水稻条斑病菌hrcC、hpa3和hrpE基因表达不依赖hrpG和hrpX基因调控

摘要：【目的】决定水稻条斑病菌（Xanthomonas oryzae pv. oryzicola）在非寄主植物上激发过敏反应（hypersensitive response, HR）和在寄主水稻上致病性（pathogenicity）的hrp基因簇是受hrpG和hrpX基因调控的,但还不清楚hrpG和hrpX基因是否共同决定着所有hrp基因的表达。【方法】本文通过基因敲除方式获得了水稻条斑病菌的hrpG和hrpX基因的双突变体。【结果】烟草和水稻上测定结果显示,双突变体与单突变体一样,均在烟草上失去HR激发能力和丧失在水稻上的致病性;相应地,功能互补后双突变体恢复至野生表型。细菌在水稻悬浮细胞、hrp诱导培养基XOM3和营养丰富的培养基NB中生长后的RT-PCR结果显示,NB中hrp基因低水平表达,XOM3和水稻细胞能够高水平诱导hrp基因表达。无论何种生长条件,hrpG单突变体中hrcC、hrcT、hpa3和hrpE基因表达,而hpa1、hpa2、hpaB、hrcJ和hrpG基因不表达;hrpX单突变体中hpa2、hrcC、hpa3、hrpE和hrpG基因表达,而hpa1、hrcT、hpaB和hrcJ基因不表达;hrpG和hrpX双突变体中hrcC、hpa3和hrpE基因表达,而hpa1、hpa2、hpaB、hrcT、hrcJ和hrpG基因不表达。【结论】这提示,水稻条斑病菌的hrcC、hrpE和hpa3基因不受hrpG和hrpX基因单独或同时调控,而hrcT基因受HrpG调控。由此推测,水稻条斑病菌III型分泌系统关键组份的表达有可能通过另外的信号途径进行调控,这为进一步分析III型分泌途经的形成提供了线索。     

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。     

水稻白叶枯病菌T3SS基因表达及其调控网络

植物病原细菌III型分泌系统(T3SS)在其毒性表达及与寄主互作中具有重要的功能。水稻白叶枯病菌(Xanthomonas oryzae pv.oryzae,Xoo)hrp基因簇编码了T3SS装置,将毒性效应子蛋白分泌到水稻细胞内,抑制和破坏寄主免疫反应,或诱导感病基因表达,以达到成功侵染的目的。hrp基因表达受到严格调控,在模拟水稻内环境的贫瘠营养培养基中被诱导表达;hrp和效应子基因均受调控蛋白HrpG和Hrp X的调控。此外,hrp基因还受到其它毒性调控网络重要因子的调控,包括双组分调控因子、转录调控因子、DNA/RNA结合蛋白、糖代谢和c-di-GMP信号因子。结合本实验室的研究结果,综述了Xoo T3SS表达调控及其致病机理的研究进展,以期为水稻细菌病害发生机理的解析及其有效防控措施提供一些新见解、思路和途径。     

人黑色素瘤相关抗原Restin的克隆和表达

目的:通过对原核表达载体、表达条件和宿主菌的优化,利用原核表达系统有效表达可溶性的人Restin融合蛋白.方法:应用PCR方法扩增获得Restin编码区,克隆入原核表达载体pET44a(+),分别转化E.coli BL21(DE3)和E.coli Rosetta-gamiTM2(DE3),不同的IPTG浓度诱导表达Restin重组融合蛋白,SDS-PAGE和Western Blot分析表达产物.结果:成功构建了重组载体pET44a(+)-Restin,并在E.coli Rosetta-gamiTM2(DE3)中获得了可溶性表达,表达量占菌体总蛋白的8.9%.结论:可溶性Restin融合蛋白的获得为后续的功能研究、蛋白制备及其抗体研制奠定了基础.     

Arthrobacter ureafaciens CZ31丙氨酸脱氢酶可溶性表达及产酶条件优化

【目的】克隆Arthrobacter ureafaciens CZ31丙氨酸脱氢酶的编码基因(alanine dehydrogenase),转化至Escherichia coli Rosetta(DE3)中构建可溶性表达alanine dehydrogenase(ald)的工程菌CZR07并优化产酶条件。【方法】提取A.ureafaciens CZ31菌株的全基因组DNA,设计引物扩增出ald基因,与pET-28a连接后导入E.coli Rosetta中表达并纯化重组蛋白,以单因素实验结果为依据,响应面法优化发酵条件。【结果】ald全长为1119 bp,编码含372个氨基酸残基的蛋白质,分子量约为40 kDa,酶活为2.65 U/mg。响应面分析温度、诱导时间及诱导剂浓度的影响强度为IPTG浓度温度温度×IPTG浓度温度×诱导时间IPTG浓度×诱导时间诱导时间。CZR07摇瓶发酵最佳条件为温度22°C、IPTG 0.7 mmol/L、诱导时间7 h,此条件下重组酶酶活达到15.23 U/mg,与响应面优化的预测值相似,较优化前提高5.75倍。【结论】克隆并实现了CZ31中ald基因的可溶性表达,采用BBD法优化产酶的诱导条件,获得显著的优化效果,为其他工程菌株产酶条件优化提供借鉴。     

