Motilin and ghrelin gene experienced episodic evolution during primitive placental mammal evolution

Motilin and ghrelin, members of a structure-function-related hormone family, play important roles in gastrointestinal function, regulation of energy homeostasis and growth hormone secretion. We observed episodic evolution in both of their prehormone gene sequences during primitive placental mammal evolution, during which most of the nonsynonymous changes result in radical substitution. Of note, a functional obestatin hormone might have only originated after this episodic evolution event. Early in placental mammal evolution, a series of biology complexities evolved. At the same time the motilin and ghrelin prehormone genes, which play important roles in several of these processes, experienced episodic evolution with dramatic changes in their coding sequences. These observations suggest that some of the lineage-specific physiological adaptations are due to episodic evolution of the motilin and ghrelin genes.

Influence of a fish bacterium Lactobacillus sp. on the production of swordtail xiphophorus helleri (Heckel 1848)

The influence of a fish gut bacterium Lactobacillus sp on the production of swordtail Xiphophorus helleri was studied for a period of one year. The Lactobacillus sp P21 produced bacteriocin-like inhibitory substance and exhibited wide spectrum of action against Aeromonas hydrophila, Bacillus spp, Pseudomonas spp and Citrobacter freundi in vitro. The growth performance of X. helleri reared in the presence of Lactobacillus P21 at 106/ml rearing water was better than the control. The total plate counts, total MRS agar counts and the counts of motile aeromonads, presumptive pseudomonads, lactose fermenters and lactose non-fermenters in the gut of probiotic group were comparatively low than the control. On day 60 the count of Lactobacillus sp P21 was observed to be log 5.28/g in the gut of X. helleri indicating colonization of this bacterium in the gastrointestinal tract. The fecundity of X. helleri was in the range of 9-134. On average, it produced from 39.42±18.72 fry/female in control group to 53.00±23.57 fry/female in probiotic group. The increase in average fecundity in probiotic group over the control group was about 25%. There existed significant difference between probiotic group and control in respect of average fecundity/female (p<0.02), average number of fry survived /female (p<0.006) and average number of fry dead/female (p<0.029). The results of the present study demonstrated that the rearing of X. helleri in probiotic-enriched water have growth inducing ability and favourably influenced the reproductive performance in terms of high fecundity, high fry survival, reduced fry mortality and reduced fry deformity.

Abundance and composition of benthic fauna in Penaeus monodon Fabricius culture pond on the west coast of Malaysia peninsular

This paper reports a study on the benthic faunal abundance and diversity of tiger shrimp P. monodon culture ponds in Perak, west coast of Malaysia Peninsular. Sampling was carried out at three weeks interval throughout the 116 days culture period. In addition, water temperature, dissolved oxygen, salinity, transparency, pH and organic matter of soil were also measured. Results showed that the major groups of macro-benthos comprised of gastropod, foraminifera, polychaetes, bivalve and insects; whereas the meio-benthos comprised of harpacticoid copepods, ostracods, nematodes, gastropods, foraminifera, bivalve, insects, crustacean nauplii and polychaetes. In macro-benthos, the abundance of different sizes of Gastropods increased throughout the culture duration. This consisted of 37-98.20% for <1cm length, 1.80-61.50% for 1-2cm length and 1.18—1.30% for >2cm length. Other macro and meio-benthic organisms decreased linearly with the culture period. The depletion symptom indicates that the culture species may have intensively preyed upon the consumable (<0.5cm in size) benthic fauna together with detritus and artificial diet; or could have been caused by pond bottom deterioration via uneaten feed, faces and toxic gases.

Study on effects of extracted probiotics from microbial flora of digestive system on growth indices in beluga (Huso huso) and Persian sturgeon (Acipenser persicus)

Isolation of Lactic Acid Bacteria (LAB) was carried out from gastro intestinal tract of beluga and Persian sturgeon at international sturgeon research institute and PCR has been used for bacteria Identification. Two species of LAB including Enterococcus seriolicida and Leuconostoc mesenteroides were isolated from Gastrointestinal tract (GI) of persian sturgeon in this study and the counts of Leu. mesenteroides (4.63×102 CFU/gr of GI) was significantly higher than other species. Lactobacillus curvatus, Lactococcus raffinolactis, Lactococcus lactis and Streptococcus sp. were also isolated from GI of beluga and maximum counts was belonged to Lb. curvatus (4.63×102 CFU/gr of GI) in this species. Dominant species were lyophilized and adding to the water since start of mix feeding of sturgeon with different counts including 2×109, 5×109 and 9×109 CFU/gr of live food, 4 times a day. The results revealed that the maximum and minimum growth rate and protease, amylase, and lipase activity in beluga was gained by using of Lb. curvatus with total viable count of 9×10 9 CFU/gr of live food and Leu. mesenteroides with total viable count of 9×109 CFU/gr of live food. According to the results of this study, the maximum and minimum growth rate and protease, amylase, and lipase activity in Persian sturgeon was gained by using of Leu. mesenteroides with total viable count of 2×10 9 CFU/gr of live food and Lb. curvatus with total viable count of 9×109 CFU/gr of live food. Histological study showed that gastrointestinal development was same during larva rearing in control and other treatments but the size of liver was bigger in treatments that received nonspecific LAB in both species. According to the results, positive effects of using dominant specific LAB bacteria for larviculture of sturgeon has been proved in this study.

