我国不同森林立地带Bt分离株杀虫晶体蛋白及基因分析

利用苏云金芽孢杆菌cry基因的PCR RFLP鉴定体系和SDS PAGE方法分析了来自我国不同森林立地带土壤中的 72株苏云金芽孢杆菌的cry1、cry2、cry3、cry4、cry5、cry8、cry9、cry10、cry11、cry1I等 10类基因类型和表达蛋白 ,并进行了杀虫活性的生物测定。研究表明 :同时含有cry1,cry2 ,cry1I 3类基因的有 2 1株菌 ,6株菌含有cry1,cry2类基因 ,4株菌含有cry1和cry1I类基因 ,只含有cry1基因的有 1株 ,cry2类基因的 4株 ,36株菌不含所鉴定的 10类cry基因。同时证明 ,绝大多数含有cry1基因的菌株表达了 130kDa蛋白 ,含有cry2基因的菌株表达了 6 0kDa蛋白。生物活性测定表明 ,共同含有cry1Aa、cry1Ac和cry2基因的菌株对棉铃虫幼虫具有较强的杀虫活性 ,只含有单一基因和不含上述基因的菌株杀虫活性较弱。     

对美国白蛾高毒Bt的筛选和杀虫蛋白基因鉴定

通过生物测定,从山东省不同地区分离和本实验室保存的122株苏云金芽孢杆菌Bacillus thuringiensis(简称Bt)中筛选到16株对美国白蛾Hyphantria cunea具有高毒力的菌株,用SDSPAGE和PCR-RFLP方法对16株Bt的杀虫蛋白和杀虫基因类型进行鉴定.结果表明,有11株Bt菌株中同时含有130 kDa和60或70 kDa的蛋白,1株同时舍有130和140 kDa的蛋白,3株只含有130 kDa的蛋白,1株含有30 kDa的蛋白;有14株Bt同时舍有cryl和cry2类杀虫蛋白基因,其中4株还包括cry9类基因,2株Bt中没有鉴定到已知基因类型,可能含有新的杀虫基因.16株Bt菌株均不舍cry3、cry4、cry8和cry10类基因.     

表达苏云金杆菌δ－内毒素蛋白基因大肠杆菌对四种林业害虫作用的生物测定

四株表达苏云金杆菌δ－内毒素蛋白基因的大肠杆菌对林业主要害虫杨扇舟蛾Ｃｌｏｓｔｅｒａａｎａｃｈｏｒｅｔａ（Ｆａｂｒｉｃｉｕｓ），马尾松毛虫Ｄｅｎｄｒｏｌｉｍｕｓｐｕｎｃｔａｔｕｓ（Ｗａｌｋｅｒ），舞毒蛾Ｌｙｍａｎｔｒｉａｄｉｓｐａｒ（Ｌｉｎｎａｅｕｓ），杨尺蠖Ａｐｏｃｈｅｉｍａｃｉｎｅｒａｒｉｕｓ（Ｅｒｓｃｈｏｆｆ）幼虫作用进行了室内生物测定，结果表明：该四株菌对上述四种昆虫幼虫均表现出较高的杀虫活性，而且能显著抑制昆虫幼虫的生长发育。     

我国四个自然保护区森林土壤中苏云金芽孢杆菌的分布

从我国四个不同立地带——长白山、百花山、鼎湖山、尖峰岭自然保护区,采集到林下0～5cm土层土壤样品75个,分离到苏云金芽孢杆菌39株。其中芽孢杆菌的总数量和苏云金芽孢杆菌的出土率和分离率有所差异,pH偏中性和含水量稍高的土壤,菌数较高;而与所测的各种土壤养分无明显相关。对39株苏云金芽孢杆菌的鉴定:17株属库尔斯塔亚种,6株属松蠋亚种,16株未定名。毒力测定表明:对杨扇舟蛾和马尾松毛虫幼虫的致死率在50％以上的菌株约占70％;对榆蓝叶甲和黄粉甲幼虫均无明显杀虫活性。     

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。     

表达苏云金杆菌δ—内毒素蛋白基因大肠杆菌对四种林业害虫作用的生…

四株表达苏云金杆菌δ－内毒素蛋白基因的大肠杆菌对林业主要害虫杨扇舟蛾Ｃｌｏｓｔｅｒａ ａｎａｃｈｏｒｅｔａ（Ｆａｂｒｉｃｉｕｓ），马尾松毛虫Ｄｅｎｄｒｏｌｉｍｕｓｐｕｎｃｔａｔｕｓ（Ｗａｌｋｅｒ），舞毒蛾Ｌｙｍａｎｔｒｉａｄｉｓｐａｒ（Ｌｉｎｎａｅｕｓ），杨尺蠖Ａｐｏｃｈｅｉｍａ ｃｉｎｅｒａｒｉｕｓ（Ｅｒｓｃｈｏｆｆ）幼虫作用进行了室内生物测定，结果表明：该四株对上述四种昆虫均表现较高的杀虫活性     

