豚草种带内生真菌及其对种子发芽和幼苗生长的影响

内生真菌是一类共生于植物体内,能够不同程度影响宿主植物生态适应性和竞争能力的微生物。分析内生真菌在豚草种子中的分布、种群结构,以及内生真菌发酵液对种子发芽和幼苗生长的作用。结果显示:发生于6个地区的豚草种子均能分离获得内生真菌,分离率在19%—92.63%之间,不同地区之间差异极显著(P0.01)。内生真菌主要存在于种子的总苞部位,分离率达到65.52%。发生于福建省长乐市松下镇的豚草种带内生真菌种群包含5个属,以链格孢属(Alternaria)真菌为优势菌群,占82.26%;其次为镰孢属(Fusarium)真菌,占9.68%;其它3个属的真菌发生较少,均低于5%。内生真菌主要以水平传播方式在豚草不同世代之间传播。供试的7个内生真菌菌株的发酵液均不同程度地抑制豚草种子发芽,降低幼苗地上部高度、根长度、根数量和总生物量,但不同菌株发酵液之间抑制程度差异明显,显示不同菌株对豚草种子发芽和幼苗生长产生不同的影响。内生真菌发酵液处理后的种子仍然保持较高程度的活力;不同内生真菌发酵液处理后,有活力的种子维持在50%—87.5%之间,均高于(或等于)清水处理的种子,说明内生真菌代谢产物只是抑制种子的发芽,但并不导致种子的腐烂和死亡。这些研究结果初步显示种子携带的内生真菌可能在豚草入侵生物学中发挥重要的作用。     

喜树内生真菌抗水稻纹枯病菌活性的研究

从喜树健康组织叶、茎以及果实中经过分离、纯化共得到26株内生菌株。利用生长速率法测定这26株菌株对植物病原菌——水稻纹枯病病原菌的抑制作用。抑菌试验结果显示：26株菌株中有22株喜树内生真菌的发酵液对植物病原菌菌丝生长均有不同程度的抑制作用;其中,菌株XSY10的发酵液抑菌效果最强,达到了74.97%。水稻盆栽试验结果表明：喷施菌株XSY10次生代谢产物7d后,对水稻纹枯病的防治效果为34.77%。以上试验结果说明喜树内生真菌XSY10对植物病害的防治具有一定的功效。     

黄连内生真菌的分离鉴定及抑菌活性研究CSCD

为研究四川省峨眉地区黄连内生真菌多样性及菌株抑菌活性和机制,发掘黄连内生真菌资源,本研究从川黄连植株分离鉴定14株黄连内生真菌,分属于2纲,5目,7科,11属。根状茎、茎、叶和须根中的内生真菌定殖率分别是27.8%、56.7%、52.5%和39.6%。14株黄连内生真菌对金黄色葡萄球菌均有抑菌效果,Cop S01、Cop L01、Cop FR01的抑菌圈直径超过35 mm,纸片扩散法测得3株内生真菌发酵液对金黄色葡萄球菌均有效,Cop L01抑菌圈直径大于20 mm,抑菌效果最好,其发酵产物最低抑菌浓度(MIC)为10 mg/mL,最低杀菌浓度(MBC)为11 mg/mL。初步探究Cop L01抑菌机制发现该菌株发酵产物破坏金黄色葡萄球菌细胞膜,引起胞内核酸及蛋白外泄,造成金黄色葡萄球菌死亡。本研究表明川黄连内生真菌具有丰富的多样性,部分菌株及其发酵产物对金黄色葡萄球菌均表现出较好的抑菌效果,预示着黄连内生真菌Cop L01具有产生新型抗菌活性物质的潜力,为后续深入开发这一资源宝库提供了新的菌种和思路。     

绞股蓝内生真菌抗大肠杆菌抗菌机制的研究

【目的】药用植物内生真菌是一种新型抗生素的微生物资源,研究绞股蓝内生真菌JY25的抗菌机制对内生真菌的研发具有重要意义。【方法】以二倍稀释法测定发酵液的最小抑菌浓度(MIC)和最小杀菌浓度(MBC);以MIC测定发酵液对大肠杆菌生长的抑制作用;扫描电镜观察发酵液作用下的大肠杆菌形态变化;同时,以β-半乳糖酶、碱性磷酸酶和电导率测定发酵液对细胞膜和细胞壁的损伤效果;采用考马斯亮蓝法检测发酵液对蛋白质合成的影响。【结果】JY25发酵液对大肠杆菌MIC为7 g/L、MBC为14 g/L;MIC浓度的发酵液使细菌对数生长期延迟12 h,菌体形态发生严重的畸形和破损;随着抑菌作用时间的延长,β-半乳糖酶含量增加、电导率增加,同时,实验发现大肠杆菌蛋白质合成异常,未检测到碱性磷酸酶。【结论】绞股蓝内生真菌JY25主要以破坏细菌的细胞膜及影响细菌蛋白质合成而抑制细菌生长。     

