该研究在对六妹羊肚菌(Morchella sextelata)原生质体单核化再生菌株分离及交配型鉴定的基础上,对单交配型原生质体再生菌株进行对峙培养杂交实验制备杂交菌株.以菌株交配型、生长势、抗鞣酸能力和菌核产生能力为技术指标,对单核化再生菌株及杂交菌株的多态性进行分析,并对杂交菌株的交配型进行检测.结果表明,20株MAT1-1-1单交配型及17株MAT1-2-1原生质体再生菌株表现较好,其菌丝带宽度均≤10 mm,褐变圈直径分别为25～34 mm、23～34 mm;从63组杂交菌株中得到40株(占63.50％)双交配型(MAT1-2-1/MAT1-2-1)杂交菌株,杂交菌株的菌丝带宽度均≥10 mm,褐变圈直径为32～54 mm,杂交菌株的交配型电泳结果表明,成功获得40株具有双交配型的杂交菌株(两条扩增带).因此,双交配型杂交菌株在生长势、抗鞣酸能力方面存在杂交优势,而菌核产生能力与菌株交配型没有相关性.

为选育纳豆激酶(Nattokinase,NK)高活性菌株,从4种不同风味食品中分离得到26株菌株,通过酪蛋白平板初筛和液体发酵复筛,得到2株纳豆激酶高产菌株,利用分子生物学技术鉴定为2株枯草芽孢杆菌(Bacillus subtilis).采用氯化锂-紫外与亚硝基胍(NTG)复合诱变的方式,最终获得1株纳豆激酶活性达到2338.01 U/mL的高产菌株SD3-3-6,该突变菌株纳豆激酶酶活相较于初始菌株SD3提高了1.93倍,是1株具有应用前景的高产菌株.

为了探讨新型快速秸秆还田促腐菌剂对小麦秸秆还田在水稻田的施用剂量和促生效果,进行了相关的大田试验.本试验在常规施肥和小麦秸秆粉碎还田的基础上设置了M1(CK),M2(30 kg/hm2),M3(75 kg/hm2)和M4(120 kg/hm2)4个梯度的促腐菌剂施用剂量,研究了不同施用剂量对水稻成产因素以及水稻产量的影响.研究表明,施用新型快速秸秆还田促腐菌剂不仅可以促进秸秆降解,且对每公顷水稻的穗数、穗粒数及产量影响显著.与对照组M1相比,M3(75 kg/hm2)组促产效果最好,其中每公顷穗数为466.35万穗,比对照提高了16.09％;穗粒数为86.03比对照提高了12.12％.在实际产量方面M3组为7510.35 kg/hm2,比对照提高了12.94％.土壤理化性质表明,促腐菌剂显著提高了土壤中碱解氮、有效磷、速效钾的含量,与对照组相比分别提高了93.33％、18.13％和46.93％.该试验评价了施用不同剂量新型快速秸秆还田促腐菌剂对小麦秸秆在水稻田降解及水稻产量的影响,为新型快速秸秆还田促腐菌剂的研发奠定了理论基础.

在体外培养的人非小细胞肺癌A549细胞、人胃腺癌BGC-823细胞、人结肠癌HCT 116细胞、人肝癌Hep 3B细胞、人宫颈癌Hela细胞中加入不同浓度的酵母异源表达的LZ-8或FIP-tvc,培养24 h后显微镜下观察细胞形态变化,并用CCK8试剂盒检测细胞存活率,以用来检测灵芝免疫调节蛋白LZ-8及云芝免疫调节蛋白FIP-tvc对几种癌细胞生长抑制能力.结果显示,LZ-8和FIP-tvc在5～10μg/mL浓度时使A549、BGC-823、Hela和Hep 3B细胞贴壁性变差,且CCK8结果显示前3种细胞的存活率有不同程度的降低(80％～95％存活率),而在20μg/mL浓度时FIP-tvc对Hep 3B细胞的存活率开始有明显抑制效果(89％存活率),但对HCT 116细胞的形态无影响,对其存活率也无抑制作用.

