茶树中富含茶氨酸、儿茶素和咖啡碱等重要功能成分,具有较高的价值功效,茶树在生命周期中经常遭受逆境胁迫,维生素B6(VB6)在植物体内参与逆境应答,吡哆醛激酶(pyridoxal kinase,PLK)是VB6补救途径中的关键酶.为进一步了解PLK在茶树生物合成中的功能和作用机理,该研究基于茶树基因组数据库,以龙井43为材料,采用逆转录PCR(RT-PCR)的方法从茶树中克隆出CsPLK的基因.结果表明:该基因序列长为1179 bp,编码393个氨基酸;CsPLK蛋白和已知物种中PLK蛋白具有较高的同源性,都是核糖激酶超家族成员;通过构建pET-CsPLK载体进行原核表达,并鉴定出重组蛋白有很强的催化活性;组织表达特异性分析表明,叶中的表达量比茎、根的高,在根中最低;荧光定量PCR表示,低温诱导CsPLK上调表达,干旱诱导CsPLK下调表达,发现该基因在茶树中有明显的逆境应答,推测CsPLK在茶树的生长发育、逆境胁迫发挥重要作用.

Plants are the main producers of vitamin B6 in nature. Among six B6 vitamers, pyridoxal 5′-phosphate is the biologically active form. In plants, de novo biosynthesis of vitamin B6 takes place in cytosol, in which pyridoxal 5′-phosphate is directly synthesized from pentose phosphate, triose phosphate, and glutamine precursors. On the other hand, the major B6 vitamer present in plant-derived foods is usually pyridoxine, either free or glycosylated. Up to now, the formation of pyridoxine in plants is still unclear. In this study, cDNA encoding pyridoxal reductase (PLR) converting pyridoxal into pyridoxine, was cloned from tobacco plants, and analyzed from multi-angle. Our results show that the NtPLR was mainly located in chloroplast, and the highest transcription level was identified from leaves, where the gene transcription was induced by pyridoxal. When tobacco plants were subjected to abiotic stress, the gene transcription showed dynamic changes, with different responses to different stresses. Down-regulation of NtPLR by RNA interference had a big impact on the transcription level of other vitamin B6 metabolic enzymes. Our results suggest that the conversion from pyridoxal to pyridoxine catalyzed by PLR mainly occurs in chloroplast, and that the chloroplast is an important place for vitamin B6 salvage pathway. The results also suggest that there is a linkage mechanism to keep a dynamic balance in the regulation of vitamin B6 metabolism in tobacco plants, and that the dynamic balance of vitamin B6 metabolism may be regulated by genetic networks.

PNP氧化酶是VB6代谢途径中一个重要的转化酶.该研究以茶树‘龙井43'为材料,采用RT-pCR方法克隆PNP氧化酶基因,以pET22b(+)为载体构建原核表达载体,通过IPTG进行诱导表达并进行功能鉴定;采用荧光定量PCR方法分析茶树不同组织中CsPNPO基因的表达差异以及在低温和干旱胁迫下的表达特征,为进一步解析茶树VB6的生理生化功能奠定基础.结果 表明:(1)茶树CsPNPO编码框长度为1 503 bp,编码501个氨基酸,分子量为48.5 kD,理论等电点5.82,不含信号肽,属于亲水性的非分泌蛋白,定位于叶绿体;氨基酸序列分析结果显示,其有叶绿体转运肽区域、YjeF-N功能域和PNP氧化酶功能域.(2)成功构建pET22b(+)-CsPNPO原核表达载体,并在pH8.5、37℃时测定重组蛋白有较强的PNP氧化酶活性.(3)荧光定量PCR检测结果显示,CsPNPO基因在茶树叶中的表达量最高,其次是茎,根的表达量最低仅为叶的十分之一,表明CsPNPO基因具有组织表达特异性;在低温和干旱条件下CsPNPO基因的表达量下降明显,推测CsPNPO基因可能参与了茶树对低温和干旱的逆境应答.

