南方部分地区烟粉虱及其寄生蜂内共生菌Cardinium的检测及其系统发育关系

【目的】研究我国南方四个省份6个烟粉虱Bemisia tabaci种群和2个蚜小蜂种群内共生菌Cardinium的感染率及其系统发育关系。【方法】利用PCR技术与DNA测序技术检测来自广州、肇庆、南宁、昆明、厦门烟粉虱种群以及广州2个蚜小蜂种群内Cardinium的感染情况,通过基于mt COⅠDNA序列和16S r RNA序列建立烟粉虱和Cardinium系统进化树,分析烟粉虱、寄生蜂和共生菌Cardinium的协同进化关系。【结果】广州的Asia II1与Asia II7烟粉虱种群、肇庆与南宁的MEAM1烟粉虱种群、昆明与厦门的Mediterranean烟粉虱种群以及广州的丽蚜小蜂Encarsia formosa、双斑蚜小蜂Encarsia bimaculata种群体内都有Cardinium的感染,且以Mediterranean与土著的Asia II1、Asia II7感染率较高(65.2%~92.5%),寄生蜂体内的感染率较低(16.7%~18.6%)。通过进化树对比分析发现,Cardinium与烟粉虱寄主之间无协同进化现象,但是Cardinium在烟粉虱不同地理种群之间,烟粉虱与寄生蜂之间的同源性超过98%。【结论】Cardinium在两种寄主昆虫之间进行相互感染与传播的概率较大,寄生蜂可能是Cardinium在不同烟粉虱种群之间水平传播的桥梁之一。     

三种抗生素对B型和Q型烟粉虱内共生菌的去除效果比较研究

利用烟粉虱Bemisia tabaci(Gennadius)内共生菌特异性引物,研究了内共生菌在B、Q型烟粉虱种群中的分布和感染率,同时评价了3种不同的抗生素利福平、氨苄青霉素和硫酸卡那霉素分别在3种不同的浓度下(100.0、50.0及25.0μg/mL)对烟粉虱内共生菌的去除效果。结果表明:B、Q型烟粉虱原生内共生细菌Portiera的带菌率均为100.0%;B、Q型烟粉虱次生内共生菌Hamiltonella的带菌率分别为91.7%和100.0%;B型烟粉虱次生内共生菌Rickettsia的带菌率为87.5%,Q型为0;其它次生内共生菌在B、Q型烟粉虱中均未检测到。利福平、氨苄青霉素和硫酸卡那霉素在3种不同的浓度下均不能去除B、Q型烟粉虱Portiera;利福平、氨苄青霉素在3种不同的浓度下均能完全去除B型烟粉虱Rickettsia,硫酸卡那霉素在不同浓度下去除Rickettsia的效果不同;3种抗生素去除Hamiltonella的能力受抗生素种类以及浓度的影响。同一抗生素在不同浓度下去除Hamiltonella的效果均是100.0μg/mL>50.0μg/mL>25.0μg/mL;不同浓度的抗生素去除Hamiltonella的效果均是利福平>氨苄青霉素>硫酸卡那霉素,各浓度与各抗生素之间的去除Hamiltonella的效果均具有显著性差异。     

不同寄主植物上烟粉虱次生共生菌感染与性比相关性

【目的】烟粉虱Bemisia tabaci(Gennadius)体内次生共生菌感染受寄主植物的影响,一些共生菌会引起害虫的雌性化,明确田间不同寄主植物上害虫种群中共生菌与性比的相关性,可有利于进一步了解烟粉虱田间种群暴发机制。【方法】采集田间不同寄主植物上烟粉虱成虫,观察其性比,并对其中次生共生菌进行分子检测,分析共生菌携带率与性比相关性。【结果】江苏南京地区棉花、番茄、黄瓜和红薯4种寄主植物上烟粉虱次生共生菌Hamiltonella和Rickettsia感染均存在显著差异,其中Hamiltonella为优势共生菌,感染率依次为:棉花>黄瓜>番茄>红薯。寄主植物间Wolbachia和Cardinium的感染率均无显著差异。各寄主植物上烟粉虱雌性比均高于60%,其中黄瓜上高达75.6%,但不同寄主植物间无显著差异。进一步分析表明,Hamiltonella和Rickettsia感染率均与烟粉虱雌性比呈显著的二次多项式相关性。当Hamiltonella和Rickettsia感染率分别低于69%和5%时,随着感染率提高,烟粉虱雌性比上升,当感染率高于上述值时,则随着感染率增加,雌性比下降。【结论】棉花、番茄、黄瓜和红薯4种寄主植物上烟粉虱均表现出雌性化,但不同寄主植物间性比无差异,烟粉虱体内次生共生菌与性比存在相关性。     

