水稻条斑病菌gacA基因的克隆及功能初析

根据黄单胞菌gacA基因的同源性设计简并引物,采用PCR方法从水稻条斑病菌(Xanthomonas oryzae pv.oryzicola,Xooc)中克隆了gacA同源基因,命名为gacA_Xooc。序列比较显示,该基因在黄单胞菌中是相对保守的。通过同源重组的方法,构建了gacA_Xooc的插入突变株。对0.1% Tryptone的趋化应答能力检测发现,gacA突变株的趋化能力明显降低,证明gacA与Xooc的趋化性相关。     

红纹黄单胞菌a-氨基酸酯水解酶的克隆、序列分析及热稳定性提高

【目的】克隆红纹黄单胞菌α-氨基酸酯水解酶基因全序列,对序列进行生物信息学分析,并提高酶的热稳定性。【方法】利用多聚酶链式反应(PCR)克隆α-氨基酸酯水解酶基因全序列;应用生物信息学软件对获得的基因序列及编码的蛋白序列进行分析;通过同源建模,预测红纹黄单胞菌α-氨基酸酯水解酶的三维结构;通过定点突变替换氨基酸序列中高度柔性的位点,提高该酶的热稳定性。【结果】从红纹黄单胞菌(Xanthomonas rubrillineans)中扩增得到α-氨基酸酯水解酶基因aeh(GenBank登录号JF744990),核苷酸序列长度1 917 bp,编码638个氨基酸。序列比对和同源性分析显示,该酶与白纹黄单胞菌Xanthomonas albilineans str.GPE PC73的肽酶及地毯草黄单胞菌Xanthomonas axono-podis pv. citri str. 306的戊二酰-7-氨基头孢烷酸酰化酶氨基酸序列相似性最高,分别为91%和83%,系统进化分析表明,该酶与白纹黄单胞菌Xanthomonas albilineans str. GPEPC73的肽酶亲缘性最高。基于预测的三维模型,对高度柔性的位点进行饱和突变,从282株突变体中筛选得到3株T50较野生型高5°C以上的突变体。【结论】对红纹黄单胞菌AEH的氨基酸序列分析有助于探索同源蛋白的进化过程。对高度柔性位点进行饱和突变的策略可以用于提高热稳定性。     

茎瘤固氮根瘤菌甲基化受体蛋白Tlp1的功能

【目的】考察茎瘤固氮根瘤菌中趋化基因簇上游的受体蛋白Tlp1编码基因的突变表型,初步探究其功能机理。【方法】利用同源重组和三亲本接合转移的方法构建突变株,在TY培养基中测定生长情况,半固体平板法观察趋化圈,刚果红固体培养基观察胞外多糖和次生代谢产物的分泌,乙炔还原法测定菌株的固氮酶活性。【结果】与野生型菌株相比,tlp1突变株的生长速率没有影响。在以甘油为碳源的L3半固体平板上突变株的趋化圈变小,其回补菌株能部分回补趋化能力。突变株的胞外多糖分泌与野生型没有区别,但其次生代谢产物黑色素出现的时间比野生型稍早。在固氮酶活性测定中,发现突变株酶活性明显比野生型降低,回补菌株能够部分回补。【结论】茎瘤固氮根瘤菌Tlp1蛋白对甘油表现出一定的趋化能力,并且影响细菌的次生代谢产物和固氮能力。     

假单胞菌gacA插入突变对藤黄绿脓菌素和吩嗪-1-羧酸合成代谢的差异性调控

假单胞菌株M18分泌藤黄绿脓菌素 (Pyoluteorin ,Plt )和吩嗪 1 羧酸 (Phenazine 1 carboxylicacid ,PCA)并抑制多种植物病菌的生长。从M18中克隆双基因调控系统gacS gacA的组成基因gacA ,并构建了该基因抗性插入突变株M18G。在KMB培养基中 ,M18G合成Plt的能力受到完全抑制 ,而PCA的积累约比野生型提高 31倍左右。Plt合成基因簇突变株M18T和在M18G基础上构建的PCA合成基因簇突变株M18GA的Plt和PCA合成的动力学变化表明 ,在M18G菌株中 ,Plt合成的抑制并不引起PCA的过量积累 ,PCA的过量积累也不引起Plt合成的抑制。由此推测 ,gacA在基因表达的水平上全局性地执行着调控功能     

