GDSL脂肪酶作为一种脂质水解酶具有广泛的底物特异性,在植物的油脂代谢、生长发育等众多生理过程中发挥着重要作用。利用RT—PCR技术从发育的陆地棉种子中克隆得到棉花的GDSL脂肪酶基因的cDNA,该基因开放读码框为1068bp,编码含有356个氨基酸的蛋白质。对棉花种子发育不同时期进行半定量PCR分析的结果表明,在种子发育15—20d时,GhGLIP基因的表达量较高。将该基因构建到真核表达载体pCAMBIA2300中,转入农杆菌GV3101后转化甘蓝型油菜野油19,通过筛选和鉴定获得转基因阳性油菜株系。转基因油菜的GDSL脂肪酶活力获得了显著提高,转基因油菜种子的油脂含量比野生型油菜提高了8.68%,提高比率为24.3%。
GDSL lipase (GLIP), a lipid hydrolase with broad substrate specificity, exists in plant tissues, playing important roles in plants'lipid metabolism, growth and development and many other physiological processes. In the study, a cotton GDSL lipase named GhGLIP has been isolated from cotton ovules by RT -PCR method. The full open reading frame was 1 068 bp, containing a protein of 356 amino acids. Semi - quantitative PCR analysis of dif- ferent cotton ovule development periods showed that GhGLIP gene expressed higher values in 15 -20 d ovules. The plant over expression vector pCAMBIA2300 - GhGLIP was obtained then transformed into Brassica napus L. (wild -oil 19) by agrobacterium mediated method. Transgenic oilseed rape plants were obtained after screening and identification with a significant promotion of GDSL lipase enzyme activity ; the oil content of transgenic Brassiea napus seeds was significantly enhanced by 8.68% higher and a increase ratio of 24.3% than wide -type.