褐飞虱体内Arsenophonus、Wolbachia、Spiroplasma和Cardinium四种次生共生菌感染分析

【目的】Arsenophonus、Wolbachia、Spiroplasma和Cardinium是广泛分布于昆虫体内的次生共生菌,能对寄主昆虫的生殖和发育生理产生影响,明确不同地理种群褐飞虱体内4种共生菌的感染和系统发育关系可为利用共生菌防治害虫提供技术依据。【方法】利用Arsenophonus特异性23S rRNA基因引物,Wolbachia、Spiroplasma和Cardinium各自的16S rRNA基因引物PCR检测广西10个地市、海南海口及上海、江苏南京、湖南长沙共14个地理种群褐飞虱雌、雄虫体内共生菌感染情况,并对扩增到的基因片段进行测序、构建系统发育树。【结果】不同地区2014年和2015年褐飞虱体内Arsenophonus感染率最高的分别为河池种群(30%)和长沙种群(20%)、PWolbachia感染率最高为河池种群(20%)和南京种群(30%),而Spiroplasma和Cardinium在褐飞虱各种群内均未检测到。序列比对和发育分析表明,褐飞虱体内Arsenophonus与同翅目昆虫白蜡虫Ericerus pela Chavannes体内感染的该菌关系最近;而感染Wolbachia菌的系统发育分析表明,除海南种群有一个个体感染Wolbachia菌A群外,其余种群感染的全部为B群;同时在所有感染两类共生菌的个体中并未发现共感染现象。【结论】不同地理种群褐飞虱感染Arsenophonus和Wolbachia两类共生菌的感染率并不一致;总体上褐飞虱雌虫Wolbachia感染率要高于雄虫,褐飞虱体内感染的两类共生菌与其它昆虫体内的共生菌序列高度一致或类似,且在所有检测的地理种群中均未发现有Spiroplasma和Cardinium感染。     

内共生菌Cardinium对烟粉虱MED隐种耐药性的影响

【目的】次生内共生菌感染可影响烟粉虱Bemisia tabaci对杀虫剂的敏感性。本研究旨在揭示内共生菌Cardinium感染对烟粉虱B. tabaci MED隐种耐药性的影响。【方法】室内测定海南陵水与山东寿光烟粉虱MED隐种种群内遗传背景一致的Cardinium感染与未感染品系对不同浓度新烟碱类杀虫剂噻虫嗪和吡虫啉的耐药性。【结果】各浓度噻虫嗪和吡虫啉处理后,烟粉虱陵水种群感染Cardinium品系死亡率比未感染品系显著降低;100, 150, 175和200 mg/L噻虫嗪以及175和200 mg/L吡虫啉处理后,烟粉虱寿光种群感染Cardinium品系死亡率比未感染品系显著上升。相对于未感染品系,烟粉虱陵水种群感染Cardinium品系对噻虫嗪、吡虫啉的抗性倍数(RR)分别是1.355和1.847,烟粉虱寿光种群感染Cardinium品系的RR分别是0.790和0.847。【结论】内共生菌Cardinium感染能影响烟粉虱MED隐种对噻虫嗪和吡虫啉的耐药性,且这种影响在不同种群间存在差异。研究结果对于揭示烟粉虱MED隐种的种群适应性及扩张机制具有重要参考价值。     

一种含特异性侵染内共生菌Rickettisa massilia的烟粉虱种群建立方法

【目的】烟粉虱Bemisia tabaci(Gennadaius)广泛分布于全球除南极洲外各大洲的90多个国家和地区,其寄主范围广,危害严重,属于农业上的重要害虫。烟粉虱体内的内共生菌有助于其扩散传播,提高其寄主适合度,但其体内的内共生菌大都为次生内共生菌,很难进行体外培养,因此,加大了其内共生菌研究的难度。【方法】采集我国部分地区的烟粉虱种群,然后利用基于mt DNA COⅠ基因的种特异性引物进行种群生物型鉴定,再用内共生菌的特异性引物来鉴定含有特异性侵染内共生菌种群,确定个体含内生菌类型后,采取继代饲养方法建立种群。【结果】经鉴定烟粉虱的生物型为Q型(MED隐种),经过5代种群筛选后,可以建立稳定遗传的含特异性侵染内共生菌Rickettisa massilia菌的烟粉虱种群。【结论】通过本方法可以获得稳定的含有特异性侵染内共生菌Rickettisa massilia菌的烟粉虱种群。该方法对研究烟粉虱与体内内共生菌、植物三者之间以及烟粉虱体内某几种内共生菌之间的互作有重要意义,同时也为明确烟粉虱体内Rickettisa菌的作用研究打下基础。     

