本期重点推介

<正>家蚕核型多角体病毒(Bombyx mori nucleopolyhedrovirus,BmN PV)是家蚕Bombyx mori最重要的病原之一,对蚕业生产危害极大。为了评估热激对家蚕的抗病毒能力的影响并探索其抗病毒免疫反应分子机制,云南农业大学植物保护学院唐芬芬和陈斌及云南省农业科学院蚕桑蜜蜂研究所白兴荣等观察测定了家蚕5龄幼虫经42℃热激15 min后感染BmNPV的存活率,同时利用qRT-PCR方法检测了热激后家     

家蚕抗核型多角体病毒病育种研究进展

家蚕核型多角体病毒(Bombyx mori nuclear polyhydrosis virus,BmNPV)病是蚕业界上最常见、危害比较严重的病害;遗传学研究和抗性分析表明,家蚕对核型多角体病毒抗性由主效基因和微效基因协同控制。在蚕病防治方面,除了加强消毒以外,选育抗病毒新品种无疑是更加经济有效的办法。近年来,随着基因组学和新一代测序技术的迅猛发展,覆盖深度达8.5倍家蚕基因组图谱完成和40个家蚕品种和野生种重测序的完成,为家蚕的抗核型多角体病毒病育种提供了理论依据和基因资源。本文综述了BmNPV基因组研究、家蚕基因组研究以及家蚕抗性品种培育方面取得的进展。     

云南家蚕核型多角体病毒分离株的毒力测定及系统进化分析

【目的】由家蚕核型多角体病毒(Bombyx mori nucleopolyhedrovirus, BmNPV)侵染家蚕Bombyx mori导致的核型多角体病(血液型脓病)在云南蚕区普遍发生,危害严重,给云南蚕业带来严重的经济损失。本研究旨在测定云南不同蚕区BmNPV分离株的毒力及了解BmNPV在云南的病害流行趋势,为监测和控制云南家蚕血液型脓病打下基础。【方法】通过BmNPV对家蚕5龄幼虫致死率和对家蚕BmN细胞毒力的测定评价了云南蚕区19个BmNPV分离株的致病力;对19个云南BmNPV分离株的bro-d基因进行克隆、测序及系统进化分析。【结果】BmNPV对家蚕5龄幼虫的致死率测定表明,云南不同地区BmNPV毒株对家蚕的口服感染力差异较大;细胞毒力测定结果显示不同BmNPV分离株的出芽型病毒粒子(budded virus, BV)滴度差异较大。进化分析表明,所获取来自云南的19个BmNPV分离株主要分为两个亚群,亚群I在云南省的东部、中部和西部地区均有分布,而亚群II主要集中分布于云南省北部地区。从地理分布图可以看出,亚群I所在地区多为亚热带,温度偏高;而亚群II所处的云南北部地区多为典型高原季风气候,温度相对偏低。【结论】云南蚕区存在丰富的BmNPV株系,各分离株对家蚕的毒力差异较大;云南BmNPV分离株的进化关系在一定程度上与地理气候密切相关。     

家蚕核型多角体病毒的PCR检测及其部分序列分析

为研究应用PCR技术进行家蚕核型多角体病毒广东株的敏感性检验以及探讨不同地理株系的基因水平的相互关系,本文通过对家蚕核型多角体病毒BmNPV广东株的人工繁殖与纯化,引用了一对根据多角体蛋白基因设计的引物phy35/phy36,对BmNPV的基因组模板DNA进行了PCR扩增,并对其产物进行测序分析.结果显示,PCR技术均可扩增检测出3×108个/mL至3×102个/mL不同浓度的BmNPV模板DNA,特异目标片段大小约为680 bp,且扩增带的亮度随着病毒液浓度的降低而减弱,说明应用引物phy35/phy36进行PCR方法可以有效地应用于检测BmNPV病毒感染的家蚕.同时,测序获得了BmNPV广东株多角体蛋白polyhedrin基因674 bp大小的片段,GC含量为46.4%.经过BLAST比对分析,与BmNPV泰国株的相似性为99%,暗示家蚕BmNPV广东株与泰国株的BmNPV (登录号AY779044)亲缘关系非常相近,两者可能属于BmNPV的不同地理株系.通过系统发育树的进一步分析发现,家蚕核型多角体病毒广东株polyhedrin基因部分序列与家蚕NPV分离株S9多角体蛋白基因(DQ231336)关系很近.     