A note on some parasites of Merluccius capensis (Pisces) and their zoogeographical significance

During the Southeastern Atlantic Expedition of the German fishery research vessel "Walther Herwig" in 1967 the main emphasis lay on selective fishing of the South African hake Merluccius capensis (von BRANDT 1967). Some of the fish were found to be infested by ecto-and endoparasites both of which were collected whenever possible. Large plerocercoids of Dibothriorhynchus grossum whose adult stage lives in the South Atlantic Ocean in Lamna cornubica (L.SZIDAT, personal communication) were quite common as were cysticercoids of a Tetrarhynchus sp., which had also been reported in Cynoscion striatus off the Argentinian coast (MACDONAGH 1927, cited in Szidat, personal communication). Brownish nematodes were infesting the ovaries of several fish, but could not be identified. The most common ectoparasite to be observed was the parasitic isopod Livoneca raynaudii (fam. Cymothoidae) whose early larval stages were also found.

Molecular characteristics of Camallanus spp. (Spirurida : Camallanidae) in fishes from China based on its rDNA sequences

In the,paper, we explored the intra- and interspecific evolutionary variation among species of Camallanus collected from different fish species in various regions of China. We determined the internal transcribed spacers of ribosomal DNA (ITS rDNA) sequences of these nematodes. The divergence (uncorrected p-distance) of ITS 1, ITS2, and ITS rDNA data sets confirmed 2 valid species of Camallanus in China, i.e., C. cotti and C. hypophthalmichthys. The 2 species were distinguished not only by their different morphologies and host ranges but also by a letranucleotide microsatellite (TTGC)n present in the ITS I region of C cotti. Phylogenetic analyses of the nematodes disclosed 2 main clades, corresponding to different individuals of C cotti and C. hypophthalmichthys from different fish species in various geographical locations, although the interior nodes of each clade received poor support.

Population dynamics and maturation cycle of Camallanus cotti (Nematoda : Camallanidae) in the Chinese hooksnout carp Opsariichthys bidens (Osteichthyes : Cyprinidae) from a reservoir in China

The seasonal population dynamics and maturation cycle of the nematode Camallanus cotti in the posterior intestine of Chinese hooksnout carp Opsariichthys bidens have been studied in the Danjiangkou Reservoir of the Hubei Province in central China from September 2004 to November, 2005. The overall prevalence, mean abundance and intensity of C cotti among fish sampled (n = 700 fish) were 47%, 2.29 +/- 12.38 ( +/- S.D.) and 1-307 (average 4.89 +/- 17.74), respectively. The overall sexual ratio of female to male nematodes (excluding L3 and L4 juveniles) was 1.17:1. Statistical results showed weakly positive correlations betweerl fish length and the number of nematodes per host. The dynamics of infection of the nematode exhibited significant seasonal pattern in changes in mean abundance. A similar pattern was found for changes in nematode prevalence, although this was not statistically significant. Higher levels of infection were observed among fish sampled in summer months and the lower in the winter. Neither the prevalence nor the abundance of the parasite was significantly different between male and female hosts. The pattern of frequency distribution of the parasite in the host was found to be over-dispersed throughout the sampling period. In addition, studies on the development and maturation of the parasite in O. bidens revealed that development (maturation), recruitment of the next generation, and reproduction may be continuous year-round, although reproduction may peak during the winter. (c) 2007 Elsevier B.V. All rights reserved.

Distribution of the meiofaunal community in a eutrophic shallow lake of China

In a Chinese eutrophic shallow lake, the spatial, temporal and vertical distributions of meiofauna in different lake zones along a eutrophic gradient were analyzed. The spatial distribution of meiofauna among sampling stations changed with nutrient levels. Nematoda were most abundant at the majority of sampling stations comprising 70.6 - 93.2 % meiofaunal abundance except for a hypereutrophic station. The seasonal patterns in abundance of nematodes, oligochaetes, rotifers, chironomids and different nematode feeding groups differed among stations, which revealed that the temporal variations of these meiofaunal groups and the nematode feeding groups may vary with different nutrient loadings. The vertical distributions of meiofaunal groups, nematode species, and nematode trophic groups in the upper and lower sediment layers were similar, suggesting a consistent vertical distribution pattern across different trophic conditions. Nematode species richness, Shannon-Wiener species diversity index, trophic diversity and Maturity Index were significantly correlated with nutrient levels (total phosphorus and nitrogen in lake water and total phosphorus in sediment). Our results suggest the importance of nematode community analyses in the assessment of freshwater eutrophication.

