球毛壳菌降解天然木质纤维素能力差异及酶系基因分析简

目的：以不同植物中分离到的4株内生球毛壳菌NK102、NK103、NK104和NK105为对象,研究不同生态来源球毛壳菌降解木质素和纤维素的能力。方法：首先采用羧甲基纤维素和纤维素刚果红平板检测各菌株的纤维素降解能力,并利用Bavendamm平板反应检测各菌株的木质素降解能力;将4株菌分别培养在以微晶纤维素、杨树叶和木屑为惟一碳源的液体培养基中,通过检测培养液中纤维素酶和漆酶的酶活力,比较各菌株分解利用天然木质纤维素材料的能力,连续培养12d后检测培养液中次级代谢产物的合成情况;利用已测序的球毛壳菌CBS148．51的基因组信息,寻找编码木质纤维素降解酶类的基因,为球毛壳菌分解利用木质纤维素提供分子生物学依据。结果：NK102、NK103、NK104和NK105在羧甲基纤维素培养基和纤维素刚果红培养基上都能够生长并形成水解圈;Bavendamm平板反应显示4株菌降解木质素的能力由强到弱依次是NK103、NK102、NK105和NK104。4株菌都能分解利用微晶纤维素、杨树叶和木屑,分泌纤维素酶和漆酶,其中NK102在以木屑为碳源的培养基上纤维素酶活力最强,达到0．76U/mL发酵液,NK103在以杨树叶为碳源的培养基上漆酶活力最强。与此同时,4株菌在发酵培养过程中都能够稳定地合成球毛壳甲素（ChA）,ChA产量受到碳源影响,在以杨树叶为碳源的培养基上,NK104的ChA产量最高,可达到14．88mg/L发酵液。利用已测序的球毛壳菌CBS148．51的基因组信息,寻找到119个编码纤维素半纤维素酶的基因、8个编码漆酶的基因和2个编码锰过氧化物酶的基因,球毛壳菌具有完整的降解纤维素半纤维素的酶体系,在木质纤维素降解真菌的开发过程中具有重要的研究价值。结论：本研究为球毛壳菌木质纤维素降解过程的研究及该菌种的开发利用奠定了基础。     

银杏内生菌Chaetomium globosum ZY-22次生代谢产物分离鉴定

采用柱层析方法从银杏叶内生真菌Chaetomium globosum ZY-22的培养菌丝体提取物中分离得到脑苷脂B(1)、脑苷脂C(2)、尿囊素(3)、9(11)-去氢麦角甾醇过氧化物(4)以及4,6,8,22-四烯-3-酮-麦角甾烷(5)和球毛壳甲素(6)共6个次生代谢物;经波谱分析确定了6个化合物的结构,其中脑苷脂B、脑苷脂C和尿囊素是首次从内生真菌中得到;海虾致死试验结果显示,化合物1～6在10 μg/mL浓度下对丰年虾的致死率分别为1.6%、4.2%、7.4%、16.9%、12.8%、83.6%、表明球毛壳甲素对海虾表现出很强的毒性作用.     

球毛壳菌循环肽HC-毒素基因的序列分析

为了验证Phrap软件是否适合在EST分析中应用,对球毛壳菌循环肽HC-毒素基因进行了序列分析。根据EST分析的结果,从cDNA文库中挑取循环肽HC-毒素基因的克隆进行了测序并序列分析。结果表明cDNA文库中循环肽HC-毒素基因的克隆插入片断大小为1217bp;用Phrap软件拼接出来的循环肽HC-毒素的表达序列标签拼接序列与实际序列不完全一致,因此Phrap软件不适合在EST分析中应用。     

纤维素降解球毛壳菌NK-102的高效遗传转化方法

摘要:【目的】在球毛壳菌(Chaetomium globosum)NK-102 中,建立菌株特异性转化体系。【方法】构建新的抗性标记pUCATPH-Pgap,转化效率优于pUCATPH 和pCM768。建立了PEG-原生质体和根癌农杆菌(Agrobacterium tumefaciens)EHA105 介导的两种转化方法。【结果】原生质体转化效率为30－50 个转化子/10 μg DNA,抗性标记pUCATPH-Pgap 效率最高。EHA105 介导转化率达到3.2×102 转化子/107 孢子。Sout     