家蚕ADP/ATP转运酶(BmANT)抑制BmNPV增殖作用机制研究

【目的】家蚕核型多角体病毒(Bombyx mori nucleopolyhedrovirus,BmNPV)隶属于杆状病毒,需要借助宿主细胞能量代谢进行自身增殖复制。家蚕ADP/ATP转运酶(Bombyx mori ADP/ATP translocase,BmANT)是线粒体转运蛋白,在BmNPV感染条件下和家蚕热休克蛋白60(heatshockprotein60,BmHSP60)具有直接的相互作用。因此,鉴定Bmant基因在BmNPV感染过程中的功能特征,有助于解析杆状病毒劫持宿主细胞因子促进自身增殖复制机制,完善杆状病毒和宿主相互作用网络。【方法】通过结构域预测BmANT蛋白的结构特征,荧光定量PCR分析Bmant基因在BmNPV感染后的变化特征;并过表达BmANT检测其对病毒DNA复制和病毒蛋白表达变化影响;进一步在转录水平分析Bmant和Bmhsp60基因的调控关系;最后通过流式细胞术等技术鉴定Bmant和Bmhsp60基因共同调控BmNPV增殖复制的机制。【结果】SMART软件预测显示BmANT包含3个线粒体载体结构域,BmNPV感染24 h后Bmant基因持续下调表达。过表达Bmant基因能够显著抑制BmNPV DNA的复制和VP39蛋白表达。荧光定量PCR分析显示Bmant和Bmhsp60基因具有相互拮抗作用,能够相互抑制转录。Bmant和Bmhsp60共同过表达分析显示,BmANT和BmHSP60共同作用BmNPV能够抑制病毒的增殖复制。【结论】结果表明,BmANT是一个线粒体载体蛋白,具有显著的抗病毒作用,能够下调Bmhsp60基因表达,并抑制BmNPV增殖复制。     

家蚕核型多角体病毒可视化快速核酸检测方法

家蚕核型多角体病在广西多批次的养蚕生产中非常普遍,严重影响蚕农的经济效益。根据家蚕核型多角体病毒(Bm NPV)广西分离株的多角体蛋白基因(polh)序列设计4条用于环介导核酸等温扩增(LAMP)特异性引物,以感染Bm NPV的5龄家蚕血淋巴的多角体DNA为模板,通过优化LAMP反应体系,确定Mg2+、d NTPs、甜菜碱最适终浓度分别为8 mmol/L、1.4 mmol/L和0.8 mol/L,外引物和内引物浓度为0.2μmol/L和1.6μmol/L,在64℃恒温条件下反应1 h;在反应管中加钙黄绿素,通过肉眼观察颜色变化直接判定结果实现可视化检测。该检测方法的Bm NPV最低模板DNA为25 fg/μL,其敏感度为常规PCR l00倍,兼具简便、快速、灵敏等优点,可用于家蚕核型多角体病的早期诊断及流行病学调查。     

基于细胞转基因平台创制抗BmNPV的细胞素材

家蚕核型多角体病毒(Bombyx mori nucleopolyhedrovirus,BmNPV)是影响蚕业生产最为严重的病原之一,每年对我国蚕业生产方面造成很大的经济损失。通过RNA干扰技术(RNAi)干扰BmNPV的增殖关键基因可以有效地抑制病毒的增殖复制。本研究以BmNPV侵染关键基因gp64和lef-1为靶标基因,并利用病毒诱导型启动子LEF3P和39KP共构建了4种RNAi干扰表达载体,分别命名为:piggyBacA3-EGFP-39KP-gp64、piggyBacA3-EGFP-39KP-lef-1、piggyBacA3-EGFP-LEF3P-gp64、piggyBacA3-EGFP-LEF3P-lef-1。经瞬时转染和筛选稳定表达的家蚕细胞系抗病毒检测结果表明,通过RNAi技术能够有效的抑制病毒增殖复制,并确定了39KP启动效果优于LEF3P启动子,干扰gp64基因的抗病毒效果优于lef-1基因。这些研究结果为后期转基因品系培育和家蚕抗病毒研究提供了基础。     

