凝结芽孢杆菌对奥尼罗非鱼消化酶活性、消化率及生长性能的影响

在基础饲料中分别添加不同剂量的凝结芽孢杆菌(Ⅰ:1.0×1011cfu/kg饲料,Ⅱ:3.0×1011cfu/kg饲料,Ⅲ:6.0×1011cfu/kg),室外水族箱中饲养奥尼罗非鱼(Oreochromis niloticus×O.aureus)(34.50±0.25 g),用基础饲料投喂作为对照,每饲料组设三个重复,每水族箱随机放养16尾鱼,投喂率为3%。采用静水饲养以避免各箱之间水的交换。56 d后测定鱼体的生长和消化酶活性。结果显示:不同添加量的凝结芽孢杆菌均能显著提高奥尼罗非鱼胃、肝胰脏和肠道蛋白酶活性(P<0.05),但酶的活性随添加量的提高呈下降趋势。凝结芽孢杆菌的添加对胃、肝胰脏和肠道淀粉酶及脂肪酶活性没有显著影响(P>0.05)。Ⅰ组和Ⅱ组的干物质表观消化率、蛋白质消化率、相对增重率、饵料系数和蛋白质效率均显著高于对照组(P<0.05),而Ⅲ组和对照组之间差异不显著(P>0.05)。结果提示,饲料中添加1.0×1011cfu/kg饲料的凝结芽孢杆菌就能显著促进奥尼罗非鱼的生长和饲料营养物质的利用,满足最佳生长。     

1株黄曲霉颉颃菌的鉴定及其抑菌活性成分分析

【目的】 筛选黄曲霉颉颃菌,探究其最佳培养条件及有效抗菌成分,为实际生产中应用生物防治方法抑制黄曲霉污染提供新的理论依据。【方法】 通过平板对峙法及分子生物学鉴定方法对待测菌株进行筛选鉴定。以抑菌率作为衡量标准,判断所得黄曲霉颉颃菌的最佳培养温度、培养时间、接种量、转速、pH及装液量。通过硫酸铵沉淀法从颉颃菌无菌发酵上清液中提取蛋白,观测粗提物对黄曲霉菌丝生长的影响。通过离子交换色谱和凝胶过滤色谱进一步纯化粗提蛋白,并进行质谱测序及序列对比分析,筛选鉴定颉颃菌产生的有效抑菌成分。【结果】 筛选到1株解淀粉芽孢杆菌B10(菌种保藏号CCTCCNO:M2018353)。最佳培养条件为温度40 ℃,培养时间36 h,接种量3%,转速120 r/min,pH 6.0,装液量30 mL,最大抑菌率可达46.56%。其粗提蛋白能有效抑制黄曲霉生长,破坏菌丝正常形态,并从中筛选出几丁质结合蛋白、壳聚糖酶、葡聚糖酶等8种可能的有效抑菌成分。【结论】 本试验分离鉴定出1株抑制黄曲霉正常生长发育的解淀粉芽孢杆菌B10,其产生的多种蛋白成分对黄曲霉有显著抑菌活性。     

