泡桐丛枝植原体16S rDNA和延伸因子基因序列分析

对采自陕西、山西、甘肃、河南、河北、山东各省的泡桐丛枝病材料,利用巢式扩增,均得到16S rDNA基因片段约1.2kb;扩增得到植原体延伸因子(EF-Tu)tuf基因,长度约为850bp.。通过将16S rDNA基因片段和延伸因子(EF-Tu)tuf基因序列与已知植原体16Sr各组成员进行同源性比较,确定我国大陆泡桐主栽区陕西、山西、甘肃、河南、河北、山东各省与已经报道的台湾省泡桐丛枝植原体基本一致,均为同一个种,没有株系的分化,全部归属于植原体16SrI-D组,从而确定了其分类地位。     

假臭草从枝病植原体鉴定与多样性分析

研究假臭草丛枝病植原体的多样性,并确定其分类地位,对于利用假臭草丛枝病植原体对假臭草进行生物防治具有重要的意义.本研究采集了海南省8个地区的假臭草丛枝病样品,采用PCR以及巢式PCR方法扩增了假臭草丛枝病植原体的16S rDNA序列片段,进一步选用AfaⅠ、Alu Ⅰ、EcoR Ⅰ、HaeⅢ、HpaⅡ、HhaⅠ、HinfⅠ、Kpn Ⅰ、Sau3A Ⅰ、Taq Ⅰ和Xsp Ⅰ等11种限制性内切酶对巢式PCR产物进行酶切分析(RFLP),并对16S rDNA序列进行测序,确定假臭草丛枝病植原体多样性和生物学地位.结果发现:假臭草丛枝病的病原确为植原体;8个地区的假臭草丛枝病植原体的酶切图谱基本一致,与已知植原体的相似度为0.26～0.97;8个地区假臭草丛枝病植原体的序列同源性均在99.4％以上,应为同种植原体;假臭草丛枝病植原体与16S rRNAⅡ-A组的花生丛枝病(PnWB)的同源性最高达到99.1％,说明假臭草丛枝病植原体在分类学地位上应归属于植原体16S rRNAⅡ-A组.     

链状亚历山大藻共附生细菌多样性

采用非分离培养分析方法,直接提取链状亚历山大藻（Alexandrium catenella）共附生细菌总DNA,以之为模板进行PCR扩增获得细菌16S rDNA基因片段,并构建16S rDNA克隆文库。通过16S rDNA限制性酶切片段长度多态性（restriction fragment length polymorphism,RFLP）和测序方法对链状亚历山大藻共附生细菌的多样性进行了研究。84个16S rDNA克隆片段经限制性内切酶HaeⅢ酶切分析,得到30种不同的酶切指纹类型。挑选50个克隆子进行测序获得其16S rDNA部分序列,并对16S rDNA序列进行聚类分析构建了系统进化树。结果表明,链状亚历山大藻共附生的细菌多样性较强,优势细菌类群为变形菌α亚群（α-Proteobacteria）和拟杆菌（Bacteroidetes）,其中玫瑰杆菌（Roseobacter sp.）在α-Proteobacteria中占绝对优势。     

柳树黄化病植原体的分子分类

[目的]柳树黄化病是一种重要的植原体病害,本研究旨在明确柳树黄化病植原体(Willow Yellow phytoplasma,WY)的分类地位,为进一步开展致病性和防治研究奠定基础.[方法]采用植原体特异引物通过PCR方法从患病植株DNA中扩增植原体16S rDNA基因和核糖体蛋白基因(ribosomal proteins gene,rp),对所得的序列进行分析,构建同源进化树,并用限制性片段长度多态性(RFTJP)对巢式PCR产物进行分析.[结果]首次从柳树黄化病植原体中分离出了16S rDNA基因和rp基因,大小分别为1246 bp和1212 bp.通过对植原体16S rDNA和rp基因的核苷酸同源性比较和RFLP分析,发现该分离物与16S rI组的核苷酸同源性均在99%以上,与16S rI-C亚组中的小麦蓝矮病植原体同源性高达99.8%(16Sr DNA)和99.6%(rp),且RFLP分析与16SrI-C亚组的植原体有相同的酶切条带.[结论]柳树黄化植原体应划分于16SrI-C亚组.     

