利用荧光mRNA差异显示技术克隆短芒大麦盐胁迫相关基因的ESTs

Cloning ESTs of salt-stress related genes from Hordeum brevisubulatum(Trin.) link with fluorescence DDRT-PCR

目的 :获取短芒大麦盐胁迫相关基因的 ESTs。 方法 :应用荧光 m RNA差异显示技术进行筛选 ,并利用反向 North-ern杂交方法对差异片段进行快速鉴定以排除假阳性。结果 :共获得 32个与盐胁迫相关的 c DNA序列 ,并获得 Gen Bank注册号。BL AST同源性分析结果表明 ,1 3个与已知基因序列有同源性 ,1 9个为未知 ESTs。其类型分别为 :诱导表达型 ,2 2个 ;增强表达型 ,7个 ;抑制表达型 ,3个。 结论 :通过荧光 m RNA差异显示技术获得了短芒大麦盐胁迫相关基因的 ESTs。

Objective:To clone ESTs of salt stress related genes from Hordeum brevisubulatum (Trin.)Link. Methods: In order to avoid false positive results in mRNA differential display reverse transcription polymerase chain reaction (DDRT PCR) method, we replaced radioactive reagents with fluorescent reagents and confirmed the results with reverse Northern hybridization. Results: Thirty two cDNAs sequences related with salt stress were revealed, which all obtained accession number in GenBank. BLAST analysis revealed that 13 cDNAs were homologous with the reported genes and the other 19 cDNAs were new ESTs. Those cDNAs were of 3 types: 22 were induced expression; 7 enhanced expression and 3 depressed expression. Conclusion: Fluorescence DDRT PCR in this paper is important for studying the salt tolerant molecular mechanism of Hordeum brevisubulatum (Trin) Link.