鸡蛋壳蜡样芽孢杆菌的分离鉴定及耐药性分析

【目的】 分析不同环境下鸡蛋外壳携带蜡样芽孢杆菌的情况和分离菌株的基本特征。【方法】 从养殖场和超市各采集9种新鲜鸡蛋,对鸡蛋表面携带的蜡样芽孢杆菌进行分离鉴定、基因分型、毒力基因筛查和耐药性分析。采用基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）技术对分离菌株进行初步鉴定,全基因组测序后进行菌种鉴定并确定基因分型、筛选毒力基因和耐药基因,同时采用微量肉汤稀释法测定分离株对头孢曲松、红霉素、万古霉素等12种抗菌药物的耐药性。【结果】 经MALDI-TOF MS初步鉴定和基因组菌种鉴定,确定从养殖场3种未经清洁加工的鲜蛋中分离出的6个菌株为蜡样芽孢杆菌,其序列分型（ST）较为多样,其中有2株新的ST型。在毒力基因筛查方面,6株蜡样芽孢杆菌均检出了非溶血型肠毒素nheB基因、腹泻型毒力因子hlyⅡ基因和侵袭免疫系统的inhA基因;nheA和nheC基因携带率为83.3%,编码溶血型肠毒素HBL的hblA、hblC和hblD基因携带率均为66.7%;cytK和hlyⅢ基因携带率分别为33.3%和50.0%;肠毒素基因BceT、entFM和致吐毒素基因ces均未检出。耐药性方面,6株菌均对庆大霉素、四环素、氯霉素、利福平、利奈唑胺和环丙沙星敏感,对氨苄西林、头孢曲松和泰妙菌素耐药;对红霉素、克林霉素的中介率分别为83.3%和100%;33.3%的菌株对万古霉素耐药。6株菌均携带有磷霉素耐药基因FosB和万古霉素耐药基因簇vanA的部分基因vanRSYZ,其中5株菌携带大环内酯类药物耐药基因mphL,部分菌株携带β-内酰胺类药物耐药基因或核苷类药物耐药基因。【结论】 本试验从养殖场未经消毒处理的鸡蛋外壳上成功分离到了蜡样芽孢杆菌,并发现其携带有多种毒力基因,存在多重耐药性,有潜在致病性和公共安全风险。     

地衣芽孢杆菌发酵条件优化和抑菌活性测定

【目的】探究不同培养及发酵条件对地衣芽孢杆菌19148抑菌作用的影响及地衣芽孢杆菌19148的耐药性。【方法】设计单因素试验,分别在不同温度、酸碱度、金属离子、摇床转速、发酵时间和接种量的条件下培养地衣芽孢杆菌19148菌液,将培养后的菌液离心、过滤制成无菌滤液,并通过牛津杯抑菌试验探究无菌滤液对大肠杆菌K88、鼠伤寒沙门氏菌和金黄色葡萄球菌1882的抑制能力。通过药敏纸片法探究地衣芽孢杆菌19148对27种抗菌药的耐药性。【结果】地衣芽孢杆菌19148在4和75 ℃下生长受到抑制,25、37和45 ℃能正常生长,其中在37 ℃抑菌活性最好。在pH 5.0~9.0的条件下能正常生长,在pH为6.0条件下抑菌活性最好,对铜离子不耐受,但对镁离子表现出了良好的耐受性,且不同浓度镁离子处理组间抑菌活性无显著差异（P＞0.05）。体外发酵试验中,地衣芽孢杆菌19148在3%种子液接种量、150 r/min发酵28 h时,体外发酵的抑菌活性最好。耐药性评价试验结果显示,地衣芽孢杆菌19148对青霉素、四环素、头孢他啶、呋喃唑酮和多黏菌素B 5种抗菌药中介,对头孢哌酮和红霉素等其他22种抗菌药敏感。【结论】地衣芽孢杆菌19148具有较好的稳定性,能应用于工业发酵饲料生产,对抗菌药不具有耐药性。     

三株不同形状伴孢晶体的苏芸金杆菌对几种害虫的毒力测定

我们从落叶松毛虫(Dendrolimus sibiricus)、印度谷斑螟(Pyralis farinali)幼虫和森林土壤中分离到产生不同形状伴孢晶体的苏芸金杆菌(Bacillus thuringiensis),对三株菌进行了形态特征的电子显微镜观察、生理生化特性的测定及营养细胞的酯酶型分析,并对几种昆虫进行了室内生物测定,表现出不同的毒力。     

抗光肩星天牛苏云金芽胞杆菌Bt886菌株的分离及对毒蛋白编码基因的初步鉴定

从拟谷盗虫尸中分离到一株对鞘翅目害虫光肩星天牛幼虫毒杀作用明显的Bt菌株Bt886。幼虫死亡率高达 6 0 % ,存活者生长明显受到抑制。经过晶体形态特征和杀虫谱分析 ,初步确认该菌株属于cry3类。比较cry3类基因的保守序列 ,利用一对cry3特异引物成功地从Bt886菌株中克隆到了一段基因片段并克隆至pGEM -T载体上。测序分析发现该片段与cry3Aa1基因具有很高的同源性 ,确定来自毒蛋白编码基因。Southern杂交分析表明该基因位于该菌株的质粒上。     