假单胞菌gacA插入突变对藤黄绿脓菌素和吩嗪-1-羧酸合成代谢的差异性调控

假单胞菌株M18分泌藤黄绿脓菌素(Pyoluteorin,Plt )和吩嗪1羧酸（Phenazine1carboxylic acid, PCA）并抑制多种植物病菌的生长。 从M18中克隆双基因调控系统gacS/gacA的组成基因gacA,并构建了该基因抗性插入突变株M18G。在KMB培养基中,M18G合成Plt的能力受到完全抑制,而PCA的积累约比野生型提高31倍左右。Plt合成基因簇突变株M18T和在M18G基础上构建的PCA合成基因簇突变株M18GA的Plt和PCA合成的动力学变化表明,在M18G菌株中,Plt合成的抑制并不引起PCA的过量积累,PCA的过量积累也不引起Plt合成的抑制。由此推测,gacA在基因表达的水平上全局性地执行着调控功能。     

荧光假单胞菌2P24中opgGH基因影响抗生素2,4-二乙酰基间苯三酚的产生

【目的】抗生素2,4-二乙酰基间苯三酚(2,4-diacetylphloroglucinol,2,4-DAPG)是生防菌株荧光假单胞菌(Pseudomonas fluorescens) 2P24防治植物病害的关键因子,然而对2,4-DAPG生物合成的调控通路并未完全解析。【方法】前期利用Tn5随机突变的方法获得一株对棉花立枯丝核菌(Rhizoctonia solani)拮抗能力完全丧失的突变菌株W3,本研究利用基因互补等方法研究该突变体中被破坏的基因对菌株2P24分泌2,4-DAPG和其他生防相关性状的影响。【结果】Tn5插入位点及其序列分析表明突变菌株W3中Tn5破坏了opgG基因。鉴于opgG和opgH基因组成操纵子,利用同源重组技术构建了opgGH内缺失突变菌株。与野生菌株2P24相比,opgGH突变菌株中2,4-DAPG的产量显著降低。对其他生防相关性状的检测发现,突变opgGH基因并不影响群体感应系统(quorum sensing,QS)信号分子的产生、氢氰酸的产生以及生物膜的形成,但可抑制菌株2P24的游动性。转录融合实验进一步表明opgGH基因并不调控gacA基因及其调控...     

茎瘤固氮根瘤菌ORS571鞭毛马达基因fliN与fliM的功能分析

【目的】考察茎瘤固氮根瘤菌ORS571中鞭毛马达蛋白FliN、FliM的编码基因分别缺失的突变体表型,初步探究其功能机理。【方法】本研究采用同源重组和三亲本接合转移的方法构建突变体,测定野生型及突变株的生长曲线、趋化性、胞外多糖的分泌、生物膜的形成及细胞絮凝等表型。【结果】三种菌株的生长速率基本无差,与野生型菌株相比突变株鞭毛结构丧失,趋化能力、分泌的胞外多糖和生物膜形成能力均下降,但相同时间内细胞絮凝程度比野生型明显。【结论】实验表明,鞭毛基因fliN、fliM对茎瘤固氮根瘤菌ORS571鞭毛的形成、趋化运动、胞外多糖的分泌、生物膜的形成及细胞絮凝能力等均有调控作用。     

