文献类型：期刊文献

中文题名：油茶cDNA-AFLP技术反应体系建立的研究

英文题名：Establish a cDNA-AFLP Technology System in Camellia oleifera

作者：谢一青[1,2] 黄勇[2] 卓仁英[1] 李志真[2] 姚小华[1]

第一作者：谢一青

机构：[1]中国林业科学研究院亚热带林业研究所;[2]福建省林业科学研究院

年份：2013

卷号：11

期号：5

起止页码：611-616

中文期刊名：分子植物育种

外文期刊名：Molecular Plant Breeding

收录：CSTPCD;;北大核心:【北大核心2011】；CSCD:【CSCD_E2013_2014】；

基金：国家科技支撑项目(2009BADB1B01);福建省科技厅项目(2009R10008-5);福建省林木种苗科技攻关项目(闽林科(2013)1号)共同资助

语种：中文

中文关键词：油茶;cDNA—AFLP;体系优化;引物筛选

外文关键词：Camellia oleifera, cDNA-AFLPR, System optimization, Primer selection

分类号：S513

摘要：以小果油茶和普通油茶的嫩叶及种仁为材料,对影响其cDNA-AFLP反应体系的关键因子进行优化分析,筛选出适合油茶的cDNA-AFLP反应体系。结果表明:分别用EASYspin Plus植物RNA快速提取试剂盒和Trizol法能成功获得高质量的油茶嫩叶和种仁总RNA;从总RNA中分离的mRNA经合成双链cDNA后,用限制性内切酶EcoRⅠ和MseⅠ在37℃下5 h内酶切完全,经5 U T4连接酶16℃连接的产物稀释10倍后可直接用于预扩增;在20μL选择性扩增体系中,以预扩增产物稀释30倍为模版、dNTP(10 mmol/L)1.5μL、引物(10μmol/L)1.0μL、Taq酶用量1.25 U时的扩增效果较好。利用优化体系成功筛选出61对可以获得带型丰富且重复性好的引物组合。差异片段回收及PCR检测的结果表明,优化后的cDNA-AFLP分析体系适用于油茶相关差异基因的表达研究。
Using the tender leaf, kernel of Camellia meiocarpa and C. oleifera as materials, a suitable cDNAAFLP reaction system for C. meioearpa and C. oleifera was established after optimizing several key factors, including enzyme digestion system, pre-amplification and selective amplification, which may affect cDNA-AFLP analysis. The results showed that using the quick extraction kit of EASYspin Plus plant RNA and Trizol method could extract ideal total RNA from the tender leaf and kernel of C. meiocarpa and C. oleifera, respectively. After mRNA was isolated from total RNA, it was synthesized into double-stranded cDNA. And then the double-stranded eDNA was digested completely by the combination of restriction endonuclease EeoR Ⅰ and Mse Ⅰ at 37℃ for 5 hours. The digested product was connected with 5 U T4 ligase at 16℃ for an overnight. Then reducible preamplification result was obtained when ligation product were diluted by 10 times for pre-mplification. And the optimal conditions for a 20 μL volume of selective amplification were obtained when the template of preexpanded product diluted by 30 times and with dNTP （10 mmol/L） 1.5 μL, primer （10 μmol/L） 1.0 μL, Taq enzyme 1.25 U. Moreover, 61 out of 210 primer pairs, producing repeatable and prolific bands, were successfully selected by this improved method. According to the PCR detected results of recovery different gene fragment, the optimized cDNA-AFLP technique was suitable for further expression study on the differential gene in C. oleifera.