苹果砧木SH40 MdNCED1基因克隆与表达分析

9-顺式-环氧类胡萝卜素双加氧酶基因是脱落酸合成途径的关键基因。本试验通过RT-PCR结合RACE技术从苹果SH40(Malus domestica×Malus Honanensis)茎尖组织中克隆了1条NCED基因,命名为MdNCED1(GenBank登录号为KC816734)。该cDNA序列全长2179 bp,包含1个编码606个氨基酸的开放阅读框。氨基酸同源性分析表明,MdNCED1与已报道的其他植物物种的氨基酸序列具有63.7%~93.0%的相似性。构建MdNCED1基因的原核表达载体pDEST15-MdNCED1,转入大肠杆菌(DE3),用IPTG诱导。SDS-PAGE分析表明,MdNCED1基因在大肠杆菌中被诱导表达的蛋白质分子量与预期结果一致。荧光定量结果表明,MdNCED1基因在SH28、M26、SH40及其嫁接品种嘎啦的表达趋势均呈先上升后下降的趋势,同时与其矮生程度呈正相关。     

天祝白牦牛IGF-2基因的cDNA克隆

旨在克隆天祝白牦牛胰岛素样生长因子2(IGF-2)基因编码区全长cDNA序列,为研究该基因的生理功能奠定基础。运用cDNA末端快速扩增(RACE)技术获得天祝白牦牛IGF-2基因全长cDNA序列。扩增获得天祝白牦牛IGF-2基因全长cDNA序列为1 060 bp(GenBank登录号:KF682139),ORF长540 bp,编码179个氨基酸。其编码的氨基酸与已报道哺乳动物IGF-2氨基酸序列同源性在80%-92%之间。天祝白牦牛IGF-2基因的成功克隆为进一步研究该基因的功能奠定了基础。     

银杏EPSPS基因克隆及表达分析

利用RACE技术,克隆到银杏EPSPS合酶基因(GbEPSPS)的cDNA序列并对其进行生物信息学分析.结果表明:银杏GbEPSPS cDNA长1 403 bp(GenBank登录号GU084139),其包含一个1 035 bp的ORF框,编码344个氨基酸;预测该基因编码蛋白质分子量为36.87 kD,其等电点为5.75.系统进化树分析表明,银杏EPSPS蛋白质序列与其他物种的EPSPS同源性较高.半定量RT-PCR分析结果显示,EPSPS基因在银杏的叶和果实中表达量最高,其次为茎,根中表达水平最低.草甘膦处理能显著诱导银杏EPSPS基因表达量升高;紫外光对银杏EPSPS基因的表达具有诱导作用;ABA诱导GbEPSPS表达量先升后降;GbEPSPS转录水平受到42℃高温显著诱导.     

荔波连蕊茶GA20氧化酶基因的克隆及表达分析

根据植物GA20ox基因编码区的保守序列设计引物,以山茶属荔波连蕊茶幼嫩茎段为材料,提取总RNA,进行RT-PCR。采用RACE技术扩增获得1 567 bp的GA20氧化酶基因全长cDNA序列,命名为ClGA20ox2(GenBank登录号KF823787)。序列分析表明,ClGA20ox2开放阅读框(ORF)为1 146 bp,编码382个氨基酸,5'非编码区115 bp,3'非编码区303 bp。预测的蛋白质分子量为43.56 kD,等电点为7.02,所推导的蛋白氨基酸序列与夹竹桃和杨树GA20ox蛋白的同源性分别为73%和72%。ClGA20ox2与其它植物GA20ox蛋白比较,构建系统进化树,结果显示山茶GA20ox蛋白与夹竹桃和杨树的GA20ox蛋白的亲缘关系最为密切。实时定量PCR结果显示,该基因在荔波连蕊茶的根、茎、叶和种子中均有表达,其表达模式却不同:ClGA20ox2基因在二年生茎段中的表达丰度最高,在顶端分生组织中表达丰度最低,在嫩叶和根中表达丰度较高,成熟叶片和种子表达丰度较低。     

