【作者】 李佳；

【导师】 杜林方；

【作者基本信息】 四川大学 ， 生物化学与分子生物学， 2007， 博士

【摘要】 病程相关蛋白（PR蛋白）是由宿主编码的具有种属特异性的植物抗逆蛋白，通常因病毒、真菌或细菌等病原侵染或生理因素、化学因素和物理因素而被诱导表达，它们被认为在防御病原物和抵抗不利环境中具有重要作用。烟草酸性PR-1a蛋白是第一个被纯化和描述的PR蛋白，被作为PR-1蛋白的代表性成员。PR-1蛋白家族是PR蛋白中非常重要的一个家族，通常作为植物系统获得性抗性的标志，但其明确功能和作用机制尚不清楚。为了建立一个高效的PR-1a蛋白原核表达系统及纯化方法来获得重组PR-1a蛋白，本研究将烟草成熟PR-1a蛋白编码区克隆到大肠杆菌表达载体pGEX-5X-3中，使其与谷胱甘肽S-转移酶（Glutathione S-transferase，GST）融合表达（GST-PR1a）。采用设计的两端分别引入BamH1和XhoI酶切位点的引物，以带有烟草PR-1a基因的克隆载体pBluescript SK为模板，成功扩增了烟草成熟PR-1a蛋白的编码序列cDNA（0.43kb）。TA连接，筛选阳性重组子经BamH1和XhoI双酶切后，与同样经BamH1和XhoI双酶切的pGEX-5X-3载体相连并转化E.coli JM109。经双酶切和DNA双向测序，证明成功构建了pGEX-PR1a表达载体。首次将烟草PR-1a蛋白在异源系统中重组表达。选择表达量最高的单菌落为后续实验的表达菌种，优化诱导条件为IPTG终浓度1.0mM，29℃诱导表达4h。GST-PR1a融合蛋白主要存在于上清中，溶解性较好，可能高表达、易溶的谷胱甘肽S-转移酶促进了烟草PR-1a的表达及溶解。诱导表达菌体经超声波破菌，离心，回收上清。由于GST-PR1a融合蛋白含有GST标签，可以通过Glutathione-Sepharose 4B亲和介质层析一步纯化，得到分子量为42 kDa电泳纯的融合蛋白。1L发酵液可获得大约6mg纯化重组蛋白，收率为1.47％。重组蛋白可作为配体用于结合实验和亲和层析，以证明并分离PR-1a蛋白的受体，来研究PR-1a蛋白与其它蛋白质之间的相互作用。用Factor Xa酶切融合蛋白去除GST标签，通过Xarrest^TM琼脂糖将Factor Xa移除，纯化得到分子量为15 kDa的PR-1a蛋白。PR-1a是发现最早的PR蛋白及PR-1家族的代表性成员，但其三维结构以及活性位点的相关信息仍未见报道，而蛋白质的三维结构在很大程度上决定了蛋白质的功能。本研究利用同源模建和分子动力学模拟方法搭建了烟草病程相关蛋白PR-1a的三维结构，以PR-1a序列为探针，搜索得到了同源性为58.7％的病程相关蛋白P14a，由Modeler自动模建程序构建得到的初始模型，经过能量最小化和分子动力学模拟后，得到了PR-1a的最佳构象，选择500皮秒时的构象作为最终构象。优化后得到的三维结构模型用Profile—3D进行了氨基酸残基的兼容性考察，Prostat用来检查所模拟的结构的几何特征，检验结果表明所得理论模型是合理可信的。利用PR-1蛋白家族中一段高度保守的序列GHYTQVVW，使用Binding-Site模块在此基础上分析预测出2个可能的活性位点。从序列分析和几何形状角度分析，推断出位点1（分别是Leu44，His46，His48，Trp72，Asn84，Cys86，His94，Thr96，Gln97，Trp100，Asn129，Tyr130，Pro135，Tyr136）是比位点2更可能的活性位点。获得PR-1a的三维结构信息对深入探讨PR-1a的抗病作用是很有意义的，对病程相关蛋白家族催化机理的深入研究提供了重要的参考信息。对GST-PR1a蛋白和切标签后得到的PR-1a蛋白随温度变化（20℃～100℃保温30min）的荧光发射光谱进行了研究，结果表明，GST-PR1a和切标签后得到的PR-1a分别在20℃～90℃和20℃～60℃处理时的荧光强度与天然状态时相比变化较小，说明分别在以上温度范围中GST-PR1a和PR-1a的构象与天然状态时比较接近。证明PR-1a蛋白稳定性较好，能够抵抗60℃的高温，而且GST标签的加入使稳定性进一步提高。用纯化的GST-PR1a蛋白免疫新西兰大白兔，获得了对GST蛋白和GST-PR1a蛋白效价分别为1：64和1：32的兔抗GST-PR1a抗血清。免疫双扩散和western免疫印迹分析均说明制备得到的pGEX-PR1a抗血清具有较好的特异性。制得的兔抗GST-PR1a抗血清不仅能与GST-PR1a和GST蛋白进行免疫印迹，还能和从天然烟草提取的酸性病程相关蛋白PR-1a、-1b、-1c进行免疫印迹。说明重组GST-PR1a融合蛋白与天然烟草酸性PR-1蛋白（PR-1a、-1b和-1c）具有一致的免疫化学特性。本研究所获得的GST-PR1a融合蛋白和相应抗血清可用于以后GST pull-down分析来探测PR-1a蛋白与其靶蛋白之间的相互作用，为了解PR-1a与其它蛋白质的相互关系提供线索。对GST-PR1a融合蛋白和切标签后得到的PR-1a蛋白进行了体外抗真菌活性初步研究。体外实验结果表明GST-PR1a和切标签后得到的PR-1a蛋白都对烟草疫霉菌丝体的生长没有抑制作用。通过对马铃薯致病疫霉孢子萌发的抑制实验，证明所得到的重组蛋白与天然烟草PR-1蛋白有一致的体外抑菌活性。当GST-PR1a蛋白浓度为600μg／mL时，孢子萌发部分被抑制，芽管长度也有所减短。当切去GST标签即PR-1a浓度在250μg／mL时就几乎能完全阻止孢子的萌发。根据实验数据绘制了GST-PR1a蛋白和切去标签后即PR-1a蛋白对马铃薯致病疫霉的孢子萌发抑制率—蛋白质浓度曲线。PR-1a蛋白的IC₅₀和IC₉₀值分别是100μg／mL和250μg／mL。GST-PR1a融合蛋白对马铃薯疫霉孢子萌发的抑制活性比切去标签后得到的PR-1a蛋白低的多，当GST-PR1a蛋白浓度为600μg／mL时，对孢子萌发抑制率还不到50％。这说明融合蛋白中的GST-tag可能对PR-1a的抗真菌活性有干扰。然而PR-1a蛋白的N-末端有GST-tag时仍然对马铃薯致病疫霉有弱的抑制作用，同时也说明在其抗真菌机制中，PR-1a的N-末端游离并不是必须的。更多还原

