诺卡氏菌与假丝酵母的跨界融合及对退化养殖生态的修复

运用溶菌酶和蜗牛酶破细胞壁，PEG和Ca^2 促融的方法完成了诺卡氏菌和假丝酵母的跨界融合，得到具有亲本优势的融合细胞二株：GW01和GW02，通过菌落形态比较，抗生素抗性鉴定，耐热性和耐酸性比较，生长速率对比的分析，可确定其为真正的融合细胞，融合细胞较亲本细胞对水体和水质的适应范围更广，降解速率更快，能大幅去除养殖水体环境中的COD，NH4^ ,NO2^-,PO4^3-等，稳定pH，提高水中DO及水体生态环境中微生物和浮游生物的生物多样性，减少换水量，从而改善养殖生态环境，促进养殖生产。     

Bacteriolytic activity and growth of marine isolates of labyrinthulids on dead bacterial cells

ABSTRACT:   Thirteen strains of labyrinthulids were isolated from the coastal area of Kagoshima Bay, Kagoshima Prefecture, Japan and from Batan Bay, Panay Island, the Philippines by using diatom double-layer agar plates. The cells of labyrinthulid isolates grew axenically on the dead bacterial cells and extract plus egg yolk agar medium (NSBEY agar). Three representative isolates were demonstrated to belong to the labyrinthulid phylogenetic group (LPG) based on 18S  rDNA sequence analysis. As bacteriolytic activity of labyrinthulid isolates was examined, it was found that they could lyze only the dead cells of gram-negative bacteria and not those of gram-positive bacteria during incubation in both agar and liquid media. The optimum temperature range for bacteriolysis was 25–31°C. Orange carotenoid pigment was accumulated during the stationary growth phase of strain 00-Bat-05, Philippine isolate, cultured in an L-shaped tube containing a bacterial dead cell suspension. Concomitant rapid cell movement of developing zoospores was observed.     

响应面法优化丹参中抗无乳链球菌活性成分的提取工艺

在单因素提取工艺优化的基础上,选取液料比、浸润时间、提取时间和提取温度进行四因素三水平的Box-behnken实验设计,并结合响应面分析法进一步优化丹参中抗无乳链球菌(Streptococcus agalactiae,SA)活性成分提取工艺。优化所得最佳提取工艺为液料比37.8:1.0(ml:g)、浸润时间104 min、提取温度86.7℃、提取时间182 min。响应面模型预测在该提取工艺下抗SA活性成分提取得率极值为25.95%,经4次平行验证实验的实际提取得率为(25.87±0.03)%,为优化前的2.59倍,表明优化方法高效可靠。     

实验条件下1龄锦绣龙虾的生长特性

通过对不同生长时期锦绣龙虾(Panulirus ornatus)月生长指标的测定,建立了1龄锦绣龙虾的生长模型。锦绣龙虾的体长(L)—体质量(W)的相关式为:W=0.034 8L3.066 3;体长(L)和头胸甲长(CL)的相关式:L=2.772 2CL+0.337 9,得出锦绣龙虾的体长生长方程:Lt=37.498×(1-e-0.076 77(t-0.055 84)),体质量生长方程:Wt=2 333.24×(1-e-0.076 7 7(t-0.055 84))3.066 3,体长生长速度方程为:dL/dt=2.879 8×e-0.076 77(t-0.055 84),体质量生长速度方程为:dW/dt=549.244 3×e-0.076 77(t-0.055 84)(1-e-0.076 77(t-0.055 84))2.066 3。计算出其拐点年龄(Tr=ln3/k+t0)为14.65个月。说明锦绣龙虾体长在前期生长较快,而后期生长缓慢;体质量前期生长较慢,后期较快;体质量增长速度曲线呈不对称的S曲线,并具拐点,在拐点处其体质量增长速度能达到最大值79.24 g/月。本研究旨在为锦绣龙虾的合理开发利用提供科学参考。     