新疆小叶白蜡丛枝病植原体的鉴定及16S rRNA基因序列分析

【目的】对新疆小叶白蜡丛枝病植原体进行检测,通过其16S rRNA基因分析确定其分类地位。【方法】利用苯胺蓝和4′,6-二脒基-2-苯基吲哚(DAPI)染色,在荧光显微镜下观察新疆小叶白蜡嫩茎横切片;采用植原体16S rRNA基因的通用引物对P1/P7和R16F2n/R16R2进行直接和巢式PCR扩增,对得到的16S rRNA基因的序列进行RFLP和构建系统进化树分析。【结果】表现丛枝病症状的新疆小叶白蜡中存在植原体,暂命名为Fraxinus sogdianaBunge witches’broom phytoplasma(Fraxinus sogdianaBunge WB);其16S rRNA基因的序列GenBank登录号为KF061042,RFLP图谱与16Sr V-B亚组的枣疯病植原体相同,系统进化地位与枣疯病菌株AB052876相同。【结论】新疆小叶白蜡丛枝病植原体为16Sr V-B亚组成员。     

坡柳(Dodonaea viscosa L.)丛枝植原体新病原的分子鉴定

从四川攀枝花芒果园中表现为丛枝、小叶和黄花等症状的坡柳植株发病样品中,利用植原体16S rDNA基因的通用引物R16m F2/R16m R1和R16F2n/R16R2,对发病植株总DNA进行巢式PCR检测,同时设计植原体抗原膜蛋白基因(AntMP)的保守引物AntMP-F/AntMP-R进行PCR验证。结果显示,坡柳样品巢式PCR的第一轮、第二轮DNA条带大小分别为1 400 bp和1 200 bp左右,经NCBI序列相似性比较均为植原体16S rDNA序列,Gen Bank登录号为KT957205和KT957206;PCR结果显示抗原膜蛋白基因大小约600 bp,与目标基因大小一致。将测得的16S rDNA基因序列与Gen Bank数据库中登录的16SrⅠ~ⅩⅤ组植原体16S rDNA序列进行同源性比对,构建系统进化树,结果显示四川坡柳丛枝植原体(DOVI-SC)属于16SrⅠ组(即翠菊黄花组),与已报道的5个16SrⅠ组(AY101386,AY566302,AY389822,L33760和KP662119)同属一个组。利用植原体亚组分类鉴定软件iPhy Classfier对获得的2条植原体16S rDNA序列进行虚拟RFLP分析,结果显示KT957205,KT957206与16SrⅠ-B亚组洋葱黄化植原体(NC-005303)相似度分为1.0、0.97,归属于16SrⅠ-B亚组。本研究对引起四川坡柳丛枝的植原体病原进行检测,为坡柳植原体病害的早期诊断、快速检测以及预防措施制定提供科学线索。     

新疆罗布泊地区可培养嗜盐细菌多样性

采用纯培养方法从新疆罗布泊盐湖中分离得到168株嗜盐细菌, 采用核糖体DNA扩增片段限制性酶切分析(ARDRA)研究了罗布泊嗜盐细菌的群落结构和多样性。通过限制性内切酶HinfI对108个菌株的16S rDNA进行酶切分型, 根据ARDRA的酶切图谱, 将其划分为12个操作分类单元。16S rDNA序列测定和系统发育分析结果显示, 分离菌株分布于喜盐芽孢杆菌属(Halobacillus)、芽孢杆菌属(Bacillus)、短杆菌属(Brevibacterium)、嗜盐单胞菌属(Halomonas)、色盐杆菌属(Chromohalobacter)、盐水球菌属(Salinicoccus)、库克菌属(Kocuria)、葡萄球菌属(Staphylococcus)、微球菌属(Micrococcus)等9个属及1个可能的新分类单元。其中Halomonas为优势菌群, Chromohalobacter、Halobacillus为次优势菌群。采用ERIC-PCR分析优势菌群Halomonas各菌株的基因组特征, 显示出有21种指纹图谱。研究结果揭示, 罗布泊嗜盐细菌不仅具有丰富的多样性, 还蕴藏着具有地域特点的新菌种资源。     

