黑龙江凉水自然保护区苏云金芽孢杆菌的收集与鉴定

黑龙江凉水自然保护区是我国现有保存下来的较大片原始红松林基地之一,总面积6394hm^2,森林覆盖率95％以上。本研究从小兴安岭凉水自然保护区采集土样782份,采用醋酸钠培养基结合高温方法筛选土壤中的芽孢杆菌,通过光学显微镜观察鉴定产生伴胞晶体的苏云金芽孢杆菌（Bacillus thuringiensis．Bt）。总计分离得到芽孢杆菌和苏云金芽孢杆菌分别为1735株和33株,Bt菌株的分离率和出菌率分别为1．90％和4．22％。利用SDS-PAGE和PCR—RFLP方法对筛选获得的成分离株进行了杀虫晶体蛋白和基因型分析,结果表明14株产菱形伴胞的＆菌株,SDS-PAGE电泳分析芽孢后期产生分子量大小130kD蛋白带,PCR-RFLP分析初步鉴定为crylAc基因,其它产圆形或其它不定形晶体蛋白的＆分离菌中,芽孢后期主要蛋白大小为20～150kD不等,PCR—RFLP方法鉴定结合PCR片段测序分析这些菌株含有新型cry4、cry39和cry40基因等。本研究是“中国助资源收鉴与利用”项目组成部分之一,凉水自然保护区苏云金芽孢杆菌的收集与鉴定目的是要对整个东北地区成资源分布和多样性作一个初步评估,实验结果表明东北森林地区具有丰富多样的威菌株和杀虫基因资源。     

广西大王岭和大明山自然保护区苏云金芽孢杆菌收集与鉴定

从广西大王岭和大明山两个自然保护区共采集到土样264份，共分离出597株芽孢杆菌，通过光学和电子显微镜检观察，16株分离株观察到伴胞晶体蛋白，初步确定为苏云金芽胞杆菌（Bacillus thuringiensis，简称Bt），出菌率为6．06％。在16株肪分离株中，有4株在芽孢形成过程中能产菱形晶体蛋白，其余12株能产圆形和其他形状的晶体蛋白。利用PCR—RFLP方法和SDS．PAGE方法对16株助分离菌进行了蛋白和基因型的鉴定，结果表明，16株分离株中含有4株crylAc基因，表达约130kD的晶体蛋白，其中含有cry30基因和cry40基因的菌株分别1株和3株，表达大约75kD的晶体蛋白；另外8株戤菌株表达蛋白大小不一，其基因型尚不能确定，有待进一步分析。生物测定表明，产菱形晶体含有crylAc基因的4株励分离株对鳞翅目小菜夜蛾幼虫有很强的毒杀活性，而其它分离株对小菜夜蛾没有毒杀活性。     

苏云金芽孢杆菌S1478—1分离株的特性鉴定及cry1Ac同源基因克隆

本研究对从海南岛尖峰岭热带雨林自然保护区的土壤样品中分离出的＆菌株S1478～1进行了特性鉴定,研究表明S1478—1分离株菌落形态和生长特征和励参照菌株HD73极其相似。16SrDNA序列分析表明,S1478—1分离株与其它＆thuringiensis、B．cereus和B.anthracis的16S rDNA序列相似性达到99％。分离株能产菱形伴胞晶体,SDS-PAGE蛋白电泳分析表明,菌株在生长后期,形成芽孢同时分泌130kD大小的晶体蛋白。生物测定表明S1478—1分离株对小菜蛾具有很高的毒杀活性,LC50值高达5．159×10^8cfu／mL。初步显示S1478—1分离株可作为防治鳞翅目害虫的生物农药菌株。利用PCR—RFLP方法鉴定S1478—1分离株含有cry1Ac同源基因,以PCR粘性端克隆方法扩增全长基因,序列测定表明该基因ORF为3537bp,编码1178个氨基酸,推定的编码蛋白分子量为133．3kD,与其它cry1Ac基因序列最高达到99％同源,因此,该基因可作为杀虫工程菌及培育转基因抗虫作物的候选基因。     

