Cloning and prodaryotic expression of a wheat β-1,3-glucanase gene induced by Puccinia striiformis Westend f.sp tritici Eriks in E. coli
2007
Liu Bo | Cui Suping | Wang Xiaojie
Chinese. 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Hind Ⅲ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- BL 21（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/L IPTG，37 ℃诱导4 h。_
Show more [+] Less [-]English. 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.
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This bibliographic record has been provided by Institute of Agricultural Information, Chinese Academy of Agricultural Sciences