太平洋牡蛎与近江牡蛎的种间杂交

为了评估太平洋牡蛎与近江牡蛎能否产生远缘杂种优势,于2010年5月,以成熟的两种牡蛎亲本为材料开展了2×2远缘杂交研究,由长牡蛎自繁组GG(Crassostrea gigas♀×C.gigas♂)、近江牡蛎自繁组AA(C.ariakensis♀×C.ariakensis♂)、正交组GA(Crassostreagigas♀×C.ariakensis♂)、反交组AG(C.ariakensis♀×C.gigas♂)4个实验组组成。分析了子代早期表型性状和杂种优势,并对杂交子代进行了遗传鉴定。结果表明:GA杂交组与AG组的受精强度具有不对称性。幼虫浮游期间,表型性状的中亲杂种优势几乎为0,GA组生长与存活性状表现出积极的单亲杂种优势,而AG组则具有明显的远交衰退现象;幼虫早期表型性状受到母本效应影响,而后减弱。变态期间,GA组变态率较高,得到了大量杂交稚贝;而AG组变态率极低,仅获得了72个杂交稚贝。稚贝培育期间,稚贝表现出中亲生长劣势与存活优势;GA组具有明显的单亲生长与存活优势,而AG组则表现出生长劣势并具有一定程度的存活优势。利用复合COI及ITS2鉴定了种间杂交子结果表明:正反交组杂交子均为真正意义上的两性融合杂交子。实验获得了具有显著杂种优势的GA组杂交子,为现有牡蛎的遗传改良提供了新的方向。     

普通变形杆菌TWN-3株基因组文库构建及免疫印迹筛选

在从患病大口鲇中分离到普通变形杆菌TWN-3株的基础上,将该菌总DNA用EcoRI不完全酶切后,分离5kb左右的片段,连接入pUC18质粒,转化E.coli BL21,构建基因组文库,得到3.6×103个重组子,远大于理论计算的1831个重组子,随机挑选重组子经EcoRI酶切鉴定重组率为100%,文库构建成功;将该菌裂解液与佐剂混合,免疫雄性新西兰大白兔,免疫程序结束后,抽血并分离血清,对用异丙基-β-D-硫代半乳糖苷(IPTG)诱导后的文库进行免疫印迹筛选,最终得到8个阳性克隆子。本实验为该菌的DNA疫苗以及重要基因表达研究打下基础。     

不同来源琼胶提取琼脂糖的比较

主要研究以条状江蓠琼胶、粉末状江蓠琼胶、条状紫菜琼胶为原料提取琼脂糖的品质差异。研究结果表明，用EDTA－Na2法提取琼脂糖，以条状江蓠琼胶为原料时，去除硫琼胶效果最好，产率较大；以条状紫菜琼胶为原料时，产品凝胶强度最大，结晶紫电泳性能最好，产率最高；以粉末状江蓠琼胶为原料时，产品质量较差，产率最低。     

Antagonistic activity of marine Streptomyces sp. S073 on pathogenic Vibrio parahaemolyticus

Fisheries Science - Marine Streptomyces sp. isolate S073 was shown to have strong antagonistic activity towards the pathogenic Vibrio parahaemolyticus using the agar diffusion method. The...     

Reducing bacterial density in the semen of rainbow trout (Oncorhynchus mykiss) by applying gradient centrifugation and swim‐up washing methods

The objective of this study was to demonstrate the effectiveness of disinfection procedures to reduce bacterial load of rainbow trout, Oncorhynchus mykiss (Walbaum 1792), semen. Fresh semen was obtained from 3–4‐year‐old male species by abdominal sampling of sperm into pre‐cooled test tubes. After sperm cryopreservation and thawing, experiments were accomplished at 4–9°C. ‘Swim‐up’ and gradient centrifugation were used as a sperm washing method with commercial kits. Phosphate buffered saline was also used as washing solution. Bacterial growth tests were employed before and after washing the semen samples. Samples were inoculated on tryptic soy agar (TSA), modified Anacker and Ordal agar (MAOA) as well as brain heart infusion (BHI) agar. After using ‘swim‐up’ method for washing the semen, many bacterial colonies were observed. However, after semen washing with gradient centrifugation, lower bacterial growth was observed on TSA, MAOA and BHI. Some motile (40%) spermatozoa were obtained doing gradient washing procedure. Although sperm motility was not satisfactory, apparently the gradient centrifugation method reduced bacterial contamination as known from the mammalians.     

