Research of 1,3-Dihydroxyacetone Production by Overexpressing Glycerol Transporter and Glycerol Dehydrogenase

1,3-Dihydroxyacetone(DHA), a natural ketose, is widely used in the chemical, cosmetic, and pharmaceutical industries. The current method for DHA production is Gluconobacter oxydans( G. oxydans) fermentation, but the high concentration of glycerol in the fermentation broth inhibits cells growth. To overcome this obstacle, in this study, we overexpressed the glycerol transporter(Glp Fp) by the use of promoters P_(tuf B), P _(gmr), P_(glp1), and P_(glp2) in G. oxydans 621H. The results show that the glycerol tolerances of strains overexpressing Glp F were all much better than that of the control strain. The glycerol dehydrogenase gene( Gdh) was overexpressed by the promoters P_(tufB) and P_(gdh), which increased the DHA titer by 12.7% compared with that of the control group. When Glp F and Gdh genes were co-overexpressed in G. oxydans 621 H, the OD600 value of the engineered strains all increased, but the DHA titers decreased in different degrees, as compared with strains that overexpressed only Gdh. This study provides a reference for future research on DHA production.     

Using oligonucleotide suspension arrays for laboratory identification of bacteria responsible for bacteremia

The aim of this study was to develop and validate an oligonucleotide suspension array for rapid identification of 15 bacterial species responsible for bacteremia, particularly prevalent in Chinese hospitals. The multiplexed array, based on the QIAGEN LiquiChip Workstation, included 15 oligonucleotide probes which were covalently bound to different bead sets. PCR amplicons of a variable region of the bacterial 23S rRNA genes were hybridized to the bead-bound probes. Thirty-eight strains belonging to 15 species were correctly identified on the basis of their corresponding species-specific hybridization profiles. The results show that the suspension array, in a single assay, can differentiate isolates over a wide range of strains and species, and suggest the potential utility of suspension array system to clinical laboratory diagnosis.     

犬冠状病毒TN449株纤突蛋白基因的克隆、序列分析

通过对犬冠状病毒TN449株纤突蛋白基因的克隆测序,将获得的序列与GeneBank中登陆的犬冠状病毒不同毒株S基因序列进行比较,分析它们的同源性、差异程度,同时绘制核苷酸序列及其氨基酸序列的系统进化树.有助于了解犬冠状病毒的抗原变异、血清型和毒力变化规律.     

原位RT-PCR检测Ig A样新基因SNC66在消化系统肿瘤组织中的表达

目的：探讨原位检测内源性基因表达产物的灵敏方法，研究IgA样新基因SNC66在消化系统肿瘤组织中的表达及其与肿瘤之间的相关性。方法：在组织切片原位逆转录反应后进行原位PCR扩增，在扩增过程中掺入地高辛标记的尿核苷酸，再用免疫组织化学的方法加以检测。比较SNC66在大肠癌、胃癌、肝癌组织与相应的正常配对组织中的表达情况。结果：10个循环时，37例大肠癌标本中有14例呈阴性，18例弱阳性，5例强阳性；20例胃癌标本中有6例呈阴性，9例弱阳性，5例强阳性；所有肝脏组织和肝癌标本都呈阴性表达。25个循环时，大肠癌10例阴性，27例强阳性；胃癌4例阴性，16例强阳性；所有肝脏组织和肝癌组织标本都呈阳性表达。相应大肠粘膜和胃粘膜标本则无论是25个循环，还是10个循环，都呈强阳性表达，但着色程度前者高于后者。结论：原位RT-PCR是一种检测内源性基因低拷贝转录产物mRNA的灵敏方法。基因SNC66是一个消化道肿瘤相关性基因，在大肠癌和胃癌组织中存在明显的表达降低或缺陷，但与肝癌之间不存在相关性。SNC66可作为侯选的消化道肿瘤的抑癌基因加以进一步研究。     

A molecular biological study on identification of common septicemia bacteria using 16s–23s rRNA gene spacer regions

