油茶种子总RNA提取及Cod8FAD基因的鉴定

Extraction of Total RNA and Identification of Cod8FAD Gene in Camellia oleifera seeds

油茶种子胚乳中富含脂肪、多糖和酚类公合物,要获得高质量的RNA难度较大。本研究以油茶优良无性系湘林1号近成熟种子的胚乳为实验材料,在试剂盒的基础上应用改良的CTAB法提取总RNA。结果表明,使用改良后的CTAB法配合试剂盒提取RNA操作简单,且能够有效去除油茶种子中多糖和多酚类等次生物质,从而得到高质量的RNA。以获得的RNA为模板,设计简并引物,通过RT-PCR,成功从油茶种子中鉴定出了油脂合成的关键酶基因之一△8脂肪酸脱饱和酶基因,命名为Cod8FAD(Camellia oleifera delta 8 fatty acid desaturase),该基因与茶树△8脂肪酸脱饱和酶基因相似性最高,为97%,与耧斗菜△8脂肪酸脱饱和酶基因相似性最低,为62%。

The isolation of high quality RNA from Camellia oleifera seeds had been very difficult because of rich polysaccharide and phenol compounds in endosperm.Using maturescent seeds of Xianglin No.1,an improved clone as test materials,the total RNA was isolated by modified CTAB combined with RNA purification Kit.Purity and concentration of isolated RNA indicated that polysaccharide and phenol compounds could be effectively eliminated in the endosperm by this method.Using the high-quality RNA as template,a key gene for lipid synthesis was successfully identified by degenerate RT-PCR,that was named Cod8FAD（C.oleifera delta 8 fatty acid desaturase）,similar（97%） with delta 8 fatty acid desaturase of C.sinensis.