根癌农杆菌介导的球毛壳菌遗传转化及T-DNA插入突变体的获得

根癌农杆菌介导的遗传转化系统是植物基因工程常用方法,目前已将这一转化系统应用到酵母、丝状真菌以及人类细胞的转化。利用这一转化系统,成功地实现了丝状真菌球毛壳菌（Chaetomium globosum）的遗传转化,转化率约为60～180个转化子/10.7个孢子 。通过对转化子的PCR检测和Southern 杂交分析表明,TDNA已整合进毛壳菌基因组中,而且在所检测的转化子中都是以单拷贝的形式整合,转化子都能够稳定遗传。根癌农杆菌介导的遗传转化具有转化率高、低拷贝、遗传稳定、操作简便等优点,因此有可能成为丝状真菌遗传转化和功能基因组研究的有力工具。     

Aureonitols A and B,Two New C13‐Polyketides from Chaetomium globosum,an Endophytic Fungus in Salvia miltiorrhiza

Two new C₁₃‐polyketides, aureonitols A and B ( 1 and 2 ), along with five known compounds ( 3 – 7 ), were isolated from the solid fermentation culture of the plant endophytic fungus Chaetomium globosum from the aerial parts of Salvia miltiorrhiza. The structures and absolute configurations of 1 and 2 were determined by comprehensive spectroscopic data analysis and computed methods. Compound 5 was found to display the remarkable antimicrobial activities against four multidrug‐resistant bacteria (Enterococcus faecalis, Enterococcus faecium, Staphylococcus aureus, and Staphylococcus epidermidis) with MIC values of 3.13–6.25 μg/mL (ciprofloxacin: 0.78–1.56 μg/mL), and also against all tested fungal strains with MIC values of 3.13–25 μg/mL (ketoconazole: 0.78–12.50 μg/mL).     

Antifungal activity against plant pathogenic fungi of chaetoviridins isolated from Chaetomium globosum

The Chaetomium globosum strain F0142, which was isolated from barnyard grass, showed potent disease control efficacy against rice blast (Magnaporthe grisea) and wheat leaf rust (Puccinia recondita). Two antifungal substances were purified from broth from this organism and identified as chaetoviridins A and B. Chaetoviridin A exhibited higher antifungal activity than chaetoviridin B against plant pathogenic fungi both in vitro and in vivo. Treatment with chaetoviridin A at 62.5 microg/mL suppressed the development of rice blast and wheat leaf rust by over 80%. The molecule also exhibited moderate control of tomato late blight, resulting in 50% control following the application of 125 microg/mL chaetoviridin A.     

球毛壳菌甘油醛-3-磷酸脱氢酶基因克隆及特性分析

用粗糙脉孢菌(Neurospora crassa,XP_327967)和菜豆炭疽病菌(Colletotrichum lindemuthianu,P35143)的甘油醛_3_磷酸脱氢酶基因(Glyceraldehyde 3_phosphatedehydrogenase,GAPDH)氨基酸序列对球毛壳菌(Chaetomium globosum)菌丝ESTs序列本地数据库进行tBlastn检索,获得了球毛壳菌GAPDH全长cDNA序列。该序列长1240bp,开放阅读框1014bp,编码337个氨基酸组成的多肽,蛋白分子量为36.1kD。用PCR方法克隆了该基因的DNA序列,序列长为1556bp,由2个内含子和3个外显子组成。BlastP同源性分析表明该基因与鹅掌柄孢壳(Podosporaanserine)同源性最高为95%;与米曲霉(Aspergillusoryzae)同源性最低为87%。GAPDH酵母转化子生物功能分析表明转化子对Na2CO3和高温有高的耐受性,证明GAPDH为抗胁迫基因。该基因的cDNA序列、DNA序列及推测的氨基酸序列在GenBank登录(登录号分别为AY522719,AY593253,AAS01412)。     

Identification of a Unique Azaphilone Produced by Chaetomium globosum Isolated from Polygonatum sibiricum

A new azaphilone, chaephilone E, eight azaphilone derivatives, and three chaetoglobosins were isolated from endophytic fungi Chaetomium globosum. The structures of the compounds were elucidated by 1D and 2D NMR as well as HR‐ESI‐MS data, and the absolute configuration of chaephilone E was established on the basis of electronic circular dichroism and NOESY spectrum. The activity of chaephilone E was evaluated via the cytotoxic assay (human hepatoma cell lines HepG‐2) and brine shrimp (Artemia salina) bioassay.     