耐辐射奇球菌recA基因的克隆、表达及其对recA缺损大肠杆菌辐射抗性的影响

将耐辐射奇球菌(Deinococcus radiodurans)recA基因克隆到表达质粒pET15b中,并在Escherichia coli HMS中高效表达了可溶性的RecA重组蛋白。同时将recA基因通过穿梭质粒pRADZ3导入recA缺损E.coli TG2细胞中,Western印迹实验显示RecA蛋白能够在不需要诱导剂IPTG的条件下稳定表达。辐射抗性实验表明,D.radiodurans的recA基因在E.coli细胞中的表达能够完全补偿recA缺损E.coli辐射抗性能力。     

人源抗TNF—α抗体Fab基因的单链化及其表达和纯化

构建人源抗TNF－α单链抗体基因,并尝试其在E.coli中的表达和纯化,采用人工接头,按VH－linkerVL的结构将人源抗TNF－a的VH,VL基因拼接成单链抗体（ScFv)基因,并将构建好的ScFv基因插入表达载体pQE30,转染E.coli M15,以IPTG诱导表达,Ni-NTA树脂纯化表达产物,构建的ScFv基因长723bp, 列分析表明,该序列拼接正确,SDS－PAGE显示,用重组的pQE30转化的M15菌经诱导后,有相对分子质量（M）约为52000的外源蛋白表达,Ni-NTA树脂纯化的表达产物纯度大于90％,因此,成功地构建了人源抗TNFa的ScFv基因,并在E.coli DH5a中表达和纯化的该基因的产物。     

青枯菌hrp基因的研究进展

青枯菌的hrp基因可诱发植物的超敏反应.对其基因组全序列测定表明:hrp基因簇位于基因组的大质粒上,共有20多个基因组成.从青枯菌中分离得到的可直接诱发植物超敏反应的效应蛋白主要为pop基因编码,它由hrp基因编码的类型Ⅲ蛋白分泌通道释放.目前的研究表明:(1)在hrp基因簇中,hrpY、hrpX及hrpV与分泌通道的一种纤毛的组装有关;(2)hrpB是整个类型Ⅲ蛋白分泌通道基因的转录激活子并作用于基因组中的其它效应基因;(3)hrpG是植物信号对hrp,基因的表达进行级联调控的组分之一.     

法夫酵母β-胡萝卜素转化酶(Asy)基因的克隆及可溶性表达研究

[目的]从高产虾青素的法夫酵母(Phaffia rhodozyma) 7B12菌株中克隆β-胡萝卜素转化酶基因(β-Carotene converting enzyme gene,asy),并将该基因在大肠杆菌(Escherichia coli)中进行可溶性表达,为深入研究该酶的性质及应用提供基础.[方法]采用cDNA末端快速扩增技术,克隆得到asy基因全长cDNA序列,将其克隆到表达载体pET32a中,通过优化温度和IPTG浓度提高其可溶表达量;进一步在E.coli BL21(DE3)中共表达携带asy基因的重组质粒和pACCAR 16△crtx质粒(携带由乙酰辅酶A合成β-胡萝卜素的基因链),用液相色谱分析共转化菌株中类胡萝卜素种类的变化,鉴定可溶性表达的Asy酶活性.[结果]法夫酵母7B12菌株asy基因的cDNA序列与GenBank能检索到的唯一一条asy基因mRNA序列(Accession No.DQ002007.1)一致性达到97％,该序列总长1 971 bp,最大开放阅读框为1 614 bp,编码538个氨基酸,在E.coli BL21 (DE3)中表达的Asy融合蛋白分子量约为70 kD;条件优化后转化asy基因的重组菌在26℃、0.5 mmol/L IPTG的条件下诱导时,可溶性融合蛋白比例达85％.与pACCAR16△crtx单质粒转化相比,共转化pACCAR 16△crtx及asy基因的菌株类胡萝卜素产物的成分发生了明显的变化,其中α-胡萝卜素的含量明显减少,伴随出现了3种新的色素,根据出峰时间判断其中一种为β-胡萝卜素和虾青素的中间产物——β-隐黄质.[结论]从法夫酵母中克隆得到asy基因,通过优化诱导温度及IPTG浓度等条件提高了该基因在E.coli BL21(DE3)中的可溶性表达量,经鉴定可溶性的Asy融合蛋白具有转化β-胡萝卜素的活性.     