【Abstract】 A group of plant-coded proteins induced by different stress stimuliincluding viruses, fungi, bacteria, physiological, chemical or physicalfactor, named "pathogenesis-related proteins"（PRs） is assigned animportant role in plant defense against pathogenic constraints and ingeneral adaptation to stressful environment. Tobacco PR-1a was the firstpurified and characterized pathogenesis-related protein and was regardedas the type member of PR-1 family.In order to establish an effective method of procaryotic expressionand purification for PR-1a protein, coding sequence of tobacco maturePR-1a was sub-cloned into Escherichia coli expressing vectorpGEX-5X-3 for Glutathione S-transferase-fusing expression. Using thedesigned forward and reverse primers containing BamH1 and XhoI site,respectively, cDNA（0.43kb） for coding tobacco mature PR-1a proteinwas amplified successfully by PCR, with pBluescript SK vectorcontaining tobacco PR-1a gene as the template. Then PCR productionswere directly inserted into T/A cloning vector and the recombinants weredigested with restriction enzymes BamH1 and XhoI. The PR-1a fragmentwas ligated with pGEX-5X-3 which was digested with the same enzymes,and then transformed E.coli JM109. The construction of pGEX-PR1a expressing vector was validated by double enzyme digestion andbidirectional DNA sequencing, and tobacco PR-1a proteins were expressedin heterologous systems for the first time.The clone with the highest expression was chose for subsequentexperiments. The optimized condition for induction was at IPTG finalconcentration 1.0 mmol/L, 29℃for 4 h. A majority of fusion protein,named GST-PR1a was soluble perhaps owing to Glutathione S-transferasetag.Induced cells were homogenized by sonic disruption and then thesupernatant was collected by centrifugation. Because fusion proteincontaining GST-tag, GST-PR1a（42 kDa） was isolated and purified byGlutathione Sepharose 4 Fast Flow affinity column. The yield was about6mg of recombinant proteins/1L of bacterial culture. The N-terminalGST moiety was removed from the recombinant protein by Factor Xa,then Factor Xa was captured by agarose Xarrest^TM. At last, purifiedPR-1a protein（15 kDa）was acquired.Although PR-1a was the first discovered PR protein and the type ofPR-1 family, its three dimensional structure and active site had not beenreported. The 3D-structure of the protein determines its function to ahigh degree. The 3D-structure of tobacco PR-1a was modeled by usinghomology and molecular dynamics methods. P14a protein was searchedout by probing with tobacco PR-1a coding sequence and percentagesimilarity to tobacco PR-1a was 58.7％. Initial model was constructed byauto-modeling program named Modeler, then optimum conformation oftobacco PR-1a was acquired after energy-minimum and moleculardynamics simulation. The conformation at 500ps was chose to be thefinal conformation. Compatibility of amino acid residues was reviewedby Profile—3D program, and geometry characteristic of the model wasexamined by Prostat program. Results showed that the 3D-structure weconstructed was reasonable and creditable.On the base of the modeling and a highly conserved sequence GHYTQVVW in PR-1 families, two possible active sites were predictedby Binding-Site module. Analyses from sequences and geometric figureand angle showed that Site 1 which comprised Leu44, His46, His48,Trp72, Asn84, Cys86, His94, Thr96, Gln97, Trp100, Asn129, Tyr130,Pro135, Tyr136 