Salmonella pathogenesis and processing of secreted effectors by caspase-3

The enteric pathogen Salmonella enterica serovar Typhimurium causes food poisoning resulting in gastroenteritis. The S. Typhimurium effector Salmonella invasion protein A (SipA) promotes gastroenteritis by functional motifs that trigger either mechanisms of inflammation or bacterial entry. During infection of intestinal epithelial cells, SipA was found to be responsible for the early activation of caspase-3, an enzyme that is required for SipA cleavage at a specific recognition motif that divided the protein into its two functional domains and activated SipA in a manner necessary for pathogenicity. Other caspase-3 cleavage sites identified in S. Typhimurium appeared to be restricted to secreted effector proteins, which indicates that this may be a general strategy used by this pathogen for processing of its secreted effectors.     

两种黄单胞病菌诱导水稻一氧化氮产生和防卫基因表达的比较研究

 【目的】阐明在不同黄单胞病菌诱导的水稻过敏性细胞死亡(HCD)中的一氧化氮(NO)信号途径及其作用机制。【方法】比较分析了在水稻细胞－番茄斑点病菌(Xanthomonas campestris pv. vesicatoria, Xcv)不亲和互作、水稻－白叶枯病菌(X. oryzae pv. oryzae, Xoo)亲和互作中NO的发生以及NO对水稻防卫基因诱导表达的影响。【结果】Xcv不仅诱导了NO的迸发、NOS活性及其NO合成相关基因(nos和nr)的表达，而且诱导了防卫基因(苯丙氨酸解氨酶基因pal、过氧化物酶基因pox和谷胱苷肽转硫酶基因gst)的表达；Xoo不诱导NO的迸发，抑制了NOS的活性、NO合成相关基因和防卫基因的表达；NO清除剂PTIO和Xoo显著地抑制了Xcv 对防卫基因表达的诱导作用。【结论】Xcv通过NO信号途径诱导了水稻防卫基因的表达和HCD；Xoo 通过NO抑制或解毒机制抑制了由NO介导的防卫基因的表达及其HCD的发生。     

杜仲EuCHIT1基因表达载体构建及在大肠杆菌中的表达

构建了T7启动子驱动杜仲几丁质酶基因EuCHIT1的原核表达载体pET-EuCHIT1和pMCSG-EuCHIT1,其表达产物分别含6个组氨酸(6His)标签和6个组氨酸连接的麦芽糖结合蛋白(his6-tag–maltose-binding protein,MBP)标签,分别将重组载体遗传转化大肠杆菌细胞BL21(DE3),在37℃、150 rpm条件下培养至菌液OD值为0.4~0.8后,转到16℃、150 rpm条件下以1 m MIPTG诱导培养12 h,表达产物经SDS-PAGE分析,p ET-EuCHIT1/BL21(DE3)表达以包涵体形式存在36.03 kD融合蛋白,pMCSG-EuCHIT1/BL21(DE3)成功表达可溶形式存在的77.21 kD融合蛋白。故可以使用pMCSG-EuCHIT1/BL21(DE3)获得可溶的融合蛋白,为之后的EuCHIT1的多克隆抗体的制备和及功能研究奠定基础。     

T.Spelta 1Bs染色体育性基因Rf3和rfk1的分子标记

为了建立T.Spelta1Bs染色体育性基因Rf3和rfk1的分子标记辅助选育技术体系,提高选育效率,对T.Spelta1Bs染色体上育性基因Rf3和rfk1进行AFLP分子标记研究。结果表明,利用AFLP技术对小麦进行分子标记研究可获得50～100条清晰、稳定的带纹,多态性较高,重复性好。根据T型细胞质的育性,对TSP3314/绵阳26的F2群体采用集群分类的方法构建等基因池,利用AFLP技术筛选16个Pst /Taq 引物组合,然后对F2群体进行AFLP分析,获得2个引物组合P3-T2,P4-T3,其与T.Spelta1Bs染色体有关育性片段Rf3基因和rfk1基因连锁。然后结合TSP3314/绵阳26的F2群体在T型细胞质下的育性结果,和可育株与K3315A测交后代的育性分离所得的K型细胞质下的育性结果,运用Mapmaker软件进行分析,找到了与T.Spelta1Bs染色体育性基因Rf3和rfk1连锁的分子标记。     

NBS-LRR Proteins and Their Partners:Molecular Switches of Plant Defense

Specificity of the plant innate immune system is often conferred by resistance（R）proteins.Most plant disease resistance （R）proteins contain a series of leucine-rich repeats（LRRs）,a nucleotide-binding site（NBS）,and a putative amino-terminal signaling domain.They are termed NBS-LRR proteins.The LRRs are mainly involved in recognition,and the amino-terminal domain determines signaling specificity,whereas the NBS domain presumably functions as a molecular switch.During the past years,the most important discoveries are the role of partners in NBS-LRR gene mediated defenses,mounting support for the so-called＂guard hypothesis＂of R gene function,and providing evidence for intramolecular interactions and intermolecular interactions within NBS- LRR proteins as a mode of signaling regulation.The outcome of these interactions determines whether a plant activates its defense responses.     