几种杀螺细菌的筛选与功效研究

湖北钉螺是日本血吸虫的唯一中间宿主,生物杀螺具有环保、成本较低以及对钉螺定向特异的优点。从鄱阳湖草洲土壤及病螺体内分离出4株对钉螺具有高毒性作用的细菌。革兰氏染色和显微镜观察表明,一株为芽孢杆菌,其余3株均为革兰氏阴性杆菌。16srDNA扩增及序列比对鉴定其种属类型,结果显示分别为Enterobactersp．、B．cereus、S．putrefaciens和Aeromonassp．。杀螺研究结果表明,4株细菌的发酵液、发酵上清液和菌体对钉螺均表现出不同程度的毒杀作用,其中S．putrefaciens和Aeromonassp．的杀螺效果最好,72h发酵液和发酵上清液对钉螺的毒杀作用分别达100％和95％以上。各细菌发酵上清SDS-PAGE结果发现了丰富蛋白带,推测这些蛋白中存在对钉螺具有毒杀功效的组分。     

红豆杉中产紫杉醇内生真菌分离部位的比较研究

目的 探讨红豆杉不同部位在内生真菌分离效率以及产紫杉醇菌株筛选率方面的规律性,为红豆杉产紫杉醇内生菌菌株的分离与筛选提供一定的理论依据.方法 从根、茎、叶3个器官取大小和表面积相同的太行山野生红豆杉(Taxous chinensis)材料,用组织块法分离红豆杉内生真菌,计算各部位内生真菌的分离效率;用高效液相法时分离到的内生真菌发酵液提取物进行紫杉醇含量分析,计算各部位产紫杉醇内生真菌的筛选率.结果 共分离到109株红豆杉内生真菌,根部、茎部和叶部分离效率指数分别为0.90、0.63和0.28;其中有28株产紫杉醇,紫杉醇菌株筛选率分别为31.48%、21.05%和17.65%.结论 在内生真菌的分离效率及其产紫杉醇内生真菌的筛选率上,均为根部>茎部>叶部,即根部在内生真菌分离效率和筛选产紫杉醇内生真菌效率上均具有明显的优势.     

抗植物病原真菌紫叶小檗内生真菌的筛选和鉴定

对紫叶小檗根、茎、叶和果实中分离纯化得到的28株内生真菌进行液体培养,培养物烘干研磨后用丙酮浸提,测定各提取物对5种供试植物病原真菌的抑菌活性,对抗菌谱较广的菌株进行分子鉴定,并分别测定其胞内、胞外代谢产物的抑菌活性.结果显示:(1)紫叶小檗25株内生真菌中有5株菌(R3、S4、L6、F2、F6)的抗菌谱较广,其中,茎中的内生菌S4抑菌活性最强,对5种植物病原真菌的抑菌活性均大于90％,对马铃薯干腐病菌的抑菌率最高达93.06％.(2)经鉴定S4为子囊菌门的Para phaeos phaeria属真菌.(3)S4菌丝体丙酮提取液对马铃薯干腐病菌的抑菌率随其浓度(加入体积)的增加而增大,呈正相关线性关系(y=15.334x+14.618,r=0.99),而S4发酵液对马铃薯干腐病菌的抑菌率随其浓度(加入体积)的增加而减小,呈负相关线性关系(y=-20.196x+64.984,r=0.99),即在较高浓度时促进病原菌生长.研究表明,紫叶小檗内生真菌S4菌株的抑菌活性成分主要为胞内代谢产物.     

喜树内生真菌的分离、鉴定以及抗植物病原菌活性的初步研究

对喜树植株中的内生真菌进行分离纯化,共得到126株内生菌株。对126株内生菌株进行液体培养,并对其发酵产物进行抗菌活性测定。发现在供试质量浓度为5mg／mL时,126株菌株中有24株喜树内生真菌的发酵液对水稻纹枯病菌、稻瘟病菌以及辣椒疫霉具有不同程度的抑制菌丝生长的活性作用;其中,菌株XSY09的发酵液对此三种植物病原菌有明显的抑制作用,抑制率分别为74．97％、39．63％和58．49％。在0．1mg／mL的测试浓度下,菌株XSY09发酵液的乙酸乙酯相对三种植物病原菌均有较好的抑制作用。对该菌株的ITS序列进行测序分析,初步鉴定XSY09为炭疽菌属（Colletotrichum gloeosporioides）真菌。     

