西瓜食酸菌RND蛋白家族外排转运体cusB基因抗铜功能研究

【目的】研究RND外排泵中cus B基因突变对西瓜食酸菌抗铜性的影响。【方法】采用Tn5转座子随机插入基因组制备筛选得到突变体,通过双亲杂交的方法构建功能互补菌株,并从西瓜食酸菌抗铜性、胞外纤维素酶和胞外蛋白酶分泌、胞外多糖产生、生物膜形成、致病性及过敏性反应等方面阐明RND外排泵中MFP蛋白亚基对西瓜食酸菌的影响。【结果】突变体Δcus B在含有1.25 mmol/L或2.5 mmol/L Cu SO4的KMB平板上不能生长,cus B基因的突变导致西瓜食酸菌的胞外多糖分泌和生物膜形成与野生型有差异,但不影响胞外纤维素酶、胞外蛋白酶、致病性及过敏性反应。【结论】RND外排泵相关基因cus B的突变会影响西瓜食酸菌的某些生物学特性,并导致病菌对铜十分敏感。研究以RND外排泵转运重金属为导向初步解析了西瓜食酸菌的抗铜机制。     

西瓜食酸菌与黄瓜互作转录组分析

【背景】细菌性果斑病是一种严重的种传细菌病害,其病原菌为西瓜食酸菌。截至目前对该病病原菌与寄主的互作机制认识极为有限。葫芦科的模式植物黄瓜易被西瓜食酸菌侵染发病,对西瓜食酸菌-黄瓜互作体系进行转录组分析,可以为探究西瓜食酸菌与寄主互作机制奠定重要基础。【目的】解析西瓜食酸菌-黄瓜互作时的相互响应规律。【方法】以细菌悬液注射接种6d黄瓜子叶,处理48 h的子叶作为转录组测序样本。利用RNA-Seq技术分析西瓜食酸菌FC440菌株与黄瓜9930品种互作时基因的表达特征。【结果】测序数据质量分析发现,各样品不同重复间相关性较强,与参考基因组比对率达95%以上,聚类分析发现对照组与处理组表达模式相反,样品处理达到一定效果,表明数据整体质量较高。选取6个差异表达基因进行RT-qPCR验证,结果显示6个基因的表达模式与转录组结果基本一致,表明转录组测序结果比较可靠。西瓜食酸菌和黄瓜互作48 h后,在转录组水平分别检测到1 618个和8 698个差异表达基因。Gene Ontology (GO)功能注释显示,细菌的差异基因显著富集在细胞组分中的细胞膜(37.5%)和膜部分(27.0%),生物过程中的氧化还原过程(66.7%)以及分子功能中的水解酶活性(66.5%);黄瓜的差异基因显著富集在细胞组分中的质体(22.2%)和叶绿体(21.3%),分子功能中的催化活性(70.0%)以及生物过程中的碳水化合物衍生物代谢(32.2%)。Kyoto Encyclopedia of Genes and Genomes (KEGG)分析显示,细菌中致病相关基因显著富集在群体感应及细菌趋化性途径,而且群体感应系统基因下调更显著。黄瓜中调控钙依赖蛋白激酶(Calcium-Dependent Protein Kinase,CDPK)、钙调素和类钙调素(Calmodulin and Calmodulin-Like,CaMCML)及呼吸氧暴发激酶(Respiratory Burst Oxidase Homologne,Rboh)的基因总体上调,调控苯丙氨酸裂解酶(Phenylalanine Ammonia-Lyase,PAL)的基因和谷胱甘肽S-转移酶(Glutathione S-Transferase,GST)的基因在相应代谢途径中数量最多且上调程度明显。【结论】获得较高质量的西瓜食酸菌与黄瓜互作的转录组测序结果。群体感应与西瓜食酸菌FC440菌株致病力密切相关;寄主黄瓜应对西瓜食酸菌侵染以Ca~(2+)信号激活的防御反应为主。PAL和GST在黄瓜抵抗西瓜食酸菌侵染中发挥重要作用。本研究为进一步深入解析西瓜食酸菌与寄主互作的机制奠定了基础。     