笋用竹正常植株和病害植株根际微生物的分析

以竹园采集土样分离出的根际微生物为主要研究对象,采用16s rDNA分子生物学手段和常规生理生化分析方法,对笋用竹正常生长植株及病害植株进行分析研究。有病竹植株的根际微生物可培养菌落数目比正常竹植株的多。特征分析表明:在分离纯化出的45株细菌中,其中的8株细菌初步鉴定为:菌株5-1-4为地衣芽孢杆菌Bacillus licheniformis,菌株5-1-10为苏云金芽孢杆菌Bacillus thuringiensis,4-1-17为枯草芽孢杆菌Bacillus subti-lis,菌株3-1-8为洋葱博克霍尔德菌Burkholderia cepacia,菌株2-1-9为粘金黄菌Chryseobacterium gleum,菌株4-1-14为短小芽孢杆菌Bacillus pumilus,菌株4-1-16为普通变形菌Proteus vulgaris,菌株4-1-6为产气肠杆菌Enterobacter aerogenes。     

苏云金芽孢杆菌杀虫晶体蛋白研究及应用概述

作为应用最广最有效的细菌杀虫剂,苏云金芽孢杆菌(Bacillus thuringiensis)在形成芽孢时可产生具有杀虫活性的杀虫晶体蛋白(ICPs)。迄今为止,ICP已有近800种,对鳞翅目、双翅目、同翅目、膜翅目、鞘翅目、蜱螨目等害虫都具有特异性的毒杀作用。该文概述了ICP的结构、功能及基因分类,以及其基因工程研究及应用,为Bt杀虫蛋白的推广应用及其定向改造提供参考。     

我国四个自然保护区森林土壤中苏云金芽孢杆菌的分布*

从我国四个不同立地带－长白山，百花山，鼎湖山，尖峰岭自然保护区，采集到林下０－５ｃｍ土层土壤样品７５个，分离到苏云金芽孢杆菌３９株。其中芽孢杆菌的总数量和苏云金芽杆菌的出土率和分离率所有差异，ｐＨ偏中性和含水量稍高的土壤，菌数较高；而与所测的各种土壤养分无明显相关。对３９株苏云金芽孢杆菌的鉴定：１７株属库尔斯塔亚种，６株属松Ｚｈｕ亚种，１６株未定名。毒力测定表明：对杨扇舟蛾和马尾松毛虫幼虫的致死率     

杨树烂皮病菌颉颃菌株的初步筛选与分子鉴定

对杨树烂皮病菌的平板对峙试验结果表明,6种颉颃真菌中木霉的抑菌率最高为51%,而颉颃细菌4个菌株的抑菌率均在60%左右,说明颉颃细菌的抑菌效果明显高于颉颃真菌;对B137等4株颉颃细菌的分子鉴定认为,1株为枯草芽孢杆菌(Bacillus subtilis),2株为解淀粉芽孢杆菌(Bacillus amyloliquefaciens),还有1株为苏云金杆菌(Bacillus thuringiensis,Bt);解淀粉芽孢杆菌和苏云金杆菌对杨树烂皮病菌有较好的颉颃效果。     

Experiences with tree fodders in temperate regions of Bhutan

Tree fodders are traditionally used in temperate and subtropical parts of Bhutan and provide approximately 20% of the fodder requirement of the country's livestock. Willow (Salix babylonica), Quercus semecarpifolia and Euonimus spp. were considered the most important species for temperate areas. Crude protein content of willow leaves decreased from 24.8.% in April to 11.3% in October. Bulls' voluntary intake was 77.7, 58.4, and 28.6 g DM/W^0.75 for willow, Popular robusta and Quercus semecarpifolia, respectively. Compared to temperate grasslands under the absence of phosphate inputs, tree fodders have a higher production potential and produce better quality fodder. Tree fodders may complement, but will not replace herbaceous fodder species in temperate areas. Assessment of yield potential in comparison to and in association with herbaceous temperate species is considered the most important research area.     

High level expression of glucose-6-phosphate dehydrogenase gene <Emphasis Type="Italic">PsG6PDH</Emphasis> from <Emphasis Type="Italic">Populus suaveolens</Emphasis> in <Emphasis Type="Italic">E. coli</Emphasis>

In order to investigate the functions of the gene PsG6PDH and the mechanisms underlying freezing tolerance of Populus suaveolens, the recombinant expression vector pET-G (pET30a-G6PDH), which contained full encoding region of PsG6PDH gene, was established. The recombinant was identified by lawn-PCR and double enzyme digestion and then transformed into expression host XA90 and induced by isopropyl-â-D-thiogalactoside (IPTG) to express 100 kD polypeptide of G6PDH fusion protein. The results showed that the expressed amount of the fusion protein culminated after 1 mmol&#8226;L^–1 IPTG treatment for 4 h and that pET-G product was predominately soluble and not extra-cellular secreting.     