黄脂菌素生物合成基因簇中调控基因xanR3的功能

【目的】研究黄脂菌素产生菌灰黄链霉菌中编码ArsR家族转录调控蛋白(Arsenical resistance regulator)的xanR3基因的功能。【方法】利用大肠杆菌和链霉菌双亲本接合转移的方法,构建xanR3基因缺失突变株及回补突变株。利用cDNA在相邻同方向的基因间隔区进行PCR确定黄脂菌素生物合成基因簇中的转录单元。利用荧光定量RT-PCR方法进行突变株中黄脂菌素生物合成基因簇转录水平的检测。【结果】对得到的xanR3基因缺失突变株及回补突变株进行发酵,发现xanR3基因缺失突变株产黄脂菌素能力下降,回补菌株中黄脂菌素产量相比缺失突变株有一定程度的恢复,但仍未达到野生型水平。经鉴定,黄脂菌素生物合成基因簇中共有18个共转录单元,其中4个共转录单元在?xanR3突变株中转录水平明显下降。【结论】ArsR家族转录调控基因xanR3是黄脂菌素生物合成的正调控基因。     

趋化信号转导基因cheA突变对Pseudomonas putida DLL-1甲基对硫磷的趋化性及原位降解的影响

Pseudomonas putida DLL-1是一株甲基对硫磷(MP)高效降解菌株,同时对MP具有趋化性。cheA基因是菌株趋化信号转导过程中负责编码组氨酸激酶的基因,为了研究菌株趋化性在农药原位降解中的作用,通过基因打靶的方式使P.putida DLL-1染色体上单拷贝的cheA基因失活,成功地获得了MP的趋化突变株P.putida DAK,突变株与野生菌株生长能力没有显著差异。通过土壤盆钵试验(MP浓度为50mg/kg),发现在灭菌与未灭菌土壤中趋化突变株对MP的降解能力低于原始出发菌株DLL-1约20%~30%,说明菌株DLL-1趋化性的丧失会减慢其对农药的降解,趋化性在农药的原位降解过程中发挥重要作用。     

fliY基因失活减弱沙福芽孢杆菌ST7菌株的锰氧化能力

【背景】沙福芽孢杆菌ST7菌株具有较强的锰氧化能力,但其分子机制不清楚。【目的】着重研究鞭毛马达开关蛋白基因(fliY)对沙福芽孢杆菌锰氧化能力的影响。【方法】根据同源重组原理,以沙福芽孢杆菌ST7菌株为起始菌株,构建fliY基因敲除的突变株ΔfliY,测定菌落迁徙、细菌生物膜和锰氧化率等,研究fliY基因突变后菌株的运动能力、生物膜生成和锰氧化能力是否发生变化。【结果】经克隆测序,证实突变株ΔfliY中fliY基因的后半段被卡纳霉素抗性基因取代,fliY基因失活;与野生型菌株ST7相比,突变株ΔfliY在全营养的LB培养基中生长变化不大,但在含锰的PYCM培养基中,突变株的生长速度减慢、菌落较小、生物膜生成量显著下降,运动性和锰氧化能力分别下降65%和20%。【结论】fliY基因不仅影响菌株的生长和运动,而且参与细菌的趋化和锰氧化等生物学过程。     

