Expression of Orseolia oryzae nucleoside diphosphate kinase (OoNDPK) is enhanced in rice gall midge feeding on susceptible rice hosts and its over‐expression leads to salt tolerance in Escherichia coli

The Asian rice gall midge, Orseolia oryzae, is a major dipteran pest of rice, with many known biotypes. The present investigation was initiated to understand the molecular mechanisms of infestation for developing novel integrated pest management strategies. We isolated and characterized a gene, nucleoside diphosphate kinase (OoNDPK), from the rice gall midge, encoding a protein with 169 amino acid residues and with a secretory signal sequence – an observation that assumes significance as salivary gland secretions have been implicated to play a major role in insect?plant interactions. Furthermore, up‐regulation (> 18 folds) of OoNDPK was observed in the salivary glands of maggots feeding on susceptible host in contrast to those feeding on resistant host. Phylogenetic analysis revealed similarity of OoNDPK with its dipteran orthologues. 3DLigandSite analysis, of the predicted OoNDPK and its orthologues, revealed phenylalanine and tyrosine residues to be specifically present in NDPK proteins from the plant feeders. Results suggest secretion of OoNDPK into the host plant and its probable involvement in gall midge?rice interaction. Using the coleoptile cell elongation assay, we demonstrated that the recombinant OoNDPK is capable of causing elongation of rice coleoptile cells. Additionally, heterologous expression of OoNDPK in Escherichia coli increased the tolerance of these cells to salt (NaCl; up to 1 mM), hinting at the involvement of this gene in abiotic stress response as well.     

Two new bioactive triterpene glycosides from the sea cucumber Pseudocolochirus violaceus

By activity-guided fractionation, two new triterpene glycosides, violaceusides A (1) and B (2), were isolated from the sea cucumber Pseudocolochirus violaceus as active compounds causing morphological abnormality of Pyricularia oryzae mycelia. By extensive 2D NMR techniques and chemical evidence, the structures of the two new glycosides were established as 16β-acetoxy-3-O-[3-O-methyl-β-d-glucopyranosyl-(1 → 3)-β-d-xylopyranosyl-(1 → 4)-β-d-quinovopyranosyl-(1 → 2)-4-O-sodiumsulphate-β-d-xylopyranosyl]-holosta-7,24-diene-3β-ol (1) and 16β-acetoxy-3-O-[3-O-methyl-β-d-glucopyranosyl-(1 → 3)-β-d-xylopyranosyl-(1 → 4)-β-d-glucopyranosyl-(1 → 2)-4-O-sodiumsulphate-β-d-xylopyranosyl]-holosta-7,24-diene-3β-ol (2), respectively. The two glycosides also exhibited significant cytotoxicity against HL-60 and BEL-7402 cancer cell lines.     

棕环海参化学成分的研究

目的研究中国南海棕环海参的化学成分。方法采用稻瘟霉模型生物活性追踪方法,应用多种色谱技术分离,根据波谱解析和化学手段鉴定化合物的结构。结果从棕环海参正丁醇萃取物中分离鉴定了3个三萜皂苷:pervicoside C(Ⅰ),holothurin A(Ⅱ)和DS-holoturin B(Ⅲ);从氯仿萃取物中分离鉴定了4个化合物:尿嘧啶(Ⅳ),胸腺嘧啶(Ⅴ),胸腺嘧啶脱氧核苷(Ⅵ)和尿嘧啶脱氧核苷(Ⅶ)。结论所有化合物均为首次从该种海参中获得,化合物I为新天然产物,Ⅰ和Ⅱ具有诱导稻瘟霉菌丝变形活性和肿瘤细胞毒性。     

海洋细菌Bacillus sp.发酵液中化学成分的研究

目的：研究海洋细菌Bacillus sp．菌株发酵液的化学成分．研究其成分对稻瘟霉的抑制情况．为从海洋微生物代谢产物中寻找新型抗肿瘤、抗真菌活性物质奠定基础。方法：利用各种色谱层析技术进行分离．根据光谱数据鉴定其结构。结果：从中分离得到8个成分．分别鉴定为吡咯并哌嗪2，5-二酮(Ⅰ)、3-异丁基-吡咯并哌嗪2，5-二酮(Ⅱ)、3-甲基-哌嗪-2，5-二酮(Ⅲ)、胸腺嘧啶(Ⅳ)、尿嘧啶(Ⅴ)、曲酸(Ⅵ)、丁四醇(Ⅶ)、苯丙氨酸(Ⅷ)。结论：其中化合物Ⅰ，Ⅱ，Ⅲ对稻瘟霉分生孢子或菌丝体有一定抑制作用；化合物Ⅵ为首次从本属菌中分离得到。     