一种含特异性侵染内共生菌Rickettisa massilia的烟粉虱种群建立方法

【目的】烟粉虱Bemisia tabaci(Gennadaius)广泛分布于全球除南极洲外各大洲的90多个国家和地区,其寄主范围广,危害严重,属于农业上的重要害虫。烟粉虱体内的内共生菌有助于其扩散传播,提高其寄主适合度,但其体内的内共生菌大都为次生内共生菌,很难进行体外培养,因此,加大了其内共生菌研究的难度。【方法】采集我国部分地区的烟粉虱种群,然后利用基于mt DNA COⅠ基因的种特异性引物进行种群生物型鉴定,再用内共生菌的特异性引物来鉴定含有特异性侵染内共生菌种群,确定个体含内生菌类型后,采取继代饲养方法建立种群。【结果】经鉴定烟粉虱的生物型为Q型(MED隐种),经过5代种群筛选后,可以建立稳定遗传的含特异性侵染内共生菌Rickettisa massilia菌的烟粉虱种群。【结论】通过本方法可以获得稳定的含有特异性侵染内共生菌Rickettisa massilia菌的烟粉虱种群。该方法对研究烟粉虱与体内内共生菌、植物三者之间以及烟粉虱体内某几种内共生菌之间的互作有重要意义,同时也为明确烟粉虱体内Rickettisa菌的作用研究打下基础。     

我国烟粉虱自然种群中存在广泛的Wolbachia感染现象

Wolbachia是广泛分布于节肢动物生殖组织中的细胞质遗传的一类共生菌,可以引起宿主生殖行为的改变.烟粉虱是重要的农业害虫,已有的研究表明烟粉虱中存在Wolbachia的感染,但所检测到的感染率不高,并且迄今在烟粉虱中所检出的Wolbachia均属于B组群.该研究从我国河北、新疆、北京、山东、浙江、广西、海南、广州和福建采集到18个烟粉虱地理种群,首先基于ITS1 rDNA克隆测序鉴定了烟粉虱的生物型,然后采用自行设计的Wolbachia 16S rDNA及wsp基因的专用引物对所采集到的烟粉虱种群进行了分子检测.结果表明几乎所有的烟粉虱自然种群中都存在Wolbachia感染,同时还发现B/Q生物型烟粉虱中主要感染A组群Wolbachia,而非B/Q生物型烟粉虱中存在大量的超感染现象.该研究显示烟粉虱自然种群中Wolbachia的感染率比预想的要高得多,而分子检测方法的灵敏度可能是影响Wolbachia感染率研究的关键因素之一.     

不同生物型烟粉虱体内Wolbachia共生菌的检测及其系统树分析

Wolbachia是广泛分布于节肢动物体内一类共生细菌,它能够通过多种机制调控宿主的生殖方式。近年来的研究表明,Wolbachia与许多外来生物的成功入侵相关。本文利用长PCR的方法特异扩增了不同生物型烟粉虱（共24个种群）体内Wolbachia的wsp基因,结果发现B型和Q型烟粉虱入侵种群体内均未检测到Wolbachia,而在非B/Q型的浙江种群和肯尼亚种群体内检测到了Wolbachia。对该wsp基因进行测序并和已知序列进行同源性分析发现,浙江烟粉虱种群的Wolbachia属于B组Con/Rug亚种群,而肯尼亚种群属于B组Btab1亚种群。Wolbachia的存在与否可能与烟粉虱的成功入侵有一定的关系。图2表2参23     