油藏水样细菌群落16S rRNA基因的RFLP分析

通过16S rRNA基因的限制性酶切片段长度多态性分析(RFLP)方法,考察了油藏水样中细菌群落及多样性。从水样中分离纯化微生物总DNA,选择性扩增细菌16S rRNA基因,并构建16S rDNA克隆文库。337个16S rDNA克隆片段分别用限制性内切酶HinfⅠ和HaeⅢ酶切分析,得到74个操作分类单元(OTUs),其中数量最多的4个OTUs共占克隆子总数的73.6%,另外70个OTUs的丰度均处于较低水平,有57个OTUs仅含有1个克隆子。结果表明,运用RFLP方法分析16S rDNA克隆片段能够有效评估油藏水样中的细菌群落和多样性。     

用AFLP技术和16S rDNA PCR\|RFLP分析毛苜蓿根瘤菌的遗传多样性

采用选择性扩增片断长度多态性(简称AFLP)DNA指纹技术对采自我国云南省与西藏交界的高山地区的野生型豆科植物毛苜蓿根际土样分离的291株毛苜蓿(Medicago edgeworthii)根瘤菌进行遗传多样性的研究。从AFLP图谱中,揭示出毛苜蓿根瘤菌有较显著的遗传多样性,从291株中选择出90个代表株用计算机进行树状图的分析。结果表明,所分析的菌株在79%的相似性水平上聚类成3个群。对这90个代表株进行多聚酶链反应(PCR)扩增的16S rDNA的4种限制性内切酶长度多态(简称16S rDNA PCR\|RFLP)分析,得出2个不同的16S rDNA PCR\|RFLP类型的菌株。分别选出这2个类型的代表菌株与各种根瘤菌的参比菌株进行16S rDNA PCR\|RFLP分析,再进行树状图的分析,初步得出了它们在根瘤菌系统分类中的地位。分析结果表明：毛苜蓿根瘤菌与根瘤菌属中的Rhizobium mongolense的相似性很高。     

泰安枣疯病植原体的分子鉴定

【目的】对3个枣疯病病原物泰安株系进行分子鉴定。【方法】采用植原体通用引物对R16F2n/R16R2,通过直接PCR技术,扩增枣疯病植原体16S rDNA基因,通过16S rDNA基因序列分析和在线模拟16S rDNA-RFLP分析,并将其16S rDNA基因序列提交到GenBank数据库。【结果】3个枣疯病病原物16S rDNA基因片段与16SrⅤ-B亚组中枣疯病植原体(AB052876和AF279272)、樱桃致死黄化植原体(AY197659)及杏卷叶植原体(FJ572660)的同源性高达99.5%99.7%,分别命名为枣疯病植原体泰安圆铃1号株系(Jujube witches’-broom phytoplasma strain Yuanling1,JWB-Yuanling1,TA)、枣疯病植原体泰安鲁北冬枣株系(Jujube witches’-broom phytoplasma strain Lubeidongzao,JWB-Lubeidongzao,TA)和枣疯病植原体泰安大白铃株系(Jujube witches’-broom phyto-plasma strain Dabailing,JWB-Dabailing,TA),基因登录号分别为:HM989946、HM989947和HM989948。【结论】3个枣疯病植原体泰安株系均归属于16SrⅤ-B亚组。     

白蚁与动物及微生物的共生类型及其机制

综述了白蚁螱客的主要种类、共生关系及相关机制的研究进展。白蚁螱客中,已报道的动物种类达170种。在与动物的共生关系中存在偏利共生（宾主共栖和异种共栖）、互利共生和无关共生三种;在与微生物的共生关系中,存在与内生菌（原生动物、细菌、真菌和放线菌）和外生菌（蚁巢伞菌等）间的互利关系。指出了白蚁与螱客研究中存在的问题,给出了解决方案,并提出了今后可能的研究热点或方向,为白蚁的综合利用（如纤维素酶）及今后研究物种间的协同进化提供了基础资料。     

New amino-dithiaphospholanes and phosphoramidodithioites and their rhodium and iridium complexes

