Increased accumulation of anthocyanins in transgenic potato tubers by overexpressing the 3GT gene

In order to explore a biotechnological method for improving potato tuber color and creating plants with increased anthocyanin contents, a potato UDP-glucose: flavonoid-3-O-glucosyltransferase (3GT) gene was inserted behind the GBSSI promoter of pBin19, and this construct was introduced into Solanum tuberosum L. cultivar Désirée plants by Agrobacterium-mediated transformation. Six independent transgenic lines overexpressing the 3GT gene were identified by PCR and Southern blot analysis from 18 kanamycin-resistant plants. Due to the expression of 3GT gene, the tuber color and the anthocyanin content were enhanced noticeably in the transgenic plants compared to the wild-type control plants. This result suggests that the 3GT gene can potentially be used to improve potato color and enhance levels of antioxidants in the diet.     

海南岛热带低地雨林刀耕火种弃耕地自然恢复过程中的群落构建

研究群落构建机制是群落生态学的一个重要目标, 群落动态过程中的构建规律对于了解群落演替机理有重要的作用。该文以海南岛刀耕火种干扰后自然恢复的10 hm ²热带低地雨林为研究对象, 通过比较不同恢复阶段的次生林(15年、30年和60年)和老龄林在幼苗、幼树和成年树群落的物种组成, 揭示次生演替过程中的群落构建规律。研究结果表明, 老龄林中不同径级群落的物种多样性及不同径级间的物种相似度显著高于各恢复阶段的次生林, 但优势种在群落中的比例低于各恢复阶段的次生林。随着自然恢复过程的进行, 次生林群落物种组成与老龄林的相似性也逐渐增大, 支持演替平衡理论。所有恢复阶段样地中幼苗的个体、物种丰富度和基于多度涵盖估计量(ACE)都低于幼树和成年树群落, 幼苗层物种组成与幼树、成年树也有较大差异, 说明新增到幼苗群落可能是一个难于预测的过程。研究结果说明了确定过程和随机过程共同决定了次生演替的群落构建。     

广西被子植物二新记录属

在第四次中药资源普查中采集了大量标本,经过对这些标本进行仔细鉴定并查阅相关资料,确定其中两号标本为香茜属(Carlemannia Benth.)和粘腺果属(Commicarpus Standl.)植物。这两属植物在广西尚无报道,为首次记录。香茜属植物叶对生,子房下位,无托叶,雄蕊仅有2枚,这和茜草科相似但又不同,系统位置较混乱,以前曾放于茜草科( Rubiaceae)和忍冬科( Caprifoliaceae)中,最近该属和蜘蛛花属独立成香茜科( Car-lemanniaceae)。该属植物共有3种,沿喜马拉雅山脉向东一直分布到缅甸、越南北部。我国西藏东南、云南南部、广西西北部分布一种即香茜( Carlenannia chinenesis Hook. f.)。粘腺果属是紫茉莉科( Nyctaginaceae)主产热带地区的1个属,全世界约25种分布于热带非洲和阿拉伯半岛南部,在南亚、东南亚和墨西哥至热带美洲也有少量分布。中国产2种,其中广西产1种即中华粘腺果[ Commiaicarpus chinensis ( L.) Heim.]。该种植物分布广泛,从南亚次大陆向东至中南半岛、马来半岛,向北到我国西沙群岛、海南岛以及广州附近,在广西首次记录,产凤山县和凌云县。     

A cyto-evolutional study of Campanumoea Blume (Campanulaceae) and a possible pathway for secondary karyotype formation

Campanumoea is a small genus in the family Campanulaceae, with species divided into sections Campanumoea and Cyclocodon. Sixteen accessions from Campanumoea and related genera native to China were used to study their karyotype. The results showed that chromosome characteristics were different between the two sections. For Campanumoea, the karyotypic formula was 2n = 2X = 2m + 12sm + 2st = 16,3A and for Cyclocodon it was 2n = 2X = 6m + 12sm = 18,3B. These data, combined with chromosomal length characteristics, support the restoration of section Cyclocodon as a genus. However, the incorporation of section Campanumoea into Codonopsis requires more evidence. Comparison of chromosomal length and haploid set length revealed that chromosomal segment rearrangements occurred within sections of Campanumoea and between genera, with the difference within sections being greater than that between genera. Therefore, chromosomal segment rearrangements are present in Campanulaceae, implying that chromosomal segment rearrangement plays an important role in the evolution of diversity in Campanulaceae. By comparing the chromosomal characteristic in section Campanumoea and the genus Adenophora, we concluded that the secondary chromosome type such as n = 17, 18 would be derived by autopolyploidization of n = 9, and by chromosome fusion.     