小鼠HSP60基因的克隆与表达条件的优化

目的:克隆小鼠热休克蛋白60(HSP60)基因,在大肠杆菌中表达,并进一步对其表达条件进行优化.方法:采用RTPCR技术克隆出非肥胖性糖尿病(NOD)小鼠HSP60的cDNA序列.构建重组表达载体pET28a- HSP60,以其转化大肠杆菌感受态细胞BL21( DE3),在不同的菌体浓度、不同浓度的异丙基-β-D-硫代半乳糖苷(IPTG)、不同诱导时间以及不同温度条件下诱导,检测重组蛋白的表达情况.结果:获得一个含有1 721bp的cDNA的片段,重组蛋白HSP60在E.coli BL -21 (DE3)中的最佳表达条件是菌体密度A600nm为0.6,诱导时间为5h,诱导物(IPTG)浓度为4mmol/L,诱导温度为35℃,重组蛋白大小约为60kD.结论:成功克隆了小鼠HSP60基因,并在大肠杆菌中获得高效表达,为重组蛋白的分离纯化及进一步研究其生物学功能奠定了基础.     

Transformation of Xanthomonas campestris pathovar campestris with plasmid DNA

Procedures for the introduction of plasmid DNA into Gram-negative bacteria have been adapted and optimized to permit transformation of the plant pathogen Xanthomonas campestris pathovar campestris with the cloning vector pKT230 and other broad-host-range plasmids. The technique involves CaCl2-induced competence and heat shock and is similar to that routinely used for Escherichia coli. Wild-type X. c. campestris strains appear to restrict incoming unmodified DNA, so that plasmid DNA for transformation must be prepared from X. c. campestris (into which it has previously been introduced by conjugation). To overcome this disadvantage a restriction-deficient mutant has been isolated.     

Defence-related enzymatic response in cabbage to Xanthomonas campestris pv. campestris

Black rot of cabbage caused by Xanthomonas campestris pv. campestris is one of the most important diseases of crucifers worldwide. Expression of defence-related enzymes in cabbage in response to X. campestris pv. campestris was investigated in the current experiment. Among the defence-related enzymes (phynylalanine ammonia lyase, peroxidase, polyphenol oxidase, superoxide dismutase [SOD] and chitinase) and quantity of phenolic compounds studied in the present investigation, phenylalanine ammonia lyase (PAL), the key enzyme in the phenylpropanoid pathway was the first enzyme suppressed at three days after inoculation in X. campestris pv. campestris-cabbage system. Correlation analysis indicated that PAL and phenolic compounds are the two most important compounds determining the susceptibility of cabbage to X. campestris pv. campestris. Induction of peroxidase isoform-1 (Rf value: 0.059) and SOD isoform-1 (Rf value: 0.179) three days after pathogen inoculation implicated the role of these isozymes in susceptible cabbage – X. campestris pv. campestris interaction. This study demonstrates the susceptibility of cabbage to X. campestris pv. campestris is a result of declination of PAL and phenolic contents at biochemical level as a manifestation of increase in bacterial population at the cellular level within the host tissues.     

Biological role of xanthomonadin pigments in Xanthomonas campestris pv. campestris

Previous studies have indicated that the yellow pigments (xanthomonadins) produced by phytopathogenic Xanthomonas bacteria are unimportant during pathogenesis but may be important for protection against photobiological damage. We used a Xanthomonas campestris pv. campestris parent strain, single-site transposon insertion mutant strains, and chromosomally restored mutant strains to define the biological role of xanthomonadins. Although xanthomonadin mutant strains were comparable to the parent strain for survival when exposed to UV light; after their exposure to the photosensitizer toluidine blue and visible light, survival was greatly reduced. Chromosomally restored mutant strains were completely restored for survival in these conditions. Likewise, epiphytic survival of a xanthomonadin mutant strain was greatly reduced in conditions of high light intensity, whereas a chromosomally restored mutant strain was comparable to the parent strain for epiphytic survival. These results are discussed with respect to previous results, and a model for epiphytic survival of X. campestris pv. campestris is presented.     

Qualitative and comparative proteomic analysis of Xanthomonas campestris pv. campestris 17

The bacterium Xanthomonas campestris pathovar campestris (XCC) 17 is a local isolate that causes crucifer black rot disease in Taiwan. In this study, its proteome was separated using 2-DE and the well-resolved proteins were excised, trypsin digested, and analyzed by MS. Over 400 protein spots were analyzed and 281 proteins were identified by searching the MS or MS/MS spectra against the proteome database of the closely related XCC ATCC 33913. Functional categorization of the identified proteins matched 141 (50%) proteins to 81 metabolic pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. In addition, we performed a comparative proteome analysis of the pathogenic strain 17 and an avirulent strain 11A to reveal the virulence-related proteins. We detected 22 up-regulated proteins in strain 17 including the degrading enzymes EngXCA, HtrA, and PepA, which had been shown to have a role in pathogenesis in other bacteria, and an anti-host defense protein, Ohr. Thus, further functional studies of these up-regulated proteins with respect to their roles in XCC pathogenicity are suggested.     