New sulfur derivatives of phosphoramidite ligands were synthesized and the impact of the sulfur unit on the spectroscopic properties of their rhodium and iridium complexes was investigated. The new ligands Bn₂NPSCH₂CH₂S^a(P-S^a) (Bn = benzyl, 4), Bn₂NPSCHCHS^a(CH₂)₃C^aH₂(P-S^a)(C^a-S^a) (6) and Bn₂NP(4-XC₆H₄OMe)₂ (X = S, 7a; X = O, 7b) were converted to the rhodium and iridium complexes trans-[Rh(CO)Cl(L)₂] (L = 4, 6, 7), [RhCl(COD)(L)] (L = 4, 6, 7), [IrCl(COD)(7a)] and [IrCl₂Cp∗(6)]. For comparison, some phosphoramidite complexes of these formulations also were synthesized. The new metal complexes were spectroscopically analyzed. For the carbonyl complexes, the ν_CO IR stretching frequencies were lower than for the corresponding phosphite and phosphoramidite ligands. The ¹J_PRh coupling constants for the rhodium complexes with the new ligands were also smaller than for the respective phosphoramidite and phosphite complexes. Finally, the ¹J_PSe coupling constants of the selenides of the new ligands were lower than those of the phosphoramidite ligands but higher than for PPh₃. The spectroscopic data reveal that the new thio ligands 4, 6 and 7a are more electron donating than phosphites and phosphoramidites but less electron donating than PPh₃.     

Astrocytes and Synaptosomes Transport and Metabolize Lactate and Acetate Differently

Astrocytes transport the monocarboxylate acetate, but synaptosomes do not. The reason for this is unknown, because both preparations express monocarboxylate transporters (MCT). The transport and metabolism of lactate, another monocarboxylate, was examined in these two preparations, and the results were compared to those for acetate. Lactate transport is more rapid in astrocytes than in synaptosomes, but of lower affinity (Kms of 17 and 4 mM, respectively). Lactate (0.2 mM) is metabolized to CO2 more rapidly in synaptosomes than in astrocytes (rates of 0.37 and 0.07 nmol x mg protein(-1) x min(-1), respectively). The reason for this is unclear, but cellular differences in lactate dehydrogenase isotype expression may be involved. Acetate is metabolized to CO2 more rapidly in astrocytes than in synaptosomes (rates of 0.43 and 0.02 nmol x mg protein(-1) x min(-1), respectively). This is likely due to cellular differences in the expression of monocarboxylate transporter subtypes.     

Rapporteur report: Basics and technology and metrology and standards

The first and second sessions of the Workshop focussed on the basics of ultrasound and infrasound, their applications in both industry and medicine, and metrology and protection standards for ultrasound applications.     

Relative and combined effects of ethanol and protein deficiency on strontium and barium bone content and fecal and urinary excretion

To elucidate accumulation of minerals in human iliac arteries with aging, the content of minerals was analyzed by inductively coupled plasma atomic emission spectrometry. Bilateral common, internal, and external iliac arteries of 16 men and 8 women, ranging ages from 65 to 93 yr, were examined. It was found that an extremely high accumulation of calcium and phosphorus occurred in the common iliac artery at old age, being higher than that of the internal and external iliac arteries. It should be noted that the accumulation of calcium and phosphorus is the highest in the common iliac artery among the human arteries examined to date. Regarding sexual differences, the content of calcium and phosphorus in the common and internal iliac arteries was higher in women than in men, whereas their content in the external iliac artery was lower in women than in men.     

Cytoskeleton and mitochondrial morphology and function

It has been well established that the cytoskeleton is an essential modulator of cell morphology and motility, intracytoplasmic transport and mitosis, however cytoskeletal linkage to the organelles has not been unequivocally demonstrated. Indeed, cytoskeleton appears to be essential in determining and modulating gene phenotype as a function of cellular environment. According to recent studies, the organization of the cytoskeleton network together with associated protein(s) could be essential in regulating mitochondrial function and particularly the permeability of the mitochondrial outer membrane to ADP. The aim of this chapter is to summarize the main properties of the cytoskeletal environment of mitochondria and the possible role(s) of this network in mitochondrial function in myocytes.     