内共生菌Rickettsia对烟粉虱生物学特性的影响

【目的】阐明次生共生菌Rickettsia对烟粉虱生物学特性的影响。【方法】Rickettsia阳性(B~+)和阴性(B~–)的烟粉虱在Rickettsia阳性棉花(C~+)和阴性棉花(C~–)上取食15 d,调查不同处理组烟粉虱的单雌产卵量、发育历期、存活率、成虫寿命以及F_1代雌雄比。【结果】(1)Rickettsia与烟粉虱共生可显著缩短烟粉虱的发育历期,B~+C~+及B~+C~–两处理组烟粉虱卵-成虫的世代发育历期均短于B~–C~+及B~–C~–两处理组。(2)Rickettsia可以提高烟粉虱各龄期的存活率,B~+C~+、B~+C~–、B~–C~+、B~–C~–各处理组烟粉虱世代存活率依次呈下降趋势。(3)Rickettsia对烟粉虱种群的雌雄比也有重要的影响,B~–C~+和B~–C~–处理组中烟粉虱种群雌性比显著小于B~+C~+和B~+C~–烟粉虱处理组。(4)Rickettsia可以影响烟粉虱成虫的寿命及繁殖力,Rickettsia阳性烟粉虱处理组成虫寿命及平均单雌产卵量显著高于阴性处理组。【结论】Rickettsia与烟粉虱共生以及Rickettsia在棉花植株中的存留对烟粉虱的发育、存活以及成虫雌性比、寿命和繁殖力都有有利影响,且Rickettsia与烟粉虱共生时对烟粉虱的影响力度要明显强于Rickettsia存留于棉花植株中时对烟粉虱产生的影响。     

壳聚糖酶的克隆表达与壳寡糖的制备分析

目的:克隆壳聚糖酶基因于大肠杆菌中实现高表达,制备壳寡糖。方法:以枯草芽孢杆菌总DNA为模板扩增壳聚糖酶基因(CSN),克隆至载体pET23a(+)上,转化菌株BL21(DE3)。重组子经0.5 mmol/L IPTG诱导后,SDS-PAGE和质谱检测与鉴定重组酶。酶纯化后水解壳聚糖,薄层色谱分析其水解产物。结果:质谱证明壳聚糖酶(31.5kDa)成功表达,表达量占菌体总蛋白的45%左右。纯化后重组酶浓度为900 mg/L,纯度95%、回收率85%,酶活力为10 000 U/mg。壳聚糖降解产物为壳二糖至壳四糖。结论:原核表达载体pET23a(+)-CSN构建正确,壳聚糖酶表达量与活性高,适用于水解壳聚糖制备壳寡糖。     

国内外蝗害治理技术现状与展望

本文首先概述了国内外蝗虫发生与为害的态势,总结了现阶段我国蝗虫发生与为害的主要特点:即农田飞蝗暴发频繁而且严重,草原土蝗的发生时常造成严重的经济损失,而且侵入城市干扰市民生活,我国与周边国家之间蝗虫过境迁移频繁,使用化学农药污染环境和农产品;分析了国内外蝗虫防治对策与技术的发展现状,重点介绍了应急防治和可持续治理对策、...     

Synthesis and turnover of cerebrosides and phosphatidylserine of myelin and microsomal fractions of adult and developing rat brain.

The synthesis and turnover of cerebrosides and phospholipids was followed in microsomal and myelin fractions of developing and adult rat brains after an intracerebral injection of [U-14C]serine. The kinetics of incorporation of radioactivity into microsomal and myelin cerebrosides indicate the possibility of a precursor-product relationship between cerebrosides of these membranes. The specific radioactivity of myelin cerebrosides was corrected for the deposition of newly formed cerebrosides in myelin. Multiphasic curves were obtained for the decline in specific radioactivity of myelin and microsomal cerebrosides, suggesting different cerebroside pools in these membranes. The half-life of the fast turning-over pool of cerebrosides of myelin was 7 and 22 days for the developing and adult rat brain respectively. The half-life of the slowly turning-over pool of myelin cerebrosides was about 145 days for both groups of animals. The half-life of the rapidly turning-over microsomal cerebrosides was calculated to be 20 and 40 h for the developing and adult animals respectively. The half-life of the intermediate and slowly turning-over microsomal cerebrosides was 11 and 60 days respectively, for both groups of animals. The amount of incorporation of radioactivity into microsomal cerebrosides from L-serine was greatly decreased in the adult animals, and greater amounts of the precursor were directed towards the synthesis of phosphatidylserine. In the developing animals, considerable amounts of cerebrosides were synthesized from L-serine, besides phosphatidylserine. The time-course of incorporation indicated that a precursor-product relationship exists between microsomal and myelin phosphatidylserine. The half-life of microsomal phosphatidylserine was calculated to be about 8 h for the fast turning-over pool in both groups of animals.     