球毛壳菌组蛋白H3基因克隆与特性分析

构建了球毛壳菌菌丝的cDNA文库,并获得了1410条ESTs序列,用里氏木霉(Hypocrea jecorina,AAM76068)和粗糙脉胞菌(Neurospora crassa,CAA25761)的组蛋白H3基因(Histone H3)蛋白序列对球毛壳菌(Chaetomium globosum)ESTs序列本地数据库进行tBlastn检索,获得了球毛壳菌组蛋白H3cDNA序列。cDNA序列全长739bp,开放阅读框411bp,编码136个氨基酸组成的多肽,蛋白分子量为15.4kD。BlastP同源性分析表明该基因与里氏木霉同源性最高为100%;与地钱(Marchantia polymorpha)同源性最低为95%。三级结构预测表明,该蛋白C端为球状结构域,而N端结构对其发挥调控作用起重要作用。该基因的cDNA序列及推测的氨基酸序列在GenBank登录(登录号分别为AY669068,AAT74576)。     

Production of extracellular proteins,cellulases and antifungal metabolites by Chaetomium globosum Kunze ex. Fr.

Chaetomium globosum has been well-known potential antagonist of several seed and soilborne fungus. Eight isolates of C. globosum were obtained from different sources and were identified by morphological characters. C. globosum isolates examined for the presence of extra cellular proteins, cellulases and antifungal metabolites in culture filtrate by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), thin-layer chromatography and high-performance liquid chromatography. Variation in the mycelial protein of C. globosum isolates was noted in the SDS-PAGE analysis. Different C. globosum isolates that showed more number of bands in protein profile was further screened for the production of cellulases in culture filtrate. Cellulase activity of C. globosum isolates revealed that maximum activity was observed in the isolate Cg-6 after 11?days of incubation, while Cg-2 had least activity. C. globosum isolates were tested for antibiotic production, among which three isolates viz. Cg-6, Cg-7 and Cg-5 were found to produce the antibiotic Chaetoglobosin A in the culture filtrate. The antibiotic Chaetoglobosin A appeared blue colour under UV spectrum with a wavelength of 250?nm.     

球毛壳菌60S核糖体蛋白L10a基因克隆与特性分析

用粗糙脉孢菌（Neurospora crassa）XP_322380和赤霉菌（Gibberella zeag）PH-1（EAA76971）的60S核糖体蛋白L10a基因（60S ribosomal protein L10a,RPL10a）蛋白序列对球毛壳菌（Chaetomium globosum）ESTs序列数据库进行tBlastn检索,获得了球毛壳菌RPL10a cDNA序列。cDNA序列长765bp,开放阅读框654bp,编码217个氨基酸组成的多肽,蛋白分了量为23．9kD。BlastP分析表明该基因氨基酸序列与粗糙脉胞菌相似最高为89％;与玉蜀黍黑粉菌（Ustilago maydis）相似性最低为78％。cDNA序列及推测的氨基酸序列在GenBank登录（登录号分别为AY669070,AAT74578）。     

High expression level of antioxidants and pharmaceutical bioactivities of endophytic fungus Chaetomium globosum JN711454

In order to maximize antioxidant activity of pharmaceutical bioactive endophytic fungus Chaetomium globosum JN711454 during fermentation process, designed fermentation experiments of culture media for three levels of eight culture factors were performed using a Taguchi orthogonal array (OA) design with layout L18 (2¹ × 3⁷). The agitation and the potato extract were the most significant affecting factors, and their interaction contributed significantly to fungus activity. The production of antioxidants was more favorable for static condition with 25 g potato extract/100 m. The remaining factors had no strong impact when considered individually. The validation of statistically optimized medium indicated the improvement of antioxidant activity to a level of twofold with approximately overall 40% enhancement in activity. The extract of optimized medium was investigated for various pharmaceutical bioactivities; it revealed a moderate antimicrobial activity, strong anticancer activity against HepG-2, UACC62 cell lines, an antiviral activity against HSV-2 virus, and strong inhibitory activity to butyrylcholinesterase enzyme, one of the neurohydrolase enzymes that play a major role in development of Alzheimer’s disease. As a result of applying statistical fermentation designs, the optimized conditions of endophytic fungus C. globosum JN711454 developed a cost-effective production medium by using inexpensive commercial potato extracts statically, which can lower the energy requirement and could become an efficient, economic, and viable fermentation process for production of pharmaceutical secondary metabolites.     

