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油菜MKK2基因原核表达及特性分析 被引量:1
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作者 张腾国 郭艳峰 +3 位作者 王圆圆 寇明刚 王娟 孙万仓 《兰州大学学报(自然科学版)》 CAS CSCD 北大核心 2015年第3期405-410,共6页
MAP激酶级联途径在信号传导过程中发挥着重要作用.以白菜型冬油菜"陇油6号"为材料,利用RT-PCR法克隆到全长MKK2基因cDNA,生物信息学分析表明油菜MKK2蛋白为亲水蛋白,包含有MAPKK蛋白所特有的磷酸化基序S/T-X_(3-5)-S/T.将其... MAP激酶级联途径在信号传导过程中发挥着重要作用.以白菜型冬油菜"陇油6号"为材料,利用RT-PCR法克隆到全长MKK2基因cDNA,生物信息学分析表明油菜MKK2蛋白为亲水蛋白,包含有MAPKK蛋白所特有的磷酸化基序S/T-X_(3-5)-S/T.将其与大肠杆菌表达载体pET30a连接,构建原核表达载体pET30a-MKK2,并转化大肠杆菌BL21,经IPTG诱导表达后,SDS-PAGE以及Western blotting检测结果表明该基因表达了1个约43 kD的蛋白.实时荧光定量PCR结果显示该基因表达没有组织特异性,其转录受UV-B胁迫的诱导. 展开更多
关键词 陇油6号 mkk2基因 原核表达 UV-B胁迫
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MEKK1, MKK1/MKK2 and MPK4 function together in a mitogen-activated protein kinase cascade to regulate innate immunity in plants 被引量:39
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作者 Minghui Gao Jinman Liu +3 位作者 Dongling Bi Zhibin Zhang Fang Cheng2, Sanfeng Chen Yuelin Zhang 《Cell Research》 SCIE CAS CSCD 2008年第12期1190-1198,共9页
Mitogen-activated protein kinase (MAPK) cascades play important roles in regulating plant innate immune responses. In a genetic screen to search for mutants with constitutive defense responses, we identified multipl... Mitogen-activated protein kinase (MAPK) cascades play important roles in regulating plant innate immune responses. In a genetic screen to search for mutants with constitutive defense responses, we identified multiple alleles of mpk4 and mekkl that exhibit cell death and constitutive defense responses. Bimolecular fluorescence complemen- tation (BiFC) analysis showed that both MPK4 and MEKK1 interact with MKK1 and MKK2, two closely related MAPK kinases, mkkl and mkk2 single mutant plants do not have obvious mutant phenotypes. To test whether MKK1 and MKK2 function redundantly, mkkl mkk2 double mutants were generated. The mkkl mkk2 double mutant plants die at seedling stage and the seedling-lethality phenotype is temperature-dependent. Similar to the mpk4 and mekkl mutants, the mkkl mkk2 double mutant seedlings accumulate high levels of H202, display spontaneous cell death, constitutively express Pathogenesis Related (PR) genes and exhibit pathogen resistance. In addition, activation of MPK4 by fig22 is impaired in the mkkl mkk2 double mutants, suggesting that MKK1 and MKK2 function together with MPK4 and MEKK1 in a MAP kinase cascade to negatively regulate innate immune responses in plants. 展开更多
关键词 MAPK innate immunity MPK4 MEKK1 MKK1 mkk2
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细粒棘球蚴MKK1和MKK2基因原核表达载体的构建及其诱导表达 被引量:1
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作者 张传山 杨乐 +4 位作者 王丽敏 李亮 王俊华 吕国栋 林仁勇 《中国病原生物学杂志》 CSCD 北大核心 2014年第1期43-47,共5页
目的分别构建细粒棘球蚴(Eg)丝裂原活化蛋白激酶激酶MKK1和MKK2基因原核表达载体,诱导表达并纯化EgMKK1和EgMKK2蛋白。方法采集绵羊肝脏感染的Eg原头蚴,Trizol法提取原头蚴总RNA,反转录PCR分别扩增EgMKK1和EgMKK2基因片段,克隆至原核表... 目的分别构建细粒棘球蚴(Eg)丝裂原活化蛋白激酶激酶MKK1和MKK2基因原核表达载体,诱导表达并纯化EgMKK1和EgMKK2蛋白。方法采集绵羊肝脏感染的Eg原头蚴,Trizol法提取原头蚴总RNA,反转录PCR分别扩增EgMKK1和EgMKK2基因片段,克隆至原核表达载体pET28a(+),经限制性内切酶双酶切和测序鉴定正确后转化大肠埃希菌感受态细胞BL21,异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,采用镍柱亲和层析法纯化蛋白,并进行SDS-PAGE分析。结果测序证明pET28a-EgMKK1和pET28a-EgMKK2原核表达载体构建正确,在37℃经IPTG(终浓度1mmol/L)诱导可表达EgMKK1和EgMKK2蛋白,分子质量单位分别为44.96ku和64.01ku,与理论值相符,且以包涵体形式存在。表达产物纯化后的可溶性蛋白EgMKK1和EgMKK2含量分别为0.64mg/ml和1.2mg/ml。结论成功构建了MKK1和MKK2基因原核表达载体,表达蛋白可用于动物免疫,为研究EgMKK1和EgMKK2在Eg体内的生物学作用奠定了基础。 展开更多
关键词 细粒棘球绦虫 MKK1和mkk2基因 基因表达 蛋白纯化
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A Major Role of the MEKK1-MKK1/2-MPK4 Pathway in ROS Signalling 被引量:23
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作者 Andrea Pitzschke Armin Djamei +1 位作者 Frederique Bitton Heribert Hirt 《Molecular Plant》 SCIE CAS CSCD 2009年第1期120-137,共18页
Over the last few years, it has become evident that reactive oxygen species (ROS) signalling plays an important role in various physiological responses, including pathogen defense and stomatal opening/closure. On th... Over the last few years, it has become evident that reactive oxygen species (ROS) signalling plays an important role in various physiological responses, including pathogen defense and stomatal opening/closure. On the other hand, ROS overproduction is detrimental for proper plant growth and development, indicating that the regulation of an appropriate redox balance is essential for plants. ROS homeostasis in plants involves the mitogen-activated protein kinase (MAPK) pathway consisting of the MAPK kinase kinase MEKK1 and the MAPK MPK4. Phenotypic and molecular analysis revealed that the MAPK kinases MKK1 and MKK2 are part of a cascade, regulating ROS and salicylic acid (SA) accumulation. Gene expression analysis shows that of 32 transcription factors reported to be highly responsive to multiple ROS-inducing conditions, 20 are regulated by the MEKK1, predominantly via the MEKK1-MKK1/2-MPK4 pathway. However, MEKK1 also functions on other as yet unknown pathways and part of the MEKK1-dependent MPK4 responses are regulated independently of MKK1 and MKK2. Overall, this analysis emphasizes the central role of this MAPK cascade in oxidative stress signalling, but also indicates the high level of complexity revealed by this signalling network. 展开更多
关键词 Mitogen-activated protein kinases MEKK1 MKK1 mkk2 MPK4 reactive oxygen species redox homeostasis stress signalling differential gene expression.
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