PAR4 和TFF2 在胃肠癌中的表达变异与临床关系以及 TFF2 的重组表达

胃癌和肠癌是常见的威胁人类健康的消化道恶性肿瘤，其发生发展涉及多因 子的作用及调控。其中，在胃肠道都有表达的蛋白酶激活受体（PARs）和三叶 因子蛋白（TFFs）家族都参与肿瘤发生发展的调控过程。正常生理条件下，PARs 的表达与胃肠道消化液的分泌和肌肉的收缩舒张相关。同时，在胃肠道肿瘤的发 生、浸润和转移过程中PARs和TFF2也发挥了作用。而PAR-4，除了具有凝血酶 激活后的血小板聚集功能外，还参与感染、细胞迁移和肿瘤的发生发展。在溃疡 性结肠炎，肠癌组织以及某些肠癌细胞系中都出现PAR4的异常表达，而这种异 常表达可能作为启动肠癌发生的重要环节。TFFs家族蛋白能够对抗粘膜损伤并 且参与修复以发挥保护胃肠道的功能。在肿瘤发生中，三叶因子既有报道作为肿 瘤抑制因子，又有报道作为潜在的肿瘤促进因子。含两个三叶因子结构域的 TFF2，主要表达在胃粘膜的颈细胞。在胃溃疡、慢性萎缩性胃炎及胃癌中，TFF2 的表达具有下降的趋势；而且分化程度越低的胃癌，TFF2的表达量越少，这是 因为TFF2的表达与胃粘膜细胞的增殖和恶性转移相关。在肠道，TFF2可以抑制 一氧化氮（NO）的生成以调节由NO引起的肠炎；在肠炎老鼠的模型中，TFF2 能减轻炎症和溃疡发生的程度，表明TFF2可能通过调节机体的免疫反应来抑制 肠道炎症的发生。 而本实验室前期对大蹼铃蟾皮肤分泌物中获得的新型血小板激动蛋白 -Bm-TFF2与PARs相互作用的实验，促使我们去研究人TFF2与PARs的关系。由 于免疫组化提示TFF2和PAR4在正常胃黏膜中都分布在从基底部到中间的位置， 而且TFF2第二个Loop区序列的保守性，以及和PAR4连接配体（tethered-ligand） 的高度相似性，促使我们推测PAR4和TFF2之间是否存在一种相互作用，或者 hTFF2是否能调节PAR4的生物学活性。所以该篇论文落脚于PAR4和hTFF2，着 重介绍PAR4和TFF2在胃肠道肿瘤中的表达变异以及TFF2对过表达PAR4的细胞 的趋化作用。 我们先用半定量PCR方法检测TFF2和PAR4在胃癌、肠癌及周围远癌部位组 织中mRNA的表达水平。结果提示两个基因在胃癌组织中的表达较周围远癌部位组织减弱，而在肠癌组织中的表达则较周围远癌部位组织增强。Western blotting 也得到相似的结果。为进一步明确PAR4和TFF2在胃癌和肠癌中表达的具体变化 情况，我们继而用实时荧光定量PCR对28例胃癌和38例肠癌及其周围远癌部位组 织中TFF2和PAR4的表达进行了研究。结果显示胃癌组织中两个基因mRNA的表 达都显著低于远癌部位组织（P<0.001），而肠癌组织中两个基因mRNA的表达 则显著高于远癌部位组织（P<0.001）。结合临床病理资料提示PAR4在淋巴结转 移的胃癌患者中的表达低于无淋巴结转移的患者（P<0.05），在胃窦癌中的表达 明显低于非胃窦癌（P<0.05）；而发生淋巴结转移的肠癌患者其TFF2和PAR4基 因的表达都显著高于无淋巴结转移的肠癌患者（P<0.05）；两个基因在中低分化 肠癌中的表达也显著高于高分化肠癌（P<0.001）。免疫组化结果也提示TFF2和 PAR4在胃癌中的表达显著低于周围远癌部位组织（P<0.001），而在肠癌中的表 达则显著高于周围远癌部位组织（P<0.001）。表明TFF2和PAR4在胃肠道肿瘤的 发生中可能受到某些因素的调节而协调性地一致性表达。 在细胞水平上，我们发现在同等浓度hTFF2的诱导下，过表达PAR4的Lovo 稳定株的细胞迁移能力较不表达者明显增强，并且hTFF2的促细胞迁移活性呈剂 量依赖性，同时伴随ERK1/2磷酸化的增强。同时，过表达PAR4的Lovo细胞增殖 能力强于无PAR4表达的细胞，但TFF2作用后其增强能力反而下降，表明TFF2 对过表达了PAR4的Lovo细胞具有抗增殖的能力。 总之这些结果提示PAR4和TFF2在胃肠道中协同表达的现实为两者之间产 生一定的作用提供了基础，而且这种共存为粘膜受损后的修复，组织自身平衡状 态的维持都发挥了一定的作用，同时也为临床相关疾病的诊断，治疗及预后提供 一个新的理论依据。当然，生理和病理情况下，存在于PAR4和TFF2之间的调控 和相互作用的分子机制仍不清楚，这也是进一步研究的关键所在。 为探讨其它动物体内三叶因子家族蛋白结构和功能的关系，我们进而利用原 核表达体系构建并表达纯化了Bm-TFF2以及它的两个单结构域。由于Bm-TFF2 分子中有三对二硫键，所以我们选用pET-32a表达体系表达融合的重组蛋白，并 利用融合蛋白N端引入的Xa因子酶切位点将融合蛋白中的硫氧还蛋白切除，亲和 柱及反向高压液相色谱纯化游离的重组蛋白。重组的Bm-TFF2全长具有血小板聚集活性，而第一个结构域只有诱导血小板变形的作用；三种重组蛋白都具有剂量 依赖性地诱导AGS细胞迁移的功能，但三种重组蛋白的细胞迁移活性无明显差 异。pET-32a表达体系成功表达Bm-TFF2的事实为我们研究人三叶因子家族蛋白 结构及功能关系提供一种方便而可靠的手段。