Pyridoxal 5′-phosphate (PLP), the catalytically active form of vitamin B6, is an important cofactor for many biochemical transformations. Plants are able to synthesize PLP de novo, but they also have a salvage pathway that functions to convert different vitamer forms between each other. Although the salvage enzymes are identified successively from plants, many questions remain unanswered. In the salvage pathway, PLP is synthesized by an ATP-dependent pyridoxal kinase (PLK) and an FMN-dependent pyridoxine 5′-phosphate oxidase (PNPO). In this study, cDNAs encoding PLK and PNPO were cloned from tobacco plants, compared and analyzed, and then the gene expression was down-regulated by RNA interference. Our results show that the NtPLK and NtPNPO contains highly conserved motifs involved in substrate binding or catalysis. NtPLK and NtPNPO are essential enzymes for all tissues of tobacco plants, and NtPNPO is mainly located in chloroplast. The down-regulation of NtPLK and NtPNPO has a greater impact on the transcription level of other vitamin B6 metabolic enzymes. Based on the results, we speculate that, in plants, the de novo synthesized free PLP may be hydrolyzed to PL by phosphatase in cytosol, and then the PL enter into organelles. In organelles, salvage pathway plays its role to produce PLP for maintenance of vitamin B6-mediated processes.

目的 建立用于幽门螺杆菌快速定量检测和分型的TaqMan MGB探针双重荧光定量PCR方法. 方法 采用Primer Premier 5软件分析设计引物和探针,采用双重荧光定量PCR扩增幽门螺杆菌CagA和VacA基因片段,建立循环数与拷贝数关系的标准曲线.检测临床标本中所含幽门螺杆菌的循环数,用该方法对临床胃黏膜标本进行检测并与标准曲线对比,计算所含幽门螺杆菌的拷贝数. 结果 建立的TaqMan MGB双重荧光定量PCR方法检测幽门螺杆菌质粒的线性范围是102～108拷贝/μl,CagA和VacA基因标准曲线的相关系数分别是0.977 8和0.990 4.29份临床胃黏膜标本的CagA和VacA基因循环数Ct值分别在29～35和30～35之间,幽门螺杆菌拷贝数分别在1.0×101.39～1.0×103.87和1.0×103.06～1.0×103.91之间,且临床标本中的幽门螺杆菌均为Ⅰ型. 结论 建立的TaqMan MGB双重荧光定量PCR法具有敏感、精确、快速的特点,可用于幽门螺杆菌的快速定量检测与分型鉴定.

Insect growth and development are primarily controlled by two major hormones, juvenile hormone and molting hormone. 20-Hydroxyecdysone is the most active form of the molting hormone. Although intensive studies have been performed on its biological function and action mechanism, it is still unknown how many genes are directly or indirectly regulated by the molting hormone. Here, we analyzed the genomic transcriptional response to 20-hydroxyecdysone in the fat body of silkworm, by using high-throughput Illumina sequencing technology and bioinformatics tools. In total, 606 differentially expressed genes with 347 up-regulated and 259 down-regulated were detected. The 606 differentially expressed genes were significantly enriched in 118 GO terms, i.e. biological process (68), molecular function (37) and cellular component (13). The KEGG analysis revealed that the significantly enriched pathways were mainly focused on the metabolic processes. The differentially expressed genes were further aligned to the functionally verified sequences of B. mori in the NCBI database, and a total of 43 functional sequences were identified, of which 23 genes were down-regulated and 20 genes were up-regulated. The up-regulated genes mainly relate to metamorphosis, immune response and protein synthesis. RT-qPCR analysis further validated the correctness of the digital gene expression data. Our study gives an overall view of the regulating effect of 20-hydroxyecdysone on the whole-genome transcript expression in the silkworm, provides useful dataset, and will be helpful for the further studies.