用构建的新型BmNPV载体在家蚕高效表达人β-干扰素

家蚕核多角体病毒(Bombyx mori Nuclear Polyhedrosis Virus.BmNPV)和家蚕细胞已成功地用来大量生产具有生物活性的重组蛋白。但是BmNPV的通用载体的类型较少。因此,本实验构建了BmNPV新型载体pBm92,该载体将多角体蛋白基因的起始密码ATG改变为ATT,然后在多角体蛋白基因的 12位外连接有5个外源基因的克隆位点。将HuIFN-β基因克隆在多角体蛋白基因的 12位后,构建了pBmIFN 12;同时构建HuIFN-β克隆在-3位后的转移栽体pBmIFN-3。将两种转移载体DNA分别与BmNPV基因组DNA共转染Bm-N细胞。利用重组病毒不产生多角体蛋白的特征,筛选重组病毒。用HuIFN-β基因探针与重组病毒DNA进行杂交鉴定。重组病毒BmIFN 12感染Bm-N细胞,其上清IFN活性最高时可达2.0×10~6IU/ml,将BmIFN 12注射5龄家蚕虫体,表达水平为50×10~7IU/ml,是HuIFN-β基因克隆在多角体蛋白基因的-3位后获得的重组病毒的表达量的2～4倍。家蚕体生产的rHulFN-β为糖基化蛋白具有天然HuIFN-β的抗原性。     

拯救细胞系恢复重组家蚕核型多角体病毒包涵体实现外源蛋白在家蚕中产业化生产

构建家蚕Bombyx mori肌动蛋白（BmA3）启动子驱动的家蚕核型多角体病毒（BmNPV）多角体基因（ph）和OpNPV极早期启动子（IE1）驱动的zeocin抗性筛选基因转座供体载体,与鳞翅目辅助转座质粒pie2piggyBac共转染家蚕卵巢细胞BmN,经200μg/ml zeocin抗生素筛选一个月,成功获得持续表达BmNPV多角体蛋白的稳定细胞系BmN-A3ph。多角体缺陷型重组病毒BmBac-GF P感染拯救细胞系BmN- A3ph, 细胞成功装配出病毒包涵体颗粒,其包装效率约为野生型病毒感染正常BmN细胞的8%。用拯救型包涵体病毒颗粒喂食家蚕幼虫进行复感染,结果表明稳定细胞系所包装的包涵体病毒与野生型病毒一样能够通过口服途径感染宿主,却并不在宿主体内形成包涵体,从而保证外源基因高效表达。拯救型包涵体病毒可望解决传统注射感染效率较低问题,通过喂食感染可促进杆状病毒介导的家蚕生物反应器产业化进程。     

异源多角体蛋白对家蚕核型多角体病毒粒子的包装

利用PCR方法从AcMNPV基因组DNA中分离出多角体蛋白基因 ,将该扩增片段克隆到转移载体pBacPAK8中 ,得到重组转移载体pOAc。将该质粒DNA与线性化的Bm BacPAK6病毒基因组DNA共传染BmN细胞 ,得到了能形成多角体且不产生蓝色空斑的重组病毒hp BmNPV。纯化该重组病毒的多角体颗粒 ,并对多角体蛋白、病毒核酸及多角体病毒颗粒进行分析 ,发现AcMNPV的多角体蛋白能在家蚕细胞中大量表达且能在细胞内识别家蚕核型多角体病毒并组装成多角体颗粒 ;病毒基因组DNA因部分交换 ,其酶切行为发生了相应的变化 ;电镜观察发现经AcMNPV多角体蛋白包装的家蚕核型多角体病毒的多角体颗粒大小为1 2 μm～ 2 9μm ,明显小于野生型家蚕核型多角体病毒的多角体颗粒     

Tuning of electronic properties of one-dimensional cyano-bridged Cu-Ni, Cu-Pd, and Cu-Pt bimetallic assemblies by stereochemistry of ligands

Preparations, XPS and electronic spectroscopy, and magnetism of seven new one-dimensional cyano-bridged coordination polymers, chiral [Cu(RR-chxn)₂][Pd(CN)₄] · 2H₂O (1), [Cu(trans-chxn)₂][M(CN)₄] · 2H₂O (2, 4, and 6 for M = Pd, Ni, and Pt), and [Cu(cis-chxn)₂][M(CN)₄] · 2H₂O (3, 5, and 7 for M = Pd, Ni, and Pt) (RR-chxn = cyclohexane-(1R,2R)-diamine, trans-chxn = racemic trans-cyclohexane-(1,2)-diamine, and cis-chxn = racemic cis-cyclohexane-(1,2)-diamine) have been reported in view of tuning of their electronic properties by stereochemistry of chxn ligands and metal-substitution. Comparison of Cu 2p_1/2 and 2p_3/2 peaks of XPS and broad d-d bands around 18 000 cm⁻¹ of electronic spectra are described systematically for 1-7. Variable-temperature magnetic measurement shows that complexes 1-7 indicate weak antiferromagnetic interactions via cyano-bridges. Because of semi-coordination coupled with pseudo Jahn-Teller elongation and electrostatic interaction for 1, the axial Cu-N coordination bond distances of 2.330(7) and 3.092(8) Å are considerably longer than those of equatorial ones in the range from 2.016(6) to 2.030(6) Å. The former bond distances of 1 are intermediate values among the related Ni (2.324(6) and 3.120(8) Å) and Pt (2.34(1) and 3.09(1) Å) complexes.     