Controlling Myzus persicae with recombinant endophytic fungi Chaetomium globosum expressing Pinellia ternata agglutinin: using recombinant endophytic fungi to control aphids

Aims: Sap‐sucking insect pests have become the major threats to many crops in recent years; however, only a few biopesticides have been developed for controlling those pests. Here, we developed a novel pest management strategy, which uses endophytes to express anti‐pest plant lectins. Methods and Results: The fungal endophyte of Chaetomium globosum YY‐11 with anti‐fungal activities was isolated from rape seedlings. Pinellia ternata agglutinin (pta) gene was cloned into YY‐11 mediated by Agrobacterium tumefaciens. The positive transformants, as selected by antibiotic resistance, were evaluated using PCR and Western blot assay. We found that the recombinant endophytes colonized most of the crops, and the resistance of rape inoculated with recombinant endophytic fungi significantly inhibited the growth and reproduction of Myzus persicae. Conclusions: Our results showed that the recombinant endophytes expressing Pinellia ernata agglutinin (PTA) may endow hosts with resistance against sap‐sucking pests. Significance and Impact of the Study: This research may have important implications for using endophytes to deliver insecticidal plant lectin proteins to control sap‐sucking pests for crop protection.     

Endophytic bacterium Bacillus subtilis (BERA 71) improves salt tolerance in chickpea plants by regulating the plant defense mechanisms

Plant growth-promoting endophytic bacteria can stimulate the growth, nutrient acquisition, symbiotic performance and stress tolerance of chickpea plants under saline soil conditions. The aim of this study was to investigate the stress-adaptive mechanisms of chickpea plants mediated by Bacillus subtilis (BERA 71) under saline conditions. Inoculation with BERA 71 enhanced plant biomass and the synthesis of photosynthetic pigments and reduced the levels of reactive oxygen species (ROS) and lipid peroxidation in plants under conditions of stress. Furthermore, the activities of ROS-scavenging antioxidant enzymes (superoxide dismutase, peroxidase, catalase and glutathione reductase), the levels of non-enzymatic antioxidants (ascorbic acid and glutathione) and the total phenol content were increased in stressed plants during bacterial association. The bacteria decreased sodium accumulation and enhanced the nitrogen, potassium, calcium and magnesium content in the plants. The suppression of ROS generation and of lipid peroxidation and the accumulation of proline in BERA-71-inoculated plants enhanced the membrane stability under salinity stress and non-stress conditions.     

Serum iron and copper status and oxidative stress in severe and mild preeclampsia

Our aim was to investigate parameters of iron and copper status and oxidative stress and antioxidant function in women with healthy pregnancy, mild and severe preeclampsia with a view to exploring the possible contribution of these parameters to the aetiology. Thirty healthy, 30 mild preeclamptic and 30 severe preeclamptic pregnant women were included. Serum and placental lipid peroxides, and serum vitamin E and total carotene levels were measured by colorimetric assay. Cholesterol, copper, iron, total iron binding capacity (TIBC), ceruloplasmin and transferrin concentrations were measured by commercially available procedures. Data were analysed statistically using one-way analysis of variance and Pearson correlation test. Logistic regression procedures were used to calculate odds ratios. Lipid peroxides in serum and placental tissue, and iron, copper and ceruloplasmin levels in serum were significantly increased, and transferrin, TIBC, vitamin E/total cholesterol and total carotene/total cholesterol ratios in serum were significantly decreased especially in women with severe preeclampsia. Significant correlations were detected between serum iron and lipid peroxides in serum and placental tissue and between serum iron and vitamin E/total cholesterol in severe preeclamptic pregnancy. Furthermore, there were significant correlations between serum malondialdehyde and ceruloplasmin and vitamin E/total cholesterol in women with severe preeclampsia, and changes in serum and placental lipid peroxides and serum iron concentrations were significantly associated with preeclampsia. In conclusion, ischaemic placental tissue may be a primary source of potentially toxic iron in preeclampsia and the released iron species may contribute to the aetiology and would exacerbate lipid peroxidation and endothelial cell injury, which may be abated by antioxidant supplementation.     

