条锈菌诱导的小麦β-1,3-葡聚糖酶基因编码区的克隆及原核表达  

Cloning and prodaryotic expression of a wheat β-1,3-glucanase gene induced by Puccinia striiformis Westend f.sp tritici Eriks in E.coli

作　　者：刘博[1] 崔素萍[2] 王晓杰[1] 黄丽丽[1] 康振生[1] 

机构地区：[1]西北农林科技大学植保学院,陕西杨凌712100 [2]陕西省农业分子生物学重点实验室,陕西杨凌712100

出　　处：《西北农林科技大学学报（自然科学版）》2007年第6期125-129,共5页Journal of Northwest A&F University(Natural Science Edition)

基　　金：教育部重大科技培育项目(2004-295);教育部长江学者和创新团队发展计划项目(IRT0558)

摘　　要：为研究小麦与条锈菌互作过程中β-1,3-葡聚糖酶基因的功能,根据小麦β-1,3-葡聚糖酶cDNA全长序列设计合成引物,通过RT-PCR方法获得小麦β-1,3-葡聚糖酶基因编码区,将其克隆至pGEM T-easy Vector,经HindⅢ和BamHⅠ双酶切回收目的片断,并将其定向克隆到pET-32a(+)Vector中构建表达载体GLU-pET-32a(+),转化E.coliBL21(DE3),用IPTG进行诱导表达。结果表明,小麦β-1,3-葡聚糖酶基因在大肠杆菌中得到了高效表达,获得了分子量约为49 ku的融合蛋白,表达量约占菌体总蛋白的19%。最佳诱导条件为:0.3 mmol/LIPTG,37℃诱导4 h。To determine the function of β-1,3-glucanase in wheat to stripe rust resistance,the encoding region of a wheat β-1,3-glucanase gene induced by Puccinia striiformis Westend f. sp tritici Eriks was engineered via reverse transcription polymerase chain reaction （RT-PCR）. The amplified fragments were first cloned into pGEM T-easy Vector, after restriction analysis the gene was transferred to an E. coli strain BL 21 （DE3） expression behind T7 promotor-pET-32a（＋） system. The recombinant gave rise to a 49 ku fusion protein in response to the IPTG induction; under the best inducement condition （37 ℃ ,0.3 mmol/L IPTG,4 h） its content was about 19% of the total cell protein by Gene Genius Bio Imaging System.

关 键 词：条锈菌 小麦 Β-1 3-葡聚糖酶基因 克隆 原核表达 

分 类 号：Q786[生物学—分子生物学]