海洋细菌Phaeobacter DL2的鉴定及其对致病弧菌抑制作用

从海水养殖水体中防腐钢片的微生物黏膜上分离筛选到一株细菌,命名为DL2.采用琼脂扩散法进行体外抑菌实验,测定其抑菌谱,发现菌株DL2具有较广泛的抑菌谱,并且对致病性弧菌表现出较强的抑菌作用.通过常规生理生化研究和细菌鉴定系统测试,发现菌株DL2属于革兰氏阴性菌,短杆状,具极生单鞭毛,菌落呈褐色,能产生浅褐色扩散性色素,测试结果表明,菌株DL2与Phaeobacter属细菌的特征非常相似.为了进一步确定菌株DL2的分类学地位,测定了其16S rRNA基因序列,与相关细菌种属相应序列的同源性进行比较并构建了系统关系树,结果显示,菌株DL2与Phaeobacter inhibens的亲缘关系最近,相似性达99.8%,与Phaeobacter gallaeciensis相似性达99.1%.综合上述结果,菌株DL2可鉴定为Phaeobacter inhibens,同时可以将其作为潜在的海洋有益菌应用于水产养殖中.     

华鲮烂尾病病原的分离鉴定及药敏分析

从患有烂尾病的华鲮(Sinilabeo rendahli)体内分离到两个优势菌株(编号:BB090516C-1和BB090516C-2),对分离菌株形态特征、主要理化特性等生物学性状鉴定后测定其16S rRNA序列并构建系统发育树,同时采用K-B琼脂扩散法进行药敏实验。结果显示:经人工感染试验,证实分离菌对华鲮有较强的致病性,该菌为革兰氏阴性、兼性厌氧发酵型短杆菌。经形态特征、培养特性和生理生化反应等指标分析鉴定结果显示该菌为维隆气单胞菌温和生物型(Aerom onas veronii biovar sobria)。用16S rRNA基因序列比对结果显示分离菌株与维隆气单胞菌的同源性高达99%,系统进化树中与维隆气单胞菌自然聚为一支,表明该菌为维隆气单胞菌温和生物型。用22种抗菌类药物进行药敏实验发现,该菌对头孢曲松、硫酸庆大霉素等9种抗菌类药物高度敏感,对新霉素等6种药物中敏,对氨苄青霉素等7种药物耐药。     

Inbreeding depression traits in pacific abalone Haliotis discus hannai by factorial mating experiments

ABSTRACT:   'Inbreeding depression' may be an avoidable phenomenon for abalone culture. However, only a few studies have been carried out on inbreeding depression. In the present study, using six families produced in 1994, a factorial mating system including inbreeding and outbreeding was constructed in order to demonstrate inbreeding depression traits of the Pacific abalone. In total, 24 inbreeding and 21 outbreeding crosses were produced during three years (1999–2001) and these offspring were reared for approximately one year. Significant differences in fertilization rate and growth were not observed between inbreeding and outbreeding crosses. However, the deformity rate of veliger larvae was always higher in inbreeding crosses than that of outbreeding crosses in all experiments. Moreover, a significantly high deformity rate was observed in some full-sib families of inbreeding. Alternatively, the survival rates of inbreeding crosses were much lower than for outbreeding crosses after about 4 months and one year in two rearing localities. These results indicate that inbreeding depression is observed in the traits of deformity rate and survival, but not in fertilization rate nor growth in the first generation of a full-sib family of the Pacific abalone.     

海水蛭弧菌分离纯化方法初步研究

蛭弧菌(Bdellovibrio)广泛存在于自然水体,它具有噬菌的特性,对水体中细菌数量控制具调节作用,有可能是进行生物防治的一种重要生物。本文比较不同分离方法、上层培养基营养盐浓度和富集培养与直接培养等对海水蛭弧菌分离、计数的影响,从而建立了海水蛭弧菌分离和纯化的方法。应用无营养盐的海水双层琼脂平板法,采用6种宿主菌分别对海南4份海水样品进行计数检测,发现不同宿主菌所得到的蛭弧菌数量不同,同时对分离纯化到的3株海水蛭弧菌进行液体培养增殖,进一步检验了海水双层琼脂法的可靠性。     

First report on the autochthonous gut microbiota of brown trout (Salmo trutta Linnaeus)