CO2浓度升高和氮肥管理对土壤线虫群落的影响

大气CO2浓度升高能够对农田生态系统产生一系列的影响。土壤线虫在农田生态系统腐屑食物网中占有重要的地位，能够对外界环境变化作出较迅速的响应。本文利用江苏省江都市小记镇的稻-麦轮作FACE系统研究平台，在2007-2008年小麦生长季，研究了大气CO2浓度升高和不同氮肥处理（高N和低N）对农田土壤线虫群落的影响。 研究结果表明：高氮肥施用情况下, CO2浓度升高显著降低了麦田土壤铵态氮和硝态氮含量。不同氮肥处理中CO2浓度升高条件下土壤可溶性碳的含量显著低于对照，而土壤总有机碳和微生物量碳含量高于对照。 大气CO2浓度升高条件下，麦田土壤线虫群落组成和多样性与对照相比表现出显著差异。CO2浓度升高显著增加了麦田土壤线虫总数、食细菌线虫、食真菌线虫和植物寄生线虫数量。在小麦拔节期和成熟期，低N和高N施用条件下，FACE处理中土壤线虫多样性指数（H’）、成熟度指数（MI和PPI）均低于对照处理，而结构指数（SI）高于对照处理。线虫生态指数的结果表明，大气CO2浓度升高条件下，土壤线虫群落多样性降低，土壤环境受到一定的干扰，食物网趋于结构化。

5-氟尿嘧啶-β-环糊精结肠靶向前体药物的研究

5-氟尿嘧啶(5-Fluorouracil， 5-FU)是一种抗代谢药物，广泛用于临床治疗结直肠癌、胃癌、乳腺癌等多种癌症，但其首过代谢显著、亲脂性较低，选择性差、毒副作用大。为克服这些缺点人们对5-FU进行了大量的修饰工作，包括小分子修饰以及与各种载体形成微球、微囊、纳米粒、共价前药等。 环糊精(Cyclodextrin，简称CD)，可被结肠中的糖苷酶特异性地降解成小分子糖，而胃和小肠中由于缺乏相应的酶而使环糊精不被降解，这一特性在结肠药物的靶向输送及释放中有重要应用价值。环糊精中含有丰富的羟基，易进行化学修饰，将药物与环糊精通过共价键结合制成前药，使其在胃和小肠中不降解，而在盲结肠中被特异性的酶降解释出药物，达到结肠靶向释药的目的。研究表明，环糊精作为一种前药载体为结肠靶向释药和缓释、控释系统提供了一种有效的手段。 本工作选择5-氟尿嘧啶为模型药物、β-环糊精作为载体，通过中间体5-FU羧酸衍生物的制备及其与β-环糊精的偶联，合成了系列5-FU-β-CD前体药物，并利用紫外、红外、质谱、核磁、元素分析、热分析等手段对其进行结构表征。同时，还研究了前体药物的体外释药性质。具体内容包括： 1. 含有羧基的5-FU衍生物中间体的合成：(5-氟尿嘧啶-1-基)-乙酸(FUAC)、3-(5-氟尿嘧啶-1-基)-丙酸(FUPC)、5-(5-氟尿嘧啶-1-基)-戊酸(FUVC)的合成。 2. 中间体5-FU的羧酸衍生物与β-CD的偶联：分别通过以6-OTs-β-CD为中间体的取代法和活化酯法，合成了第一面取代和第二面取代的5-FU-β-CD大分子前体药物。在二面取代的前体药物制备中，通过改变原料的比例，合成了系列不同取代度(DS)的2-[(5-氟尿嘧啶-1-基)-乙酰基] -β-环糊精结合物。 3. 对上述前体药物进行体外释放研究：分别考察了前体药物在不同pH缓冲溶液中的水解行为及其在小鼠胃肠道人工体液中的酶解行为，并通过UV-Vis及HPLC对前体药物释放情况进行检测分析。 5-Fluorouracil(5-Fu), commonly known as a broad-spectrum antineoplastic drug, has been widely used in the treatment of various kinds of cancer including colon cancer for 40 years. However, this antitumor agent exhibits serious adverse effects, such as their marrow toxicity, gastrointestinal reaction and low selectivity in their clinical use. In order to improve its antitumor activity and reduce its toxicity, the compound was modified in various ways, including the formation of conjugated prodrugs with kinds of carrier, microsphere and nanoparticles etc. Cyclodextrins(CDs) are known to be barely capable of being hydrolyzed and only slightly absorbed in passing through the stomach and small intestine; however they are fermented into small saccharides by colonic microflora and thus absorbed as small saccharides in the large intestine. This biodegradation property of CDs may be useful as a colon-targeting carrier, and thus CD prodrugs may serve as a source of site-specific delivery of drugs to colon. It was demonstrated that prodrugs of CDs can provide a versatile means for construction of not only colon targeted delivery systems, but also delayed release systems. 5-Fluorouracil was taken as a model drug and β-CD as the carrier in this study. Series prodrugs of 5-FU was prepared through the preparation of reactive 5-FU derivatives containing carboxyl group and coupling to hydroxyl groups of CD. The structures of the conjugates were charactered by using IR, UV–vis, ESI-MS, 1H, 13C-NMR spectra, elemental analyses, and thermal analysis. In vitro hydrolysis behavior in aqueous solution and in rat gastrointestinal tract contents of the conjugates were also investigated. The main content of this dissertation includes following aspects: 1. The preparation of 5-FU derivatives containing carboxyl group: 5-Fluorouracil- acetic acid(FUAC)、3-(5-FU-1)-propionic acid (FUPC)、and 5-(5-FU-1)-valeric acid(FUVC). 2. The coupling of 5-FU derivatives to β-CD: 5-FU was selectively conjugated onto the primary or secondary hydroxyl groups of β-CD through an ester linkage, by the substitution of 6-OTs-β-CD and the activated ester method respectively. For the secondary face conjugation, the degree of substitution(DS) can be controlled by changing the mole ratio of the starting materials(FUAC and β-CD). 3. In vitro release behavior of the conjugates in aqueous solution and in rat gastro- intestinal tract contents of the conjugates were investigated, and the reaction was monitored and analyzed by using UV-Vis and HPLC methods.