乙肝前S2抗原决定簇串联体与核心抗原的基因融合与表达

乙肝前Ｓ２（ＨＢＶＰｒｅＳ２）肽段由５５个氨基酸组成,其Ｎ端肽段含Ｔｈ和Ｂ细胞抗原决定簇,为增强ＰｒｅＳ２的抗原性,本实验采用将化学合成的ＰｒｅＳ２ｅｐｉｔｏｐｅ（１２０－１４５）基因以串联方式与ＨＢｃＡｇ的基因进行了融合,融合基因在大肠杆菌中获得表达,并通过ＥＬＩＳＡ比较研究了融合蛋白中ＰｒｅＳ_２ｅｐｉｔｏｐｅ单体及串联体的抗原性差异。     

应用抗病毒抗体表位筛选的多肽文库的构建

1985年,Sndth在《科学》杂志上提出构建"融合噬菌体",即在这种丝状病毒的包膜蛋白上表达融合蛋白[1]．1990年,Scott和Smith山利用这种表面表达载体建立了随机多肽文库,可以很方便地筛选出抗体及其他功能蛋白的强结合配基[2]．我们建立了十二肽的随机多肽库,将从中筛选出抗HIV-gP160抗体的抗独特型多肽．1材料和方法1．1质粒、菌株和培养墓噬菌粒成h血四、大肠杆菌XLI-Blue和辅助噬菌体VCSM13由军事医学科学院微生物流行病研究所王海涛教授惠赠．SB液体培养基：30gtmptone,20g酵母膏,10gMops溶于IL去离子水中,调pH7．0．枝…     

Characteristics of adventitious root formation in cotyledon segments of mango (Mangifera indica L. cv. Zihua): two induction patterns, histological origins and the relationship with polar auxin transport

Cotyledon segments derived from zygote embryos of mango (Mangifera indica L. cv. Zihua) were cultured on agar medium for 28 days. Depending on different pre-treatments with plant growth regulators, two distinct patterns of adventitious roots were observed. A first pattern of adventitious roots was seen at the proximal cut surface, whereas no roots were formed on the opposite, distal cut surface. The rooting ability depended on the segment length and was significantly promoted by pre-treatment of embryos with indol-3-acetic acid (IAA) or indole-3-butyric acid (IBA) for 1 h. A pre-treatment with the auxin transport inhibitor 2,3,5-triiodobenzoic acid (TIBA) completely inhibited adventitious root formation on proximal cut surfaces. A second pattern of roots was observed on abaxial surfaces of cotyledon segments when embryos were pre-treated with 2,700 μM 1-naphthalenacetic acid (NAA) for 1 h. Histological observations indicated that both patterns of adventitious roots originated from parenchymal cells, but developmental directions of the root primordia were different. A polar auxin transport assay was used to demonstrate transport of [³H] indole-3-acetic acid (IAA) in cotyledon segments from the distal to the proximal cut surface. In conclusion, we suggest that polar auxin transport plays a role in adventitious root formation at the proximal cut surface, whereas NAA levels (influx by diffusion; carrier mediated efflux) seem to control development of adventitious roots on the abaxial surface of cotyledon segments.     

