六倍体小黑麦耐盐相关基因cDNA-AFLP技术体系的建立

Contruction of cDNA-AFLP Technique for Genes Related to Salt Stress in Hexopliod Tirticale

为建立和优化六倍体小黑麦耐盐相关基因克隆cDNA-AFLP技术反应体系,并探索其应用效果,以对盐胁迫反应不同的4个六倍体小黑麦品种(系)为材料,对其在0.15mol/L氯化钠胁迫72h的cDNA-AFLP技术中DNA聚合酶浓度、模版浓度和引物筛选等过程进行了摸索和优化。结果显示,建立了以1.0UTaqDNA聚合酶为扩增酶、预扩产物稀释20倍、EcoRI和MseI为内切酶及其相应接头为引物的六倍体小黑麦耐盐相关基因cDNA-AFLP技术体系,并从64对引物组合中筛选出稳定、高效的引物组合以及236个差异片段。结果表明,该cDNA-AFLP技术体系更易分离出差异性片段,为六倍体小黑麦在盐胁迫及其它逆境环境下的基因克隆提供一种有效手段。

To construct a more efficient cDNA-AFLP system and explore its usage for gene clone related with salt stress in triticale.Four triticale varieties or lines with sensitive or tolerant to salt were used to be stressed by 0.15 mol/L NaCl for 72 hours as materials,the courses of DNA Polymerase concentration,template concentration,primer screening in the cDNA-AFLP technolyge were optimized.The result showed that we constructed the cDNA-AFLP system that 1.0 U Taq DNA polymerase for pre-amplication enzyme,the 20 times of diluted pre-amplication product,EcoR I and Mse I and their realted adapters for primers,and several stable,edible primer combinations were screened out of 64 pairs of primers and 236 differentially expressed cDNAs were obtained with the newly established cDNA-AFLP system.The result indicated that cDNA-AFLP could easily isolate more differential cDNAfragments than usual AFLP techniques and it will provide a powerful method for molecular cloning of genes in triticale related with salt stress.