核桃不同组织高质量总RNA的提取方法(英文)  

A method for isolation of high quality RNA from various Juglans regia tissues

作　　者：许锋[1] 蔡荣[1] 陈柳吉[1] 常杰[1] 程水源[2] 

机构地区：[1]长江大学园艺园林学院,湖北荆州434024 [2]黄冈师范学院生命科学与工程学院,湖北黄冈438000

出　　处：《果树学报》2008年第3期435-439,共5页Journal of Fruit Science

基　　金：教育部新世纪优秀人才支持计划(NCET-04-0746);湖北省教育厅重大项目(Z200627002)

摘　　要：利用改良CTAB法,分别从富含多糖、多酚及黄酮类物质的核桃叶、绿果、茎、种子和根中成功提取高质量的总RNA。经电泳、紫外分光光度计、RT-PCR和Northern杂交分析,结果显示,各组织的总RNA至少有2条清晰的电泳条带,产量可达174μg/g,A260/A280比值在2.0左右,通过RT-PCR在不同组织中均能扩增出核桃18S基因的cDNA片段,而且以18S基因为探针在不同组织中也分别获得清晰的Northern杂交信号,说明利用此法分离核桃各组织的RNA纯度和浓度完全符合后续分子生物学研究的要求。Isolation of high-quality RNA is a prerequisite for gene expression studies. However, RNA extraction from walnut tissues is difficult due to the presence of rich polysaccharides, flavonoids and polyphenolic compounds. In this study, we tried an improved protocol to isolate total RNA in high yield and integrity from walnut leaves, green nuts, shoots, seeds and roots, which contained high levels of flavonoids, polysaccharides and polyphenolic secondary metabolites. Polyvinylpolypyrrolidone （PVPP） at 2% and β-mercaptoethanol at 4% were added to the standard CTAB extraction buffer, and after chloroform and phenol extraction followed by ethanol/sodium acetate precipitation, a second phenol/chloroform extraction was introduced to remove coprecipitated polysaccharides. The modified CTAB method resulted in isolation of high yield （174 μg/g） and high quality A260/A280≈2.0） RNA, and at least two distinct rRNA bands were detected after electrophoresis in agarose gels. Sharp hybridization signals were obtained from Northern blots and 18S gene fragments were successfully amplified by RT-PCR, suggesting the high integrity of isolated RNA. The total RNA isolated by this protocol was of sufficient quality for subsequent molecular applications.

关 键 词：核桃 不同组织 RNA提取 CTAB 

分 类 号：S664.1[农业科学—果树学]