氮肥对黄瓜根际土壤线虫群落组成、结构及其多样性的影响

土壤线虫是土壤动物的主要功能类群之一，在土壤养分分解和循环中起到重要的作用。本研究通过施用两种形态氮肥，硝态氮(NO-3–N)和铵态氮(NH+4–N)，对黄瓜整个生长期内根际土壤线虫的群落组成、结构及其多样性等的影响进行了比较研究。为增进土壤健康，提高土壤质量以及合理施用氮肥提供科学的理论依据。研究结果如下： 1. 氮肥处理后，不同浓度NO-3–N处理提高了根际土壤线虫数量，而NH+4–N处理（202.5 kg N/ hm2）抑制了线虫数量的增加。线虫群落结构相对较稳定，营养类群变化不大。且少量优势科/属会对土壤线虫群落特征起着至关重要的作用。 2. 在整个生长季节内，非寄生线虫的群体动态变化与寄生线虫的群体的动态变化具有相反的变化趋势。其中，NO-3–N处理和NH+4–N处理后植物寄生线虫出现频率的变化趋势相近，都是由高到低；非植物寄生线虫出现频率的变化趋势则都是由低到高。这说明非植物寄生线虫数量增长和空间占位对植物寄生线虫群体有一定的抑制作用。另外，也反映了适量氮肥在一定程度上能够减轻植物寄生线虫对黄瓜的危害。 3. 由多样性指数变化可知，NO-3–N和NH+4–N在低肥（67.5 kg N/hm2）和高肥（202.5 kg N/hm2）处理较中肥（135.0 kg N/hm2）处理，更不利于提高土壤线虫多样性地提高和线虫群落的稳定性。中肥不同形态氮肥处理与对照相比，H´指数在初花期和结果期显著增加了土壤线虫的H´指数，说明施入两种形态的氮肥能够提高线虫生物多样性程度。NH+4–N处理在初花期和结果期显著降低了土壤线虫种类的丰富性。土壤线虫生物多样性变化中，H´和SR指数在一定程度上能够反映施用无机氮肥对土壤线虫的多样性的影响，而J指数和λ指数效果不明显。NO-3–N和NH+4–N处理相比较，NO-3–N处理对黄瓜土壤线虫的多样性指数影响更大，促进了土壤线虫群体的多样化和种类的丰富度，更有利于提高土壤线虫的多样性，增加其稳定性。这些结果表明适量无机氮肥特别是NO-3–N的施用对黄瓜土壤线虫的生物多样性有一定的维护和提高作用。 4. 线虫数量与土壤质量指标的相关分析表明：线虫数量与有关土壤理化生指标，如土壤NO-3–N、NH+4–N、有机质含量等的正相关程度高，与总酚含量等显著负相关；与根际土壤微生物，细菌、真菌、放线菌数量等呈显著正相关。另外，线虫数量与土壤含水量未表现出显著相关关系。 Nematodes play a major role in decomposition and nutrient cycling in soil. Nematode community analyses are useful in assessing soil ecosystem status and function. The effects of two forms of mineral nitrogenous fertilizers (NO-3–N and NH+4–N) on nematode community composition, structure and diversity in rhizosphere of cucumber were investigated during different growing seasons of cucumber. Systematically research of effects of nitrogenous fertilizers could help to obtain better undstanding of a healthy soil and using nitrogenous fertilizers in reason. The main results are as follows: 1. The total numbers of nematode were more abundant in NH+4–N treatments than other teatments. However, NH+4–N teatment（202.5 kg N/hm2）dramatically inhibited it. All the tropic groups in the soil nematode communities were stable, and the dominant family or genus had an important function in the nematode community structure. 2. There was similar trend of the frequency of plant parasitic nematodes between NO-3–N and NH+4–N treatment, the similar trend of the frequency of non-plant parasitic nematodes was also found. But the frequency of plant parasitic nematodes exhibited a contrary trend to that of plant parasitic nematodes after different nitrogenous fertilizer treatments. The results showed that the increasing trend of the frequency and the niche of non-plant parasitic nematodes inhibited the plant parasitic nematodes, and indicated that right chemical fertilizers dosage could abate plant parasitic nematodes harm to cucumber. 3. The changes of the biodiversity index showed that the nitrogen treatment（135.0 kg N/hm2）promoted the stabilization of soil nematode diversity than other nitrogen treatments(67.5 kg N/hm2 and 202.5 kg N/hm2). In the treatment（135.0 kg N/hm2）,The changes in nematode diversity between the control plots and treated plots were compared by the biodiversity index (H´, J, SR, λ). Among these tested index, H´ and SR were effective in reflecting the effects of different nitrogenous fertilizers on the diversity of soil nematodes. In comparison with the NH+4–N treatment, the NO-3–N treatment promoted the stabilization of soil nematode diversity. 4. Correlation coefficients between nematode abundance and soil quality indices indicated that the total numbers of nematode were affected positively by NO-3–N, NH+4–N and the organic matter, and negatively by total phenolic acids; the total num- bers of nematode had positive correlation with bacteria, fungi and actinomycetes nu- mbers. Soil water contents had only a weak negative influence on it.