酒糟和窖泥中苏云金芽孢杆菌的分离及鉴定

从泸州老窖酒厂的酒糟和窖泥中采集的168个样品中分离出苏云金芽孢杆菌1株,经光学显微镜观察伴孢晶体形态为菱形。以cry1/7/9、cry2、cry3、cry5、cry6、cry8、cry4/10、cry11、cry1Ⅰ的引物做PCR分析进行基因型鉴定,发现该分离株有cry1基因。同时还对该株蛋白质图谱和杀虫活性等特性分析,结果表明该分离株对小菜蛾有一定活性,死亡率可达80%,但该菌株对致倦库蚊无活性。     

煤矿中苏云金芽胞杆菌(Bt)菌株的分离及其鉴定

苏云金芽胞杆菌(Bacillus thuringiensis,Bt)是一种已被广泛用于农业害虫生物防治的昆虫病原细菌。本研究从永春天湖山矿区采集的159份样品中分离出1株Bt,光学显微镜检测发现菱形伴孢晶体。以cry1/7/9、cry2、cry3、cry4/10、cry5、cry6、cry8、cry11和cry1I的通用引物对该Bt分离菌进行Cry毒素基因分析,利用SDS-PAGE和双向电泳-质谱法(2-dimensional electrophoresis/mass spectrometry,2-DE/MS)对Bt分离菌进行蛋白鉴定。结果表明,分离菌含有cry1基因型,经克隆、测序后可知,菌株中的cry1亚基因型与cry1Ac有极高的同源性。分离株表达的晶体蛋白约为130 k D,通过蛋白质谱分析表明,杀虫蛋白为Cry1Ac,与PCR-RFLP所得出的结果一致;通过质谱分析还鉴定出分子伴侣Gro EL、F0F1型ATP-β亚基合成酶、延伸因子Tu和丙酮酸脱氢酶。生物信息学分析结果表明,这些蛋白主要参与帮助新生肽的正确折叠、能源生产和转换,以及提高细胞的应激能力以抵御外界不良刺激。本研究从煤矿中分离鉴定出Bt菌,为发现新资源、新型杀虫蛋白基因以及利用微生物治理重金属污染提供基础资料。     

苏云金芽孢杆菌Cry1Ac杀虫晶体蛋白及其分子设计

Cry1Ac编码的杀虫晶体蛋白是苏云金芽孢杆菌（Bt）产生的多种杀虫晶体蛋白中对鳞翅目昆虫有很高毒性的蛋白。第一个Cry1Ac杀虫晶体蛋白最早在库斯塔克亚种HD73中以伴胞晶体形式分离获得,其编码区为3534bp,编码蛋白分子量为133kD,含1178个氨基酸,等电点为4.84。白此以来,Cry1Ac杀虫晶体蛋白结构、功能以及应用研究一直是Bt杀虫晶体蛋白研究的重要方向。本文介绍了苏云金芽孢杆菌中应用最广泛的Cry1Ac杀虫晶体蛋白家族的结构、功能及其基因分类,并进一步就基于苏云金芽孢杆菌Cry1Ac杀虫晶体蛋白的基因工程研究做了分析,提出了持续利用Bt Cry1Ac杀虫晶体蛋白的一些见解。     