In the search for a rapid and reliable method for identification of bacteria in blood and cerebrospinal fluid , we developed a unified set of primers and used them under polymerase chain reaction(PCR) to amplify the spacer regions between the 16s and 23s genes in the prokaryotic rRNA genetic loci . Spacer regions within these loci showed a significant level of length and sequence polymorphism across most of the species lines. A generic pair of priming sequences was selected from highly conserved sequences in the 16s and 23s genes occurring adjacent to these polymorphic regions. This single set of primers and reaction conditions were used for the amplification of the 16s-23s spacer regions for 61 strains of standard bacteria and corresponding clinical isolates belonging to 20 genera and 27 species, including Listeria, Staphylococcus and Salmonella species, et al. When the spacer amplification products were resolved by electrophoresis, the resulting patterns could be used to distinguish most of the bacteria species within the test group, and the amplification products of the clinical isolates clustered at the standard species level. Some species presenting similar pattern were further analyzed by HinfI or AluI digestion or DNA clone and sequences analysis in order to establish the specific 16s-23s rRNA gene spacer regions map. Analysis of 42 blood specimens from septicemic neonates and 6 CSF specimens from suspected purulent meningitis patients by bacterial culture and PCR-RFLP(Restriction Fregament Length Polymorphism) showed that 15 specimens of blood culture were positive(35.7%) in the 42 septicemic neonates; 27 specimens were positive(64.2%) by PCR, and that the positive rate by PCR was significantly higher than that by blood culture(P<0.01). Among the 6 CSF specimens, one specimen found positive by blood culture was also positive by PCR, two found negative by blood culture showed positive by PCR; all three were S.epidermidis according to the DNA map. One C.neoformans found positive by blood culture showed negative by PCR. The remaining two specimens were both negative by PCR and blood culture. These results indicated that the method of detecting bacterial 16s-23s rRNA spacer regions using PCR and RFLP techniques was rapid, sensitive and specific in the detection of bacterial infections; and so, has very important application in the clinical diagnosis of sepsis in neonates.     

Construction of recombinant industrial Saccharomyces cerevisiae strain with bglS gene insertion into PEP4 locus by homologous recombination

The bglS gene encoding endo-1,3-1,4-β-glucanase from Bacillus subtilis was cloned and sequenced in this study. The bglS expression cassette, including PGK1 promoter, bglS gene fused to the signal sequence of the yeast mating pheromone α-factor (MFals), and ADH1 terminator with G418-resistance as the selected marker, was constructed. Then one of the PEP4 allele of Saccharomyces cerevisiae WZ65 strain was replaced by bglS expression cassette using chromosomal integration of polymerase chain reaction (PCR)-mediated homologous recombination, and the bglS gene was expressed simultaneously. The recombinant strain S. cerevisiae (SC-βG) was preliminarily screened by the clearing hydrolysis zone formed after the barley β-glucan was hydrolyzed in the plate and no proteinase A (PrA) activity was measured in fermenting liquor. The results of PCR analysis ofgenome DNA showed that one of the PEP4 allele had been replaced and bglS gene had been inserted into the locus of PEP4 gene in recombinant strains. Different endo-1,3-1,4-β-glucanase assay methods showed that the recombinant strain SC-βG had high endo-1,3-1,4-β-glucanase expression level with the maximum of 69.3 U/(h-ml) after 60 h of incubation. Meanwhile, the Congo Red method was suitable for the determination of endo-1,3-1,4-β-glucanase activity during the actual brewing process. The current research implies that the constructed yeast strain could be utilized to improve the industrial brewing property of beer.     

Cloning and GST-fused expression in E. coli of mouse β-1,4-galactosyltransferase

INTRODUCTION b-1,4-galactosyltransferase (b4Gal-T) (EC 2.4.1.38), as one of the most researched glycosyl-transferases in recent years, produces a b-1,4-linked galactosylated glycan by the transferation of ga-lactose (Gal) from an uridine diphosphate-galactose (UDP-Gal) to a monosaccharide N-acetylglucosa- mine (GlcNAc) or a GlcNAc residue located in the non-reducing terminal of glycans of glycoproteins or glycolipids. In addition to GlcNAc, the enzyme can also use other reducing su…     

向日葵TiT—DNA转化系几个生理生化特性的研究

根癌农杆菌B6S3和T37菌株Ti质粒T-DNA基因(TiT-DNA)在向日葵基因组中的导入与表达,不仅使转化细胞产生特殊的基因产物,并且使之能将D-乳糖作为唯一碳源利用和对某些代谢类似物(BUdR)具有抗性,此外还影响宿主细胞一系列生理生化活动。在无激素和含激素两种培养条件下,向日葵TiT-DNA转化系与相应正常系在过氧化物酶的活性及其同工酶基因的表达、蛋白质的种类及比例、游离氨基酸库等均存在着较大的差异。在转化系内,有关冠瘿碱生物合成的前休氨基酸具有较高的代谢水平。     

绿僵菌Argonaute基因片段原核表达载体的构建及其表达

绿僵菌是一类广泛应用于生物防治的昆虫病原真菌.研究表明小RNA能够调控基因的表达,其中Argonaute基因在整个小RNA通路中发挥重要的作用.本研究通过RT-PCR的方法从绿僵菌中获得了Argonaute基因的部分功能片段,构建了其重组原核表达载体,将重组载体转化至大肠杆菌进行诱导表达;采用镍柱亲和纯化重组的目的蛋白并通过Western blot技术鉴定.结果发现：通过RT-PCR的方法获得长度约为950bp的基因片段;重组原核表达载体经诱导表达后,SDS-PAGE检测发现分子量约为34kDa的目的蛋白条带;诱导5h后蛋白的表达量最高,采用镍柱亲和层析纯化重组蛋白,经Western blot技术检测,重组蛋白可与His-tag抗体发生特异性反应.该纯化重组蛋白的获得为将来绿僵菌Argonaute蛋白抗体的制备,并进一步通过该抗体获得绿僵菌体内的小RNA及其靶基因提供了基础.     