Identification of small clusters of divergent amino acids that mediate the opposing effects of ras and Krev-1

Krev-1 is an anti-oncogene that was originally identified by its ability to induce morphologic reversion of ras-transformed cells that continue to express the ras gene. The Krev-1-encoded protein is structurally related to Ras proteins. The biological activities of a series of ras-Krev-1 chimeras were studied to test the hypothesis that Krev-1 may directly interfere with a ras function. The ras-specific and Krev-1-specific amino acids immediately surrounding residues 32 to 44, which are identical between the two proteins, determined whether the protein induced cellular transformation or suppressed ras transformation. Because this region in Ras proteins has been implicated in effector function, the results suggest that Krev-1 suppresses ras-induced transformation by interfering with interaction of Ras with its effector.     

Oleosin与GUS融合蛋白基因的植物表达载体的构建及融合蛋白性质的预测

将油菜油脂蛋白Oleosin基因与报告基因GUS通过6个氨基酸编码的凝血酶识别位点(Gly-Val-Arg-Gly-Pro-Arg)连接起来,形成一个融合蛋白。其中编码区基因长2415bp,共编码804个氨基酸和一个终止密码子。使用蛋白质分析软件Antheprot.5.0和DNAstar对融合蛋白的理化特性,二级结构,潜在信号肽断裂位点,跨膜区进行分析表明:融合蛋白较好的保持了原来两个蛋白的理化特性和二级结构。将该融合基因克隆到中间载体pUC-121上,并用棉花LEA蛋白D-113基因启动子替换CaMV35S启动子,构建成植物表达载体pBI-LEA-O::G121。     

小麦条锈菌效应蛋白基因pst-2511的克隆、 表达和初步结构研究

对小麦条锈菌（Puccinia striiformis f.sp. tritici）效应蛋白pst-2511进行研究,分析其可能形成二级结构的条件,为以后通过核磁方法解析该效应蛋白的三维结构提供基础。构建了小麦条锈菌效应蛋白基因pst-2511的原核表达载体pET-32a-PP-pst-2511,并成功将其在大肠杆菌 BL21(DE3)中进行原核表达;表达出的蛋白用亲和层析法和高效液相色谱法进行纯化,获得可溶性的效应蛋白,用凝胶电泳检测得到单一条带,经质谱鉴定,纯化后的蛋白质分子量与理论值符合。圆二色谱表征该蛋白质的二级结构有α螺旋,且随着三氟乙醇（TFE）浓度的增加,α螺旋比例增加。对效应蛋白pst-2511的初步结构研究及三维结构解析,可以为防止小麦条锈病提供理论依据,同时为深入研究效应蛋白的调控通路以及阐明条锈菌侵染机制和更有效地防止小麦条锈病提供帮助。     

参与植物防御反应的LRR型蛋白结构与功能

植物含有多种富含亮氨酸重复（LRRs）结构的蛋白质,它们在植物生长、发育和抗病反应等方面发挥着重要作用。综述了这类具有LRRs结构蛋白质家族的结构特征及其参与植物防御反应的功能。参与植物防御反应LRR型蛋白质家族包括：抗病基因编码蛋白质、类受体蛋白激酶、多聚半乳糖醛酸酶抑制蛋白和伸展蛋白家族。这四大蛋白质家族成员主要通过LRRs结构识别并结合病原物蛋白质,参与抗病信号传递,诱导植物防卫基因的表达,使植物获得系统抗性。其中LRRs序列中氨基酸的不同和单位重复数目的差异决定了蛋白识别的特异性和结合能力。     