苏云金芽孢杆菌WB9菌株cry2Ac4基因的克隆及表达

WB9是我国分离自武夷山的对多种重要农业害虫具有高毒力的苏云金芽孢杆菌(Bacillus thuringiensis,简称Bt)菌株,经PCR-RFLP鉴定含有cry2Ac基因。根据cry2基因序列设计引物,以WB9质粒为模板扩增cry2Ac全长基因,与大肠杆菌(Escherichia coli)克隆载体pMD18-T连接获得含有cry2Ac全长基因的重组质粒pMD2Ac并测序。该基因在GenBank上登录号为DQ361267,被Bt国际命名委员会正式命名为cry2Ac4。通过亚克隆方法将cry2Ac4基因插入穿梭表达载体pHT315获得重组表达质粒pHT2Ac,将其转化大肠杆菌SCS110和Bt无晶体突变株HD73 Cry-,得到的工程菌能正常表达70 kD蛋白,形成方形晶体。生物测定结果表明,cry2Ac4基因表达产物对桔小实蝇(Bactrocera dorsalis Hendel)幼虫具有显著的毒杀作用,但对小菜蛾(Plutella xylostella)和致倦库蚊(Culex fatlgans)幼虫基本没有效果。     

苏云金芽孢杆菌cry2Aa基因的克隆、表达与活性

B-8-G和Ly30是我国自行分离的对多种重要农业害虫具有高毒力的苏云金芽孢杆菌（Bacillus thuringiensis）菌株,经PCR-RFLP鉴定均含有cry2Aa基因.根据cry2Aa全长基因序列设计特异引物,以B-8-G总DNA为模板扩增其中的cry2Aa全长基因,与大肠杆菌（Escherichia coli）表达载体pET-21b相连接,获得含有cry2Aa全长基因的重组质粒pET2Aa,该基因在大肠杆菌BL21菌株能够正常表达65 kD蛋白.通过构建Ly30总DNA文库方法从中筛选获得cry2Aa基因,将其连接至Bt-E.coli穿梭表达载体pHT315上,转化Bt无晶体突变株HD-73中,该基因能正常表达65 kD蛋白,并形成立方体状晶体.这两种基因序列已被国际Bt基因命名委员会分别正式命名为cry2Aa9和cry2Aa10.杀虫生物活性测定结果表明cry2Aa基因表达产物对黄胫小车蝗（Oedaleusinfernlis）、尖音库蚊（Culex pipiens）、黑翅伊蚊（Aedes melanopterus）、水稻二化螟（Chilo suppressalis）和小菜蛾（Plutellaxylostella）幼虫均具有显著的毒杀作用.首次报道cry2Aa10基因表达蛋白对蝗虫、库蚊具有杀虫活性.这些基因的获得,将为高效工程菌和抗虫转基因植物的研制提供了新的基因资源.     

基于Illumina测序技术挖掘Bt杀虫基因资源

苏云金芽孢杆菌作为应用最为广泛的生物农药,已经成为化学合成农药必要的补充或替代品,其杀虫蛋白基因已成功的应用到转基因抗虫作物中。本文基于海南五指山和吊罗山热带原始雨林区土壤样品筛选分离的Bt菌株资源,利用第二代测序技术Illumina对分离的特异Bt菌株的杀虫基因进行全面分析,鉴定出新型Bt杀虫蛋白基因25个,其中包括5个新型的营养期杀虫蛋白基因（vip）和3个新型cyt基因。本研究表明第二代DNA测序技术可以高效用于挖掘Bt杀虫基因资源,而获得的新型杀虫蛋白基因将进一步丰富我国能够应用的杀虫基因种类。     

