一谷两用 菌畜双兴

宜昌市科技情报研究所食用菌专家组为满足稻谷利用的问题,经潜心研究,成功地设计出先用稻谷种食用菌、在收获完食用菌后再用菌谷作饲料喂牲畜,使稻谷增值三倍以上的一谷两用、菌畜双兴的新模式,现简介如下:一。

一谷两用,菌畜双兴

宜昌市科技情报研究食用菌专家组经潜心研究,成功地设计出先用稻谷种食用菌,在收获完食用菌后再用菌谷作饲料喂畜,使用稻谷增值三倍以上的一谷两用、菌畜双兴的新模式,现简介如下:(一)用稻谷种菇的方法。

抗噬菌体产谷氨酸菌株的自然筛选

本文采用自然界直接分离法从不同来源土壤中筛选谷氨酸产生菌,经产酸测定、噬菌体抗性检查,筛选出25株抗噬菌体产谷氨酸菌株,其中有2株产酸率在3.0％以上。

噬菌体对国内谷氨酸生产菌的鉴别

利用噬菌体的宿主专一性,对收集的国内用于生产的谷氨酸生产菌进行鉴别,发现一株7338菌株对T6—13噬菌体敏感,而对7338的噬菌体不敏感。根据上述事实,工厂中为防止噬菌体侵袭,轮换使用不同菌株时,提出要了解菌株对不同噬菌体的敏感性,以免感染造成巨大经济损失。

Studies on Enhancing Yield of Embryos in Microspore Culture of Brassica napus L.

In ecological zone of Chengdu, Sichuan, microspore culture was carried out in Brassica napus L. to study the influencing factors on microspore culture. The results showed that the temperature on microspore formation stage, day and night temperature, disinfection solution of buds, cultivation concentration on microspore and strain-age were both important influencing factors on microspore culture. At a temperature below 5 ℃ or above 20 ℃, the material had a much lower embryo producing rate or even could not produce any embryo, but at the optimum temperature of 10 -15 ℃ the embryo yield was up to 300 pieces per bud; the best cultivation effect appeared when 0. 1% HgCl2 was used for disin- fection; the best density of microspore was 3 -4 buds per dish; In 2009, while strain-age was from 125 d to 150 d, the microspore embryo yield increased as strain-age increased. When stain-age was 150 days, the microspore embryo yield was up to the highest, but the yield declined after 150 days.

Genetic Study on JS399-19 Resistance in Hyphal Fusion of Fusarium graminearum by Using Nitrate Nonutilizing Mutants as Genetic Markers

Twenty-two nitrate nonutilizing （nit） mutants were recovered from five wild-type isolates of Fusarium graminearum and fifty nit mutants were recovered from three JS399-19-resistant mutants of F. graminearum cultured on MMC medium. Some biological properties were compared between nit mutants and their parental isolates. The results showed that there were no significant differences in growth rate, cultural characters or pathogenicity between JS399-19-resistant nit mutants and their parental isolates. But the conidial production and the sexual reproduction ability changed to some extent. There was no cross resistance toward chlorate and JS399-19 in F. graminearum and the resistance could be stable through 20-time subcultures. Therefore, the nit could be used as a genetic marker for studying the genetics of JS399-19 resistance in E graminearum, which was used to study JS399-19 resistance transferability in hyphal fusion. Resistance in JS399-19 could not be transferred by hyphal fusion or could be transferred with low chance between two compatible isolates, which would delay the development of JS399-19 resistance in the field.

Purification and Characterization of Glutamate Decarboxylase of Lactobacillus brevis CGMCC 1306 Isolated from Fresh Milk

A Lactobacillus brevis CGMCC 1306 isolated from fresh milk without pasteurization was found to have higher glutamate decarboxylase （GAD） activity. An effective isolation and purification procedure of GAD from a cell-free extract of Lactobacillus brevis was developed, and the procedure included four steps： 30%-90% saturation （NH4）2SO4 fractional precipitation, Q sepharose FF anion-exchange chromatography, sephacryl S-200 gel filtration, and resource Q anion-exchange chromatography. Using this protocol, the purified GAD was demonstrated to possess electrophoretic homogeneity via SDS-PAGE. The purification fold and activity recovery of GAD were 43.78 and 16.95%, respectively. The molecular weight of the purified GAD was estimated to be approximately 62 kDa via SDS-PAGE. The optimum pH and temperature of the purified GAD were 4.4 and 37℃, respectively. The purified GAD had a half-life of 50rain at 45℃ and the Km value of the enzyme from Lineweaver-Burk plot was found to be 8.22.5＇-pyridoxal phosphate （PLP） had little effect on the regulation of its activity.