绿竹BoGPIAP基因克隆与异位表达研究

糖基化磷脂酰肌醇锚定蛋白(GPIAP)因其结构和功能的多样性,决定了它在各种生物学过程中都发挥着重要作用。采用同源克隆的方法从绿竹(Bambusa oldhamii)中获得一个GPIAP同源基因,命名为BoGPIAP,cDNA全长1 772 bp,其中包括1 356 bp的开放阅读框,编码一个451 aa的的蛋白。蛋白结构分析表明,该蛋白包含1个典型的GPIAP家族保守区域(47-211)和1个CCVS结构域,在N-端和C-端分别具有1个跨膜信号肽和1个GPI锚定信号肽,属于GPIAP家族。构建BoGPIAP与GFP融合的表达载体,在洋葱表皮细胞中瞬时表达,结果显示BoCOBL::GFP融合蛋白定位于细胞膜上,证明BoGPIAP基因编码的蛋白为膜蛋白。分别构建BoGPIAP的正义、反义表达载体并转化烟草(Nicotiana tabacum)。PCR检测结果表明,BoGPIAP已转入烟草。与野生型相比,转反义基因植株细弱,纤维细胞壁明显变薄;而转正义基因植株粗壮,纤维细胞壁明显变厚。表明BoGPIAP可能对竹子纤维细胞壁的发育具有调控作用。     

中间锦鸡儿CiDR1的克隆及干旱胁迫下的表达分析

[目的]研究并了解中间锦鸡儿CiDR1基因功能及其对干旱胁迫的响应,为抗性育种提供候选基因。[方法]通过RACE技术从中间锦鸡儿中克隆CiDR1基因的c DNA全长,利用生物信息学分析软件对其基因结构及功能进行分析和预测。再通过qRT-PCR技术对干旱胁迫后的幼苗中的CiDR1表达模式进行研究。[结果]从中间锦鸡儿中克隆到CiDR1基因的c DNA全长共计4 297 bp,Gen Bank登录号为KP277100。生物信息学分析表明,预测的CiDR1蛋白序列中含有1 243个氨基酸残基,具有抗病基因特征结构域TIR、NB-ARC、LRR等,其等电点为6.35,不稳定指数为42.91,不具备信号肽,为非分泌蛋白,定位于细胞质中。定量PCR检测发现,CiDR1基因在幼年期的茎中表达量较低,在成年期的叶片中表达量较高;在干旱胁迫处理后的幼苗中,CiDR1表达水平有明显下降,表明该基因的表达受干旱抑制,可能与中间锦鸡儿适应干旱相关。[结论]中间锦鸡儿在干旱胁迫后其根、茎和叶中CiDR1的表达均明显下降,表明CiDR1的表达受干旱抑制,可能与中间锦鸡儿适应干旱相关,进一步研究发现CiDR1在根、茎、叶中的表达水平可能受发育阶段调控。     

Compression and overlap of unique vegetation system of subtropical mountain resembling tropical and temperate forests along elevation

The purpose of this study was to investigate how forests on subtropical mountains, which resembled tropical and temperate forests, were assembled, and to examine the compression and overlap of vegetations. We established 344 sample plots (400 m²) located at different altitudinal gradients ranging from 16 to 3,500 m above sea level (ASL) in Hsueshan Range northern Taiwan. Vegetation types were classified by TWINSPAN and the results of DCA were used to analyze the changes in vegetation types along elevation. Vegetation I was composed of the species of Euphorbiaceae, Moraceae, and Rubiaceae, which were taxa of tropical floristic elements, and the extent of this vegetation was limited below 1,500 m ASL. Vegetation II was defined into two subtypes, which were distributed from 1,000 to 2,500 m ASL, and were composed predominantly of Chamaecyparis and Tsuga, respectively. Our data also revealed that vegetation II contained co-dominants of species from families Lauraceae and Fagaceae. Moreover, evergreen broadleaved trees similar to tropical mountain forests were found to have narrower altitudinal ranges on subtropical mountains, whereas coniferous forests resembled temperate areas and were characterized by endemic conifer species. Vegetation III, from 2,500 to 3,500 m ASL, was characterized by endemic conifer species, Abies and Tsuga. Interestingly, deciduous forest was almost absent in this area and Fagus was the only tree type on a small mountain ridge. Data obtained from this study will help in raising conservation awareness for subtropical mountains since unique patterns of compression and overlap characteristics of tropical and temperate resembling forests were evident.     

