铁皮石斛组培苗与菌根真菌共培养过程中的相互作用

由于人为的滥采滥挖和野外生境的退化,使得铁皮石斛(Dendrobium officinale)这种名贵的中药材一直处于极度濒危的状态。为了从菌根真菌的角度给人工保育铁皮石斛提供理论指导,对铁皮石斛的组织培养苗人工接种‘GDB181’菌株(Epulorhiza sp.)。培养60d后,接菌苗平均鲜重增长率比对照苗高出了84.8%。在营养元素含量方面,接菌苗的B、Si、Fe、Cu和Mn元素含量的净增率分别为780%、533%、192%、191%和128%,均在100%以上;其他元素含量也有不同程度的增加(除Zn外),结果证明两者有效地建立了共生关系。在显微和超微结构的观察中发现:真菌菌丝随机破坏铁皮石斛的根被入侵到外皮层,并从外皮层细胞不断扩展延伸到皮层的大型细胞,最后在大型细胞中被分解消化。在真菌侵染过程中,被侵染的皮层细胞的细胞壁严重扭曲变形,菌丝在皮层细胞形成菌丝结,菌丝结常位于细胞核附近或包围细胞核。在皮层的大型细胞中,菌丝细胞被植物的溶酶体包围,部分或全部被消解,出现脱壁或失去细胞质甚至成为空腔等变化,最终形成衰败的菌丝残骸,溶酶体也随之消失。溶酶体分布越多的部位,菌丝细胞消解变形越严重。含有菌丝残骸的皮层细胞可被新侵染的菌丝重新定殖,这一菌丝侵染被消化再侵染的过程在铁皮石斛生长发育过程中可不断重复发生。

带叶兜兰种子原地共生萌发及有效菌根真菌的分离与鉴定

为获得带叶兜兰(Paphiopedilum hirsutissimum)种子萌发的共生真菌,采用原地共生萌发技术获得了2株自然萌发的小幼苗,并分离和筛选出了有效的种子萌发共生菌——瘤菌根菌(Epulorhiza sp.)。为验证分离菌株对带叶兜兰种子萌发的有效性,将Phs34号菌株与带叶兜兰种子在灭菌后的原生境基质上进行室内共生萌发试验,结果表明,经过6周的培养,对照组没有观察到种子的萌发;接菌的种子胚明显膨大,突破种皮,形成原球茎,平均萌发率为(58.35±3.41)%。这表明分离得到的瘤菌根菌能促进带叶兜兰的种子萌发。

Synergism Among VA Mycorrhiza, Phosphate Solubilizing Bacteria and Rhizobium for Symbiosis with Blackgram (Vigna mungo L.) Under Field Conditions

A field experiment was conducted at the G.B. Pant University Research Station, Ujhani (U.P.) in rainy (Kharif) season of the year 1994-1995 to study the effect of Rhizobium, VAM (vesicular arbuscular myc- orrhiza) and PSB (phosphate solubilizing bacteria) inoculation, with and without P, on blackgram (Vigna mungo L.) seed yield. Phosphorus application in soil with medium P content (5.4 mg kg～1) increased nodu- lation, grain yield, N and P in plant and grain over no phosphorus control. Forty kilograms of P-2O-5 each hactare recorded an increase of 20.6 % in nodule dry weight, significant increases of 0.35 g kg～(-1) in N con- centration and 1.28 g kg～(-1) in P concentration of plant over 20 kg P-2O-5 ha～(-1). Similar significant increases of 0.59 g kg～(-l) in grain yield and 0.54 and 0.23 g kg～(-1) in N and P concentrations of the grain, respectively, over 20 kg P-2O-5 ha～(-1) were also obtained with higher dose. Inoculation of Rhizobium ＋ VAM ＋ PSB at all the stages of plant growth recorded maximum increases in all the parameters studied. Dual inoculation of Rhizobium with either VAM or PSB was generally significant in the effect and better than that of VAM ＋ PSB, however, P accumulation in plant and grain was more with VAM ＋ PSB. Among single inocula tions, Rhizobium gave highest and 21.0 % more nodule number, 34.7 % more nodule dry mass, 0.73 g kg～(-1) more N in grain and 4.2 % higher grain yield over PSB. PSB, however, registered significant increases in P concentration in plant and grain over VAM and Rhizobium.