尼罗罗非鱼P2X4R基因克隆及原核表达分析

[目的]克隆尼罗罗非鱼P2X4R基因,并构建原核表达载体进行诱导表达,为深入研究P2X4R在鱼类中的生物学功能打下基础.[方法]利用PCR克隆尼罗罗非鱼P2X4R基因的3个片段(G1、G2和G3),拼接获得目的基因后连接pCold II载体构建pCold II-P2X4R重组质粒,再转化大肠杆菌BL21(DE3)感受态细胞,以IPTG进行诱导表达.分别采用SDS-PAGE和Western blotting检测分析重组蛋白P2X4R的表达情况,并运用生物信息学在线分析软件对其理化性质、糖基化位点、跨膜区域、亚细胞定位及信号肽等进行预测分析.[结果]克隆获得的尼罗罗非鱼P2X4R基因大小为1108 bp,与pCold II载体重组后转化BL21(DE3)感受态细胞获得的原核表达载体经IPTG诱导可表达获得目的蛋白,在IPTG 1.0 mmol/L、37℃诱导4 h的条件下重组蛋白表达量高于在IPTG 0.1 mmol/L、16℃过夜诱导的表达量.重组蛋白P2X4R的分子量约43.0 kD,其氨基酸数量为354个,理论等电点(pI)为6.78,不稳定指数为37.62,属于稳定蛋白,脂肪族指数为74.35;重组蛋白P2X4R具有3个N-糖基化位点和1个O-糖基位点;该蛋白未见跨膜区,其蛋白几乎100%位于细胞膜内,不含信号肽.[结论]诱导表达获得的尼罗罗非鱼P2X4R蛋白具有3个N-糖基化位点和1个O-糖基化位点,推测其存在糖基化现象,可制备相应抗体用于揭示罗非鱼巨噬细胞的抗原呈递作用机制.     

非洲猪瘟病毒(Malawi LIL 20/1株)k8R基因的表达与鉴定

蛋白多肽二级结构的电脑预测表明,非洲猪瘟病毒（ＭａｌａｗｉＬＩＬ２０／１株）ｋ８Ｒ基因编码带有多个疏水氨基酸小区的２７ｋＤａ蛋白质。该基因的ＰＣＲ产物克隆入质粒ｐＧＥＸ－２Ｔ后,在大肠杆菌中表达４２～５４ｋＤａ不溶性ＧＳＴ－ｋ８Ｒ融合蛋白。此表达蛋白能被针对不同非洲猪瘟病毒株的猪免疫血清识别。在非洲猪瘟病毒细胞适应株感染的细胞中,针对ｋ８Ｒ大肠杆菌表达产物的单抗能检测出２７ｋＤａ特异病毒蛋白。进一步鉴定表明,ｋ８Ｒ基因编码的蛋白质为病毒非结构蛋白,不发生糖基化,出现于病毒感染周期的晚期,主要集中于靠近细胞核的病毒复制部位。用大肠杆菌表达的ＧＳＴ－ｋ８Ｒ融合蛋白免疫猪未能抵抗ＭａｌａｗｉＬＩＬ２０／１强毒株攻击。     

Recognition of histone H3 lysine-4 methylation by the double tudor domain of JMJD2A

Biological responses to histone methylation critically depend on the faithful readout and transduction of the methyl-lysine signal by "effector" proteins, yet our understanding of methyl-lysine recognition has so far been limited to the study of histone binding by chromodomain and WD40-repeat proteins. The double tudor domain of JMJD2A, a Jmjc domain-containing histone demethylase, binds methylated histone H3-K4 and H4-K20. We found that the double tudor domain has an interdigitated structure, and the unusual fold is required for its ability to bind methylated histone tails. The cocrystal structure of the JMJD2A double tudor domain with a trimethylated H3-K4 peptide reveals that the trimethyl-K4 is bound in a cage of three aromatic residues, two of which are from the tudor-2 motif, whereas the binding specificity is determined by side-chain interactions involving amino acids from the tudor-1 motif. Our study provides mechanistic insights into recognition of methylated histone tails by tudor domains and reveals the structural intricacy of methyl-lysine recognition by two closely spaced effector domains.     

水稻白叶枯病菌hrpF启动子序列调控的绿色荧光蛋白基因表达体系的构建

为了阐明启动子序列对水稻白叶枯病菌效应子基因表达的调控作用及特征,通过融合PCR将报告基因GFP基因置于水稻白叶枯病菌hrpF基因启动子序列的下游,经酶切、PCR鉴定及序列测定,成功构建了受hrpF启动子序列调控的GFP基因转录单元pMF-GFP,并转入JXO I菌株中。结果发现构建的hrpF：：GFP转录单元,在hrp诱导培养基XOM2上可有效诱导表达,而在NA培养基上不能诱导表达。结果还显示,在大肠杆菌中,中间表达载体phrpF：：GFP具有GFP表达活性。