西瓜食酸菌Ⅲ型效应蛋白AopAI的鉴定及其功能初步分析

【背景】西瓜食酸菌（Acidovorax citrulli,Ac）引起的细菌性果斑病是葫芦科植物重要的病害之一,通过Ⅲ型分泌系统（type Ⅲ secreted system,T3SS）分泌至植物体内的Ⅲ型效应蛋白（type Ⅲ effector,T3E）是该菌重要的致病因子,目前对Ac T3E的认识仍然非常有限。【目的】鉴定西瓜食酸菌候选的T3E Acidovorax outer protein AI （AopAI）,分析其对Ac致病力的影响和干扰植物免疫的方式。【方法】利用生物信息学方法分析AopAI序列特征、AvrBs1无毒报告系统验证蛋白转运功能;通过荧光定量PCR技术分析aopAI基因表达的调控及其对植物病原相关分子模式（pathogen-associated molecular pattern,PAMP）激发的免疫反应（PAMP-triggered immunity,PTI）信号通路标记基因表达的影响;利用基因插入突变和基因功能互补方法,检测菌的致病力、植物组织过氧化氢和胼胝质积累量的变化;运用瞬时表达技术分析AopAI亚细胞定位和其抑制激发子诱导细胞死亡的能力。【结果】AopAI蛋白序列中不含跨膜螺旋区和信号肽,含有二磷酸腺苷（adenosine diphosphate,ADP）核糖基转移酶保守结构域;在T3SS核心基因hrpG和hrpX突变体中aopAI基因表达量显著降低;表达AopAI及AvrBs1功能区（59-445 aa）的avrBs1突变体可诱导ECW-10R辣椒叶发生过敏性坏死反应,表明AopAI具有转运功能;aopAI基因突变体在黄瓜子叶上的致病力减弱,但与其互作的黄瓜子叶组织中过氧化氢和胼胝质的含量均显著增加;AopAI在本氏烟叶瞬时表达后,显示其定位于细胞膜和细胞核,还表现抑制激发子NIP诱导的叶细胞死亡,导致叶细胞的PTI信号通路标记基因GRAS2和ACRE31的表达量显著降低。【结论】在西瓜食酸菌中具有一个定位于细胞核和细胞膜、有ADP核糖基转移酶结构域的T3E蛋白AopAI,该T3E是能够抑制NIP诱导的细胞死亡的毒性蛋白,通过抑制ACRE31调节的免疫途径降低植物过氧化氢和胼胝质的积累,以抑制植物PTI防御反应机制。     

西瓜食酸菌Ⅲ型分泌效应物基因aopW功能初步分析

【背景】细菌性果斑病(bacterial fruit blotch,BFB)是葫芦科植物上一种严重的检疫性病害,其病原菌为西瓜食酸菌(Acidovorax citrulli)。目前已知Ⅲ型分泌效应物(typeⅢsecreted effectors,T3SEs)是该病菌的关键致病因子,但对其效应物功能和作用机制的认识非常有限。【目的】鉴定西瓜食酸菌Ⅲ型分泌效应物基因aopW,分析其编码蛋白质影响植物免疫的方式,为更深入地认识该基因在西瓜食酸菌致病机制中的作用奠定基础。【方法】利用生物信息学分析其序列特征;借助荧光定量PCR技术分析aopW的表达调控及其与抗病相关基因表达间的关系;利用基因突变及基因功能互补手段,通过分析致病性、寄主活性氧积累量等解析基因功能;采用瞬时表达技术了解AopW诱导非寄主hypersensitive response (HR)能力及其亚细胞定位情况。【结果】aopW基因启动子区存在Ⅲ型分泌系统(type Ⅲ secretion system,T3SS)核心基因结合位点,其编码的蛋白不存在信号肽和跨膜螺旋区,与Ⅲ型分泌效应物harpin蛋白同源;T3SS核心基因hr...     