水稻黄单胞菌黄色素合成相关基因的克隆与鉴定

用硫酸二乙酯(DES)诱变水稻白叶枯病菌(Xanthomonas oryzae pv. oryzae,简称Xoo)和条斑病细菌(Xanthomonas oryzae pv. oryzicola, 简称Xooc),分别得到5株和13株黄色素缺失突变体,其中来自Xooc的M6和M12 还丧失了对水稻的致病性和在烟草上激发过敏反应的能力。以Xooc黄色素缺失突变体M51为受体菌交叉互补从Xoo JXOIII基因文库中筛选出一个黄色素合成相关的基因克隆pA341,以Xoo黄色素缺失突变体M1071为受体菌,从Xooc RS105基因文库中获得了一个黄色素合成相关的基因克隆pA270。功能互补显示,18株黄色素缺失突变体中的10株能分别被pA341和 pA270互补后正常产生黄色素,但这两个克隆不能同时互补同一株黄色素缺失突变体。能被pA341互补的黄色素缺失突变体M6没有恢复对水稻的致病性和在烟草上激发过敏反应,表明黄色素合成相关基因与hrp基因间不存在相关性。斑点杂交结果表明,pA270与pA341之间没有同源性。pA270亚克隆结果显示,与黄色素合成相关的基因约11.6kb大小,以基因簇的形式存在,不仅决定了黄色素的产生,还影响黄色素合成的数量和质量（吸收峰）。在紫外光条件下,黄色素能够提高菌体的存活率,提示黄色素对病原细菌有保护作用。     

水稻条斑病细菌hrp基因诱导表达系统的建立

水稻条斑病细菌(Xanthomonas oryzae pv.oryzicola,Xooc)决定在非寄主植物上激发过敏反应(hypersensitive response)和在寄主水稻上具致病性(pathogenicity)的hrp基因簇是诱导表达的。为研究hrp基因的功能,利用hpa1和hrpX基因的启动子与gfp基因进行融合,构建了hrp基因诱导表达系统。绿色荧光蛋白表达揭示,Xooc的hrp基因在营养丰富的NB培养基上不能有效表达,在hrp诱导培养基XOM3上可有效表达。以hrpX和hrpG突变体为参照,RT-PCR研究结果提示,Xooc野生型菌株hpa1基因在NB上不能有效表达,在XOM3培养基上可有效表达。相应地,hrpX突变体中hpa1基因不能被诱导表达,而在hrpG突变体中hpa1基因转录表达水平低于野生菌。研究结果还证实,水稻悬浮细胞能高效诱导Xooc的hrp基因表达。Xooc hrp基因诱导表达系统的建立为研究hrp基因功能、发掘T3SS效应分子以及开展Xooc致病性研究奠定了基础。     

水稻白叶枯病抗性基因Xa23鉴别菌株突变体库的构建及无毒基因突变体的筛选

利用in vivo转座技术构建了白叶枯病抗性基因Xa23鉴别菌株的突变体库,特异性引物PCR扩增和转座子插入位点旁侧序列分析结果表明转座子插入到白叶枯病菌的基因组中。经人工接种鉴定,筛选到4个毒力发生变化的突变体。为进一步克隆Xa23无毒基因提供了条件。     

Functional analysis of <Emphasis Type="Italic">pilQ</Emphasis> gene in <Emphasis Type="Italic">Xanthomanas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis>, bacterial blight pathogen of rice

Bacterial blight (BB) of rice, caused by Xanthomonas oryzae pv. oryzae (Xoo), is the most devastating bacterial disease in rice. A virulence-attenuated mutant strain HNU89K9 of X. oryzae pv. oryzae (KACC10331), with a transposon insertion in the pilQ gene was used for this study. The pilQ was involved in the gene cluster pilMNOPQ of the Xoo genome. Growth rate of the pilQ mutant was similar to that of wild-type. At level of amino acids, PilQ of Xoo showed that a high sequence identities more than 94% and 70% to Xanthomonas species and to Xyllela fastidiosa, respectively but a low sequence homology less than 30% to other bacterial species. The twitching motility forming a marginal fringe on PSA media was observed on colony of the wild-type strain KACC10331, but not in mutant HNU89K9. Wild-type Xoo cells formed a biofilm on the surface of the PVC plastic test tube, while the mutant strain HNU89K9 did not form a biofilm. The results suggest that the pilQ gene of X. oryzae pv. oryzae plays a critical role in pathogenicity, twitching motility, and biofilm formation.     