Cross-reactivity between storage and dust mites and between mites and shrimp

Many patients have sensitivities to multiple species of storage and house dust mites. It is not clear if this is because patients have multiple sensitivities to species-specific mite allergens or if these mites share many cross-reacting allergens. Our objective was to further define the cross-allergenicity between several species of storage and house dust mites using crossed-immunoelectrophoresis (CIE), crossed-radioimmunoelectrophoresis (CRIE), immunoblotting, and ELISA. CIE and CRIE reactions revealed that storage mites shared two cross-antigenic molecules and one of these bound IgE in a serum pool from mite allergic patients. Antibody in anti-sera built to each species of mite recognized many SDS–PAGE resolved proteins of other mite species and this suggested the potential for other cross-reactive allergens. Among patient sera, IgE bound to many different proteins but few had IgE that bound to a protein with common molecular weights across the mite species and this suggested mostly species-specific allergens. Antiserum built to each mite species precipitated one protein in shrimp extracts that bound anti-Der p 10 (tropomyosin) and IgE in the serum pool. Anti-Der p 10 showed strong binding to shrimp tropomyosin but very little to any of the mite proteins. ELISA showed the mite extracts contained very little tropomyosin. The storage and dust mites investigated contain mostly species-specific allergens and very small amounts of the pan-allergen tropomyosin compared to shrimp and snail.     

人血管能抑素基因的克隆、表达及其表达产物的纯化和生物活性测定

以人胎盘脐带组织为材料,提取组织总RNA,用netRTPCR方法合成人血管能抑素cDNA基因,将该cDNA克隆进pSP72载体获得重组质粒pSP72C, DNA序列分析结果与预期序列一致。用BamHⅠ和NdeⅠ双酶切,切下pSP72C上的血管能抑素cDNA,插入pET3c载体的相应位点获得重组表达质粒pETC, 转化E. coli BL21(DE3), SDSPAGE分析显示：在IPTG诱导下,血管能抑素基因获得了高效表达,表达量约占菌体总蛋白的 27.9 %,主要以包涵体形式存在。包涵体经过洗涤、裂解、蛋白复性以及Sephadex G75凝胶过滤层析等步骤后,获得了纯度达91.4 %的人血管能抑素。CAM实验证明10 μg纯化蛋白就能显著抑制鸡胚新生血管生成。     

Vascular volumes and hematology in male and female runners and cyclists

To examine the hypothesis that foot-strike hemolysis alters vascular volumes and selected hematological properties is trained athletes, we have measured total blood volume (TBV), red cell volume (RCV) and plasma volume (PV) in cyclists (n = 21) and runners (n = 17) and compared them to those of untrained controls (n = 20). TBV (ml x kg(-1)) was calculated as the sum of RCV (ml x kg(-1)) and PV (ml x kg(-1)) obtained using 51Cr and 125I-labelled albumin, respectively. Hematological assessment was carried out using a Coulter counter. Peak aerobic power (VO2peak) was measured during progressive exercise to fatigue using both cycle and treadmill ergometry. RCV was 15% higher (P < 0.05) in male cyclists [35.4 (1.0), mean (SE); n = 12] and runners [35.3 (0.98); n = 9] compared to the controls [30.7 (0.92); n = 12]. Similar differences existed between the female cyclists [28.2 (2.1); n = 9] and runners [28.4 (1.0); n = 8] compared to the untrained controls [24.9 (1.4); n = 8]. For the male athletes, PV was between 19% (cyclists) and 28% (runners) higher (P < 0.05) in the trained athletes compared to the untrained controls. The differences in PV between the female groups were not significant. Although the males had a higher (P < 0.05) TBV, RCV and PV than the females, no differences between cyclists and runners were found for either gender. Mean cell volume was not different between the athletic groups. VO2peak (ml x kg(-1) x min(-1)) was higher (P < 0.05) in both male [68.4 (1.5)] and female [54.8 (2.1)] runners when compared to the untrained males [47.1 (1.0)] and females [40.5 (2.1)]. Although differences existed between the genders in VO2peak for both cyclists and runners, no differences were found between the athletic groups within a gender. Since the vascular volumes were not different between cyclists and runners for either the males or females, foot-strike hemolysis would not appear to have an effect on that parameter. The significant correlations (P < 0.05) found between VO2peak and RCV (r = 0.64 and 0.64) and TBV (r = 0.82 and 0.63) for the males and females, respectively, suggests a role for the vascular system in realizing a high aerobic power.