative PCR analysis indicated that the CocoFatB1 transcript was most abundant in the endosperm from 8-month-old coconuts; the leaves and endosperm from 15-month-old coconuts had ~80% and ~10% of this level. The CocoFatB1 coding region was overexpressed in tobacco (Nicotiana tabacum L.) under the control of the seed-specific napin promoter following Agrobacterium tumefaciens-mediated transformation. CocoFatB1 transcript expression varied 20-fold between different transgenic plants, with 21 plants exhibiting detectable levels of CocoFatB1 expression. Analysis of the fatty acid composition of transgenic tobacco seeds showed that the levels of myristic acid (14 : 0), palmitic acid (16 : 0) and stearic acid (18 : 0) were increased by 25%, 34% and 17%, respectively, compared with untransformed plants. These results indicated that CocoFatB1 acts specifically on 14 : 0-ACP, 16 : 0-ACP and 18 : 0-ACP, and can increase medium chain saturated fatty acids. The gene may valuable for engineering fatty acid metabolism in crop improvement programmes.
椰子（Cocos nucifera L.）含有大量中链脂肪酸，这些中链脂肪酸大多能识别酰基-酰基载体蛋白（ACP）硫酯酶，该酶可将酰基-ACP水解为游离脂肪酸，从而在脂肪酸生物合成过程中终止酰基链的延长。从果实发育过程中椰子胚乳制备的cDNA文库中分离出一个酰基-ACP硫酯酶的全长cDNA，命名为CocoFatB1。该基因包含一个1254 bp的开放阅读框，编码一个417个氨基酸的蛋白质。CocoFatB1蛋白的氨基酸序列与CnFatB1和油棕（Elaeis guineensis Jacq.）酰基-ACP硫酯酶的序列相似性分别为100%和95%。实时荧光定量PCR分析表明，CocoFatB1转录本在8个月大的椰子胚乳中含量最高；15个月大的椰子的叶片和胚乳中该水平分别约为80%和10%。在根癌农杆菌介导的转化后，CocoFatB1编码区在种子特异性napin启动子的控制下在烟草（Nicotiana tabacum L.）中过表达。不同转基因植物中CocoFatB1转录本表达差异达20倍，有21株植物表现出可检测到的CocoFatB1表达水平。对转基因烟草种子的脂肪酸组成分析表明，与未转化植株相比，肉豆蔻酸（14∶0）、棕榈酸（16∶0）和硬脂酸（18∶0）的含量分别增加了25%、34%和17%。这些结果表明，CocoFatB1特异性作用于14∶0-ACP、16∶0-ACP和18∶0-ACP，并能增加中链饱和脂肪酸。该基因在作物改良计划中对脂肪酸代谢工程可能具有重要价值。