黑曲霉NRRL3135 bgl基因的克隆及序列分析

利用RT-PCR技术从黑曲霉菌株NRRL3135中扩增了β-葡萄糖苷酶(BGL)基因的全长cDNA序列,被命名为bgl3135(GenBank登录号:FN430671)。对该基因编码的BGL进行了同源性分析并预测了其三维构象。试验结果表明,bgl3135的全长cDNA为2598bp,含有一个2583bp的开放阅读框(ORF),编码一个长度为860个氨基酸残基的蛋白质。同源性分析表明,该基因编码的蛋白与来自黑曲霉As3.350和CBS513.88的BGL(GenBank登录号分别为DQ655704和XM_001398779)的相似性最高,其氨基酸序列同源性分别达99%。参照已发表的β-葡萄糖苷酶(BGL)的氨基酸保守序列,判断本研究所克隆的bgl基因属于糖苷水解酶基因家族3的成员。三维结构预测表明,该BGL的立体构象中存在20个α-螺旋和15个β-折叠,对构成和维持酶的底物识别和催化活性位点可能起着重要作用。     

八肋游仆虫核糖体蛋白L11的基因克隆与序列分析

以单细胞真核生物八肋游仆虫Euplotes octocarinatus为实验材料,采用PCR、RT-PCR方法克隆了核糖体蛋白L11基因(EoRPL11).将该序列与GenBank中其他物种的RPL11基因序列进行同源性比对,采用DNAStar软件进行聚类分析.结果显示,成功克隆到游仆虫RPL11基因,大核中该基因全长709 bp,开放阅读框(ORF)为531 bp,编码176个氨基酸;cDNA序列与大核中ORF序列一致,表明该基因具有转录活性;所推导的氨基酸序列与嗜热四膜虫Tetrahymena thermophila的RPL11同源性最高,为66%.     

黄瓜(Cucumis stavius L.)CSEBF1基因的克隆及其表达特性分析

通过对乙烯利诱导黄瓜茎尖SSH文库的筛选,采用RT-PCR和电子克隆技术,从黄瓜中克隆到一个EIN3-Binding F box protein 1基因,命名为CSEBF1基因(GenBank登录号为KF366911)。结果表明:CSEBF1基因的cDNA全长1 964bp,编码640个氨基酸,含有F-box蛋白保守区域和蛋白质泛素化作用底物识别必需结构—LRRs(leucine-rich repeats),氨基酸序列与拟南芥的同源性为60.47%。实时荧光定量RT-PCR法分析了CSEBF1基因在乙烯利诱导后植株不同部位的表达情况,表明该基因在叶片和根部处理8 h达到最高值,而在茎部16 h达到最高值。同时通过RTPCR方法检测茎、叶部乙烯信号转导相关基因的表达情况发现,CSEIN3基因于处理4 h在茎、叶诱导表达;CSCTR1基因在处理后8 h的茎和16 h的叶片有表达量;CSACS2基因在处理后2 h的叶片被诱导表达,并且表达量随后增加。CSACS1G基因(即F基因)于处理后4 h的叶片增强表达,随后持续较微弱的水平。     

猪源冠状病毒聚合酶基因的克隆及特性分析

参照GenBank中Purdue株序列对TGEVTS株聚合酶基因(ORF1)设计特异性引物,经RT-PCR扩增获得了20054bp的片段,与预期大小相符。TS株与Pur46-MAD株的ORF1核苷酸和氨基酸序列同源性分别为98.8%和99.0%,与FIPV、PEDV、HCV299E及SARS氨基酸同源性分别为88%、57%、57%和45%。不同冠状病毒比较结果显示RdRp有很高的保守性而且有重要的作用,序列分析标明TS株在ORF1a和ORF1b重叠区有核糖体剪切位点UUUAAAC,且相应区域RNA二级结构形成3个茎环结构。     