小麦抗禾谷孢囊线虫（ Heterodera avenae）相关基因的研究

禾谷孢囊线虫严重影响禾谷类作物的产量，在小麦中由禾谷孢囊线虫引起的产量损失可达30-100%。尤其在澳大利亚、欧洲、印度和中东危害严重，目前禾谷孢囊线虫已成为危害我国作物的主要病源。控制禾谷孢囊线虫的方法主要有：作物轮作、杀线虫剂、寄主抗性等等，其中基因工程方法培育抗线虫小麦品种被认为是最经济有效的方法。分离抗禾谷类孢囊线虫基因对揭示抗性基因结构与功能及其表达调控具有重要意义。 尽管小麦是重要的粮食作物，在小麦中已发现的抗禾谷孢囊线虫的基因很少，而比其近缘属如节节麦、易变山羊草、偏凸山羊草中含有丰富的抗源。目前已鉴定出禾谷孢囊线虫抗性位点Cre，并发现了9个禾谷孢囊线虫抗性基因(Cre1,2, 3, 4, 5, 6, 7, 8, and R) ，其中只有Cre1和Cre8直接从普通小麦中获得。从节节麦中获得的Cre3基因能最有效的控制线虫数量，其次是Cre1和Cre8。这些基因的克隆对于了解禾谷孢囊线虫抗性机制及进一步的育种应用都是非常关键的。然而，目前为止仅有Cre3基因通过图位克隆的方法从节节麦中被分离得到。该基因已被克隆得到的多数线虫抗性基因一样均属于核苷酸结合位点区（NBS）－亮氨酸重复序列区（LRR）基因家族。目前，已有很多抗性基因被分离，这些已知的NBS-LRR类抗性基因的保守序列为应用PCR的方法克隆新的抗性基因提供了可能。 因此本课题的目的是采用保守区同源克隆、3′RACE 和5′RACE 等方法从抗禾谷孢囊线虫小麦-易变山羊草小片段易位系E10 中克隆小麦抗禾谷孢囊线虫基因全序列，进而通过半定量PCR 和荧光定量PCR 研究该基因的表达模式。同时通过mRNA 差别显示技术和任意引物PCR（RAP-PCR）技术分离克隆植物禾谷孢囊线虫抗性基因及其相关基因，为阐明植物抗病性分子机制以及改良作物抗病性和作物育种提供基础，为通过分子标记辅助育种和基因工程方法实现高效、定向转移抗病基因到优良小麦品种奠定了重要的理论和物质基础。主要研究结果： 1． 本实验根据此前从抗禾谷孢囊线虫材料E-10 扩增得到的与来自节节麦的抗禾谷孢囊线虫Cre3 基因及其他的NBS-LRR 类抗性基因的NBS 和LRR 保守区序列设计了两对特异性引物，从E10 中扩增到532bp 和1175bp 的两个目标条带，它们有一个32bp 的共同序列，连接构成总长为1675bp 的NBS-LRR 编码区（命名为RCCN）。根据RCCN设计引物，利用NBS-LRR区序列设计引物，通过5′RACE 和3′RACE 技术采用3′-Full RACE Core Set(TaKaRa)和5'-Full RACE Kit (TaKaRa)试剂盒，反转录后通过嵌套引物GSP1 和GSP2 分别进行两轮基因特异性扩增，分别将NBS_LRR 区向5′端和3′端延伸了1173bp 和449bp，并包含了起始密码子和终止密码子。根据拼接的得到的序列重新设计引物扩增进行全基因扩增的结果与上面获得的一致。拼接后得到全长2775 bp 的基因序列（记作CreZ, GenBank 号：EU327996）。CreZ 基因包括完整的开放阅读框，全长2775 bp，编码924个氨基酸。序列分析表明它与已知的禾谷孢囊线虫抗性基因Cre3的一致性很高，并且它与已经报到的NBS-LRR 类疾病抗性基因有着相同的保守结构域。推测CreZ基因可能是一个新的NBS-LRR 类禾谷孢囊线虫抗性基因，该基因的获得为通过基因工程途径培育抗禾谷孢囊线虫小麦新品种奠定了基础，并为抗禾谷孢囊线虫基因的调控表达研究提供了参考。 2． 通过半定量PCR和SYBR Green荧光定量PCR技术对CreZ基因的相对表达模式进行了研究。以α-tubulin 2作为参照，采用半定量PCR 分析CreZ 基因在不同接种时期1d, 5d, 10, 15d 的E-10的根和叶的的表达情况。在内参扩增一致的条件下，CreZ 在E-10的根部随着侵染时间的增加表达量有明显的增加，在没有侵染的E-10的根部其表达量没有明显变化，而在叶中没有检测表达，说明该基因只在抗性材料的根部表达。SYBR Green定量PCR分析接种前后E10根部基因CreZ基因的表达水平为检测CreZ基因的表达建立了一套灵敏、可靠的SYBRGreen I 荧光定量PCR 检测方法。接种禾谷孢囊线虫后E10根内CreZ基因的相对表达水平显著高于接种前。随接种时间的延长持续增加，最终CreZ基因的相对表达量达到未接种的对照植株的10.95倍。小麦禾谷孢囊线虫抗性基因CreZ的表达量与胁迫呈正相关，表明其与小麦的的禾谷孢囊线虫抗性密切相关，推测CreZ基因可能是一个新的禾谷孢囊线虫候选抗性基因。 3． 针对小麦基因组庞大、重复序列较多，禾谷孢囊线虫抗性基因及其相关基因的片断难以有效克隆的问题，通过mRNA 差别显示技术及RAP-PCR 技术分离克隆植物禾谷孢囊线虫抗性及其相关基因。试验最终得到154 条差异表达条带，将回收得到的差异条带的二次PCR 扩增产物经纯化后点到带正电的尼龙膜上，进行反向Northern 杂交筛选，最终筛选得到102 个阳性差异点。将其中81 个进行测序，并将序列提交到Genbank 中的dbEST 数据库，分别获得登录号(FE192210 -FE192265，FE193048- FE193074 )。序列比对分析发现，其中26 个序列与已知功能的基因序列同源；有28 条EST 序列在已有核酸数据库中未找到同源已知基因和EST，属新的ESTs 序列；另外27 个EST 序列与已知核酸数据库中的ESTs 具有一定相似性，但功能未知。其所得ESTs 序列补充了Genbank ESTs 数据库，为今后进一步开展抗禾谷类孢囊线虫基因研究工作打下了基础。结合本试验功能基因的相关信息，对小麦接种禾谷孢囊线虫后产生的抗性机制进行了探讨。接种禾谷孢囊线虫后植物在mRNA 水平上的应答是相当复杂的，同时植物的抗病机制是一个复杂的过程，涉及到多个代谢途径的相互作用。 