苏云金芽孢杆菌cry1Aa14基因的分离、克隆及其表达

Bt25是中国自行分离的对小菜蛾(Plutella xylotella)具有高毒力的苏云金芽孢杆菌(Bacillus thuringiensis),经PCR-RFLP鉴定含有cry1Aa基因。以全长基因PCR产物的粘端定向克隆的方法,设计1对特异引物,以Bt25质粒DNA为模板扩增cry1Aa全长基因。序列测定结果表明,该基因编码区为3552bp,编码1183个氨基酸,分子量为133．7kD,pI4．755。该基因序列已在GenBank注册,登记号为AY197341,并获得正式命名cry1Aa14。在氨基酸序列918～1180间,和已知的11种cry1Aa存在22-23个氨基酸的差异(其中1094～1097的4个氨基酸无对应序列),而这段区域和Cry1Ab氨基酸序列的对应区域无差异。cry1Aa14全长基因插入Bt表达载体,获得了重组表达质粒pBYB1,转化Bt无晶体突变株HD73cry-,经过抗性筛选、DNA酶切分析和PCR检测,证实转化成功。SDS-PAGE分析表明,该基因在上述受体中能正常表达133kD蛋白。杀虫生物测定结果表明,cry1Aa14表达产物对小菜蛾幼虫具有显著的毒杀作用,与cry1Aa12进行比较,毒力无明显差异。这种单基因菌株的发现及其基因的获得,为害虫抗性研究和高效工程菌的构建提供了重要实验材料。     

Plant litter quality influences the contribution of soil fauna to litter decomposition in humid tropical forests, southwestern China

The aim of this field experiment was to quantify the contribution of soil fauna to plant litter decomposition in three forest sites differing in C/N ratio under natural conditions in Xishuangbanna, southwestern China. We conducted a survey of soil fauna communities, the forest floor litter and investigated mass loss of mixed tree species leaf litter for two years in a tropical secondary forest, an evergreen broad-leaf forest and a tropical rain forest. Exclusion treatments of different sized soil fauna from the leaf litter by using varying mesh size litter bags (2 mm and 0.15 mm) were also performed. Mass loss and C and N concentrations in litter bag leaf materials were determined at monthly intervals. We found that: (1) the three forests differed in floor litter biomass and nutrient contents but not in soil fauna richness and abundance; (2) litter mass loss and decomposition rate were slower when soil macrofauna and most of mesofauna were excluded; and (3) greatest soil fauna contribution to plant litter decomposition occurred in the rain forest, where leaf litter C/N ratio was also highest (41.5% contribution: 54.8 C/N ratio), in comparison to 8.69% in the broad-leaf forest and 19.52% in the secondary forest, both with low leaf litter C/N ratios (<32). Our results suggested that, soil fauna played a more pronounced role in the decomposition of mixed leaf litter in tropical rain forest, and significantly bigger effects from fauna were ascribed to the enhancement of N concentration and decrease of C concentration of the initially high C/N ratio litter in this forest site.     

A Comparative Study of the Use of Organic Carbon and Loss on Ignition in Defining Tropical Organic Soil Materials

Organic soils or Histosols or peats as they are commonly referred to, are characterized by the presence of large amounts of organic soil materials (OSM), which is commonly quantified by the Walkley and Black (1934) (WB) method to determine the soil organic matter (SOM) using a correction factor of 1.724. SOM of Histosols is also identified through a combustion (loss on ignition, LOI) or elemental C-analysis (with a carbon-nitrogen-sulfur (CNS) analyzer with combustion and gas density detector). These methods were established using temperate and boreal peat deposits and here we demonstrate that tropical peat deposits require a modified approach. Typical SE-Asian tropical lowland peat pedons from rain forest and oil palm settings were sampled and the material analysed using a CNS analyzer, WB-C and LOI. The ratios for LOI:CNS-C for the 20 samples yielded values between 2.00–3.09 with a mean of 2.50 while the LOI:WB-C ratio yielded values from 1.75 to 2.58 with a mean of 1.94. A comparison of these values for topsoils and subsoils showed mean ratios (LOI:WB-C) of 1.94 and 1.89 for topsoils and subsoils, respectively. The forest samples had higher LOI:WB-C ratios than the subsoils from oil palm settings (1.94 vs 1.84). These values suggest that the standard factor of 1.724 to correct OSM to SOM for tropical soils is untenable. The values to convert CNS and WB-C values of tropical topsoils/subsoils to SOM or LOI should be 2.5 or 1.9, respectively. Our results indicate a significant difference in the soil organic carbon (SOC) of tropical lowland peats depending on the method used.     