中国不同地带性森林乔木叶片热值特征及其影响因素

[Objective]To study the leaf calorific value of different forest types.[Method]This study focused on four common forest types widely distributed across China, including boreal coniferous forest, warm temperate deciduous broad-leaved forest, subtropical evergreen broad-leaved forest, and tropical monsoon forest. The leaf calorific values of 175 dominant (or common) tree species were measured. The leaf calorific value characteristics of the trees found in the different forest types were analyzed, and the primary factors affecting leaf calorific value were investigated, including leaf morphological traits, nutrient elements, climate, and soil traits. [Result]The results showed that in these forest types, the leaf calorific values ranged from 14.84 to 21.98 KJ·g^-1, with an overall mean of 19.06 KJ·g^-1. The presence of organisms appeared to affect the leaf calorific value, which differed among forest types as follows: coniferous trees > broadleaf trees or evergreen trees > deciduous trees. [Conclusion]The latitudinal pattern of tree leaf calorific value, ordered from north to south, is as follows: warm temperate deciduous broad-leaved forest > subtropical evergreen broad-leaved forest > tropical mountain rainforest. The mean leaf calorific value of boreal coniferous forest was slightly lower than those of warm temperate deciduous broad-leaved forest and subtropical evergreen broad-leaved forest. The leaf calorific values were significantly correlated with leaf carbon content (R²= 0.89, P<0.001). A multiple regression equation was established to describe the relationships among leaf calorific value, leaf carbon content, leaf nitrogen content, and leaf thickness.     

五莲峰市级自然保护区种子植物区系研究

选择样线和样地调查法对五莲峰自然保护区进行系统的标本采集和考察,从科、属、种方面对保护区种子植物区系特征进行研究。结果表明,保护区植物种类丰富,共记录有维管束植物138科、435属、814种;保护区地处云南高原的北部,北邻四川盆地,东临华中植物区系,气候类型总体上或大部分处于亚热带的北缘,植物区系性质由亚热带逐渐过渡到温带;植物区系具有明显的温带性质,同时也表现出与云南热带植物区系的过渡性。另外,大量华中地区常见植物区系成分的出现,表明其在区系起源上与云南其他地区的不同,与华中植物区系间具有较为紧密的联系。     

巨桉EXP基因家族的生物信息学分析

[目的]本研究有助于了解EXP基因家族的基本特征,为深入研究其功能搭建平台。[方法]本研究对从巨桉(Eucalyptus grandis Hill)中筛选出35个EXP基因家族成员(Egr EXP1 Egr EXP35),利用生物信息学方法对其基因特征与表达模式进行综合分析。[结果]巨桉EXP基因分布在8条染色体之上,EXP蛋白均定位在细胞质膜上发挥作用,大多数的家族成员具有信号肽。巨桉EXP编码的蛋白质由α-螺旋、延伸链、无规卷曲、β-转角组成。进化分析结果表明,巨桉EXP蛋白与毛果杨(Populus trichocarpa) EXP蛋白的进化关系接近。35个巨桉EXP基因在巨桉未成熟木质部、成熟叶片、韧皮部、茎尖、木质部以及幼叶组织中表达模式存在显著差异。[结论]EXP基因家族各成员的表达模式不同,Egr EXP17、Egr EXP18可能在巨桉木材形成过程中起重要作用。     

Spread of white hypovirulent strains of Cryphonectria parasitica on grafted American chestnut trees exhibiting a high level of blight control

In 1982 and 1983, natural blight cankers, located in a zone extending from the ground to 183 cm on the main stem of grafted American chestnut trees, were inoculated with a mixture of dsRNA-containing, white (European) and pigmented hypovirulent strains of Cryphonectria parasitica (H-inoculated zone). In 1996, white isolates (34% of 156 C. parasitica isolates) were recovered from superficial cankers throughout the grafts and as far as 564 cm from the H-inoculated zone. Lloyd's index of patchiness value (1.36) indicated that white isolates were slightly aggregated in cankers on the grafts. Forty-five percent of 95 C. parasitica isolates, recovered 5–50 months after inoculating the grafts with pigmented virulent strain WK, were white and some converted strain WK to the white phenotype in vitro. All cankers and bark cores yielding only pigmented isolates, vegetatively compatible with virulent strain WK, had superficial necrosis 5 and 11 months after inoculation with strain WK. All white isolates of C. parasitica assayed contained a 12.7 kbp dsRNA in high concentrations, and were hypovirulent in pathogenicity trials. Twenty-eight vegetative compatibility groups were identified among 65 pigmented graft area isolates; none of 48 pigmented isolates contained dsRNA. In addition to host resistance factors, spread of white strains may be responsible for the unusually high level of blight control on the grafts.