过表达OsMPK17激酶蛋白质增强了水稻的耐旱性
Overexpression of OsMPK17 protein enhances drought tolerance of rice
A: 水稻OsMPK17基因的PCR扩增: 以带有OsMPK17全长序列的质粒DNA为模板, 用上游引物5′-GCGGTACCATGGG CGGCCGCGCCCGCTC-3′, 下游引物5′-GCGAGCTCGGTTTTC AGTTGAGCAAC-3′, 扩增OsMPK17基因。B: pET30a-MPK17重组质粒的双酶切验证: 将PCR产物与pET30a用Kpn I+ Sac I双酶切, 连接后转化克隆菌DH5α, 提取质粒后再进行双酶切鉴定。C: 融合蛋白质OsMPK17的诱导表达及考染检测: 取pET30a-MPK17酶切验证的质粒进行测序再验证, 将测序正确的质粒转化表达菌Codon plus诱导表达。在含50 μg mL-1卡那霉素的LB液体培养基中诱导表达, 振荡培养至OD600为0.6~0.8, 加入IPTG, 25°C过夜培养, 收菌后超声破碎, 离心取上清液(S)和沉淀(P), 0: 0时间点培养物, 用10% SDS-PAGE分离、考染。M为分子量标记; PCR为扩增产物; K+S为Kpn I+ Sac I双酶切产物。
A: PCR amplification of rice OsMPK17 gene. A plasmid containing full-length OsMPK17 gene was used as template for PCR amplification of OsMPK17 gene using primers 5′-GCGGTACCATGGG CGGCCGCGCCCGCTC-3′ and 5′-GCGAGCTCGGTTTTCAGTT GAGCAAC-3′. B: Verification of recombinant pET30a-MPK17 plasmid by double digestion using Kpn I and Sac I. The PCR products and pET30a plasmid DNA were digested by Kpn I and Sac I, the ligation product was used to transform DH5α. Recombinant plasmid was verified by double digestion. C: Induction of fusion protein OsMPK17 and Coomassie blue staining. Correct pET30a- MPK17 plasmid verified by double digestion was double checked by sequencing. Sequencing verified plasmid was transformed to Codon plus bacterial strain to express fusion protein. The bacteria was cultured in LB medium containing 50 μg mL-1 kanamycin and IPTG which was added when the OD600 reached 0.6-0.8. The bacteria was collected after over night culture at 25°C and disrupted by sonication. The supernatant (S) and pellet (P) were obtained after centrifugation and total protein was separated by 10% SDS-PAGE and stained with Coomassie blue. 0: Total protein isolated at 0 time point. M: Molecular weight marker; PCR: Amplification products; K+S: Double digestion product using Kpn I and Sac I.