利用大肠杆菌(Escherichia coli)表达系统,经异丙基-β-D-硫代半乳糖苷(IPTG)诱导,成功异源表达了一株链球菌(Streptococcus)S1的α-半乳糖苷酶基因,重组α-半乳糖苷酶经镍柱纯化后,测定其酶学性质.重组α-半乳糖苷酶最适pH值为6.5,最适温度为50℃,在碱性环境中(pH 7.5～10.0)及在40℃以下温度条件下该酶较为稳定;酶催化动力学结果显示,该酶在最适条件下水解硝基苯-α-D-半乳糖苷(pNPG)的最大水解速率(Vmax)为508.38μmol/(min·mg),米氏常数(Km)值为1.2 mmol/L;通过薄层层析(TLC)法检测到该重组α-半乳糖苷酶可以高效地水解天然底物蜜二糖、棉籽糖和水苏糖中的α-半乳糖苷键.

纳豆红曲胶囊是以保健红曲和纳豆冻干粉为原料制成的保健食品,不仅含有红曲的活性物质,如红曲色素、Monacolin K、麦角甾醇、γ-氨基丁酸等,还含有纳豆的活性成分如纳豆激酶、纳豆菌、维生素K2、异黄酮等,因此纳豆红曲胶囊具有传统红曲和纳豆的双重保健功效,具有降血脂、溶栓、抗心脑血管疾病、抗菌、抗癌等功能.本文对红曲和纳豆的主要活性成分及其保健功效作了简单的介绍,并对其发展前景进行了展望.

To further ameliorate the cultivation characteristics and improve the yield of Sparassis crispa,the condition for preparation and regeneration of protoplasts from Sparassis crispa were preliminary studied.Factors including isotonic solutions,enzyme temperature,pH,enzymolysis time were optimized based on the regeneration amount.Results showed that 2％ lysozyme was used to dissolve the mycelium cell wall of mycelium of S.crispa,and the optimal condition for preparing protoplasts were 0.6 M sucrose as isotonic solution,30 ℃,pH 6.0,3 h.Under this condition,the regeneration amount was 11 850 per mL.

本研究从实验室保存的15株枯草芽孢杆菌中选育出适合鹰嘴豆(Cicer arietinum)发酵生产纳豆激酶(Nattokinase，NK)的优良菌株，并开展了菌株固体发酵产NK的工艺参数优化研究；NK酶活检测采用血纤维蛋白平板透明圈法。结果发现，培养基含水量、pH值、发酵温度及发酵时间均对鹰嘴豆发酵产生纳豆激酶有较大影响，培养基中添加适量的无机盐和表面活性剂有利于纳豆激酶的产生。在优化条件下（培养基含水量64%，PH7.0，31℃，培养36h），鹰嘴豆固态发酵产纳豆激酶的单位酶活可达到6731FU/g，获得了较高的单位酶活。 In this study, fifteen laboratory stored Bacillus subtilus strains were tested for their NK activity, and several parameters of the solid fermentation with chickpea (Cicer arietinum) media for NK production were optimized. The NK activity was decided with the transparent zones method on the fibrin-thrombin plate. The results showed that the NK secretion ability of strains was different; and the water content, pH value, cultural temperature and cultural duration have obviously influence on NK production. The optimum condition for NK production was 31 ˚C, pH 7.0, 64% water content and 36 hrs cultural duration and the addition of inorganic salts and surface active agents was helpful for NK production with chickpea media. At the optimum condition, the NK activity of the solid fermentation could reach 6731 FU/g.

Vectors pGAPZαA and pPICZαA were utilized to construct the multi-copies expression plasmid that can be linearized to maintain the transformation rate at a high level and have a higher yield of interesting protein than the single-copy plasmid.The gene of the interesting protein was originally constructed in the constitutiveexpression vector pGAPZαA,then the expression cassette is digested out with the isocaudamers Bgl Ⅱ and BamH Ⅰ and constructed in the methanol inducible expression vector pPICZαA at BamH Ⅰ site.The multi-copies expression plasmid was constructed after several times repeating.The LZ8 which is a fungal immunomodulatory protein from Lingzhi is used as an example.Its multi-copies expression plasmid pPICZαA-[GAPZαA-LZ8] 4 is constructed after repeating 4 times.Then the plasmid is linearized at the unique Sac Ⅰ site of pPICZαA and transformed in GS115.The yield of LZ8 expressed in the clones is transformed with pPICZαA-[GAPZαA-LZ8]4 reaches 2 or 3 times of that in the clones which is transformed with pGAPZαA-LZ8.We find the linearizable multi-copies expression plasmid bases on combination utilization of pGAPZαA and pPICZαA can express the interesting protein in GS115 at a higher level than the single-copy plasmid.