杜仲内生真菌DZJ03发酵液生物活性的研究

测定处于不同生长期的杜仲内生真菌DZJ03胞外多糖含量、发酵液中3种核苷含量,并对其菌株发酵液抑菌效果进行了研究,按50μg/mL的浓度测定了发酵液的石油醚、乙酸乙酯和甲醇等3种提取物对水稻恶苗病菌、棉花枯萎病菌2种植物病原菌以及烟草青枯病菌、金黄色葡萄球菌的抑制活性。结果表明:内生真菌DZJ03胞外多糖含量最高可达到2.0410g/L,其发酵液中所含腺苷、尿苷以及鸟苷的含量分别为1.2647 mg/g、0.8586 mg/g、1.0493 mg/g;发酵液乙酸乙酯相具有较强的抑菌活性,其对水稻恶苗病菌的抑制率为71.92%,抗烟草青枯病菌的抑菌圈直径达到19.21 mm。     

蒺藜内生真菌发酵液抗菌及抗肿瘤活性初步研究

【目的】筛选出具有抗菌和抗肿瘤活性的蒺藜内生真菌。【方法】采用牛津杯法、稻瘟菌模型及肿瘤细胞模型评价蒺藜内生真菌PDB和察氏培养基发酵产物的抗菌活性和肿瘤细胞毒性。【结果】PDB培养基发酵液和察氏培养基发酵液抑菌圈直径大于10 mm的菌株分别占总菌株数的19.05%和23.81%。PDB培养基发酵液和察氏培养基发酵液对稻瘟菌的最小抑制浓度(MIC)低于10%的菌株分别占总菌株数的19.05%和47.61%。对肿瘤细胞抑制率高于50%的PDB发酵产物占PDB发酵产物总数的52.38%, 而对肿瘤细胞抑制率高于50%的察氏发酵产物占察氏发酵产物总数的28.57%。【结论】部分蒺藜内生真菌的发酵产物具有抗菌和抗肿瘤活性。     

大叶醉鱼草内生真菌LL3026菌株杀虫活性及ITS-5.8S rDNA序列分析

从大叶醉鱼草的叶子中分离得到一株内生真菌LL3026,以卤虫模型测稀释后发酵液的杀虫活性,结果表明LL3026发酵液杀虫活性较强,且温度、光照及紫外照射对LL3026发酵液杀虫活性影响不显著;采用分子生物学方法对LL3026菌株rDNA的ITS基因(ITS-5.8S rDNA)进行PCR扩增、测序,构建系统发育树。ITS基因显示其属于刺盘孢属真菌。     

Inactivation of phosphomannose isomerase gene abolishes sporulation and antibiotic production in Streptomyces coelicolor

Phosphomannose isomerases (PMIs) in bacteria and fungi catalyze the reversible conversion of D-fructose-6-phosphate to D-mannose-6-phosphate during biosynthesis of GDP-mannose, which is the main intermediate in the mannosylation of important cell wall components, glycoproteins, and certain glycolipids. In the present study, the kinetic parameters of PMI from Streptomyces coelicolor were obtained, and its function on antibiotic production and sporulation was studied. manA (SCO3025) encoding PMI in S. coelicolor was deleted by insertional inactivation. Its mutant (S. coelicolor?manA) was found to exhibit a bld-like phenotype. Additionally, S. coelicolor?manA failed to produce the antibiotics actinorhodin and red tripyrolle undecylprodigiosin in liquid media. To identify the function of manA, the gene was cloned and expressed in Escherichia coli BL21 (DE3). The purified recombinant ManA exhibited PMI activity (K(cat)/K(m) (mM(-1) s(-1) = 0.41 for D-mannose-6-phosphate), but failed to show GDP-D-mannose pyrophosphorylase [GMP (ManC)] activity. Complementation analysis with manA from S. coelicolor or E. coli resulted in the recovery of bld-like phenotype of S. coelicolor?manA. SCO3026, another ORF that encodes a protein with sequence similarity towards bifunctional PMI and GMP, was also tested for its ability to function as an alternate ManA. However, the purified protein of SCO3026 failed to exhibit both PMI and GMP activity. The present study shows that enzymes involved in carbohydrate metabolism could control cellular differentiation as well as the production of secondary metabolites.     