Oligomeric Sensor Kinase DcuS in the Membrane of Escherichia coli and in Proteoliposomes: Chemical Cross-linking and FRET Spectroscopy

DcuS is the membrane-integral sensor histidine kinase of the DcuSR two-component system in Escherichia coli that responds to extracellular C₄-dicarboxylates. The oligomeric state of full-length DcuS was investigated in vitro and in living cells by chemical cross-linking and by fluorescence resonance energy transfer (FRET) spectroscopy. The FRET results were quantified by an improved method using background-free spectra of living cells for determining FRET efficiency (E) and donor fraction {f_D = (donor)/[(donor) + (acceptor)]}. Functional fusions of cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) variants of green fluorescent protein to DcuS were used for in vivo FRET measurements. Based on noninteracting membrane proteins and perfectly interacting proteins (a CFP-YFP fusion), the results of FRET of cells coexpressing DcuS-CFP and DcuS-YFP were quantitatively evaluated. In living cells and after reconstitution of purified recombinant DcuS in proteoliposomes, DcuS was found as a dimer or higher oligomer, independent of the presence of an effector. Chemical cross-linking with disuccinimidyl suberate showed tetrameric, in addition to dimeric, DcuS in proteoliposomes and in membranes of bacteria, whereas purified DcuS in nondenaturing detergent was mainly monomeric. The presence and amount of tetrameric DcuS in vivo and in proteoliposomes was not dependent on the concentration of DcuS. Only membrane-embedded DcuS (present in the oligomeric state) is active in (auto)phosphorylation. Overall, the FRET and cross-linking data demonstrate the presence in living cells, in bacterial membranes, and in proteoliposomes of full-length DcuS protein in an oligomeric state, including a tetramer.The DcuSR (dicarboxylate uptake sensor and regulator) system of Escherichia coli is a typical two-component system consisting of a membranous sensor kinase (DcuS) and a cytoplasmic response regulator (DcuR) (11, 26, 48). DcuS responds to C₄-dicarboxylates like fumarate, malate, or succinate (19). In the presence of the C₄-dicarboxlates, the expression of the genes of anaerobic fumarate respiration (dcuB, fumB, and frdABCD) and of aerobic C₄-dicarboxylate uptake (dctA) is activated. DcuS is a histidine protein kinase composed of two transmembrane helices with an intermittent sensory PAS domain in the periplasm (PAS_P) that was also termed the PDC domain (for PhoQ/DcuS/DctB/CitA domain or fold) (7, 20, 32, 48). The second transmembrane helix is followed by a cytoplasmic PAS domain (PAS_C) and the C-terminal transmitter domain. PAS_C functions in signal transfer from transmembrane helix 2 (TM2) to the kinase domain (9). The C-terminal part of the transmitter domain consists of a catalytic or HATPase (histidine kinase/ATPase) subdomain for autophosphorylation of DcuS (16). The N-terminal part of the transmitter contains two conserved α-helical regions, including a conserved His residue which is the site for autophosphorylation. The α-helices serve in dimerization and form a four-helix bundle in the kinase dimer (dimerization and histidine phosphotransfer [DHp] domain) (25, 35, 42, 44).The dimeric sensor kinases have been supposed to phosphorylate mutually, by the catalytic domain of one monomer, the His residue of the partner monomer (10). The oligomeric state of the membrane-bound sensor kinases EnvZ and VirA was also deduced from in vivo complementation studies (31, 46). In addition, signal transduction across the membrane and along cytoplasmic PAS domains appears to be a mechanical process requiring oligomeric proteins (9, 40). Therefore, His kinases are supposed to be dimeric in the functional state, but a higher oligomeric state has not been tested and is conceivable. Only a limited number of membrane-bound sensor kinases have been studied for their oligomerization in their membrane-bound state. Thus, the oligomeric state of the KdpD and TorS sensor kinases of E. coli have been shown to prevail in the detergent-solubilized state as oligomers, presumably dimers (14, 29). There was indirect information that functional DcuS is a dimer as well. Purified DcuS shows kinase activity only after reconstitution into liposomes, and phosphorylation is stimulated by C₄-dicarboxylates (16, 19). Detergent-solubilized DcuS, on the other hand, shows no kinase activity, and it was assumed that reconstituted DcuS prevails as a dimer, whereas the inactivation of the detergent-solubilized form is due to monomerization. Recently, it was suggested that autophosphorylation in a sensor kinase of Thermotoga maritima proceeds by a cis mechanism on DHp and catalytic kinase domains within the same monomer (6). The sensor kinase is supposed to prevail as a dimer for reasons of signal transfer to the sensor domain, but the presence of cis phosphorylation principally brings into question the need for dimers for sensor kinase function.Overall, it appears that sensor kinases are oligomers for functional reasons. There is, however, no clear evidence for an oligomeric state of full-length sensor kinases in their membrane-embedded state. Moreover, the studies do not address the question of whether the sensor kinases are dimers or higher oligomers. Therefore, several aspects of the oligomeric state of sensor kinases in vivo in bacterial membranes, that is, before solubilization by detergent, are not clear. In this study, the oligomerization of full-length DcuS was examined in vivo in growing bacteria and in bacterial membranes and in vitro after isolation and reconstitution in liposomes by chemical cross-linking and fluorescence resonance energy transfer (FRET) spectroscopy. FRET techniques have been used widely to study intermolecular interactions of biological molecules (1, 4, 18, 21, 23, 34). The sensitivity of fluorescence allows experiments at low concentrations of native proteins, and genetically generated fusions of DcuS with fluorescent proteins ensure site-specific labeling of DcuS for noninvasive and nondestructive measurements in living cells. In particular, it was investigated whether dimers or higher oligomeric states can be detected for DcuS and whether the oligomerization state depends on function-related parameters.     