枣树肌动蛋白基因cDNA片段的克隆及其表达分析

根据GenBank中登录的植物肌动蛋白基因同源核苷酸保守序列设计引物,以壶瓶枣(Ziziphus jujuba Mill.Hupingzao)刚萌发结果枝构建的cDNA文库为模板,利用PCR技术得到了一个478 bp的cDNA片段.与其它植物同源序列进行分析表明,其核苷酸序列的同源性在70%以上,氨基酸序列的同源性在85%以上,证明它是肌动蛋白基因在枣树中的同源cDNA片段,命名为ZjAT1(Ziziphus jujuba actin1),GenBank登录号为EU251882.DNA印迹分析结果表明,ZjAT1在枣树基因组中以单拷贝的形式存在.对不同发育阶段的不同器官组织进行了RT-PCR分析,结果显示ZjAT1基因只在花和幼果中有明显表达,在毛根、幼茎、叶片、成熟茎尖、成熟茎段以及成熟果实的果肉和种仁中都没有检测到表达.     

家蝇气味结合蛋白基因cDNA片段的克隆与序列分析

根据同源性设计特异性引物,采用RT-PCR方法扩增出了家蝇Musca domestica 2种气味结合蛋白基因cDNA片段,大小分别为381 bp和353 bp,分别命名为MdomOBP1(GenBank登录号：AY730350)和MdomOBP2(GenBank登录号：AY730351)。测序分析结果表明,它们具有气味结合蛋白的标志性结构域。对这2个片段推导的氨基酸序列进行同源性分析,结果表明两者与已报道的双翅目昆虫的气味结合蛋白的同源性分别达57%～88% 和52%～91%。     

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.     

Prostaglandin and thromboxane production by human and guinea-pig macrophages and leucocytes

The ability of partially purified human and guinea-pig haematogenous cell populations, when cultured in vitro, to metabolise arachidonic acid (AA) has been studied. Supernatants from 24 hour cell culture have been subjected to analysis for products of AA metabolism by gas chromatography with electron-capture detection.The cell types studied were human peripheral blood monocytes (both glass adherent and non-adherent), neutrophils, eosinophils and leukemic leucocytes; thoracic duct lymphocytes and lung alveolar macrophages. From the guinea-pig, induced and non-induced macrophage or neutrophil enriched peritoneal exudate populations, lymph node cells, peritoneal eosinophils and peripheral blood platelets were examined. Supernatants were assayed for the presence of PGE₂, PGD₂, PGF_2α, TXB₂ and 6-keto-PGF_1α. In all types studied PGE₂ and TXB₂ were the major products formed. The identification of PGE₂ and TXB₂ was confirmed by GC/MS with multiple ion monitoring.The results have been compared with other reports and their possible significance discussed in relation to the proposed role of prostaglandins as mediators and modulators in immunopathology.     

Chemokines and cytokines: axis and allies in asthma and allergy

Allergic asthma can be precipitated by many factors. For the atopic person, fungus, pollen, dust mites, cockroach antigens, and diesel exhaust are all agents that may trigger an allergic attack. Cytokines and chemokines are integral mediators of fungal asthma. From the earliest time points, they recruit and activate the cells required for the clearance of fungus as well as being critical factors involved in the immunopathology of this disease. In the final analysis, it is clear that these mediators can act to the benefit or the detriment of the host.     

Oxidation and nitration of ribonuclease and lysozyme by peroxynitrite and myeloperoxidase

In spite of the many studies on protein modifications by reactive species, knowledge about the products resulting from the oxidation of protein-aromatic residues, including protein-derived radicals and their stable products, remains limited. Here, we compared the oxidative modifications promoted by peroxynitrite and myeloperoxidase/hydrogen peroxide/nitrite in two model proteins, ribonuclease (6Tyr) and lysozyme (3Tyr/6Trp). The formation of protein-derived radicals and products was higher at pH 5.4 and 7.4 for myeloperoxidase and peroxynitrite, respectively. The main product was 3-nitro-Tyr for both proteins and oxidants. Lysozyme rendered similar yields of nitro-Trp, particularly when oxidized by peroxynitrite. Hydroxylated and dimerized products of Trp and Tyr were also produced, but in lower yields. Localization of the main modified residues indicates that peroxynitrite decomposes to radicals within the proteins behaving less specifically than myeloperoxidase. Nitrogen dioxide is emphasized as an important protein modifier.