干旱胁迫下新疆野生沙生冰草的银染mRNA差异显示分析  

Silver-staining mRNA Differential Display Analysis ofAgropyron desertorum Schult at Drought Stress

作　　者：李莉[1] 贾纳提[1] 朱昊[1] 王希东[2,3] 

机构地区：[1]新疆畜牧科学院草业研究所,新疆乌鲁木齐830000 [2]新疆农业大学农学院,新疆乌鲁木齐830052 [3]新疆农业大学农业技术重点实验室,新疆乌鲁木齐830052

出　　处：《草食家畜》2010年第4期26-28,32,共4页Grass-Feeding Livestock

基　　金：新疆自然科学基金项目(200721109);科技支疆项目"新疆荒漠逆境植物抗旱功能基因的筛选与应用"(200840102-05)

摘　　要：运用非同位素银染mRNA差异显示方法,分析新疆野生沙生冰草抗旱基因表达差异。用10%(-1.0Mpa)PEG-6000溶液处理三叶一心期冰草,以叶片mRNA为模板采用锚定引物和随机引物组合,进行反转录差异显示PCR扩增,经6%变性聚丙烯酰胺凝胶电泳后用银染方法差异显示DNA条带,在直观下,从凝胶中回收差异带并进行再扩增,2%琼脂糖电泳得到了DNA的单一条带。通过分离与冰草抗旱性状相关的基因片段,为进一步从分子水平上认识冰草抗旱机理、进行抗旱相关基因的遗传操作奠定基础。mRNA differential display polymerase chain reaction（DD-PCR） method with silver-staining,was used to isolate the differentially expressed gene of Agropyron desertorum（Fisch.） Schult.With 10%（-1.0Mpa） PEG-6000 solution treatment with wheatgrass,Total RNA was extracted from Wheatgrass at the three-leaf stage.The cDNA copies of differentially expressed mRNA were identified by means of reverse transcription with anchor primer and amplified by subsequent PCR with sets of anchor primer and arbitrary primer.The DNA bands on gel were displayed by silver stain method after electrophoring in urea-denaturing sequencing gel.The differentially expressed bands were visually retrieved and reamplified,most of which obtained one band when visualized in 2 % agarose gels.Isolated the drought-related gene fragments from wheatgrass,in order to further understand the drought resistance mechanism in molecular level,and lay the foundation for the genetic operation of wheatgrass.

关 键 词：银染方法 差异显示 锚定引物 MRNA 

分 类 号：Q789[生物学—分子生物学]