Deinococcus radiodurans is one of the most radiation-resistant organisms that was found on the earth,and has very high resistance to UV,drought,and mutagenic agents. D. radiodurans has drawn attentions from scientists around the world,and there are many reports about its radiation-resistant mechanisms. In this paper the recent progresses on radiation-resistant mechanisms of D. radiodurans are summarized from DNA-repair pathway,antioxidative defense system,special survival approaches,and cellular purification system. The development trends and future prospects of radiation-resistant mechanisms of D. radiodurans are also discussed,the aim of this paper is to provide some foundations for further study of radiation-resistant mechanisms of the bacteria.

This work aims to analyze the reason that resulted in the failure of cloning and to check if there are some microorganisms strongly degrading DNA in the environment of electrophoresis chamber. The colony morphology,gram staining and the 16S rDNA sequence were utilized to identify the strain degrading DNA,and agarose gel electrophoresis was for analyzing the DNA-degrading activity. As results, a strain was isolated from DNA electrophoresis chamber,and identified as gram-negative bacterium. Then it was cultured in YPD medium, and the capacity of the fermentation broth degrading DNA was detected using plasmid pUC19 as substrate. The strain quickly and sufficiently degraded DNA at the optimal temperature 45℃,thus this strain was designated DD(DNA degrading). The sequence alignment of the DD by the 16S rDNA showed that this DD had 100% identity with the that of Serrtia marcescens. Conclusively,the bacterial strain DD isolated from DNA electrophoresis chamber is a Serrtia marcescens strain possessing significant DNA-degrading activity based on the results of colony morphology,gram staining,and 16S rDNA sequence.

为对比观察国产蛋白酶酶解和益生菌发酵降解鹰嘴豆浆液蛋白的效果,比较酶解法、发酵法降解鹰嘴豆浆液蛋白为小分子蛋白肽的能力,采用碱性蛋白酶、木瓜蛋白酶分别酶解,纳豆芽孢杆菌、植物乳酸菌、蛹虫草分别发酵鹰嘴豆浆液蛋白,测定不同处理时间时的可溶性氮、DH、电泳条带等的变化情况.研究结果发现,国产碱性蛋白酶酶解鹰嘴豆浆液蛋白240 min,上清液的可溶性氮含量可达到0.1389％,DH为6.43％;木瓜蛋白酶酶解240 min时的上清液可溶性氮含量达到0.0972％,DH为4.41％;而发酵法处理以纳豆芽孢杆菌效果最显著,纳豆芽孢杆菌发酵处理鹰嘴豆浆液蛋白8h时的上清液可溶性氮含量为0.0895％,DH为4.03％.本研究表明,酶解法降解鹰嘴豆浆液蛋白的效果要比微生物发酵法处理时间短,降解效果更好.

In this paper,the influence of several single factors on the Cordyceps militaris mycelia cordycepin and exo?polysaccharides in liquid cultures were studied,It was found that the media constitutes have a excellent effect on the yield of C.militaris mycelia cordycepin and exo?polysaccharides,and the maize meal、soybean meal and the 6%-8%corn steep liquor powder(CSL,dry)were better than others;The Vitamin B1 concentration 10 mg/100 mL were suited,and 0.01%MnSO4 was helpful for C.militaris mycelia cordycepin and exo?polysaccharides secretion;Meanwhile, the media optimal pH value 5.5,the cultural temperature 22-24℃,and 1%inoculum proportion,50 mL liquid media per 250 mL flask for 5-7days culture were suitable for C.militaris mycelia cordycepin and exo?polysaccharides production.