Effect of cadmium on the defense response of Pacific oyster Crassostrea gigas to Listonella anguillarum challenge

Heavy metal pollution can affect the immune capability of organisms. We evaluated the effect of cadmium （Cd） on the defense responses of the Pacific oyster Crassostrea gigas to Listonella anguillarum challenge. The activities of several important defensive enzymes, including superoxide dismutase （SOD）, glutathione peroxidase （GPx）, acid phosphatase （ACP）, Na＋, K＋-ATPase in gills and hepatopancreas, and phenoloxidase-like （POL） enzyme in hemolymph were assayed. In addition, the expression levels of several genes, including heat shock protein 90 （IrtSP9~））, metallothionein （MT）, and bactericidal/permeability increasing （BPI） protein were quantified by fluorescent quantitative PCR. The enzyme activities of SOD, ACP, POL, and GPx in hepatopancreas, and the expression of HSP90 were down-regulated, whereas GPx activity in the gill, Na＋, K＋-ATPase activities in both tissues, and MT expression was increased in Cd- exposed oysters post L. anguillarum challenge. However, BPI expression was not significantly altered by co-stress of L. anguillarum infection and cadmium exposure. Our results suggest that cadmium exposure alters the oysters＇ immune responses and energy metabolism following vibrio infection.

Engineering of Corynebacterium glutamicum to Enhance L-ornithine Production by Gene Knockout and Comparative Proteomic Analysis

Engineered Corynebacterium glutamicum was constructed for L-ornithine production by disrupting genes of argF and proB to prevent the flux away from L-ornithine.Effect of the inactivation of 2-oxoglutarate de-hydrogenase complex(ODHC) on L-ornithine production was also investigated.It was found that the inactivation of ODHC by knockout of the kgd gene enhanced L-ornithine production.The engineered C.glutamicum ATCC13032(ΔargFΔproBΔkgd) produced L-ornithine up to 4.78 g·L-1 from 0.24 g·L-1 of the wild-type strain.In order to understand the mechanism of L-ornithine production in C.glutamicum ATCC13032(ΔargFΔproBΔkgd) and find out new strategies for further enhancing L-ornithine production,the comparative proteome between the wild-type and the engineered strain was analyzed.L-Ornithine overproduction in the engineered strain was related to the up-regulation of the expression levels of enzymes involved in L-ornithine biosynthesis pathway and down-regulation of the expression levels of proteins involved in pentose phosphate pathway.The overexpression of genes in the upstream pathway of glutamate to increase the availability of endogenous glutamate may further in-crease ornithine production in the engineered C.glutamicum and the ornithine synthesis enzymes(ArgCJBD) may not be the limiting enzymes in the engineered C.glutamicum.

Plant Diseases in Globally Changing Russian Climate

Across all Russia global climate change is observed. Consequences of climatic changes, undoubtedly, will be reflected in distribution of harmful organisms, their injuriousness and will demand development of new approaches in plant protection. Over the last 10 years, the spread of cereal crop diseases in the Northwest Russia has been monitored. The purpose of researches is to find new diseases in the Northwest region of Russia. Disease progression was mainly monitored 3 or 4 times during the growing season, from germination to crop maturity. As a result in this region the new diseases were found. In 2005-2007 the causal agent of yellow leaf spot Pyrenophora tritici-repentis was found on wheat. Fusarium graminearum historically has two areas in Russia： the North Caucasus and the Far East. However, since 2003 F. graminearum appeared on the territory of the North-West of Russia. Septoria tritici became the main pathogen of wheat in the North-Western Region.. In 2013 Ramularia collo-cygni was found in Arkhangelsk region. These observations suggest that global warming of climate leads to an expansion south species pathogen to the north regions of Russia.