Pan Proteome of Xanthomonas campestris pv. campestris Isolates Contrasting in Virulence

Fully sequenced genomes of Xanthomonas campestris pv. campestris (Xcc) strains are reported. However, intra‐pathovar differences are still intriguing and far from clear. In this work, the contrasting virulence between two isolates of Xcc ‐ Xcc51 (more virulent) and XccY21 (less virulent) is evaluated by determining their pan proteome profiles. The bacteria are grown in NYG and XVM1 (optimal for induction of hrp regulon) broths and collected at the max‐exponential growth phase. Shotgun proteomics reveals a total of 329 proteins when Xcc isolates are grown in XVM1. A comparison of both profiles reveals 47 proteins with significant abundance fluctuations, out of which, 39 show an increased abundance in Xcc51 and are mainly involved in virulence/adaptation mechanisms, genetic information processing, and membrane receptor/iron transport systems, such as BfeA, BtuB, Cap, Clp, Dcp, FyuA, GroEs, HpaG, Tig, and OmpP6. Several differential proteins are further analyzed by qRT‐PCR, which reveals a similar expression pattern to the protein abundance. The data shed light on the complex Xcc pathogenicity mechanisms and point out a set of proteins related to the higher virulence of Xcc51. This information is essential for the development of more efficient strategies aiming at the control of black rot disease.     

Light signaling mediated by PAS domain-containing proteins in Xanthomonas campestris pv. campestris

Per-ARNT-Sim (PAS) domains are important signalling modules that possibly monitor changes in various stimuli such as light. For the majority of PAS domains that have been identified by sequence similarity, the biological function of the signalling pathways has not yet been experimentally investigated.Thirty-three PAS proteins were discovered in Xanthomonas campestris pv. campestris(Xcc) by genome/proteome analysis. Thirteen PAS proteins were identified as contributing to light signalling and Xcc growth, motility or virulence using molecular genetics and bioinformatics methods. The PAS domains played important roles in light signalling to regulate the growth, motility and virulence of Xcc. They might be regulated by not only light quality (wavelength)but also quantity (intensity) as potential light-signalling components. Evaluating the light wavelength, three light-signalling types of PAS proteins in Xcc were shown to be involved in blue light signalling, tricolour (blue, red and far red)signalling or red/far-red signalling. This showed that Xcc had evolved a complicated light-signalling system to adapt to a complex environment.     

Extracellular proteases from Xanthomonas campestris pv. campestris, the black rot pathogen

Two proteases (PRT1 and PRT2) were fractionated from culture supernatants of wild-type Xanthomonas campestris pv. campestris by cation-exchange chromatography on SP-5PW. Inhibitor experiments showed that PRT 1 was a serine protease which required calcium ions for activity or stability or both and that PRT 2 was a zinc-requiring metalloprotease. PRT 1 and PRT 2 showed different patterns of degradation of beta-casein. The two proteases comprised almost all of the extracellular proteolytic activity of the wild type. A protease-deficient mutant which lacked both PRT 1 and PRT 2 showed considerable loss of virulence in pathogenicity tests when bacteria were introduced into mature turnip leaves through cut vein endings. This suggests that PRT 1 and PRT 2 have a role in black rot pathogenesis.     

Identification of Xanthomonas campestris pv. campestris galactose utilization genes from transcriptome data

A 70 mer oligonucleotide microarray was constructed to analyze genome-wide expression profiles of Xanthomonas campestris pv. campestris B100, a plant-pathogenic bacterium that is industrially employed to produce the exopolysaccharide xanthan gum which has many applications as a stabilizing, thickening, gelling, and emulsifying agent in food, pharmaceutical, and cosmetic industries. As an application example, global changes of gene expression were monitored during growth of X. campestris pv. campestris B100 on two different carbon sources. Exponential growing bacterial cultures were incubated either for 1h or permanently in minimal medium supplemented with 1% galactose in comparison to growth in minimal medium supplemented with 1% glucose. Six genes were identified that were significantly increased in gene expression under both growth conditions. These genes were located in three distinguished chromosomal regions in operon-like gene clusters. Genes from these clusters encode secreted glycosidases, which were predicted to be specific for galactose-containing carbohydrates, as well as transport proteins probably located in the outer and inner cell membrane. Finally genes from one cluster code for cytoplasmic enzymes of a metabolic pathway specific for the breakdown of galactose to intermediates of glycolysis.