圆红冬孢酵母高效电转化条件的优化

【目的】建立圆红冬孢酵母的高效电转化方法,为利用圆红冬孢酵母重组表达功能性基因提供方法基础。【方法】以圆红冬孢酵母作为受体菌,将具有潮霉素抗性标记的改造质粒p CAMBIA1301-KCS通过电转化的方式导入到受体菌中,研究酵母细胞培养时间(2、8、14、20、26 h)、电击电压(1、1.5、2.0、2.5、3.0 kV)、前处理剂种类(DTT、LiAc、CaCl2、H2O)对转化效率的影响。【结果】当培养时间为20 h [培养液D(600 nm)约为1.6]、电击电压为2.0 kV、前处理试剂为DTT时,该系统转化率最高可达到20.8×104 cfu/μg。【结论】采用电击法将环状质粒转化入圆红冬孢酵母,能够成功实现外源基因的遗传转化,采用优化的转化条件可以获得较高的转化效率,是过去相关报道最高转化率的100倍。     

罗非鱼源无乳链球菌培养基及培养条件的优化

研究无乳链球菌(Streptococcus agalactiae)ZQGY08的培养与生长特性,探讨脑心浸液(Brian Heart Infusion,BHI)培养基的营养因子、初始pH值、溶氧、温度等因素对无乳链球菌生长的影响,并采用正交实验法对无乳链球菌培养基营养因子添加量进行优化。结果表明,无乳链球菌生长的最适初始pH为7～8,培养温度为28～37℃,振荡转速为180～200 r/min。在BHI培养基中添加20 mg/mL的酵母粉和2 mg/mL的葡萄糖即可在对数期获得最大细菌量。     

红笛鲷NCCRP-1基因原核表达条件的优化及纯化

通过克隆编码红笛鲷(Lutjanus sanguineus)非特异性毒性细胞受体蛋白-1(nonspecific cytotoxic cell receptor protein-1,NCCRP-1)成熟肽基因序列,然后与载体连接构建pET21a-NCCRP融合蛋白表达载体,将其转入大肠杆菌BL21进行诱导表达并优化条件。SDS-PAGE分析表明,在异丙基-β-D-硫代半乳糖苷(IPTG)浓度0.8 mmol/L、37℃条件下培养3 h后表达量最大,分子大小与预期值相符,融合蛋白主要以包涵体形式高效表达,通过HisTrap HP柱子使其得到进一步纯化;Western blot分析表明,该融合蛋白可与鼠抗His-tag单克隆抗体发生特异性结合,说明获得该表达产物。     

培养条件对破囊壶菌生长及其产DHA的影响

探讨了培养温度、pH、光照、种龄、接种量以及培养时间等培养条件对破囊壶菌ThraustochytriumroseumMF2生长及其产DHA的影响。结果表明 ,T roseumMF2生长及其产DHA的适宜条件为 :培养温度 2 5℃、初始pH值 6～ 7、种龄 4 8h、接种量 4 %、培养时间 4d ,生物量达到 1 0 4g L ,DHA产量达到1 2 6 6mg L。     

酵母培养物对凡纳滨对虾肠道菌群结构的影响

在基础饲料中分别添加质量分数0(对照)和0.30%的酵母培养物,配制2种等氮等脂饲料(分别记为Y0、Y0.3组),投喂初始体质量为(1.20±0.01)g的凡纳滨对虾(Litopenaeus vannamei)56 d。提取凡纳滨对虾肠道总DNA,采用Illumina Mi Seq高通量测序比较样品菌群组成和多样性,研究饲料中添加酵母培养物对凡纳滨对虾肠道菌群结构的影响。结果表明,Y0.3组对虾肠道分类单元(OTUs)数量、丰富度指数(Chao1、ACE值)、多样性指数(Shannon、np Shannon指数)与Y0组差异无统计学意义(P0.05);从物种分类树发现,两组的主要细菌类群均为变形菌门(Proteobacteria),其中,γ-变形菌纲是对虾肠道的最主要菌群,所占比例达69.48%;从科分类水平看,Y0.3组对虾肠道菌群链球菌科(Streptococcaceae)比Y0组提高4.98%;从优势属水平来看,Y0.3组对虾肠道的乳球菌属(Lactococcus)高于Y0组,差异有统计学意义(P0.01);Y0.3组对虾肠道的发光杆菌属(Photobacterium)低于Y0组,差异有统计学意义(P0.05)。饲料中添加质量分数0.30%酵母培养物可改善凡纳滨对虾肠道菌群多样性。     