The cereal cyst nematode (CCN), Heterodera avenae Woll, causes severe yieldreductions in cereal crops. The losses caused by CCN can be up to 30-100% in somewheat fields. At present, cereal cyst nematode has become the major disease sourcein China and it also damaged heavily in Australia, Europe, India and Middle East.The damage caused by CCN can be mitigated through several methods, includingcrop rotation, nematicide application, cultural practice, host resistance, and others.Of these methods, incorporating resistance genes into wheat cultivars and breedingresistant lines is considered to be the most cost-effective control measure forreducing nematode populations. Although wheat is an economically important crop around the world, far fewergenes resistant to CCN were found in wheat than were detected in its relatives, suchas Aegilops taucchi, Aegilops variabilis and Aegilops ventricosa. Cloning these genesis essential for understanding the mechanism of this resistance and for furtherapplication in breeding. Because of the huge genome and high repeat sequencescontent, the efficient methods to clone genes from cereal crops, are still lacking. A resistance locus, Cre, has been identified and 9 genes resistant to CCN (designatedCre1, 2, 3, 4, 5, 6, 7, 8, and R) have been described, in which Cre1 and Cre8 werederived directly from common wheat. The Cre3 locus, which was derived from Ae.tauschii, has the greatest impact on reducing the number of female cysts, followed byCre1 and Cre8. Cloning these genes is essential for understanding the mechanism ofthis resistance and for further application in breeding. However, to this point, only Cre3, a NBS-LRR disease resistance gene, has been obtained through mappingcloning in Ae. tauschii. The majority of nematode resistance genes cloned so far belong to a super familywhich contains highly conserved nucleotide-binding sites (NBS) and leucine-richrepeat (LRR) domains. To date, many NBS-LRR resistance genes have been isolated.The conserved sequences of these recognized NBS-LRR resistance genes provide thepossibility to isolate novel resistance genes using a PCR-based strategy. The aim of the present study was to clone the resistance gene of CCN fromWheat/Aegilops variabilis small fragment chromosome translocation line E10 whichis resistant to CCN and investigate the espression profiles of this gene withsemi-quantitative PCR and real-time PCR. Another purpose of this study is cloningthe relational resistance gene for CCN by mRNA differential display PCR andRAP-PCR. These works will offer a foundation for disease defence of crop andbreeding and directional transferring resistance gene into wheat with geneengineering. Primary results as following: 1．According to the conversed motif of NBS and LRR region of cereal cystnematode resistance gene Cre3 from wild wheat (Triticum tauschlii) and the knownNBS-LRR group resistance genes, we designed two pairs of specific primers for NBSand LRR region respectively. One band of approximately 530bp was amplified usingthe specific primers for conversed NBS region and one band of approximately 1175bpwas amplified with the specific primers for conversed LRR region. After sequencing,we found that these two sequences included 32bp common nucleotide having 1675bpin total, which was registered as RCCN in the Genbank. Based on the conservedregions of