西瓜食酸菌Ⅲ型效应蛋白AopBF1的鉴定与功能分析

【背景】细菌性果斑病（bacterial fruit blotch,BFB）是一种发生在葫芦科作物上的检疫性病害,其病原菌为西瓜食酸菌（Acidovorax citrulli）,西瓜食酸菌通过Ⅲ型分泌系统（type Ⅲ secretion system,T3SS）将重要的致病因子Ⅲ型效应蛋白（type Ⅲ effector,T3E）转运到植物体内,从而致病。目前,对于T3E致病机制的认识非常有限。课题组前期已鉴定到西瓜食酸菌FC440菌株中的一些候选T3E。【目的】明确西瓜食酸菌FC440菌株候选T3E中AopBF1的序列特征、转运特性及其在病原菌致病过程中的作用,可以为深入解析病原菌致病机制奠定理论基础。【方法】利用生物信息学手段,预测分析AopBF1的T3E序列特征;通过RT-qPCR、无毒蛋白报告系统检测AopBF1所受调控及其转运特性;观察aopBF1突变体（插入突变）及过表达时西瓜食酸菌的致病力表型,分析AopBF1对西瓜食酸菌致病性的贡献。【结果】AopBF1具有T3E的序列特征、不含保守结构域,具有蛋白激酶序列特征;AopBF1在T3SS核心调控基因hrpX、hrpG突变株中的表达量显著降低;当aopBF1基因与AvrBs1功能区（59-445 aa）片段同时于avrBs1突变株中表达时,能够诱发含Bs1蛋白的ECW-10R辣椒叶片发生过敏性坏死反应;aopBF1突变株对寄主黄瓜的致病力显著减弱,而同时黄瓜组织中过氧化氢、超氧阴离子自由基及胼胝质的积累量表现为显著增加;AopBF1过表达菌株对寄主的致病力显著增强,其在诱导本氏烟的hypersensitive response （HR）反应发生上表现延迟;AopBF1瞬时表达时,显示定位于本氏烟细胞的细胞质、细胞核和细胞膜,可诱发本氏烟发生HR反应,促进PAMP-triggered immunity （PTI）及激素通路相关基因的表达。【结论】AopBF1是西瓜食酸菌的一个具有蛋白激酶特征的T3E,抑制寄主活性氧和胼胝质积累等PTI免疫反应以促进西瓜食酸菌的致病,激发含R抗性蛋白的本氏烟的PTI和激素相关抗病免疫反应。     

橡胶草TkCPT1基因的生物信息学分析及功能验证

TkCPT1基因是橡胶草(Taraxacum kok-saghyz)合成顺式橡胶过程中的一个重要基因.本实验运用生物信息学手段对橡胶草中顺式异戊烯基转移酶(cis isoprene transferase,CPT)进行一系列特征分析,并利用农杆菌介导的遗传转化法,通过超量表达TkCPT1基因遗传转化烟草,获得转基因烟草...     

敏捷食酸菌腈水解酶基因在大肠杆菌中的表达

以含有敏捷食酸菌的腈水解酶基因的克隆质粒为模板,通过PCR扩增获得长度为1 080 bp的腈水解酶(Nitrilase)基因片段。使用表达质粒p ET-28-a构建表达载体,获得重组质粒p ET-Nit。对所表达的基因进行测序对比发现与Gen Bank所公布的基因序列比较,相似性99%,读码框出现2 bp的突变,但并未引起相应氨基酸突变,故不影响酶的表达与特性。将重组质粒转化到表达宿主Escherichia coli Rosetta(DE3)感受态中,使用诱导剂IPTG对菌株进行诱导表达,获取菌液进行SDS-PAGE分析,得知目的蛋白分子量约为41.28 k D,与预期所想的一致。酶活力分析表明,上清的比活力为3 U/mg。进一步对诱导条件进行优化,包括诱导温度,IPTG浓度,诱导时间等一系列条件。在最优条件下,扩大培养体积,比活力可达15 U/mg,活力提高了5倍左右。     

基于生物信息学分析的肝癌核心基因的筛选及验证

本研究基于GEO数据库,选取由慢性乙型肝炎诱导的肝细胞癌芯片数据GSE121248为研究对象,利用GEO2R软件分析数据,筛选出差异表达基因,利用DAVID数据库进行GO分析和KEGG pathway富集分析.利用STRING数据库构建PPI网络,分析筛选核心基因.利用GEPIA对核心基因的表达进行验证,Kaplan ...     