Three-stage fermentation and kinetic modeling of bioflocculant by Corynebacterium glutamicum

Fermentation of bioflocculant with Corynebacterium glutamicum was studied by way of kinetic modeling.Lorentzian modified Logistic model, time-corrected Luedeking–Piret and Luedeking–Piret type models were proposed and applied to describe the cell growth, bioflocculant synthesis and consumption of substrates, with the correlation of initial biomass concentration and initial glucose concentration, respectively. The results showed that these models could well characterize the batch culture process of C. glutamicum at various initial glucose concentrations from 10.0 to 17.5 g·L-1. The initial biomass concentration could shorten the lag time of cell growth,while the maximum biomass concentration was achieved only at the optimal initial glucose concentration of16.22 g·L-1. A novel three-stage fed-batch strategy for bioflocculant production was developed based on the model prediction, in which the lag phase, quick biomass growth and bioflocculant production stages were sequentially proceeded with the adjustment of glucose concentration and dissolved oxygen. Biomass of2.23 g·L-1was obtained and bioflocculant concentration was enhanced to 176.32 mg·L-1, 18.62% and403.63% higher than those in the batch process, respectively, indicating an efficient fed-batch culture strategy for bioflocculant production.

Wheat Genetic Transformation as Efficient Tools to Fight against Fungal Diseases

Wheat ranks first among cereal crops cultivated in the world. In its production, diseases like powdery mildew, fusarium head blight and rusts caused by fungal pathogens represent a major problem. They produce different symptoms that cause severe crop damage by infecting the spikes, leaves, roots, stems and grains. They are causing losses both by reducing the quantity of the harvested crop and the quality of the product. Quality problems of the harvested product can be due to shrivelled seed, which are frequently found as a consequence of the infection by leaf pathogens, such as mildews, rusts and Septoria. Fusarium head blight is the major culprit for mycotoxin contamination from the harvested grain, causing economic losses and in the worst casing human and animal health problems. In severe epidemics, all these fungal diseases can significantly reduce yield. Resistance to fungi is beneficial not only from a commercial point of view （yield）, but also because of the reduced levels of mycotoxins. The integration of transgenic approaches offers a potential chemical-free and environment-friendly solution for controlling fungal pathogens. This is an essential asset for wheat world food security.

脱氧雪腐镰刀菌烯醇提取、纯化及含量检测

本试验通过提取和纯化方法的优化,旨在获得高纯度的脱氧雪腐镰刀菌烯醇(DON)。首先将禾谷镰刀菌孢子液接种于玉米培养基,于27℃30%湿度的条件下培养25d,收集产毒培养基,制成毒素粗提液。经过层析柱2次洗脱,分段收集洗脱液,用薄层色谱法(TLC)确定含DON的洗脱液,合并洗脱液,运用多重结晶法进行纯化,获得DON的纯品,高效液相色谱法(HPLC)进行分析确认。采用该培养方法,每千克玉米培养基可产生DON 56.65 mg,纯化后可得到DON 29.5mg,纯度达到98.1%;HPLC检测条件为:Waters C18柱(4.6mm×150mm,5μm),流动相为乙腈-水(16∶84),流速0.8mL/min,紫外波长218nm,色谱柱柱温30℃;本方法加标回收率为86.9%~100.6%,日内相对标准偏差≤9.42%,日间相对标准偏差≤5.79%。本试验建立的DON提取与纯化方法简单,不需要任何特殊的试剂与设备,且该检测方法回收率高,准确度和精密度均能满足试验要求,为DON纯品的定量提供了技术支撑。

Rapid and specific identification of Fusarium pseudograminearum by qPCR based on RPB sequence

Fusarium crown rot,mainly caused by Fusarium pseudograminearum,is a destructive disease in wheat production.To establish a rapid and reliable detection method for F.peasudeograminearum,the specific PCR primer pair(Fpg-F1;R2)was designed based on the RPB sequence,and real-time fluorescence quantitative PCR(qPCR)was used to validate the efficiency of the primer.The results showed that the primer pair had high specificity and sensitivity of 100 pg of DNA.Furthermore,the qPCR system for early and rapid detection of F.peasudeograminearum had an amplification efficiency of 87.5%and correlation coefficient of 0.99,and the pathologic threshold of F.pseudograminearum in soil was determined by using this detection system.It was found that F.pseudograminearum could cause Fusarium crown rot when the DNA concentration of F.pseudograminearum in field soil exceeded 213 pg·g^(-1).Hence,the qPCR-based method we developed for F.pseudograminearum detection has the advantages of high specificity and sensitivity,and can be used for rapid and early detection of F.pseudograminearum even in field soils.