Loss of FHIT expression in gastric mucosa of patients with family histories of gastric cancer and Helicobacter pylori infection

AIM: To answer the question whether FHIT gene expression is affected by the family history of gastric carcinoma and the presence of Helicobacter pylori (Hpylori) in the gastric mucosa of patients with dyspepsia.METHODS: FHIT gene expression in two different topographic sites of the gastric mucosa of twenty-one patients with dyspepsia and with or without familial gastric carcinoma, infected or not infected with H pylori, was evaluated by reverse transcription-PCR (RT-PCR) and IMAGE QUANT methods. A rapid urease test and histopathological examination were used to determine H pylori colonization.RESULTS: In the gastric mucosa of patients with family histories of gastric carcinoma, the amount of FHIT protein mRNA was reduced down to 32%, and for patients with H pylori colonization, to 24% in comparison to controls with dyspepsia and without cancer in the family. FHIT expression was independent of the topography of specimens (corpus vsantrum), and for the control patients it was less sensitive to infection with H pylori. A considerable statistical difference in FHIT levels was observed in the gastric mucosa from the corpus of patients with family histories of gastric carcinoma in respect to H pylori colonization (P = 0.06). Macroscopic evaluation of the gastric mucosa demonstrated that pathologic changes classified according to the Sydney system had no significant influence on FHIT expression within each tested group of patients.CONCLUSION: Loss of FHIT expression was observed in patients with dyspepsia and family histories of gastric carcinoma, especially those infected with H pylori. Such results may constitute an early indication of the development of gastric carcinoma, which is associated with family factors including heredity and H pylori infection. The loss of the FHIT gene may serve as a marker for early diagnosis and prevention of gastric carcinoma, especially in context of early monitoring of H pylori infection in individuals with a record of familial stomach cancer.

Screening of Highly Effective Sinorhizobium meliloti Strains for 'Vector' Alfalfa and Testing of Its Competitive Nodulation Ability in the Field

Seventeen Sinorhizobium meliloti strains from seven provinces in China were used to screen highly effective strains for alfalfa cultivar in a greenhouse study and their symbiotic relationship and competitive ability were studied in the field. CCBAU30138 was the most effective strain, as evidenced by increase in dry weights. A field experiment showed that the inoculation of alfalfa with CCBAU30138 resulted in increases of 11.9% and 19.6% of dry matter production and crude protein production, respectively, in forage of monocultured plants. The total dry matter yields of alfalfa and tall fescue in binary culture were increased by 16.3% by inoculation of alfalfa with this strain. These results showed that S. rneliloti strain CCBAU30138 was an effective inoculant both in the greenhouse and in the field. The analysis of randomly amplified polymorphic DNA （RAPD） by polymerase chain reaction （PCR） from nodule extracts showed that the strain CCBAU30138 had high competitiveness in the field. It occupied 47.5% of nodules in alfalfa monoculture and 44.4% of nodules in alfalfa-tall fescue binary culture after 20 weeks of growth. In conclusion, a simple system to select highly effective and competitive symbiotic strains specific to alfalfa was established. Using this system, a s.train suitable for the alfalfa cultivar ‘Vector＇ grown in Wuqiao County of Hebei Province was obtained.

与菌根真菌共生的兰科福建金线莲差异表达基因的筛选

研究菌根真菌促进药用植物生长发育的分子作用机制在农业上有着十分重要的理论意义和实践意义.本研究从野生福建金线莲(Anoectochilus roxburghii)根中分离出一株内生真菌AR-18,经鉴定为瘤菌属(Epulorhizasp.)真菌;将AR-18菌与福建金线莲组培苗接菌培养60天,发现其根中能形成内生菌根并能显著地促进福建金线莲苗的生长.为从与AR-18菌共生的福建金线莲中分离出差异表达基因,本实验采用差异显示PCR(DDRT-PCR)方法,使用3条锚定引物和5条随机引物设计15个引物对,从接菌组和对照组中共分离出52条差异显示条带,其中有9条带能通过双引物重扩增显现.再运用反式Northern点杂交进一步筛选,发现其中5条上调表达基因,包括:编码尿磷酸核苷转移酶基因(UPRTs;EC2.4.2.9)和1条假蛋白基因;1条下调表达基因,编码氨基酸跨膜转运子;1条在接菌组中特异表达基因,编码突变酶K(matK).同时,讨论了上述基因可能的功能,尤其是UPRTs基因和matK基因与菌根真菌互作的功能.本研究是福建金线莲植物在该领域的首次报道.