A study was conducted to characterize the autochthonous gut microbiota present in the pyloric caeca (PC), anterior mucosa (AM) and posterior mucosa (PM) of brown trout (Salmo trutta). Total viable counts (TVC) bacterial populations were enumerated using tryptone soy agar, lactic acid bacteria (LAB) levels were enumerated on de Man, Rogosa & Sharpe agar and PCR‐DGGE was employed as a culture‐independent method to assess the total communities. No significant differences were observed between the different gut regions for TVC or LAB levels. 16S rRNA sequencing identified all LAB isolates as Carnobacterium maltaromaticum. In contrast, the TVC community was more diverse; Firmicutes and Bacteroidetes were present but all gut regions were dominated by Proteobacteria, accounting for 88.4–92.6% of the communities. Citrobacter freundii was the dominant species and accounted for 51.0–57.8% of the isolates. Complex bacterial communities were observed using PCR‐DGGE and a trend towards the reduction in the number of operational taxonomic units (OTUs), microbial richness and microbial diversity was observed from the PC to the PM. The similarity between regions was low (52–68%) and cluster analysis revealed that the communities grouped into two distinct clusters; one dominated by the PM samples and the other contained the AM and PC samples. OTUs from the DGGE were identified as members of the phyla Proteobacteria and Firmicutes. Many OTUs were detected in all gastrointestinal regions, however, some OTUs showed regional specialization. Further studies are required to elucidate the activity of these genera in situ and how their actions impact the host.     

罗非鱼无乳链球菌LrrG-Sip融合基因原核表达载体的构建及表达

LrrG和表面免疫原性蛋白（Sip）是无乳链球菌（Streptococcus agalactiae）的2种表面蛋白,具有良好的免疫原性。为获得罗非鱼无乳链球菌表面蛋白LrrG和Sip蛋白的融合蛋白,该试验采用基因拼接技术中的双酶切法分2步逐个将Sip和LrrG基因插入pColdⅡ载体中,构建原核表达载体pColdⅡ-LrrG-Sip。将成功构建的融合基因原核表达载体转化感受态细胞BL21（DE3）,进行诱导表达条件的优化。结果显示,15℃、IPTG 0.5 mmol·L-1诱导9 h,目的蛋白呈可溶状态的表达量最高。Western Blot检测结果显示LrrG-Sip融合蛋白大小与预测一致（162kDa）,说明成功构建了融合基因,为罗非鱼源无乳链球菌亚单位疫苗的研制奠定了基础。     

氨氮胁迫对锦绣龙虾代谢酶和抗氧化酶活力的影响

为了研究急性氨氮胁迫对锦绣龙虾(Panulirus ornatus)抗氧化系统及氨氮代谢机制的影响,通过设置对照[(0.24±0.07)mg/L]、低浓度[(1.04±0.08)mg/L]、中浓度[(9.75±0.21)mg/L]和高浓度[(19.87±0.46)mg/L]氨氮胁迫方法对锦绣龙虾进行48 h急性实验,测...     

2株海鞘共附生真菌的次生代谢产物研究

分别对采集自防城港白龙的皱瘤海鞘(Styela plicata)和北海南汅的冠瘤海鞘(Styela canopus)样品的共附生真菌Purpureocillium sp. FBZ-1和Penicillium sp. BNG-1进行研究。通过显微镜观察鉴定其形态,并通过分析生物学特征鉴定其种属。通过对两种真菌的次级代谢产物进行分离和结构鉴定,共分离鉴定28个小分子化合物,从中未发现新的化合物,主要的化合物类型是芳香胺类、生物碱类和二酮哌嗪类,分别为:lumichrome、 1,2,3,4-四氢-β-咔啉-3-羧酸、β-咔啉、环(异亮氨酸-脯氨酸)、环(亮氨酸-羟基脯氨酸)、环(色氨酸-缬氨酸)、环(亮氨酸-脯氨酸)、环(苯丙氨酸-脯氨酸)、环(异亮氨酸-丙氨酸)、环(色氨酸-亮氨酸)、苯乙胺、N-乙酰苯乙胺、吲哚乙胺、N-甲基色胺、N-(4-羟基)丁酰苯乙胺、对羟基苯乙胺、 5,7,4-三羟基异黄酮、 7,4-二羟基异黄酮、 5,7,3,4-三羟基异黄酮、邻苯二甲酸异辛酯、 2,4-N,N-dimethyl-lumichrome、 3,4-二氢-3-甲基-β-咔啉-1-酮、色氨酸、 2-羟基-3-吲哚丙酸、环(缬氨酸-脯氨酸)、环(亮氨酸-丙氨酸)、环(苯丙氨酸-5-甲基-3,4-脱氢脯氨酸)、环(色氨酸-天冬酰胺)。研究结果对利用我国丰富的海鞘及其共附生微生物资源并研究其次级代谢产物结构多样性,发现其中可能含有的结构新颖的化学物质以开发抗菌、抗病毒及抗肿瘤新药,具有十分重要的理论意义和潜在的应用价值。     

