异子蓬异型种子萌发基因差显的cDNA-AFLP技术体系优化  

Establishment of cDNA-AFLP Analysis System in Differential Display of Gene Expression of Suaeda aralocaspica during the Process of Seed Germination

作　　者：邢佳佳[1,2] 陈莎莎[3] 吕秀云[1] 兰海燕[1] 

机构地区：[1]新疆大学生命科学与技术学院,新疆生物资源与基因工程重点实验室,乌鲁木齐830046 [2]新疆巴州环境监测站,新疆库尔勒841000 [3]清华大学生命科学学院,北京100000

出　　处：《西北农业学报》2013年第8期65-71,共7页Acta Agriculturae Boreali-occidentalis Sinica

基　　金：国家自然科学基金(31060027,31260037);新疆生物资源基因工程重点实验室开放基金(XJDX0201-2011-03)

摘　　要：以异子蓬种子为材料,建立并优化其萌发过程中基因表达差显的cDNA-AFLP技术体系。分别以总RNA和mRNA反转录的双链cDNA为模板,对25μL PCR扩增体系中的退火温度、引物量、DNA聚合酶种类及电泳条件进行优化。结果显示,以分离的mRNA为模板可以反转录得到品质较好的双链cDNA;PCR扩增选择较高退火温度(第1轮为56℃,第2轮为65～56℃)、引物量为1μL(10μmol/L)、使用高效PremixLA Taq Hot Start DNA聚合酶、在约800V电压下进行60g/L变性聚丙烯酰胺凝胶电泳,可得到条带清晰、多态性好的cDNA-AFLP结果。The cDNA-AFLP analysis system in differential display of gene expression of Suaeda aralocaspica during the process of seed germination was established after optimizing several key factors.In the study,double-strand cDNA was synthesized with mRNA or total RNA,then different concentrations of primer,annealing temperature,DNA polymerase and conditions of polyacrylamide gel electrophoresis were studied.Results indicated that mRNA isolated from total RNA was better for synthesizing double-strand cDNA.The distinct bands could be obtained when the primer was added to 1μL（10 μmol/L）,the annealing temperature should be higher（first run of amplification：56 ℃,second run of amplification：65-56℃）,Premix LA Taq Hot Start was chosen as DNA polymerase in 25μL PCR system.Furthermore,60g/L denatured polyacrylamide gel electrophoresis was appropriate at a voltage of 800V.

关 键 词：异子蓬 异型种子 萌发 CDNA-AFLP技术 体系优化 

分 类 号：Q943.2[生物学—植物学]