Strategies used for genetically modifying bacterial genome: site-directed mutagenesis, gene inactivation, and gene over-expression

With the availability of the whole genome sequence of Escherichia coli or Corynebacterium glutamicum, strategies for directed DNA manipulation have developed rapidly. DNA manipulation plays an important role in understanding the function of genes and in constructing novel engineering bacteria according to requirement. DNA manipulation involves modifying the autologous genes and expressing the heterogenous genes. Two alternative approaches, using electroporation linear DNA or recombinant suicide plasmid, allow a wide variety of DNA manipulation. However, the over-expression of the desired gene is generally executed via plasmid-mediation. The current review summarizes the common strategies used for genetically modifying E. coli and C. glutamicum genomes, and discusses the technical problem of multi-layered DNA manipulation. Strategies for gene over-expression via integrating into genome are proposed. This review is intended to be an accessible introduction to DNA manipulation within the bacterial genome for novices and a source of the latest experimental information for experienced investigators.

A GATA-type transcription factor AcAREB for nitrogen metabolism is involved in regulation of cephalosporin biosynthesis in Acremonium chrysogenum

In filamentous fungi,nitrogen metabolism is repressed by GATA-type zinc finger transcription factors.Nitrogen metabolite repression has been found to affect antibiotic production,but the mechanism is still poorly understood.AcareB,encoding a homologue of fungal GATA-type regulatory protein,was cloned from Acremonium chrysogenum.Gene disruption and genetic complementation demonstrated that AcareB plays a key role in utilization of ammonium,glutamine and urea.In addition,significant reduction of cephalosporin production in the AcareB disruption mutant indicated that AcareB is important for cephalosporin production.In consistence with it,the transcriptional level of cephalosporin biosynthetic genes was significantly decreased in the AcareB disruption mutant.Electrophoretic mobility shift assay showed that AcAREB directly bound to the intergenic regions of pcbAB-pcbC,cefD1-cefD2 and cefEF-cefG.Sequence analysis showed that all the AcAREB binding sites contained the consensus GATA elements.AcareB is negatively autoregulated during cephalosporin production.Moreover,another GATA zinc-finger protein encoded by AcareA positively regulates the transcription of AcareB.However,AcareB does not regulate the transcription of AcareA.These results indicated that AcAREB plays an important role in both regulation of nitrogen metabolism and cephalosporin production in A.chrysogenum.

Metabolic engineering and flux analysis of Corynebacterium glutamicum for L-serine production

L-Serine plays a critical role as a building block for cell growth, and thus it is difficult to achieve the direct fermentation of L-serine from glucose. In this study, Corynebacterium glutamicum ATCC 13032 was engineered de novo by blocking and at- tenuating the conversion of L-serine to pyruvate and glycine, releasing the feedback inhibition by L-serine to 3-phosphoglycerate dehydrogenase （PGDH）, in combination with the co-expression of 3-phosphoglycerate kinase （PGK） and feedback-resistant PGDH （PGDHr）. The resulting strain, SER-8, exhibited a lower specific growth rate and significant differ- ences in L-serine levels from Phase I to Phase V as determined for fed-batch fermentation. The intracellular L-serine pool reached （14.22_＋1.41） ~trnol gcoM-1, which was higher than glycine pool, contrary to fermentation with the wild-type strain. Furthermore, metabolic flux analysis demonstrated that the over-expression of PGK directed the flux of the pentose phosphate pathway （PPP） towards the glycolysis pathway （EMP）, and the expression of PGDHr improved the L-serine biosynthesis pathway. In addition, the flux from L-serine to glycine dropped by 24%, indicating that the deletion of the activator GlyR re- sulted in down-regulation of serine hydroxymethyltransferase （SHMT） expression. Taken together, our findings imply that L-serine pool management is fundamental for sustaining the viability of C. glutamicum, and improvement of C1 units genera- tion by introducing the glycine cleavage system （GCV） to degrade the excessive glycine is a promising target for L-serine pro- duction in C. glutamicum.