was more likely to be the active site than Site 2.Purified GST-PR1a and PR-1a proteins were incubated at 20℃～100℃for 30min, fluorescence spectra indicate that the fluorescenceintensity of GST-PR1a at 20℃～90℃and PR-1a at 20℃～60℃hadsmall changes. Results showed that the conformations of GST-PR1a andPR-1a were similar to natural state at above temperature range. PR-1aprotein was stable under 60℃and GST-tag improved the stability of thefusion protein.The purified GST-PR1a was used to immunize New Zealand rabbitsand the polyclonal antibody was obtained, whose titer against GST andGST-PR1a were 1:64 and 1:32 respectively. Double-immunodiffusionand western blotting both confirmed the preparation of anti-rabbitGST-PR1a polyclonal antibody of high specificity. The anti-rabbitGST-PR1a polyclonal antibody we obtained can immuno-react not onlywith recombinant GST-PR1a protein, but also with native tobacco PR-1a,-1b and-1c. We can conclude that the recombinant GsT-PR1a protein isof identical immunochemical properties with native tobacco acid PR-1.Studies on antifungal activities of recombinant GST-PR1a proteinsbefore and after removement of GST-tag were carried out. The results invitro showed that neither GST-PR1a nor cleaved PR-1a could inhibitmycelium growth of Phytophthora nicotianae. The inhibitory tests ofPhytophthora infestans zoospore germination in vitro revealed thatrecombinant proteins had consistent antifungal activities with naturetobacco PR-1. The germination of the zoospores and germ-tube lengthwere reduced in the presence of GST-PR1a at a concentration of 600μg/ml, and the germination of the zoospores was almost stoppedcompletely in the presence of cleaved PR-1a at a concentration of 250μ g/ml. Dose-response curve of inhibition of Phytophthora infestanszoospore germination by cleaved PR-1a proteins showed that IC₅₀ andIC₉₀ of cleaved PR-1a were 100μg/ml and 250μg/ml respectively. It wasnoted that GST-PR1a protein had much more lower inhibition againstPhytophthora infestans than cleaved PR-1a protein. When theconcentration of GST-PR1a protein reached up to 600μg/ml, it causedno more than 50％growth inhibition. These results suggested that theGST-tag in recombinant protein probably interfere in antifungal activity.However, the effectiveness of PR-1a With N-terminal GST-tag ininhibiting P. infestans suggests that the free N-terminus of PR-1a is notnecessary in the antifungal mechanism.The fusion GST-PR1a protein and the corresponding antiserum weobtained could be used for GST pull-down assay to detect interaction ofPR-1a with its targetsin further studies, in order to provide some cluesfor finding out the relationships of PR-1a and other proteins. Moreover,the recombinant proteins will be used as ligand for binding studies andaffinity chromatography to identify and isolate the PR-1 receptors.Finally, the three dimensional structure and the possible active sites oftobacco PR-1a offer very significant and important information forprobing into the antifungal mechanism further.更多还原