红笛鲷重组激活基因rag1原核表达条件的优化及纯化

克隆编码红笛鲷(Lutjanus sanguineus)RAG1蛋白(recombination activating protein1)活性核心区的基因序列,并与pET-28a(+)载体连接,构建原核表达载体pET-28a-RAG1,将其转入大肠杆菌BL21(DE3)菌株,利用IPTG进行诱导表达。为提高融合蛋白的表达效率,运用传统的实验方法对诱导条件进行优化。SDS-PAGE分析表明,在37℃条件下,利用0.1 mmol/L IPTG诱导8 h后,RAG1重组融合蛋白的表达量最大,相对分子质量与预测值相符,该蛋白主要以包涵体形式高效表达,利用His Trap HP亲和柱使其得到进一步纯化;Western blot分析显示,该融合蛋白可与鼠抗His-tag单克隆抗体发生特异性结合,说明表达蛋白为目的蛋白。     

酵母培养物替代鱼粉对凡纳滨对虾生长性能、血清生化指标、免疫力和抗病力的影响

【目的】探讨高蛋白酵母培养物替代鱼粉对凡纳滨对虾(Litopenaeusvannamei)生长性能、血清生化指标、非特异性免疫力和抗病力的影响。【方法】在基础饲料中分别添加质量分数0、2.5%、5.0%和7.5%酵母培养物(记为Y0、Y2.5、Y5.0和Y7.5组),分别替代质量分数0、10%、20%和30%的鱼粉,配制4种等氮等脂饲料,饲喂凡纳滨对虾(初始体质量0.70±0.03 g)56 d后,测定对虾生长性能、血清生化及免疫酶指标;用哈维氏弧菌(Vibrioharveyi)攻毒,测定对虾累计死亡率。【结果】Y2.5和Y5.0组凡纳滨对虾增重率和特定生长率与Y0组的差异无统计学意义(P> 0.05),Y5.0组凡纳滨对虾摄食率显著高于Y0和Y2.5组(P <0.05);Y5.0组凡纳滨对虾血清甘油三酯含量显著低于Y0组(P<0.05),Y7.5组凡纳滨对虾血清谷丙转氨酶含量显著高于Y0、Y2.5组(P <0.05),血清总胆固醇显著低于Y0、Y2.5组(P <0.05);酵母培养物替代饲料中20%和30%的鱼粉可显著提高对虾攻毒前肝胰腺超氧化物歧化酶、过氧化物酶活性(P <0.05),Y7.5组对虾肝胰腺丙二醛含量在哈维氏弧菌(Vibrio harveyi)攻毒后显著低于Y0组(P <0.05);攻毒7 d后,Y7.5组凡纳滨对虾累积死亡率显著低于Y0组(P<0.05)。【结论】酵母培养物替代鱼粉比例小于20%时,不会显著影响凡纳滨对虾生长性能;替代比例为30%时,生长受抑制。当替代量比例为30%时可显著提高凡纳滨对虾非特异性免疫力和抗病力。     

生物采油解堵剂中产蛋白酶菌株的筛选及培养条件优化

为研究生物酶采油解堵剂中产蛋白酶菌株的初、复筛选及培养条件优化,从大庆原油样品中筛选菌种,通过水解酪素的透明圈实验及福林酚测蛋白酶酶活的方法进行菌株的初、复筛选;以蛋白酶酶活为优化指标,采用单因素实验对筛选的产蛋白酶菌株的培养基及培养条件进行优化,优化最适培养基:可溶性淀粉为15g/L,蛋白胨为20g/L,酵母膏为20g/L,NaCl为1.0g/L,CaCl2为0.02g/L,Na2HPO4为0.2g/L,NaH2PO4为0.1g/L;在初始pH为6.0、接种量为5%(体积分数)、温度为31℃、摇床转速为160r/min的条件下,培养72h后,菌株的蛋白酶酶活为551.0U/mL,为复筛选菌株的蛋白酶酶活的22.92倍,即为菌株生长繁殖及代谢的最佳条件,能够获得更高的蛋白酶酶活,有利于后续实验的进行.结果表明:菌株产蛋白酶对原油作用效果为发酵液表面张力从作用前的56.2mN/m降低到作用后的30.5mN/m,表面张力显著降低,还有降解降黏原油等效果,具有一定的研究价值.     

Enhanced Carotenoid Production by a Mutant of the Marine Yeast Rhodotorula sp. hidai

1 Introduction In recent years, carotenoids have received increasing attention as they have extensive use in medicine, cosmetic, chemistry, food industry and feed industry. In industry, carotenoid and astaxanthin can be used as the additives of food or feeds. Carotenoids can also serve as the precursor of vitamin A in mammals. In recent years, many types of carotenoid have aroused extensive interest because of their many beneficial effects on human health. For in-stance, lycopene and astaxant…     

罗非鱼海豚链球菌的培养及其培养基的优化

研究了海豚链球菌(Streptococcus iniae)ZJMX04的培养与生长特性,探讨培养基的pH值、气体环境、温度等因素对海豚链球菌生长的影响,并采用正交实验法对海豚链球菌培养基生长因子添加量进行优化。结果表明,海豚链球菌培养基的最适pH为7～8,最适培养温度为30℃,振荡培养对海豚链球菌的生长影响不明显。培养基优化研究表明,在BHI培养基中添加10mg/mL的酵母粉和2mg/mL的葡萄糖即可在对数期获得最大细菌量。     