Many virulence genes have been reported to play important roles in Helicobacter pylori pathogenesis. However the detailed mechanisms of many of them have not been completely clear. In this study, we found gene hp0169, encoding a putative collagenase (HpPrtC), was involved in pathogenesis of H. pylori. Recombinant HpPrtC shows activities to both native and heat-denatured collagens. This result indicated that HpPrtC may act as a virulence factor to help the bacterium colonize in their host stomach by degrading surrounding collagens. hp0169 was deleted by homologous recombination to study its function in bacterium-host cell interaction. For the pathogenic functions on the host cells, the hp0169 mutant exhibits no significant changes on inducing apoptosis of GES-1 cells. However, the viability and proliferation rate of GES-1 cells infected with mutant strain were higher than the cells infected with wild-type strain. These results indicated that except for its collagenolytic activity, HpPrtC might participate in H. pylori pathogenesis through an additional pathway. Functional studies on hp0169 involved in pathogenesis would shed light on deep understanding of the pathogenic mechanism of H. pylori.

Helicobacter pylori is a Gram-negative, microaerophilic bacterium associated with human gastric diseases. Further investigations on virulence genes are still required to clarify the pathogenic mechanism of H. pylori and the heterogeneous problem of infection. In order to develop an efficient and accurate method to study gene functions in H. pylori pathogenesis, an unmarked deletion method for both a single gene and a large fragment was established based on the FLP-FRT recombination system. Using this method, the gene hp0788, encoding an outer membrane protein (HofF), was deleted. Deletion of hp0788 did not affect growth or motility of H. pylori, but reduced the adherence of the bacteria to gastric epithelial cells. The apoptosis of GES-1 cells caused by H. pylori infection was also reduced by the defection of hp0788. These suggest that hp0788 takes part in the bacterium-host interaction and plays an important role in H. pylori infection. Furthermore, a large genomic fragment deletion from hp0541 to hp0547 in cag pathogenicity island was also successfully achieved using FLP-FRT method. The innovative application of the FLP-FRT recombination system in H. pylori to construct unmarked deletion would provide a helpful tool for further function research of putative pathogenic genes and contribute to the understanding of H. pylori pathogenesis.

Only a small percentage of people infected with Helicobacter pylori (H. pylori) will develop overt chronic gastric diseases. To understand the pathological mechanism, the action of H. pylori on monocyte apoptosis was detected. H. pylori co-culturing with peripheral blood monocytes, THP-1 or U937 cells result in early apoptosis at 6, 12, and 24 h after infection. The phosphorylated Bad and JNK were increased, and Bcl-2 was declined at 6, 12, and 24 h in peripheral blood monocytes after H. pylori infection. The phosphorylated Akt was augmented at 6 and 12 h post-infection. A slow apoptotic response was induced by H. pylori via Bad and Bcl-2 regulators, activated caspase-8 and caspase-9, and JNK at 24 h in THP-1 cells. Meanwhile, only Bad and JNK were involved in regulating U937 cells apoptosis at 24 h after infection. These results supported a novel mechanism of H. pylori escaping from monocytes by upregulation of early apoptosis and inhibition of late apoptosis. The differences among the three cells may reveal why H. pylori-derived disease occurs in relatively few people and provide a pathological mechanism whereby a treatment for H. pylori-derived disease may be developed.

目的研究LAIR-1基因沉默对幽门螺杆菌（Helicobacter pylori）诱导单核细胞THP-1凋亡的影响。方法以Hp ATCC26695分别感染THP-1细胞6、12、24和48h,采用流式细胞术检测细胞凋亡及LAIR-1蛋白的表达。应用Lipofectamine RNAiMAX转染3对LAIR-1siRNAs入单核细胞THP-1中,采用半定量RT-PCR及流式细胞术检测沉默效应,筛选出的较有效的一对siRNA再转染入单核细胞THP-1,48h后加入Hp培养24h,采用流式细胞术检测细胞凋亡率。结果 Hp感染THP-1细胞6、12、24和48h后的细胞凋亡率分别为（84±1.77）%、（69.13±4.29）%、（48.3±3.37）%和（21.1±4.67）%,LAIR-1蛋白表达相对荧光强度分别为14 934±178、14 369±244、12 259±523和7438±539;转染后48h较有效的siRNA对LAIR-1mRNA及蛋白沉默效率分别为（39.1±4.67）%和（38.4±3.18）%;将筛选出的siRNA转染细胞48h后,用Hp感染24h,流式细胞仪检测显示转染组与阴性对照组细胞凋亡率分别为（56.1±9.8）%和（36.9±3.2）%,转染后感染组与空白组LAIR-1蛋白表达相对荧光强度分别为12 181±219和10 228±136。结论在Hp感染细胞THP-1 48h内,随着感染时间延长LAIR-1的表达及THP-1细胞凋亡率显著降低即LAIR-1siRNA能抑制LAIR-1的表达及THP-1细胞的凋亡。