The host defense peptide LL‐37 activates the tumor‐suppressing bone morphogenetic protein signaling via inhibition of proteasome in gastric cancer cells

The human cathelicidin LL‐37, a pleiotropic host defense peptide, is down‐regulated in gastric adenocarcinomas. We therefore investigated whether this peptide suppresses gastric cancer growth. LL‐37 lowered gastric cancer cell proliferation and delayed G₁‐S transition in vitro and inhibits the growth of gastric cancer xenograft in vivo. In this connection, LL‐37 increased the tumor‐suppressing bone morphogenetic protein (BMP) signaling, manifested as an increase in BMP4 expression and the subsequent Smad1/5 phosphorylation and the induction of p21^Waf1/Cip1. The anti‐mitogenic effect, Smad1/5 phosphorylation, and p21^Waf1/Cip1 up‐regulation induced by LL‐37 were reversed by the knockdown of BMP receptor II. The activation of BMP signaling was paralleled by the inhibition of chymotrypsin‐like and caspase‐like activity of proteasome. In this regard, proteasome inhibitor MG‐132 mimicked the effect of LL‐37 by up‐regulating BMP4 expression and Smad1/5 phosphorylation. Further analysis of clinical samples revealed that LL‐37 and p21^Waf1/Cip1 mRNA expressions were both down‐regulated in gastric cancer tissues and their expressions were positively correlated. Collectively, we describe for the first time that LL‐37 inhibits gastric cancer cell proliferation through activation of BMP signaling via a proteasome‐dependent mechanism. This unique biological activity may open up novel therapeutic avenue for the treatment of gastric cancer. J. Cell. Physiol. 223: 178–186, 2010. © 2010 Wiley‐Liss, Inc.     

水稻miRNA3026启动子及硫氧还蛋白基因OsTxnDC9的克隆与分析

硫氧还原蛋白基因 OsTxnDC9 是水稻miRNA3026的宿主基因。克隆出了水稻miRNA3026启动子,其总长度为1 477bp;构建出4个缺失片段,瞬时表达表明这个启动子为弱启动子。在此基础上克隆出水稻硫氧还原蛋白 OsTxnDC9 基因,其长度为480bp。生物信息学表明 OsTxnDC9 基因编码的氨基酸序列与二穗短柄草硫氧还原蛋白基因编码的氨基酸(XP_003573612.1)序列同源性最高。荧光定量PCR发现OsTxnDC9在水稻花粉一核中表达量最高,亚细胞定位表明其主要在细胞质中表达。这为探索miRNA3026启动子和宿主基因的关系奠定了基础。     

High light intensity protects photosynthetic apparatus of pea plants against exposure to lead.

The electron transport rates and coupling factor activity in the chloroplasts; adenylate contents, rates of photosynthesis and respiration in the leaves as well as activity of isolated mitochondria were investigated in Pisum sativum L. leaves of plants grown under low or high light intensity and exposed after detachment to 5 mM Pb(NO(3))(2). The presence of Pb(2+) reduced rate of photosynthesis in the leaves from plants grown under the high light (HL) and low light (LL) conditions, whereas the respiration was enhanced in the leaves from HL plants. Mitochondria from Pb(2+) treated HL-leaves oxidized glycine at a higher rate than those isolated from LL leaves. ATP content in the Pb-treated leaves increased to a greater extend in the HL than LL grown plants. Similarly ATP synthase activity increased markedly when chloroplasts isolated from control and Pb-treated leaves of HL and LL grown plants were subjected to high intensity light. The presence of Pb ions was found inhibit ATP synthase activity only in chloroplasts from LL grown plants or those illuminated with low intensity light. Low light intensity during growth also lowered PSI electron transport rates and the Pb(2+) induced changes in photochemical activity of this photosystem were visible only in the chloroplasts isolated from LL grown plants. The activity of PSII was influenced by Pb ions on similar manner in both light conditions. This study demonstrates that leaves from plants grown under HL conditions were more resistant to lead toxicity than those obtained from the LL grown plants. The data indicate that light conditions during growth might play a role in regulation of photosynthetic and respiratory energy conservation in heavy metal stressed plants by increasing the flexibility of the stoichiometry of ATP to ADP production.     