The aim was to explore the toxic effects of lead and chromium stresses on wheat and the influence of them on wheat proline. Treating wheat with different concentrations of lead and chromium was used to study the effects of single lead and chromium stress or combination of them on the proline content in wheat seedlings and wheat seed germination. The results showed that the seed germination rate, seedling height, root length, fresh weight and dry biomass all decreased under lead and chromium stresses in both single and combination. The proline content in each treatment group increased comparing with that in the control group at 1 d after treatment, especially under combined 100 mg/L Pb + 100 mg/L Cr stress, the proline content increased 32.81% compared with the control. The proline content in the wheat seedlings treated with 100 mg/L Cr, 50 mg/L Cr, 200 mg/L Pb + 100 mg/L Cr and 100 mg/L Pb + 100 mg/L Cr showed a significant increase by 3 d, 5 d, 7 d after treatment when compared to the untreated control. No significant changes were observed for proline content in the other treatment groups. In conclusion, lead and chromium stresses inhibited wheat seed germination and seedling growth, and increased proline content in wheat. Therefore, it is suggested that the proline content in wheat may be used as one of the indicators to monitor lead and chromium stress.

在河南省汝阳县伏牛山区首次发现了蛹虫草的生长与分布.调查了蛹虫草的生长环境并对蛹虫草菌株进行了分离纯化、形态学观察和分子鉴定.结果表明,野生蜗虫草主要分布在山体的背阴面,毒生于针、阔叶混交林地表土层鳞翅目昆虫的蛹体上,子座呈1个或多个分支,长3～8 cm,颜色呈橘黄至棕黄色,子囊壳丰富.分子鉴定表明,蛹虫草的ITS1-5.8SrDNA-ITS2序列长度为478 bp,GC含量为56.4％.经GenBank数据库同源性检索比对,与蛹虫草有高度同源性,达到100％.分离自野生蛹虫草的菌株ycc-ry-01在大米综合培养基上只能形成原基,不能长出子座,而对照菌株ycc-jzc可以培养出子座;经液体发酵培养后的菌株ycc-ry-01与对照菌株ycc-jzc均表现出纤溶活性,菌丝体提取液的活性大于发酵液的活性.

通过对蛹虫草(Cordyceps militaris)菌丝液体发酵产纤溶酶的实验研究,获得了蛹虫草纤溶酶液体发酵的适宜参数.结果表明,蛹虫草菌丝液体发酵产纤溶酶的适宜碳源为玉米粉和蔗糖,豆饼粉和较高浓度的玉米浆(8％～12％)作为氮源是适宜的,培养基中适量添加MgSO4(0.05％)、KH2PO4(0.1％)和CaCl2(0.05％)、MnSO4(0.05％)等无机盐有利于蛹虫草纤溶酶产生;发酵pH值以4.5～5.5为宜,温度以22～24℃为宜,培养时间以6～7 d为宜.

The strains Aspergillus niger with higher protease production,Aspergillus oryzae with higher α-amylase production were screened from fungal strains in laboratory,through the preliminary screening with Casein plate and Soluble starch plate solid culture medium,the second screening with liquid fermentation enzyme production medium of shake flash fermentation. It was studied on producing enzymes by solid-state fermention. The results showed that the protease activity was 3760 U/g for fermentating 24 h,while the fungal α-amylase activity was 0.405 U/g for fermentating 32 h.

To isolate and identify Empedobacter brevis strains in rhizospheric soil of Chinese cabbage soft rot,15 aroma bacterial strains were isolated with selected medium,from the rhizospheric soil where the Chinese cabbages were planted.The isolated strains were identified according to their 16S rRNA gene sequences analysis as well as their morphological features,physiological and biochemical analysis.Their taxonomic statuses were confirmed by their 16S rRNA gene sequences homologous analyses and phylogenetic trees.The result showed that 12 aroma strains exhibited the highest levels(99.86%) of similarity to Empedobacter brevis LMG 4011T(AM177497).

In this paper,the protoplast preparation conditions of Agrocybe cylindracea strain were studied,these conditions include different media,different temperatures,different pH,different reaction time,different mycelial age and different osmotic stabilizers.The results showed that:after the mycelium cultured for 5 days in liquid oscillation shaking,with 0.6 mol/L mannitol or MgSO4 as osmotic stabilizer,and a concentration of 2% of the fungal wall dissolving enzyme(lywallzyme) in the pH6.0,temperature 31 ℃ for 2 hours reaction treatment,the yield of protoplast of Agrocybe cylindracea strain was highest and can reach 2.31×106 /mL.