| 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.coli BL21（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。 |
| 关键词: 条锈菌 小麦 β-1,3-葡聚糖酶基因 克隆 原核表达 |
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| 基金项目: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） |
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| Cloning and prodaryotic expression of a wheat β-1,3-glucanase gene induced by Puccinia striiformis Westend f. sp tritici Eriks in E. coli |
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| Abstract: |
| 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. |
| Key words: Puccinia striiformis wheat β-1,3-glucanase gene cloning prokaryotic expression |
