以山葡萄品种左山一的叶片和花芽为材料,对cDNA-AFLP体系的几个关键因素进行摸索,建立了适宜山葡萄的cDNA-AFLP反应体系.结果表明,提取山葡萄的花芽RNA适宜用改良的CTAB法,提取叶片RNA适宜用SDS-酸酚法.在2x Y/T Buffer的作用下,500 mg的cDNA利用MseⅠ与EcoRⅠ双酶切4 h即可酶切完全,将16℃连接过夜的产物稀释倍作为预扩增的模板,预扩增产物稀释20倍作为选扩模板能获得带型稳定可重复性好的结果.
The cDNA-AFLP systemfor Vitis amurensis Rupr was established after optimizing several key factors affecting cDNA-AFLP analysis.The result indicated that CTAB method could be used for extracting ideal RNA from leaf and inflorescence of Vitis amurensis Rupr,while SDS-acidic phenol method for extracting RNA from bud;500 ng cDNA was digested completely by EcoR I and Mse I for 6 hours.The reducible results was obtained when the ligation products were diluted to 5 times for preamplification template and preamplification productsthe were diluted to 20 times for selective amplification.
[1] Bachem C W B,Van der Hoeven R S,De Bruijn S M,et al.Visualization of differential gene expression using a novel method of RNA fingerprinting based on AFLP:analysis of gene expression during potato tuber development[J].Plant J,1996,9(5):745-753.
[2] 范淑英,乐建刚,成广杰,等.用eDNA.AFLP技术构建白菜转录图谱[J].中国农业科学,2008,46(6):1735-1741.
[3] Yan Y X,Li M,Liu Z,et al.A novel gene,screened by cDNA-AFLP appreach,contributes to lowering the acidity of fruit in apple[J].Plant Physilo Biochem,2007,45(2):139-145.
[4] Polesani M,Desario F,Technolngies,et al.cDNA-AFLP analysis of plant and pathogen genes expressed in grapevine infected with Plasmopara viticola[J].BMC Genomics,2008,9:142-156.
[5] Zheng Xianwu,Chen Xuewei,Zhang Xiao-hong,et al.Isolation and identification of a gene in response to rice blast disease in rice[J].Plant Molecular Biology,2003:1-11.
[6] 李驰,卢新雄,陈晓玲,等.利用cDNA-AFLP技术分析老化棉花种子基因差异表达[J].棉花学报,2007,19(4):138-143.
[7] 郭丽琼,林俊芳,杨丽卿,等.应用cDNA-AFLP技术分离草菇冷诱导表达基因[J].园艺学报,2005,32(1):54-59.
[8] 刘涛,徐平珍,胡运乾,等.板栗花序与叶的cDNA-AFLP的差异表达分析[J].中国农学通报,23(2):81-84.
[9] 张玉刚,成建红,韩振海,等.小金海棠总RNA提取方法比较及cDNA的LD-PCR扩增[J].生物技术通报,2005,4:50-53.
[10] 王壮伟,渠慎春,章镇,等.苹果属RNA高效快速提取新方法[J].果树学报,2004,21(4):385-387.
[11] Karen E,Niclas Olsson,James Schlossor,et al.An optimized grapevine RNA isolation procedure and statistical determination of reference genes for real-time RT-PCR during berry development[J].BMC Plant Biology,2006,6:27-37.
