Expression in yeast and tobacco of plant cDNAs encoding acyl CoA:diacylglycerol acyltransferase
Expression in yeast and tobacco of plant cDNAs encoding acyl CoA:diacylglycerol acyltransferase
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DOI：
10.1046/j.1432-1327.2000.00961.x
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发表时间：
2000-01-01
期刊：
影响因子：
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通讯作者：
Schaller, H
中科院分区：
文献类型：
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作者：
Bouvier-Navé, P;Benveniste, P;Schaller, H
在寻找编码植物甾醇酰基转移酶的cDNA的过程中，使用与酵母和动物酰基CoA：胆固醇酰基转移酶具有实质性同一性的表达序列标签克隆来筛选来自拟南芥和烟草的cDNA文库。这导致分离出两种分别编码520和532个氨基酸的蛋白质的全长cDNA。试图弥补酵母双突变体的酰基辅酶A：胆固醇酰基转移酶的缺陷是不成功的，这表明两个基因都不编码酰基辅酶A：胆固醇酰基转移酶。他们推导的氨基酸序列，然后被证明有40和38%的同一性，分别与鼠酰基CoA：二酰基甘油酰基转移酶和他们的表达在are 1 are 2或野生型酵母导致在一个强大的增加油酰CoA到三酰基甘油的掺入。在来自用这些植物cDNA转化的酵母的微粒体中的掺入比在用空载体转化的酵母中的掺入高2-3倍，清楚地表明这些cDNA编码酰基CoA：二酰基甘油酰基转移酶。此外，在从拟南芥DGAT转化的酵母制备微粒体的过程中，在100 000 g上清液的顶部观察到漂浮层。该馏分富含三酰基甘油，并表现出很强的酰基辅酶A：二酰基甘油酰基转移酶活性，而几乎没有检测到相应的清晰的对照酵母馏分中的活动。由于使用该活性级分和二己酰甘油作为底物，可以证明通过AtDGAT从头合成1，2-二己酰3-油基甘油。还进行了用AtDGAT转化烟草。19个初级转化体的分析允许检测，在几个人中，与AtDGAT mRNA表达相关的三酰甘油含量的显着增加（高达7倍）。此外，用脂质特异性染料染色的叶表皮细胞的光学显微镜观察显示，在三酰甘油过度生产植物的细胞中存在脂滴，从而说明了酰基CoA：二酰甘油酰基转移酶转化植物的潜在应用。
During the course of a search for cDNAs encoding plant sterol acyltransferases, an expressed sequence tag clone presenting substantial identity with yeast and animal acyl CoA:cholesterol acyltransferases was used to screen cDNA libraries from Arabidopsis and tobacco. This resulted in the isolation of two full-length cDNAs encoding proteins of 520 and 532 amino acids, respectively. Attempts to complement the yeast double-mutant are1 are2 defective in acyl CoA:cholesterol acyltransferase were unsuccessful, showing that neither gene encodes acyl CoA:cholesterol acyltransferase. Their deduced amino acid sequences were then shown to have 40 and 38% identity, respectively, with a murine acyl CoA:diacylglycerol acyltransferase and their expression in are1 are2 or wild-type yeast resulted in a strong increase in the incorporation of oleyl CoA into triacylglycerols. Incorporation was 2-3 times higher in microsomes from yeast transformed with these plant cDNAs than in yeast transformed with the void vector, clearly showing that these cDNAs encode acyl CoA:diacylglycerol acyltransferases. Moreover, during the preparation of microsomes from the Arabidopsis DGAT-transformed yeast, a floating layer was observed on top of the 100 000 g supernatant. This fraction was enriched in triacylglycerols and exhibited strong acyl CoA:diacylglycerol acyltransferase activity, whereas almost no activity was detected in the corresponding clear fraction from the control yeast. Thanks to the use of this active fraction and dihexanoylglycerol as a substrate, the de novo synthesis of 1,2-dihexanoyl 3-oleyl glycerol by AtDGAT could be demonstrated. Transformation of tobacco with AtDGAT was also performed. Analysis of 19 primary transformants allowed detection, in several individuals, of a marked increase (up to seven times) of triacylglycerol content which correlated with the AtDGAT mRNA expression. Furthermore, light-microscopy observations of leaf epidermis cells, stained with a lipid-specific dye, showed the presence of lipid droplets in the cells of triacylglycerol-overproducer plants, thus illustrating the potential application of acyl CoA:diacylglycerol acyltransferase-transformed plants.