known resistance genes, a NBS-LRR type CCN resistance gene analog wasisolated from the CCN resistant line E-10 of the wheat near isogenic lines (NILs), by5′RACE and 3′ RACE.designated as CreZ (GenBank accession number: EU327996) .It contained a comlete ORF of 2775 bp and encoded 924 amino acids. Sequencecomparison indicated that it shared 92% nucleotide and 87% amino acid identitieswith those of the known CCN-resistance gene Cre3 and it had the same characteristic of the conserved motifs as other established NBS-LRR disease resistance genes. 2． Usingα-tubulin 2 as exoteric reference, semi-quantitative PCR and real-timePCR analysis were conducted. The expression profiling of CreZ indicated that it wasspecifically expressed in the roots of resistant plants and its relative expression levelincreased sharply when the plants were inoculated with cereal cyst nematodes. therelative expression level of the 15days-infected E10 is the 10.95 times as that ofuninfected E10,ultimately. It was inferred that the CreZ gene be a novel potentialresistance gene to CCN. 3．We cloned the relational resistance gene for CCN by mRNA differentialdisplay PCR and arbitrarily primed PCR fingerprinting of RNA from wheat whichpossess huge and high repeat sequence content genomes. Total 154 differentialexpression bands were separated and second amplified by PCR. The products werenylon membrane. The 102 positive clones were filtrated by reverse northern dot blotand 81 of those were sent to sequence. The EST sequences were submitted toGenbank (Genbank accession: FE192210 - FE192265, FE193048 - FE193074). Thesequences alignment analysis indicated 26 of them were identical with known genes;28 were not found identical sequence in nucleic acid database; another 27 ests wereidentical with some known ests, but their functions were not clear. These ESTsenriched Genbank ESTs database and offered foundation for further research ofresistance gene of CCN.

施用农用化学品及生防制剂对土壤线虫群落动态影响的研究

Dynamics of soil nematode communities amended with agrochemicals and bio control preparations were investigated in a soybean field. The results showed that the frequency of plant non parasitic nematodes were obviously higher in soil amended with bio control preparations (Doufeng 1) than with urea and herbicide, however, that of plant parasitic nematodes exhibited an inverse trend.

Studies on the Biotransformation of Arctigenin Using Electrospray Ionization Mass Spectrometry

To study the biotransformation of arctigenin, arctigenin was anaerobically incubated with Eubacterium sp. ARC-2 of human intestinal bacteria in vitro. Arctigenin formed a molecular ion [M-H](-) in negative ion mode. The arctigenin and its metabolites were investigated directly by the electrospray ionization tandem mass spectrometry ion trap and Fourier transform ion cyclotron resonance. Arctigenin was transformed to 4',4 ''-dihydroxylenterolactone by E sp. ARC-2 through 3 types of demethylation products.