Identification and Characterization of a New Type of Holin-Endolysin Lysis Cassette in Acidovorax oryzae Phage AP1

Phages utilize lysis systems to allow the release of newly assembled viral particles that kill the bacterial host. This is also the case for phage AP1, which infects the rice pathogen Acidovorax oryzae. However, how lysis occurs on a molecular level is currently unknown. We performed in silico bioinformatics analyses, which indicated that the lysis cassette contains a holin (HolAP) and endolysin (LysAP), which are encoded by two adjacent genes. Recombinant expression of LysAP caused Escherichia coli lysis, while HolAP arrested growth. Co-expression of both proteins resulted in enhanced lysis activity compared to the individual proteins alone. Interestingly, LysAP contains a C-terminal region transmembrane domain, which is different from most known endolysins where a N-terminal hydrophobic region is found, with the potential to insert into the membrane. We show that the C-terminal transmembrane domain is crucial for protein localization and bacterial lysis in phage AP1. Our study characterizes the new phage lysis cassette and the mechanism to induce cell disruption, giving new insight in the understanding of phage life cycles.     

Two novel trichothecenes from <Emphasis Type="Italic">Myrothecium roridum</Emphasis>

Two new trichothecenes, Roridin P and Isororidin P, and two known trichothecenes, Verrucarin A and Verrucarin J, were isolated from liquid cultures of Myrothecium roridum Tale ex Fr. The stereo-structures of Roridin P and Isororidin P were established on the basis of one- and two-dimensional nuclear magnetic resonance (NMR) spectral analyses. Roridin P and Isororidin P induced morphological changes in Pyricularia oryzae, with a minimum concentration (MMCC) value of 6.2 ± 1.1 μmol/L and 7.9 ± 1.5 μmol/L.     

Directed deletions in the aflatoxin biosynthesis gene homolog cluster of Aspergillus oryzae

To investigate the structure of the aflatoxin gene cluster in Aspergillus oryzae, 39 strains belonging to this species were examined for the existence of pksA, fas-1A, aflR and vbs, and the results compared with those for ver-1 obtained previously. These five genes are involved in aflatoxin biosynthesis in Aspergillus parasiticus. The strains examined were categorized into three groups; group 1, having the five homologs; 2, having ver-1 and vbs; and 3, having vbs homologs. Long-PCR analysis of the regions between the five homologs in A. oryzae IFO 4135, coupled with Southern-hybridization analysis, shows that those homologs are clustered with the same arrangement as in A. parasiticus. These results suggest that directed deletions of the cluster occur in A. oryzae strains. The possible breakpoints of the deletions in the strains of group 2 and 3 were estimated. Received: 23 April / 8 November 1999     

链霉菌JXJ-402产生的抗生素JXJ-402-1的研究

在筛选微生物来源抗生素的过程中，从500余株链霉菌中发现十余株抗真菌阳性菌株，其中JXJ-402菌株显示出较强的抗稻瘟病菌活性。从该菌株固体发酵粗提取物中分离纯化到化合物JXJ-402-1。经^13C-NMR数据分析，确定其化学结构与文献报道的尼日利亚菌素（nigericin）结构一致。生物学活性研究发现，化合物JXJ-402-1具有抗稻瘟病菌、赤星病菌、白念珠菌、藤黄微球菌和枯草芽孢杆菌等真菌和细菌活性，其中抗稻瘟病菌和赤星病菌活性未见文献报道。     

黑黏座孢霉二次代谢产物的两个活性成分

目的研究云南省西双版纳采集的土样中黑黏座孢霉(Myrothecium roridumTode ex Fr.)发酵液的活性成分。方法通过硅胶柱色谱、制备薄层色谱、HPLC等手段进行分离纯化,并根据理化性质和光谱数据鉴定其化学结构。结果从黑黏座孢霉发酵液乙酸乙酯提取物中共分离得到2个化合物,经鉴定分别为黏液霉素A(verrucarin A)和黏液霉素J(verrucarin J),并利用稻瘟霉(Pyricu-laria oryzae)菌丝体形态变化作为活性指标,测定了它们的活性。结论黏液霉素A和J为首次从该菌种中分离得到。