过表达OsMPK17激酶蛋白质增强了水稻的耐旱性

马金姣,兰金苹,张彤,陈悦,郭亚璐,刘玉晴,燕高伟,魏健,窦世娟,杨明,李莉云,刘国振

Overexpression of OsMPK17 protein enhances drought tolerance of rice

Jin-Jiao MA,Jin-Ping LAN,Tong ZHANG,Yue CHEN,Ya-Lu GUO,Yu-Qing LIU,Gao-Wei YAN,Jian WEI,Shi-Juan DOU,Ming YANG,Li-Yun LI,Guo-Zhen LIU

图3 水稻OsMPK17基因过表达载体的构建与鉴定 A: 水稻OsMPK17基因的PCR扩增; B: Hind III + Xba I双酶切鉴定pEASY-MPK17-3HA重组质粒; C: Kpn I+ Spel I双酶切鉴定pUBI-C4300-MPK17重组质粒。以带有目的基因cDNA的质粒为模板, 用上游引物5′-GCGGTACCATGGGCGGCCGCGCCC GCTC-3′ (下画线为Kpn I限制性内切酶位点)和下游引物5′-GCGAGCTCGGTTTTCAGTTGAGCAAC-3′ (下画线为Sac I限制性内切酶位点)进行PCR扩增。将扩增的OsMPK17片段插入中间载体pEASYT1-3HA, 双酶切验证。将测序正确的中间载体切胶回收目的片段, 再用Kpn I+Spel I双酶切插入转化载体pUBI-C4300, 获得重组质粒DNA双酶切验证。M: 分子量标记; PCR: 扩增产物; H+X: Hind III+ Xba I双酶切; K+S: Kpn I+ Spel I双酶切。

Fig. 3 Construction and identification of rice OsMPK17 overexpression vector A: PCR amplification of rice OsMPK17 gene; B: Hind III+ Xba I restriction enzyme digestion of recombinant pEASY-MPK17-3HA plasmid; C: Kpn I+ Spel I restriction enzyme digestion of recombinant pUBI-C4300-MPK17 plasmid. PCR amplification of OsMPK17 gene using plasmid containing full-length OsMPK17 cDNA as template, the primers used were 5′-GCGGTACCATGGGC GGCCGCGCCCGCTC-3′ (Kpn I restriction site was underlined) and 5′-GCGAGCTCGGTTTTCAGTTGAGCAAC-3′ (Sac I restriction site was underlined). The amplified fragment was inserted into pEASY-3HA vector and verified by double digestion. Sequence verified pEASY-MPK17-3HA was digested by Kpn I+ Spe I, the fragment was inserted into pUBI-C4300 and verified by double digestion. M: Molecular weight marker; PCR: PCR amplification product; H+X: Hind III+ Xba I restriction enzyme digestion; K+S: Kpn I+ Spel I restriction enzyme digestion.