Recent development of national parks in Nicaragua

Nicaragua, a Central American country, has established two national parks recently and more reserves are in the planning stage. Saslaya National Park, 11,800 ha of virgin tropical rain forest and cloud forest, was established in 1971. Management of the park is as yet minimal, and few data have been collected. Masaya Volcano National Park, 4400 ha, contains an active volcano, a crater lagoon, and lava flows supporting successional stages to the dry deciduous forest. A Master Plan prepared for the area will soon be implemented. An inventory of other potential wildlands is being conducted, from which other reserves will be established. Obstacles to the management of a wildlands system include lack of a responsible government agency, principally, and also lack of trained manpower and funds.     

Nitrification activity in tropical rain forest soils of the Coastal Lowlands and Atherton Tablelands,Queensland, Australia

Intact soil cores from a montane tropical rain forest site in the Atherton Tablelands (Kauri Creek) and from a lowland tropical rain forest site in the Coastal Lowlands (Bellenden Ker), Queensland, Australia were investigated during different hygric seasons for the magnitude of gross nitrification rates using the Barometric Process Separation technique (BaPS). Pronounced seasonal variations of gross nitrification rates were found at both sites with highest values during the transition period between dry and wet season (montane site: 24.0 mg N (kg SDW)^—1 d^—1; lowland site: 13.1 mg N (kg SDW)^—1 d^—1) and significantly lower rates of gross nitrification during the dry and wet season. Rates of gross nitrification were always higher at the montane site than at the lowland site, but the opposite was found for N₂O emissions. The results indicated that the high losses of N₂O at the lowland tropical rain forest site may be contributed largely by high denitrification activity due to its wetter and warmer climate as compared to the dryer and colder climate at the montane tropical rain forest site. This conclusion was supported by analysis of cell numbers of microbes involved in N‐cycling. Higher numbers of denitrifiers were present at the lowland site, whereas higher numbers of nitrifiers were found at the montane site.     

禾谷镰刀菌拮抗菌株的筛选及鉴定

禾谷镰刀菌引起的小麦赤霉病能够导致小麦产量的大幅降低和品质的严重损失。为探索赤霉病生物防治方法,本试验从扬花期被禾谷镰刀菌侵染的麦穗上分离得到28株芽孢杆菌,利用平板对峙法对其进行筛选,其中20株菌对禾谷镰刀菌表现出明显的拮抗作用。根据生理生化特性和16S rDNA序列分析,这20株拮抗菌分别被鉴定为甲基营养型芽孢杆菌(11株)、解淀粉芽孢杆菌(2株)、枯草芽孢杆菌(2株)、暹罗芽孢杆菌(3株)和特基拉芽孢杆菌(2株)。这些菌株能够有效降低小麦赤霉病的发病率、发病程度和病情指数,其中菌株JS62N、JS15E和JS29I能够将赤霉病发病率降低80%以上;菌株JS29I、JS62N、JS39C和JS39D能够将赤霉病发病程度降低80%以上。16株菌能将病情指数降低80%以上;所有菌株均能极显著增加小麦的百粒重,JS12Q效果最好(+101.4%)。本研究结果为小麦赤霉病的防治提供了生物防治材料。     

Nitrogenase activity associated with leaf,root, and rhizosphere soils in an Indian tropical forest

Summary N₂ fixation (acetylene reduction assay) by phylloplane microorganisms was measured in dominant and co-dominant plant species growing in a tropical rain forest. No significant acetylene reduction was recorded with intact leaf samples. Azotobacter sp., Beijerinckia sp., Derxia sp., and Klebsiella pneumoniae were isolated as phylloplane N₂-fixing bacteria. Azospirillum lipoferum was only isolated from soil samples containing the roots of Poaceae. Nitrogenase activity was recorded in culture derived from the roots and rhizosphere soil samples, although low acetylene reduction activity indicates that these associations did not provide large amounts of N to the systems studied.