水稻纹枯病菌Rspg1基因的克隆、表达及其编码产物生物信息学分析

【目的】为克隆、表达水稻纹枯病菌Rspg1基因,明确其编码产物的生物学特性,为研究该基因在病菌致病过程及与寄主互作中的作用提供理论依据。【方法】据GenBank提供的相关序列设计特异引物扩增目的基因,并对之进行原核表达和生物信息学分析。【结果】从水稻纹枯病菌基因组DNA中扩增出一1395 bp的目的片段。RT-PCR分析表明,该片段为Rspg1基因的完整开放阅读框,含有5个内含子(278-334,57 bp;545-601,57 bp;657-715,59 bp;1090-1155,66 bp;1244-1304,61 bp),编码区为1 095 bp,编码一含有364个氨基酸的蛋白。原核表达Rspg1基因,表达产物大小约为40 kDa,具明显的多聚半乳糖醛酸酶(Polygalacturonase,PG)活性,活性值为277.78 U/mg。生物信息学分析表明,RsPG1中含有所有生物PG特有的严格保计序列180NTD、202DD、223GHG和255RIK,存在一18个氨基酸的信号肽分子;二级结构以α-螺旋、β-折叠和随机卷曲为其基本结构单元,6个半胱氨酸残基形成3个二硫键,跨膜结构预测以从胞内向胞外分泌为主;三级结构为10个重复的β-折叠环按右手螺旋规则排列形成的螺旋结构,形成一个开放的有活性的裂隙结构。【结论】Rspg1基因编码产物为一40 kDa蛋白,具明显的PG(Polygalacturonase,PG)活性,以胞外分泌为主,且有一个开放的有活性的裂隙结构。     

盘基网柄菌早衰蛋白结构与功能生物信息学分析

早衰蛋白(presenilin, PS)是γ分泌酶的组成成分,其突变可造成阿尔兹海默病(Alzheimer disease,AD)的形成。因PS所造成AD的发病机理较复杂,因而我们想探究一下模式生物盘基网柄菌(Dictyostelium discoideum)是否能够作为研究早衰蛋白的模型。从NCBI网站中得到盘基网柄菌早衰蛋白DdPsenA和DdPsenB氨基酸序列,随后利用ProtScale、ProtParam理化性分析工具、保守结构域、TMHMM2跨膜区分析网站、进化树软件MEGA6.0、二级结构PredictProtein以及三级结构SWISS-Model服务器对上述蛋白进行生物信息学分析。DdPsenA的氨基酸长度为622,是亲水性蛋白,其相对分子质量为69 502.79,等电点为4.53。DdPsenB的氨基酸长度为473,属于疏水性蛋白,其相对分子质量为52 558.74,等电点为4.49,与DdPsenA同属于Peptidase_A22B超家族。跨膜分析出DdPsenA和DdPsenB均有8个跨膜区。DdPsenA与人类presenilin-2序列相似性为67%,Dd PsenB与人类presenilin-1序列相似性分别为60%。盘基网柄菌可以作为研究早衰蛋白功能和作用机制的模型。     

Identification of a Cupin Protein Gene Responsible for Pathogenicity,Phage Susceptibility and LPS Synthesis of Acidovorax citrulli