目的 分析幽门螺杆菌感染胃上皮细胞过程中整合素β1亚基表达的变化,观察整合素β1对幽门螺杆菌感染胃上皮细胞GES-1凋亡及增殖的影响.方法 将幽门螺杆菌与胃上皮细胞GES-1共培养,采用流式细胞术检测整合素β1的表达量变化;应用iRNA技术降低胃上皮细胞GES-1中β1亚基的表达;幽门螺杆菌感染胃上皮细胞及iRNA降低β1的表达后,采用流式细胞术检测细胞的凋亡,采用CCK-8法检测细胞增殖情况.结果 幽门螺杆菌感染胃上皮细胞12、24、48h后空白对照组凋亡率分别为(10.80±0.71)％、(12.23±0.06)％和(12.30±2.12)％,Hp感染组凋亡率分别为(24.20±0.14)％、(30.05±2.47)％和(26.40±1.91)％,差异均有统计学意义(t值分别为-26.28、-7.701和-12.855,P＜0.05);与幽门螺杆菌共培养6、24、48 h胃上皮细胞增殖受到抑制,抑制率分别为(35.00±3.22)％、(40.96±2.45)％和(8.00±3.33)％;幽门螺杆菌与胃上皮细胞GES-1共培养24、48 h流式细胞术检测整合素β1表达,空白对照组平均荧光强度(MFI)分别为(1616.33±24.70)和(1834.67±17.01),Hp感染组为(1484.00±60.89)和(1376.00±14.11),差异有统计学意义(t值分别为3.488和35.95,P＜0.05);应用iRNA技术降低胃上皮细胞GES-1整合素β1亚基表达24、48 h,对照组凋亡率分别为(18.75±3.59)％和(39.75±3.68)％,iRNA组凋亡率分别为(26.25±4.11)％和(55.30±5.57)％,差异有统计学意义(t值为-2.746和-4.656,P＜0.05);胃上皮细胞中β1亚基的表达降低后细胞增殖受到抑制,24、36、48、72 h抑制率分别为(6.16±1.07)％、(17.15±2.76)％、(10.84±1.34)％和(21.87±1.72)％.结论 幽门螺杆菌能降低胃上皮细胞GES-1整合素β1亚基的表达,而且可能通过降低整合素β1亚基的表达促进上皮细胞GES-1的凋亡并抑制其增殖.

目的 检测并分析幽门螺杆菌(Hp)尿素酶β亚基(UreB)与Hsp60之间的相互作用. 方法 克隆Hp 26695的尿素酶β亚基基因(UreB)融合GST标签和Hsp60基因融合His标签,分别在大肠埃希菌中进行异源表达.提取两种蛋白,采用pull-down方法检测两者间的相互作用.利用Modeller 9v2软件,以E.coli的GroEL为基础模建HpHsp60的三维结构,再与已知的UreB结构利用AutoDock 4.2软件进行分子共模拟,分析二者的相互作用面和关键氨基酸. 结果 构建了Hp Hsp60和尿素酶β亚基(UreB)的大肠埃希菌异源表达体系并获得纯化蛋白;Pull-down试验显示部分未融合GST标签的Hsp60出现在GST-UreB的洗脱液中,表明Hsp60与GST-UreB之间存在相互作用;以E.coli的GroEL为基础模建了Hp Hsp60的三维结构,利用docking技术构建UreB与Hp Hsp60的相互作用模型,发现其主要的相互作用面位于Hsp60的α9与UreB的α2之间,其可能形成氢键的关键位点为UreB的α2上的T147氨基酸与Hsp60的α9上的E237、K238. 结论 Hp Hsp60能直接与尿素酶β亚基(UreB)相互作用,Hsp60可能通过这种相互作用在UreB成熟过程中发挥分子伴侣功能.这为阐明Hp尿素酶的组装与成熟机制奠定了基础.