Identification and composition of the tonsillar and anal enterococcal and streptococcal flora of dogs and cats

Enterococcus faecalis was the most frequently isolated enterococcal species from anal swabs and tonsils of dogs and cats, although in the anal samples from dogs Ent. hirae was found almost as often as Ent. faecalis. Most Ent.faecium strains from dog tonsils differed from those associated with humans and other animals in that they fermented sorbitol. Typical Ent. avium as well as atypical Ent. avium -like strains were seen in dogs, while the related species Ent. raffinosus was associated with cat tonsils. Enterococcus cecorum also occurred mainly in cats. Certain atypical strains, presumptively identified as Ent. cecorum , shared characteristics with Ent. columbae.
The most frequent streptococcal species in tonsils of cats and dogs were Streptococcus suis and Strep. canis. Streptococcus canis and Strep. bovis predominated in anal swabs. The canine Strep. suis differed from the common porcine strains in fermenting mannitol.
Forty-seven of the 288 isolates examined could not be identified or related to known species. The characteristics of two groups of these bacteria, provisionally called 'Ton 31 group' and 'O7 group' are described.     

Molecular characterisation of species and genotypes of Cryptosporidium and Giardia and assessment of zoonotic transmission

The molecular characterisation of species and genotypes of Cryptosporidium and Giardia is essential for accurately identifying organisms and assessing zoonotic transmission. Results of recent molecular epidemiological studies strongly suggest that zoonotic transmission plays an important role in cryptosporidiosis epidemiology. In such cases the most prevalent zoonotic species is Cryptosporidium parvum. Genotyping and subtyping data suggest that zoonotic transmission is not as prevalent in the epidemiology of giardiasis. Molecular characterisation of Cryptosporidium and Giardia is a relatively recent application that is evolving as new genes are found that increase the accuracy of identification while discovering a greater diversity of species and yet unnamed taxa within these two important genera. As molecular data accumulate, our understanding of the role of zoonotic transmission in epidemiology and clinical manifestations is becoming clearer.     

白术同源四倍体的诱导和鉴定及其与二倍体过氧化物酶的比较

以白术（Atractylodes macrooephala Koidz.）二倍体组培苗为材料,对其四倍体诱导方法进行研究,共获得45个白术同源四倍体株系,为优良株系的选育提供了材料。此外,还分析比较了其中8个白术四倍体株系与二倍体的过氧化物酶同工酶（POD）的酶谱差异,发现四倍体各株系过氧化物酶同工酶谱比二倍体的均多了Rf0.310的谱带,且总过氧化物酶比活力也发生了很大改变,对探讨白术四倍体优良株系的生理生化机理具有一定的参考价值。     

Kinetics and thermodynamics of peroxidase- and laccase-catalyzed oxidation of N-substituted phenothiazines and phenoxazines

N -substituted phenothiazines (PTs) and phenoxazines (POs) catalyzed by fungal Coprinus cinereus peroxidase and Polyporus pinsitus laccase were investigated at pH 4–10. In the case of peroxidase, an apparent bimolecular rate constant (expressed as k _cat/K _m) varied from 1 ×10⁷M⁻¹s⁻¹to 2.6×10⁸M⁻¹s⁻¹ at pH 7.0. The constants for PO oxidation were higher in comparison to PT. pH dependence revealed two or three ionizable groups with pK _a values of 4.9–5.7 and 7.7–9.7 that significantly affected the activity of peroxidase. Single-turnover experiments showed that the limiting step of PT oxidation was reduction of compound II and second-order rate constants were obtained which were consistent with the constants at steady-state conditions. Laccase-catalyzed PT and PO oxidation rates were lower; apparent bimolecular rate constants varied from 1.8×10⁵M⁻¹s⁻¹ to 2.0×10⁷M⁻¹s⁻¹ at pH 5.3. PO constants were higher in comparison to PT, as was the case with peroxidase. The dependence of the apparent bimolecular constants of compound II or copper type 1 reduction, in the case of peroxidase or laccase, respectively, was analyzed in the framework of the Marcus outer-sphere electron-transfer theory. Peroxidase-catalyzed reactions with PT, as well as PO, fitted the same hyperbolic dependence with a maximal oxidation rate of 1.6×10⁸M⁻¹s⁻¹ and a reorganization energy of 0.30 eV. The respective parameters for laccase were 5.0×10⁷M⁻¹s⁻¹ and 0.29 eV. Received: 20 September 1999 / Accepted: 24 February 2000