Isolation,Screening, and Identification of Proteolytic Lactic Acid Bacteria from Indigenous Chao Product

The aim of the study was to isolate, select, and identify proteolytic lactic acid bacteria (LAB) from chao, a traditional fermented fish from Pangkajene and Kepulauan Regency, South Sulawesi, Indonesia. LAB was isolated by poured plate method. Proteolytic LAB were selected using agar skim milk media. Protease activity of LAB was determined based on the amount of tyrosine released in unit/mL. Proteolytic LAB were identified using API 50 CHL kit and 16S rRNA gene sequence analysis. The result showed that a total of 60 isolates were obtained from chao, and 57% of them were cocci-shape. Fifteen isolates were halotolerant proteolytic LAB. Their R values and protease activity were 2.11–3.39 and 0.267–0.304 U/mL, respectively. Identification by API 50 CHL kit showed that four rod-shape isolates were Lactobacillus plantarum, and two others were Lactobacillus curvatus. Cocci-shape isolates could not be identified as cocci bacterium. Rep-PCR results showed that there were two kinds of bands, namely thick and thin. Two isolates were selected from two types of bands that had the highest R for the analysis of 16S rRNA gene sequences, namely Ags1-3 and Ags7-3. The results showed that Ags1-3 isolate was identified as Lactobacillus plantarum and Ags7-3 as Pediococcus acidilactici.     

双列杂交分析比较两个养殖群体杂交牙鲆抗病力

采用双列杂交设计对牙鲆Paralichthys olivaceus两个养殖群体：日本群体（J）和中国抗病选育群体（C）进行群体间杂交和群体内自繁,获得C（♀）×C（♂）、J（♀）×J（♂）、J（♀）×C（♂）和C（♀）×J（♂）4个交配组合的子一代。针对受精率、孵化率、白化率和存活率4个性状进行了组合间的比较,计算了各项性状的杂种优势率,对组间各性状进行了方差分析,并进行了相关分析。结果表明,杂交组在受精率、孵化率及存活率上存在着不同程度的杂种优势,其中C（♀）×J（♂）后代在孵化率和存活率两个性状上的杂种优势率分别为33.99%和31.37%。对4个性状的表型相关分析表明,受精率和孵化率的相关系数为0.657,受精率和存活率相关系数为0.432,分别达到了极显著水平（P〈0.01）。分析结果认为,两个不同群体间的种内杂交是牙鲆优良品种培育的有效途径。     

Offspring Growth in a Diallel Crossbreeding with Three Strains of Nile Tilapia Oreochromis niloticus

Abstract— A complete diallel crossing of three strains, Stirling, Korean and Local, was used to evaluate heterosis of intraspecific crossbreeding on early growth of Nile tilapia Oreochromis niloticus . All possible F-1 hybrid offspring were obtained and their growth rates to 90 d were compared. The specific heterosis for F-1 hybrids in Stirling × Korean, Stirling × Local and Korean × Local crosses were 7.61, 5.63 and 7.70 for weight, and 0.47, 0.26 and 0.55 for length, respectively. The purebred Stirling strain showed better growth than the other two strains (P < 0.05). When growth of F-1 hybrids was compared between sexes, males grew more rapidly than females (P < 0.05). However, no differences were observed in growth of male or female between hybrid groups. Purebred males in the Stirling strain were slightly heavier than those in the Korean and Local strains, and Local × Stirling, Korean × Local and Local × Korean crosses. However, in purebred females Stirling was superior to Korean, but not the Local strain and all F-1 hybrid crosses. Implications to choose the appropriate strains for developing a breeding program of tilapia are also discussed.