Bacteriophages infecting Acidovorax citrulli, the causal agent of bacterial fruit blotch, have been proven to be effective for the prevention and control of this disease. However, the occurrence of bacteriophage-resistant bacteria is one of hurdles in phage biocontrol and the understanding of phage resistance in this bacterium is an essential step. In this study, we aim to investigate possible phage resistance of A. citrulli and relationship between phage resistance and pathogenicity, and to isolate and characterize the genes involved in these phenomena. A phage-resistant and less-virulent mutant named as AC-17-G1 was isolated among 3,264 A. citrulli Tn5 mutants through serial spot assays and plaque assays followed by pathogenicity test using seed coating method. The mutant has the integrated Tn5 in the middle of a cupin protein gene. This mutant recovered its pathogenicity and phage sensitivity by complementation with corresponding wild-type gene. Site-directed mutation of this gene from wild-type by CRISPR/Cas9 system resulted in the loss of pathogenicity and acquisition of phage resistance. The growth of AC-17-G1 in King’s B medium was much less than the wild-type, but the growth turned into normal in the medium supplemented with D-mannose 6-phosphate or D-fructose 6-phosphate indicating the cupin protein functions as a phosphomannos isomerase. Sodium dodecyl sulfa analysis of lipopolysaccharide (LPS) extracted from the mutant was smaller than that from wild-type. All these data suggest that the cupin protein is a phosphomannos isomerase involved in LPS synthesis, and LPS is an important determinant of pathogenicity and phage susceptibility of A. citrulli.     

Comparative Proteomic Analysis for a Putative Pyridoxal Phosphate-Dependent Aminotransferase Required for Virulence in Acidovorax citrulli

Acidovorax citrulli (Ac) is the causative agent of bacterial fruit blotch disease in watermelon. Since resistant cultivars have not yet been developed, the virulence factors/mechanisms of Ac need to be characterized. This study reports the functions of a putative pyridoxal phosphate-dependent aminotransferase (PpdaAc) that transfers amino groups to its substrates and uses pyridoxal phosphate as a coenzyme. It was observed that a ppdaAc knockout mutant had a significantly reduced virulence in watermelon when introduced via germinated-seed inoculation as well as leaf infiltration. Comparative proteomic analysis predicted the cellular mechanisms related to PpdaAc. Apart from causing virulence, the PpdaAc may have significant roles in energy production, cell membrane, motility, chemotaxis, post-translational modifications, and iron-related mechanisms. Therefore, it is postulated that PpdaAc may possess pleiotropic effects. These results provide new insights into the functions of a previously unidentified PpdaAc in Ac.     

改良DAS-Dot-ELISA检测西瓜细菌性果斑病菌

以硝酸纤维素膜为载体,对Dot-ELISA法的封闭条件、包被抗体浓度、点样量等反应条件进行优化,建立改良DAS-Dot-ELISA法快速检测西瓜细菌性果斑病菌。研究发现,以含乙二胺四乙酸二钠(EDTA)的脱脂奶粉液高温处理后用于封闭,可有效降低背景;轻微振荡可提高杂交效率,减少非特异性结合。改良DAS-Dot-ELISA可快速、经济的检测西瓜果斑病菌,灵敏度达1.9×105CFU/mL。在对两批次种子样品的检测中,改良DAS-Dot-ELISA法检测带菌率分别为8.0%和6.0%,与微孔板ELISA结果完全一致;对每粒种子的检测结果,改良DAS-Dot-ELISA法与微孔板ELISA吻合率平均达99.0%,显示较好的实用前景,同时为快速检测西瓜果斑病菌提供一种新途径。     

Nicotiana species as surrogate host for studying the pathogenicity of Acidovorax citrulli,the causal agent of bacterial fruit blotch of cucurbits