Acquired tolerance of tomato (Lycopersicon esculentum cv. Micro‐Tom) plants to cadmium‐induced stress

The effects of varying concentrations of cadmium (Cd) on the development of Lycopersicon esculentum cv. Micro‐Tom (MT) plants were investigated after 40 days (vegetative growth) and 95 days (fruit production), corresponding to 20 days and 75 days of exposure to CdCl₂, respectively. Inhibition of growth was clearly observed in the leaves after 20 days and was greater after 75 days of growth in 1 mM CdCl₂, whereas the fruits exhibited reduced growth following the exposure to a concentration as low as 0.1 mM CdCl₂. Cd was shown to accumulate in the roots after 75 days of growth but was mainly translocated to the upper parts of the plants accumulating to high concentrations in the fruits. Lipid peroxidation was more pronounced in the roots even at 0.05 mM CdCl₂ after 75 days, whereas in leaves, there was a major increase after 20 days of exposure to 1 mM CdCl₂, but the fruit only exhibited a slight significant increase in lipid peroxidation in plants subjected to 1 mM CdCl₂ when compared with the control. Oxidative stress was also investigated by the analysis of four key antioxidant enzymes, which exhibited changes in response to the increasing concentrations of Cd tested. Catalase (EC 1.11.1.6) activity was shown to increase after 75 days of Cd treatment, but the major increases were observed at 0.1 and 0.2 mM CdCl₂, whereas guaiacol peroxidase (EC 1.11.1.7) did not vary significantly from the control in leaves and roots apart from specific changes at 0.5 and 1 mM CdCl₂. The other two enzymes tested, glutathione reductase (EC 1.6.4.2) and superoxide dismutase (SOD, EC 1.15.1.1), did not exhibit any significant changes in activity, apart from a slight decrease in SOD activity at concentrations above 0.2 mM CdCl₂. However, the most striking results were obtained when an extra treatment was used in which a set of plants was subjected to a stepwise increase in CdCl₂ from 0.05 to 1 mM, leading to tolerance of the Cd applied even at the final highest concentration of 1 mM. This apparent adaptation to the toxic effect of Cd was confirmed by biomass values being similar to the control, indicating a tolerance to Cd acquired by the MT plants.     

Morphological changes and antioxidant defence systems in soybean genotypes as affected by salt stress

Abstract

The present investigation was conducted to evaluate salt tolerance in nine genotypes of soybean (Glycine max L.). Ten-day-old seedlings, grown hydroponically, were treated with 0, 25, 50, 75, 100, 125 and 150 mM NaCl for five days. Growth, lipid peroxidation and antioxidant enzyme activities were evaluated. Growth, measured in terms of length, fresh weight and dry weight of plants, was drastically reduced in PK-416, while there was little effect of NaCl treatment on Pusa-37 genotype of soybean. A high level of lipid peroxidation was observed in PK-416 as indicated by increased level of malondialdehyde. Activities of superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase were maximum in Pusa-37 where 9-fold, 1-fold, 5-fold and 6-fold increases over control were observed, respectively. The results suggested that PK-416 and Pusa-37 are salt-sensitive and salt-tolerant genotypes of soybean, respectively, and antioxidant defence systems involved in conferring the sensitiveness and tolerance in these genotypes.     

Membrane stabilization by abscisic acid under cold aids proline in alleviating chilling injury in maize (Zea mays L.) cultured cells

Previous studies of maize suspension‐cultured cells showed that abscisic acid (ABA) treatment at warm temperatures improved the tolerance of cells to subsequent chilling. In the present study, it is shown that both ABA‐treated and untreated maize cells accumulated proline in response to chilling. However, ABA‐treated cells displayed less lipid peroxidation during chilling, and thus, unlike untreated cells, were able to retain the accumulated proline intracellularly. Proline application experiments indicate that an intracellular proline level higher than 2 µmole (g FW)^?1 prior to chilling was needed to meaningfully reduce chilling‐enhanced lipid peroxidation and significantly improve chilling tolerance. The results suggest that total proline accumulation in ABA‐treated as well as untreated cells during chilling was enough to potentially improve chilling tolerance, but proline leakage rendered the control cells unable to benefit from the endogenous synthesis of proline in relation to the alleviation of chilling injury. Proline participated in chilling tolerance improvement in ABA‐treated maize cells, as evidenced by: (1) the inhibition of proline accumulation by l ‐methionine‐d , l ‐sulphoximine (MSO), an inhibitor of glutamine synthetase, reduced ABA‐improved chilling tolerance, and (2) the addition of glutamine into the medium prevented the MSO‐induced reduction in chilling tolerance. The revised relationship between proline accumulation and membrane stability at cold is discussed in the light of these current findings.