【目的】克隆84K杨水杨酸结合蛋白2(Salicylic acid-binding protein 2,SABP2)基因并预测其功能。【方法】以生长至5片小叶的84K杨组培苗为材料,提取其叶和茎的总RNA。根据毛果杨SABP2基因(GenBank序列号:XM_002310718.2)的完整CDs序列...【目的】克隆84K杨水杨酸结合蛋白2(Salicylic acid-binding protein 2,SABP2)基因并预测其功能。【方法】以生长至5片小叶的84K杨组培苗为材料,提取其叶和茎的总RNA。根据毛果杨SABP2基因(GenBank序列号:XM_002310718.2)的完整CDs序列,设计84K杨SABP2基因的引物,采用RT-PCR技术扩增84K杨SABP2基因全长,然后连接到pGM-T克隆载体,转化大肠杆菌TOP10感受态细胞,对84K杨SABP2基因进行克隆,获得该基因的全长序列。通过各种在线软件对84K杨SABP2基因及其编码的蛋白质进行生物信息学分析。【结果】84K杨基因的cDNA序列全长822bp,开放阅读框789bp,编码263个氨基酸。生物信息学分析表明,84K杨SABP2与毛白杨SABP2(GenBank序列号:JQ086570.1)的同源性最高,达98%;84K杨SABP2基因位于微体中;SABP2蛋白有11个蛋白质结合位点,属于α/β折叠水解酶家庭成员中的酯酶,为亲水性蛋白。【结论】成功克隆了84K杨的SABP2基因,其功能与前人对草本植物中SABP2部分功能的研究结果一致。展开更多
Aphids represent a major threat to crops.Hundreds of different viruses are aphid-borne.Upon aphid attack,plants release volatile organic compounds(VOCs)as airborne alarm signals to turn on the airborne defense(AD)of n...Aphids represent a major threat to crops.Hundreds of different viruses are aphid-borne.Upon aphid attack,plants release volatile organic compounds(VOCs)as airborne alarm signals to turn on the airborne defense(AD)of neighboring plants,thereby repelling aphids as well as reducing aphid fitness and virus transmission.This phenomenon provides a critical community-wide plant protection to fend off aphids,but the underlying molecular basis remains undetermined for a long time.In a recent article,Gong et al.established the NAC2-SAMT1 module as the core component regulating the emission of methyl-salicylate(MeSA),a major component of VOCs in aphid-attacked plants.Furthermore,they showed that SABP2 protein is critical for the perception of volatile MeSA signal by converting MeSA to Salicylic Acid(SA),which is the cue to elicit AD against aphids at the community level.Moreover,they showed that multiple viruses use a conserved glycine residue in the ATP-dependent helicase domain in viral proteins to shuttle NAC2 from the nucleus to the cytoplasm for degradation,leading to the attenuation of MeSA emission and AD.These findings illuminate the functional roles of key regulators in the complex MeSA-mediated airborne defense process and a counter-defense mechanism used by viruses,which has profound significance in advancing the knowledge of plant-pathogen interactions as well as providing potential targets for gene editing-based crop breeding.展开更多
目的:从拟南芥叶中克隆水杨酸结合蛋白(SA binding protein 2,SABP2,也称水杨酸受体)基因sabp2进行异源表达并测定其活性。方法:从拟南芥叶RNA中通过反转录PCR扩增sabp2,将PCR产物克隆至载体pMD-19T simple中,经测序验证后,再基于pET28...目的:从拟南芥叶中克隆水杨酸结合蛋白(SA binding protein 2,SABP2,也称水杨酸受体)基因sabp2进行异源表达并测定其活性。方法:从拟南芥叶RNA中通过反转录PCR扩增sabp2,将PCR产物克隆至载体pMD-19T simple中,经测序验证后,再基于pET28a构建重组表达载体,转化至大肠杆菌BL21(DE3)并表达,检测重组蛋白的活性。另一方面,对sabp2在拟南芥中转录水平进行了研究。结果:PCR获得792bp的sabp2基因,并成功构建异源表达载体pET28a-sabp2。优化结果表明,在0.4mmol/L IPTG诱导下20℃培养8h,表达产物活性较强,具天然SABP2的特征性酯酶活性。该基因在拟南芥叶中转录模式呈SA应激性和组织特异性。结论:sabp2成功表达,不仅为筛选SA受体拮抗剂提供新的原核体系,而且为探讨SA与SABP2相互作用在植物防御过程中时空变化奠定基础。展开更多
文摘【目的】克隆84K杨水杨酸结合蛋白2(Salicylic acid-binding protein 2,SABP2)基因并预测其功能。【方法】以生长至5片小叶的84K杨组培苗为材料,提取其叶和茎的总RNA。根据毛果杨SABP2基因(GenBank序列号:XM_002310718.2)的完整CDs序列,设计84K杨SABP2基因的引物,采用RT-PCR技术扩增84K杨SABP2基因全长,然后连接到pGM-T克隆载体,转化大肠杆菌TOP10感受态细胞,对84K杨SABP2基因进行克隆,获得该基因的全长序列。通过各种在线软件对84K杨SABP2基因及其编码的蛋白质进行生物信息学分析。【结果】84K杨基因的cDNA序列全长822bp,开放阅读框789bp,编码263个氨基酸。生物信息学分析表明,84K杨SABP2与毛白杨SABP2(GenBank序列号:JQ086570.1)的同源性最高,达98%;84K杨SABP2基因位于微体中;SABP2蛋白有11个蛋白质结合位点,属于α/β折叠水解酶家庭成员中的酯酶,为亲水性蛋白。【结论】成功克隆了84K杨的SABP2基因,其功能与前人对草本植物中SABP2部分功能的研究结果一致。
基金supported by the US National Science Foundation(MCB-1906060 and MCB-2145967 to Y.W.)。
文摘Aphids represent a major threat to crops.Hundreds of different viruses are aphid-borne.Upon aphid attack,plants release volatile organic compounds(VOCs)as airborne alarm signals to turn on the airborne defense(AD)of neighboring plants,thereby repelling aphids as well as reducing aphid fitness and virus transmission.This phenomenon provides a critical community-wide plant protection to fend off aphids,but the underlying molecular basis remains undetermined for a long time.In a recent article,Gong et al.established the NAC2-SAMT1 module as the core component regulating the emission of methyl-salicylate(MeSA),a major component of VOCs in aphid-attacked plants.Furthermore,they showed that SABP2 protein is critical for the perception of volatile MeSA signal by converting MeSA to Salicylic Acid(SA),which is the cue to elicit AD against aphids at the community level.Moreover,they showed that multiple viruses use a conserved glycine residue in the ATP-dependent helicase domain in viral proteins to shuttle NAC2 from the nucleus to the cytoplasm for degradation,leading to the attenuation of MeSA emission and AD.These findings illuminate the functional roles of key regulators in the complex MeSA-mediated airborne defense process and a counter-defense mechanism used by viruses,which has profound significance in advancing the knowledge of plant-pathogen interactions as well as providing potential targets for gene editing-based crop breeding.