Pigment and virulence deficiencies associated with mutations in the aroE gene of Xanthomonas oryzae pv. oryzae

Xanthomonadins are yellow, membrane-bound pigments produced by members of the genus Xanthomonas. We identified an ethyl methanesulfonate-induced Xanthomonas oryzae pv. oryzae mutant (BXO65) that is deficient for xanthomonadin production and virulence on rice, as well as auxotrophic for aromatic amino acids (Pig(-) Vir(-) Aro(-)). Reversion analysis indicated that these multiple phenotypes are due to a single mutation. A genomic library of the wild-type strain was used to isolate a 7.0-kb clone that complements BXO65. By transposon mutagenesis, marker exchange, sequence analysis, and subcloning, the complementing activity was localized to a 849-bp open reading frame (ORF). This ORF is homologous to the aroE gene, which encodes shikimate dehydrogenase in various bacterial species. Shikimate dehydrogenase activity was present in the wild-type strain and the mutant with the complementing clone, whereas no activity was found in BXO65. This clone also complemented an Escherichia coli aroE mutant for prototrophy, indicating that aroE is functionally conserved in X. oryzae pv. oryzae and E. coli. The nucleotide sequence of the 2.9-kb region containing aroE revealed that a putative DNA helicase gene is located adjacent to aroE. Our results indicate that aroE is required for normal levels of virulence and xanthomonadin production in X. oryzae pv. oryzae.     

Functional analysis of the aroC gene encoding chorismate synthase from Xanthomonas oryzae pathovar oryzae

Xanthomonas oryzae pv. oryzae causes bacterial blight in rice, and this bacterial blight has been widely found in the major rice-growing areas. We constructed a transposon mutagenesis library of X. oryzae pv. oryzae and identified a mutant strain (KXOM9) that is deficient for pigment production and virulence. Furthermore, the KXOM9 mutant was unable to grow in minimal medium lacking aromatic amino acids. Thermal asymmetric interlaced-PCR and sequence analysis of KXOM9 revealed that the transposon was inserted into the aroC gene, which encodes a chorismate synthase in various bacterial pathogens. In planta growth assays revealed that bacterial growth of the KXOM9 mutant in rice leaves was severely reduced. Genetic complementation of this mutant with a 7.9-kb fragment containing aroC restored virulence, pigmentation, and prototrophy. These results suggest that the aroC gene plays a crucial role in the growth, attenuation of virulence, and pigment production of X. oryzae pv. oryzae.     

Plasmid and pathogenicity in Xanthomonas oryzae pathovar oryzae, the bacterial blight pathogen of Oryza sativa

Xanthomonas oryzae pv. oryzae , the causative agent for bacterial leaf blight of rice, comprises diverse groups of strains differing in biochemical and pathological characteristics. A collection of X.o . pv. oryzae strains differing in geographical origin was screened for the presence of plasmids. Out of 17 isolates of X.o. pv. oryzae , 14 harboured plasmids of which two isolates (XoP5, XoC26) had two plasmids each and one isolate (XoR20) had three. The remaining isolates contained a single plasmid of identical mobility. Finger print analysis of plasmids was carried out using Eco RI for 10 isolates. The restriction fragment pattern was distinct for each isolate. They were classified under three groups based on cluster analysis using the unweighted pair group method with averages (UPGMA). Of the 18 plasmids, the plasmid pMA36 ( X.o. pv. oryzae XoC36) was further characterized. This plasmid was cured by acridine orange at the frequency rate of 10%. The cured strain was transformed with pMA36 at a frequency of 2.3 times 10² transformants μg^-1 of plasmid DNA. The plasmid-cured strain was virulent on rice but symptom development was delayed when compared to wild and transformed strains. The wild type strain ( X.o. pv. oryzae XoC36) was resistant to ampicillin, carbenicillin and rifampicin whereas the cured strain was resistant to carbenicillin and rifampicin but sensitive to ampicillin. The transformant was resistant to the three antibiotics indicating that the plasmid pMA36 codes for ampicillin resistance. The plasmid influenced the pathogenicity of X.o. pv. oryzae.