牛奶中苏云金芽胞杆菌的分离与鉴定

采用醋酸钠-抗生素分离法对超市牛奶样品进行苏云金芽胞杆菌(Bacillus thuringiensis,Bt)的分离,获得2株Bt菌株,分别命名为BtBRC-LJ1和BtBRC-LJ2。利用PCR方法对BtBRC-LJ1和BtBRC-LJ2菌株hblD和nheC肠毒素基因的分布进行检测。结果显示,这2个菌株均含有这两种肠毒素基因。     

西文蜜蜂LOC724521基因座的cDNA序列克隆及TSS的分析

5’LongSAGE标签得自于全长mRNA分子的5’末端的前19 nt.该研究利用定位在西方蜜蜂基因组中的一个预测基因座LOC724521的2条5’LongSAGE标签序列作为5’引物,通过RT-PCR克隆了该预测基因座的2条长335 bp和337 bp的cDNA序列（GenBank登录号：GU358205,GU358204）.此cDNA编码一条长88个氨基酸残基的多肽.用所克隆的cDNA序列对基因座LOC724521进行功能注释发现,该基因含有3个外显子和2个＂GT-AG＂型内含子.5’LongSAGE标签序列的基因组定位结果显示：基因座LOC724521在雄蜂的头部中表达丰度很高,RNA PolⅡ可从5个转录起始位点（TSS）上以不同效率起始转录,该基因的59%和31%的转录是从2个优势TSS上起始.有趣的是,有一条5’LongSAGE标签序列被定位在内含子区,暗示该基因存在外显子的可变性选择现象.该研究结果不仅在转录水平上证实了软件预测的基因座LOC724521确实存在,同时揭示了该基因存在可变性转录起始位点和可变性外显子选择等转录调控机制.     

基于PCR技术的RGAP分子标记研究进展

基于PCR技术的RGAP分子标记,由于其本身所具有的特点和优越性爱到大家的广泛关注,为近年来分子生物学领域的研究开辟了一个新途径.目前主要应用在克隆和定位抗病基因、分子标记辅助育种、系统植物学研究等方面.本文就RGAP法的基本原理、优越性、目前研究的现状、进展以及研究中还存在的问题分别作以阐述,以期使人们对此标记有个系统的认识.     

Analysis of the 3' ends of tRNA as the cause of insertion sites of foreign DNA in Prochlorococcus

The purpose of this study was to investigate the characteristics of transfer RNA(tRNA) responsible for the association between tRNA genes and genes of apparently foreign origin(genomic islands) in five high-light adapted Prochlorococcus strains.Both bidirectional best BLASTP(basic local alignment search tool for proteins) search and the conservation of gene order against each other were utilized to identify genomic islands,and 7 genomic islands were found to be immediately adjacent to tRNAs in Prochlorococcus marinus AS9601,11 in P.marinus MIT9515,8 in P.marinus MED4,6 in P.marinus MIT9301,and 6 in P.marinus MIT9312.Monte Carlo simulation showed that tRNA genes are hotspots for the integration of genomic islands in Prochlorococcus strains.The tRNA genes associated with genomic islands showed the following characteristics:(1) the association was biased towards a specific subset of all iso-accepting tRNA genes;(2) the codon usages of genes within genomic islands appear to be unrelated to the codons recognized by associated tRNAs;and,(3) the majority of the 3' ends of associated tRNAs lack CCA ends.These findings contradict previous hypotheses concerning the molecular basis for the frequent use of tRNA as the insertion site for foreign genetic materials.The analysis of a genomic island associated with a tRNA-Asn gene in P.marinus MIT9301 suggests that foreign genetic material is inserted into the host genomes by means of site-specific recombination,with the 3' end of the tRNA as the target,and during the process,a direct repeat of the 3' end sequence of a boundary tRNA(namely,a scar from the process of insertion) is formed elsewhere in the genomic island.Through the analysis of the sequences of these targets,it can be concluded that a region characterized by both high GC content and a palindromic structure is the preferred insertion site.     

用5＇ LongSAGE标签分析蜜蜂Yps基因转录起始位点的多样性

使用5＇ LongSAGE标签序列确定了一个新的西方蜜蜂Ypsilon Schachtel（Yps）基因的转录起始位点,并进而预测了该基因的启动子序列．5＇ LongSAGE标签的蜜蜂基因组定位结果表明：在成年雄蜂的头部中,蜜蜂Yps基因存在23个转录起始位点,其中优势转录起始位点有4个,其起始频率分别为27．9％、23．1％、13．5％和11．5％．Yps基因TSS的碱基组成分析发现,此23个TSS第一个碱基为A、G、T、C的概率分别为52％、39％、4%、4％．这些结果暗示RNA聚合酶和调控因子在Yps基因启动子的一个较宽范围内的互作控制着不同转录本的起始效率．