大黄鱼共生菌Bacillus coagulans LL1103的代谢产物研究

[目的]研究大黄鱼共生菌Bacillus coagulans LL1103的次生代谢产物及其抑菌活性。[方法]利用色谱层析技术对B.coagulans LL1103发酵液乙酸乙酯和正丁醇提取物进行分离纯化;运用波谱技术鉴定化合物的结构;采用微量肉汤稀释法对化合物进行抗菌活性测定。[结果]从大黄鱼内生菌B.coagulans LL1103发酵液中分离并鉴定9个环二肽类化合物,分别为:(1)环(4-羟基-脯氨酸-亮氨酸)、(2)环(异亮氨酸-丙氨酸)、(3)环(脯氨酸-缬氨酸)、(4)环(脯氨酸-苯丙氨酸)、(5)环(脯氨酸-亮氨酸)、(6)环(甘氨酸-丙氨酸)、(7)环(脯氨酸-甘氨酸)、(8)环(脯氨酸-丙氨酸)和(9)环(络氨酸-甘氨酸)。活性评价显示化合物3、7和9对大肠埃希菌(Escherichia coli)具有较强抑制作用,最低抑菌浓度(minimal inhibitory concentration,MIC)分别为8.0、4.0和16.0μg/mL。[结论]从大黄鱼共生菌B.coagulans LL1103的发酵液中分离得到3个对大肠埃希菌(E.coli)具有较好抑制活性的化合物。     

Isolation and Structure of Ascochlorin and Its Analogs

Ascochlorin and its analogs were isolated from the filter cake of the fermented broth of a fungus, Ascochyta viciae Libert; the compounds obtained were ascochlorin, LL–Z1272 δ, LL–Z1272 ε, ascofuranone, ascofuranol and a new analog, C₂₃H₃₁ClO₅. Some of these prenyl phenols show hypolipidemic activity in both normolipidemic and hyperlipidemic rats. Details of the structure determination for ascochlorin are presented. The structure of the new analog was elucidated as 4′-hydroxy-5′-hydroascochIorin.     

Effect of Pb ions on superoxide dismutase and catalase activities in leaves of pea plants grown in high and low irradiance

The role of irradiance on the activity of antioxidant enzymes: superoxide dismutase (SOD) and catalase (CAT) was examined in the leaves of Pisum sativum L. plants grown under low (LL) or high (HL) irradiance (PPFD 50 or 600 μmol m⁻² s⁻¹) and exposed after detachment to 5 mM Pb (NO₃)₂ for 24 h. The activities of both enzymes increased in response to LL compared with HL and no effect of Pb ions was observed. Photosystem (PS) 1 and PS 2 activities were also investigated in chloroplasts isolated from these leaves. LL lowered PS 1 electron transport rate and changes in photochemical activity of PS 1 induced by Pb²⁺ were visible only in the chloroplasts isolated from leaves of LL grown plants. PS 2 activity was influenced similarly by Pb ions at both PPFD. This study demonstrates that leaves of HL grown plants were less sensitive to lead toxicity than those from LL grown plants. Changes in electron transport rates were the main factors responsible for the generation of reactive oxygen species in the chloroplasts and as a consequence, in induction of antioxidant enzymes.     

Effect of Z-100, an immunomodulator extracted from human type tubercle bacilli,on the pulmonary metastases of lewis lung carcinoma in attempt to regulate suppressor T cells and suppressor factor,IL-4

In the present study, anti-metastatic effect of Z-100 on the spontaneous pulmonary metastases of Lewis lung carcinoma (3LL) was examined in an attempt to regulate suppressor T cells. When Z-100 (10 mg/kg) was daily injected i.p. after 3LL inoculation, survival rate of these mice was increased significantly (p<0.05). In addition, the number of pulmonary metastatic colonies of 3LL in Z-100-treated mice were significantly decreased by 38% at 21 days, as compared with that of control mice (p<0.05). Along with the decrease of pulmonary metastases, suppressor cell activity was also gradually reduced in these mice, as compared with that of control mice. When splenic suppressor cells (5×10⁷ cells) from 3LL-bearing mice were adoptively transferred into normal mice (recipients) just before inoculation of 3LL, the development of pulmonary metastases in recipients was significantly accelerated. However, splenocytes from 3LL-bearing mice treated with Z-100 did not affect the development of pulmonary metastasis. The potential to accelerate the metastasis of splenic mononuclear cells from 3LL-bearing mice was decreased significantly by the treatment with anti-Thy 1.2 monoclonal antibody (mAb), anti-Lyt 2.2 mAb or anti-CD11b mAb followed by complement. IL-4 activity in the sera of 3LL-bearing mice was detected 15 days after tumor inoculation (13 pg/ml) and gradually increased (18 pg/ml) 20 days after tumor inoculation. However, when Z-100 (10 mg/kg) was daily injected i.p., IL-4 activity in sera was decreased significantly, and the IL-4 activity was not detected in these mice on day 20. These results suggest that Z-100 could inhibit the pulmonary metastases in 3LL-bearing mice through the inhibition of suppressor T cell activity and a possible candidate of its effector molecule, IL-4.