用噬菌体随机肽库筛选SARS冠状病毒S蛋白单克隆抗体2C5的模拟表位

SARS-CoVS蛋白特异的单克隆抗体2C5具有病毒中和作用。以单克隆抗体2C5为筛选靶分子,筛选噬菌体展示随机7肽库。经三轮淘洗后随机挑选20个噬菌体克隆进行ELISA分析和序列测定。在10个ELISAOD值大于0.2的阳性噬菌体克隆中,有8个噬菌体克隆展示有共同的7肽序列TPEQQFT。展示有该序列的噬菌体克隆能竞争抑制SARS-CoVS蛋白抗原与单抗2C5的结合。结果表明TPEQQFT为单克隆抗体2C5的模拟表位。该结果可对进一步研究S蛋白结构与功能和设计SARS疫苗有一定的参考意义。     

诺沙坦改善非胰岛素依赖型糖尿病大鼠胰岛素敏感性的作用机制

本文研究血管紧张素Ⅱ受体拮抗剂诺沙坦对非胰岛素依赖型糖尿病(non-insulin-dependent diabetes mellitus,NIDDM)大鼠胰岛素敏感性的改善作用,并探讨其作用机制。从饮水中给予正常或高脂喂养加小剂量链脲佐菌素(STZ)诱发的NIDDM大鼠诺沙坦(4 mg/kg),连续6周。分离骨骼肌,用免疫印迹法检测诺沙坦对胰岛素受体底物1(insulin receptor substrate 1,IRS-1)、蛋白激酶B(protein kinase B,PKB)和葡萄糖转运因子4(glucose transporter 4,GLUT4)的表达,以及IRS-1的磷酸化、IRS-1与磷脂酰肌醇3激酶(phosphatidylinositol(PI)3-kinase)的结合。口服葡萄糖耐量试验表明,口服诺沙坦可改善糖尿病大鼠胰岛素敏感性。在骨骼肌组织,NIDDM和正常大鼠的IRS-1、PKB和GLUT4蛋白表达无差异,且不受诺沙坦处理的影响。NIDDM大鼠胰岛素刺激后的骨骼肌IRS-1酪氨酸磷酸化水平、PI 3-kinase结合IRS-1的活性和PKB活性较对照组显著降低(P<0.01),且不能被诺沙坦改善。诺沙坦显著增加NIDDM大鼠肌细胞质膜(plasma membrane,PM)和T管(T-tubules,TT)胰岛素诱导的GLUT4的 含量(P<0.05)。与该结果一致的是,诺沙坦处理的NIDDM大鼠血糖水平较未处理NIDDM大鼠下降(P<0.05)。结果表明,诺沙坦可改善胰岛素抵抗状态,主要是通过非PI 3-kinase依赖的