藤茶叶片总RNA的提取及苯丙氨酸解氨酶基因(pal)片段的克隆  

Extraction of total RNA from leaves of Ampelopsis grossedentata and cloning of phenylalanine ammonia lyase gene fragment

作　　者：许明[1] 杨志坚[1] 郑金贵 

机构地区：[1]福建农林大学作物科学学院,福建福州350002

出　　处：《生物学杂志》2015年第2期96-99,共4页Journal of Biology

基　　金：国家科技支撑计划项目(2013BAD01B05);福建省自然科学基金(2012J01090);福建农林大学科技创新平台建设项目(ptjh12015)

摘　　要：藤茶叶片富含多酚、多糖以及黄酮类化合物，严重干扰了其总RNA的提取。实验在裂解前先用70％丙酮对藤茶叶片冷冻研磨材料进行多次抽提，然后采用试剂盒提取法、Trizol法和异硫氰酸胍法提取总RNA。结果表明，经丙酮预处理后，上述3种方法均能从藤茶叶片中成功提取到高质量的RNA，其完整性好，纯度高， D260 nm／D280 nm值介于1.8～2.0之间，产率为22.74～33.66μg／g。以提取的总RNA逆转录的cDNA为模板进行RT-PCR，克隆到一条长度为817 bp的pal片段。经Blast比对分析，该基因片段与葡萄、茶树等物种的pal之间具有较高的同源性。The leaves of Ampelopsis grossedentata are rich of flavones , polyphenols and polymeric carbohydrateds , which hinder the iso lation of RNA.The total RNA with good integrity and high purtity can be extracted from the leaves treated with 70%acetone before ly-sis by the three methods of RNA Extract Kit , Trizol and Guandine thiocyanate .The values of D260 nm/D280 nm of the extracted RNA were between 1.8 to 2.0, the yields were between 22.74-33.66 μg/g FW.A 817 bp phenylalanine ammonia lyase （pal） fragment was successfully amplified from cDNAs reverse-transcribed from the total RNA .By homology analysis ,the cloned pal fragment in Ampelopsis grossedentata is highly identical to the corresponding genes in vitis vinifera and camellia sinensis.

关 键 词：藤茶 叶片 总RNA提取 PAL 

分 类 号：Q943.2[生物学—植物学]