再论中国古代南部海洋文化的农业性特征

从经济运作的方式、历代海洋政策的变迁、古代海洋文学三个方面来分析中国古代南部海洋文化的农业性特征,认为这种特征有别于商业文化的表现形态,它受到中国内陆文化的影响,具有传统性、辅助性和保守性。因此,在考察中国古代海洋文化时,应将中国古代海洋文化作为中国古代农业文化的有机组成部分。     

一株促藻生长细菌培养条件的优化

研究分离自对虾养殖池的一株促藻生长细菌的最佳培养条件,通过单因子实验得到该菌株的最佳碳源为葡萄糖,最佳氮源为酵母膏,初始pH值为7.5,最佳氯化钠质量浓度5 g/L;在其他培养条件不变的情况下,通过对酵母膏、葡萄糖、初始pH值做三因子三水平的正交实验,得到培养基最佳组合为:酵母膏10 g/L,葡萄糖1 g/L,初始pH值7.5。     

Screening and Characterization of Nitrite-Degrading Bacterial Isolates Using a Novel Culture Medium

In this study,a novel culture medium that simulates shrimp pond conditions was established to screen nitrite-degrading isolates.The medium was supplemented with nitrite as a nitrogen source and shrimp feed as the major carbon source,to achieve the high nitrogen and low carbon nutritional status found in shrimp farming ponds.Screening using this medium identified potent denitrifying Bacillus isolates,among which Bacillus subtilis M7-1 was considered best.M7-1 was able to completely degrade nitrite-N in 24 h without much consumption of dissolved oxygen.Efficient denitrification activity took place in liquid cultures within a set of non-stringent ranges of pH(5.0–9.0),salinity(0–30)and temperature(25–35℃).The denitrifying enzyme gene was amplified,sequenced and further identified as nirS type.In biosecurity assessments,M7-1 had no negative effects on shrimps at a dose of 106 cfu mL−1.M7-1 could therefore be used in aquaculture to reduce and control the nitrogen concentration,and to promote the development of sustainable and healthy culture systems.     

Optimization of culture conditions and medium composition for the marine algicidal bacterium Alteromonas sp. DH46 by uniform design

Harmful algal blooms (HABs) have led to extensive ecological and environmental issues and huge economic losses. Various HAB control techniques have been developed, and biological methods have been paid more attention. Algicidal bacteria is a general designation for bacteria which inhibit algal growth in a direct or indirect manner, and kill or damage the algal cells. A metabolite which is strongly toxic to the dinoflagellate Alexandrium tamarense was produced by strain DH46 of the alga-lysing bacterium Alteromonas sp. The culture conditions were optimized using a single-factor test method. Factors including carbon source, nitrogen source, temperature, initial pH value, rotational speed and salinity were studied. The results showed that the cultivation of the bacteria at 28°C and 180 r min^?1 with initial pH 7 and 30 salt contcentration favored both the cell growth and the lysing effect of strain DH46. The optimal medium composition for strain DH46 was determined by means of uniform design experimentation, and the most important components influencing the cell density were tryptone, yeast extract, soluble starch, NaNO₃ and MgSO₄. When the following culture medium was used (tryptone 14.0g, yeast extract 1.63g, soluble starch 5.0 g, NaNO₃ 1.6 g, MgSO₄ 2.3 g in 1L), the largest bacterial dry weight (7.36 g L^?1) was obtained, which was an enhancement of 107% compared to the initial medium; and the algal lysis rate was as high as 98.4% which increased nearly 10% after optimization.     

一种户外开放式微藻培养体系优化

【目的】建立可用于大规模培养的户外开放式微藻培养体系。【方法】设计一种微藻平面开放浅层培养(Flat,open and shallow,FOS,简称浮法)体系,以小球藻(Chlorella sorokiniana)为模式藻种,探讨温度、光照、pH值和培养基营养成分等因素在该培养体系中对小球藻生长的影响,开展900 L体系的户外培养试验。【结果】浮法体系主要由塑料袋和垫板组成,在该培养体系中的小球藻在20~40℃范围内均可生长,最适生长温度为30~35℃。小球藻生长有明显的照度依赖性,无光时几不生长,随着照度升高,生长速度加快,在较高的照度下表现有光饱和现象。以TAP为基础培养基,在碳源或氮源缺乏时小球藻几不生长。pH 7.5左右有利于小球藻生长。900 L体系的户外培养试验的生物量(干物质得率)为0.15 g/(L·d)。【结论】在此新型户外平面开放浅层微藻培养体系中,温度、照度、pH值及营养成分等对小球藻的生长均有不同程度的影响。该体系有成本低、操作简便、容易控制、自然资源利用率高等特点,有大面积推广潜力。