Helicobacter pylori infection plays an important role in the etiology of various gastroduodenal diseases. However, pathogenic mechanism of H. pylori is not clear. More potential pathogenic factors need to be further discovered and studied. In this study, two vectors for generating double-crossover recombination gene knockout plasmids in H. pylori were designed based on the backbone of plasmid pLYL03. Genes on plasmid pLYL03 were rearranged, and the redundant sequences were reduced. Erythromycin-resistant gene on pLYL03 was replaced by aphA or catGC to generate the kanamycin-resistant plasmid pSJHK or the chloramphenicol-resistant plasmid pSJHC. The sizes of pSJHK and pSJHC are 4371 and 3949 bp, respectively. Based on plasmids pSJHK and pSJHC, double-crossover recombination gene knockout plasmids targeting hp0169 and hp0788 were constructed, and deletion mutants were achieved by electroporation of the gene-targeting plasmids. The results indicated that plasmids pSJHK and pSJHC are efficient for gene deletion in H. pylori, and the transformation efficiency of pSJHC is slightly higher than pSJHK. These plasmids provide convenient genetic tools for further research of novel pathogenic factors in H. pylori. Derivative plasmids developed by changing antibiotic-resistant genes would also provide valuable tools for the study of functional genes in other bacteria.

Helicobacter pylori infection represents a key factor in the etiology of various gastro-duodenal diseases, ranging from chronic gastritis to the development of peptic ulcer disease and end-stage gastric cancer. In the present study, the 26695 and SS1 strains of H. pylori were used to study the differential functional profiles of gastric epithelial cells infected with H. pylori. The apoptosis rates in GES-1 cells were significantly increased 3, 12 and 24 h after H. pylori 26695 and SS1 infection. Moreover, apoptosis by cells infected with the H. pylori 26695 strain was significantly higher than cells infected with the SS1 strain of H. pylori. No significant changes in the proliferation rates of GES-1 cells were observed after H. pylori 26695 or SS1 infection at any time during the experimental period. Exposure to H. pylori 26695 and SS1 induced a significant decline in the adhesion rates of GES-1 cells in a time-dependent manner. Furthermore, H. pylori 26695 infection increased migration of GES-1 cells every hour during the whole experimental period compared with control cells. However, GES-1 cells infected with the H. pylori SS1 strain exhibited migration rates almost stable and comparable to those of control cells. These results indicate that the gastric epithelial cells respond differently depending on the H. pylori strains. This study indicates that the development of different gastric-related diseases may be a H. pylori strain-specific response.

目的 通过构建小鼠感染模型,研究幽门螺杆菌(Hp)感染对白细胞相关免疫球蛋白样受体1(LAIR-1)表达的影响. 方法 将32只C57BL/6小鼠随机分为对照组和感染组.感染组通过Hp菌液灌胃建立小鼠感染模型,对照组以PBS灌胃.6周后处死小鼠,采用胃黏膜分离培养、革兰染色、PCR及生化反应检查Hp定植情况,采用流式细胞术检测小鼠外周血单个核细胞(PBMC)中单核细胞和淋巴细胞表面LAIR-1的表达. 结果 感染组小鼠Hp感染率为91.3％(21/23).感染组和对照组小鼠PBMC中单核细胞比例分别为(22.85±3.56)％和(49.85±0.35)％,淋巴细胞比例分别为(40.93±10.05)％和(30.15±0.35)％,差异均有统计学意义(t=5.845和2.137,P＜0.05).感染组和对照组小鼠PBMC表面LAIR-1表达率分别为(70.16±7.75)％和(83.4±0.1)％,淋巴细胞表面LAIR-1表达率分别为(56.81±11.37)％和(72.3±0.6)％,差异均有统计学意义(t=3.761和2.491,P＜0.05). 结论 Hp感染下调了小鼠PBMC中单核细胞及LAIR-1的表达,上调淋巴细胞比例但下调其LAIR-1的表达.