两种产纤维素酶菌株分离的初步研究

本研究对采自贵州林区中的响叶杨(Populusadenopoda Maxim)腐朽木和白蚁(Odontotermes formosanus(Shiraki))进行分离培养,分离自杨树腐朽木的产纤维素酶菌株为木霉属康宁木霉(Trichodermakoningii Oud);分离自白蚁肠的产纤维素酶菌株为芽孢杆菌属(Bacillus sp.)。对所分离的两个菌株进行滤纸降解观察与测定。所分离的菌株(Trichoderma koningii Oud.)滤纸培养30天后,有菌丝生长,培养基浑浊,滤纸呈絮状,培养7天后该菌对滤纸降解强度为7.13%(校正值)。刚果红法初步证明该菌能产纤维素酶。从白蚁肠分离的菌株(Bacillus sp.)滤纸培养15天后,滤纸呈絮状,培养基变浑浊,有细菌菌体沉积;培养7天后,对滤纸的降解强度为8.18%(校正值)。刚果红法初步证明该菌能产生纤维素酶。用五种培养基质培养芽孢杆菌属(Bacillus sp.)菌株,并测定其生长量,其中以滤纸为主要基质的培养液中,最高可检测到0.3×107cfu/ml菌株量。     

苏云金芽孢杆菌菌种保藏

采用砂土管和滤纸片两种保藏方法，对分属３１个亚种的６１株有代表性的苏云金芽孢杆菌经过１－２３年保存后的菌体存活情况，形态特征及对林业主要害虫舞毒蛾幼虫的杀虫活性进行了试验，并对苏云金芽孢杆菌５个亚种的标准菌株和保存不同年限后的１１项生理生化特性及酯酶型进行了测定。     

Plant biotechnology: A case study ofBacillus thuringiensis (Bt) and its application to the future of genetic engineered trees

Agricultural productivity may be raised in a sustainable way by many different technologies such as biological fertilizers, soil and water conservation, biodiversity conservation, improved pest control, and changes in land ownership and distribution. Of these measures, biotechnology applications probably hold the most promise in augmenting conventional agricultural productivity, because biotechnology applications give not only the need to increase production, but also protect the environment and conserving natural resources for future generations. Biotechnology applications will have the possibilities to increase productivity and food availability through better agronomic performance of new varieties, including resistance to pests; rapid multiplication of disease-free plants; ability to obtain natural plant products using tissue culture; diagnosis of diseases of plants and livestock; manipulation of reproduction methods increasing the efficiency of breeding; and the provision of incentives for greater participation by the private sector through investments. Insect resistance through the transfer of a gene for resistance fromBacillus thuringiensis (Bt) is one of the most advanced biotechnology applications already being commercialized in many parts of the world. This paper reviews the development and the status ofBt technology and application ofBt transgenic plants in current agriculture, and discusses specific issues related to the transfer of the technology to the future of genetic engineered trees with emphasis on conifers. Biography: Tang Wei (1964-), male, Ph. Doctor, Research associate, Department of Biology, Howell Science Complex, East Carelina University, Greenville, NC 27858-4353, USA. Responsible editor: Chal Ruihai     

Inheritance and cross-resistance of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> insecticidal crystal protein Cry1Ac resistance in cotton bollworm <Emphasis Type="Italic">Helicoverpa armigera</Emphasis> Hubner population from Tamil Nadu,India

Cotton bollworm, Helicoverpa armigera Hubner selected for five generations with Bacillus thuringiensis insecticidal crystal protein Cry1Ac in the laboratory developed 12.98-fold resistance. Resistance and susceptible populations were mass crossed to study the dominance of resistance. The Cry1Ac—selected (BCR) population showed 5.8-fold resistance to Cry1Aa and 5.04-fold resistance to Cry1Ab. The degree of dominance (D) was 0.34 and 0.40 for the R × S and S × R hybrids, respectively, which indicates incomplete recessive character of Cry1Ac resistance in the population. The estimated realized heritability (h ²) and response quotient (Q) of resistance for Cry1Ac were 0.52 and 0.15, respectively. This indicated the lower phenotypic variation in the selected population. The resistance risk assessment based on h ² indicated that the resistance would increased tenfold after <9 generations for Cry1Ac in the resistant population. The results show the ability of H. armigera to develop resistance against Cry1Ac and cross-resistance to Cry1Aa and Cry1Ab.