Bacterial fruit blotch (BFB) caused by Acidovorax citrulli is one of the most important bacterial diseases of cucurbits worldwide. However, the mechanisms associated with A. citrulli pathogenicity and genetics of host resistance have not been extensively investigated. We idenitfied Nicotiana benthamiana and Nicotiana tabacum as surrogate hosts for studying A. citrulli pathogenicity and non-host resistance triggered by type III secreted (T3S) effectors. Two A. citrulli strains, M6 and AAC00-1, that represent the two major groups amongst A. citrulli populations, induced disease symptoms on N. benthamiana, but triggered a hypersensitive response (HR) on N. tabacum plants. Transient expression of 19 T3S effectors from A. citrulli in N. benthamiana leaves revealed that three effectors, Aave_1548, Aave_2708, and Aave_2166, trigger water-soaking-like cell death in N. benthamiana. Aave_1548 knockout mutants of M6 and AAC00-1 displayed reduced virulence on N. benthamiana and melon (Cucumis melo L.). Transient expression of Aave_1548 and Aave_2166 effectors triggered a non-host HR in N. tabacum, which was dependent on the functionality of the immune signalling component, NtSGT1. Hence, employing Nicotiana species as surrogate hosts for studying A. citrulli pathogenicity may help characterize the function of A. citrulli T3S effectors and facilitate the development of new strategies for BFB management.     

Quorum Sensing Contributes to Seed‐to‐Seedling Transmission of Acidovorax citrulli on Watermelon

Based on the observation that Acidovorax citrulli switches from saprobic to pathogenic growth for seed‐to‐seedling transmission of bacterial fruit blotch of cucurbits (BFB), we hypothesized that quorum sensing (QS) was involved in the regulation of this process. Using aacI (luxI homologue) and aacR (luxR homologue) mutants of AAC00‐1, we investigated the role of QS in watermelon seed colonization and seed‐to‐seedling transmission of BFB. aacR and aacI mutants of AAC00‐1 colonized germinating watermelon seed at wild‐type levels; however, BFB seed‐to‐seedling transmission was affected in a cell density‐dependent manner. There were no significant differences in BFB seedling transmission between watermelon seed infiltrated with approximately 1 × 10⁶ CFU of AAC00‐1, the aacR or aacI deletion mutants (95.2, 94.9 and 98.3% BFB incidence, respectively). In contrast, when seed inoculum was reduced to approximately 1 × 10³ CFU/seed, BFB seed‐to‐seedling transmission declined to 34.3% for the aacI mutant, which was significantly less than the wild type (78.6%). Interestingly, BFB seed‐to‐seedling transmission for the aacR mutant was not significantly different to the wild‐type strain. These data suggest that QS plays a role in regulation of genes involved in seed‐to‐seedling transmission of BFB.     

1.37 Å Crystal structure of pathogenic factor pectate lyase from Acidovorax citrulli

Pectates lyase (Pel) plays an important role in bacteria pathogenicity. The crystal structure of Pel from Acidovorax citrulli (AcPel) has been solved to 1.37 Å resolution. AcPel belongs to the polysaccharide lyase family 1 (PL1), which has a characteristic right‐handed β‐helix fold. AcPel is similar with other Pels in the PL1 family, but also shows some differences at the substrate binding site. Proteins 2013; 81:1485–1490. © 2013 Wiley Periodicals, Inc.     

The type VI protein secretion system contributes to biofilm formation and seed‐to‐seedling transmission of Acidovorax citrulli on melon

The type VI protein secretion system (T6SS) is essential for the virulence of several Gram‐negative bacteria. In this study, we identified a T6SS gene cluster in Acidovorax citrulli, a plant‐pathogenic bacterium that causes bacterial fruit blotch (BFB) of cucurbits. One T6SS cluster, of approximately 25 kb in length and comprising 17 genes, was found in the A. citrulli AAC00‐1 genome. Seventeen A. citrulli mutants were generated, each with a deletion of a single T6SS core gene. There were significant differences in BFB seed‐to‐seedling transmission between wild‐type A. citrulli strain, xjl12, and ΔvasD, ΔimpK, ΔimpJ and ΔimpF mutants (71.71%, 9.83%, 8.41%, 7.15% and 5.99% BFB disease index, respectively). In addition, we observed that these four mutants were reduced in melon seed colonization and biofilm formation; however, they were not affected in virulence when infiltrated into melon seedling tissues. There were no significant differences in BFB seed‐to‐seedling transmission, melon tissue colonization and biofilm formation between xjl12 and the other 13 T6SS mutants. Overall, our results indicate that T6SS plays a role in seed‐to‐seedling transmission of BFB on melon.