Inhibition of Aspergillus parasiticus and Cancer Cells by Marine Actinomycete Strains

Ten actinomycete strains isolated from the Yellow Sea off China＇s coasts were identified as belonging to two genera by 16S rDNA phylogenetic analysis： Streptomyces and Nocardiopsis. Six Streptomyces strains （MA10, 2SHXF01-3, MA35, MA05-2, MA05-2-1 and MA08-1） and one Nocardiopsis strain （MA03） were predicted to have the potential to produce aromatic polyketides based on the analysis of the KSa （ketoacyl-synthase） gene in the type II PKS （polyketides synthase） gene cluster. Four strains （MA03, MA01, MA10 and MA05-2） exhibited significant inhibitory effects on mycelia growth （inhibition rate 〉50%） and subsequent aria- toxin production （inhibition rate 〉75%） of the mutant aflatoxigenic Aspergillus parasiticus NFRI-95. The ethyl acetate extracts of the broth of these four strains displayed significant inhibitory effects on mycelia growth, and the IC50 values were calculated （MA03： 0.275 mg mL-1, MA01：0.106 mg mL-1, MA10：1.345 mg mL-1 and MA05-2：1.362 mg mL-1）. Five strains （2SHXF01-3, MA03, MA05-2, MA01 and MA08-1） were selected based on their high cytotoxic activities. The ethyl acetate extract of the Nocardiopsis strain MA03 was particularly noted for its high antitumor activity against human carcinomas of the cervix （HeLa）, lung （A549）, kidney （Caki-1） and liver （HepG2） （IC50： 2.890, 1.981, 3.032 and 2.603 μgmL-1, respectively）. The extract also remarkably inhibited colony formation of HeLa cells at an extremely low concentration （0.5μgmL 1）. This study highlights that marine-derived actinomycetes are a huge resource of compounds for the biological control of aflatoxin contamination and the development of novel drugs for human carcinomas.

硬叶兰种子的迁地共生萌发及有效共生真菌的分离和鉴定

兰科植物的种子原地和迁地共生萌发技术是近年发展起来的开展兰科植物种子和共生真菌研究的有效方法。该研究对兰属(Cymbidium)附生植物硬叶兰(C.mannii)开展了种子的迁地共生萌发研究,试图获得其种子萌发的有效真菌。利用硬叶兰成年植株根部周围的树皮、苔藓、枯枝落叶、腐殖质等作为培养基质,进行种子的共生培养。在培养133天后,成功地获得了处于不同阶段的已萌发种子、原球茎和幼苗,并从原球茎中分离得到一种瘤菌根菌属(Epulorhiza)真菌。用所分离到的FCb4菌株和一种从兜唇石斛(Dendrobium aphyllum)分离到的胶膜菌属(Tulasnella)FDaI7菌株和硬叶兰种子在燕麦琼脂培养基上进行共生萌发,设置不接菌作为对照处理,以检验FCb4菌株对硬叶兰种子萌发的有效性。经过58天的培养,不接菌的对照处理中种子没有萌发,接种FCb4和FDaI7菌株的处理都有很高的种子萌发率,两种接菌处理在不同光照条件下的种子萌发率均无显著性差异。但暗培养条件下,种子萌发形成原球茎后,表现出生长停滞的趋势,仅有很少的原球茎继续生长达到幼苗阶段,说明原球茎发育后期与幼苗发育阶段需要光照。在光照条件下,接种FCb4菌株处理中达到幼苗阶段种子的比例为(25.67±9.27)%,显著高于接种FDaI7菌株处理的(3.04±2.27)%(W=56,p=0.026,Mann-WhitneyU-test),表明此研究中分离到的瘤菌根菌属真菌能有效地促使硬叶兰种子萌发并生长发育到幼苗阶段。