Land,livestock, and livelihoods: Changing dynamics of gender,caste, and ethnicity in a Nepalese Village

Over the past 10 years, Ghusel VDC, Lalitpur District has moved from primarily subsistence agriculture into the wider cash economy aided by the Small Farmers' Development Program (SFDP), which provides credit to farmers mainly for the purchase of buffalo for milk production, and by the National Dairy Corporation, which supports local dairy cooperatives. Analysis reveals that buffalo-keeping and milk sales are increasing the well-being of many households, while at the same time creating new inequalities in gender roles and responsibilities, greater inequities between Brahmin and Tamang residents in Ghusel, and placing pressures on the ecosystem for increased supplies of fodder and fuelwood. Evidence suggests that there is critical, need for attention to the social, and particularly gender-based, implications of maintaining livestock for milk sales and to the ecological underpinnings of this livelihood system.     

Structure and function of S-adenosylhomocysteine hydrolase

In mammals, S-adenosylhomocysteine hydrolase (AdoHcyase) is the only known enzyme to catalyze the breakdown of S-adenosylhomocysteine (AdoHcy) to homocysteine and adenosine. AdoHcy is the product of all adenosylmethionine (AdoMet)-dependent biological transmethylations. These reactions have a wide range of products, and are common in all facets of biometabolism. As a product inhibitor, elevated levels of AdoHcy suppress AdoMet-dependent transmethylations. Thus, AdoHcyase is a regulator of biological transmethylation in general. The three-dimensional structure of AdoHcyase complexed with reduced nicotinamide adenine dinucleotide phosphate (NADH) and the inhibitor (1′R, 2′S, 3′R)-9-(2′,3′-dihyroxycyclopenten-1-yl)adenine (DHCeA) was solved by a combination of the crystallographic direct methods program, SnB, to determine the selenium atom substructure and by treating the multiwavelength anomalous diffraction data as a special case of multiple isomorphous replacement. The enzyme architecture resembles that observed for NAD-dependent dehydrogenases, with the catalytic domain and the cofactor binding domain each containing a modified Rossmann fold. The two domains form a deep active site cleft containing the cofactor and bound inhibitor molecule. A comparison of the inhibitor complex of the human enzyme and the structure of the rat enzyme, solved without inhibitor, suggests that a 17° rigid body movement of the catalytic domain occurs upon inhibitor/substrate binding.     

Chiral Separation of G‐type Chemical Warfare Nerve Agents via Analytical Supercritical Fluid Chromatography

Chemical warfare nerve agents (CWNAs) are extremely toxic organophosphorus compounds that contain a chiral phosphorus center. Undirected synthesis of G‐type CWNAs produces stereoisomers of tabun, sarin, soman, and cyclosarin (GA, GB, GD, and GF, respectively). Analytical‐scale methods were developed using a supercritical fluid chromatography (SFC) system in tandem with a mass spectrometer for the separation, quantitation, and isolation of individual stereoisomers of GA, GB, GD, and GF. Screening various chiral stationary phases (CSPs) for the capacity to provide full baseline separation of the CWNAs revealed that a Regis WhelkO1 (SS) column was capable of separating the enantiomers of GA, GB, and GF, with elution of the P(+) enantiomer preceding elution of the corresponding P(–) enantiomer; two WhelkO1 (SS) columns had to be connected in series to achieve complete baseline resolution. The four diastereomers of GD were also resolved using two tandem WhelkO1 (SS) columns, with complete baseline separation of the two P(+) epimers. A single WhelkO1 (RR) column with inverse stereochemistry resulted in baseline separation of the GD P(–) epimers. The analytical methods described can be scaled to allow isolation of individual stereoisomers to assist in screening and development of countermeasures to organophosphorus nerve agents. Chirality 26:817–824, 2014. © 2014 The Authors. Chirality published by John Wiley Periodicals, Inc.     

Influence of Fluoride on Rat Kidney Antioxidant System: Effects of Methionine and Vitamin E

The aim of the study has been to determine and compare the influence upon the kidney antioxidative system, exercised by administration of vitamin E, and vitamin E in combination with methionine, under conditions of oxidative stress induced by sodium fluoride. The experiment was carried out on Wistar FL rats (adult males) that, for 35 days, were administered water, NaF, NaF with vitamin E, or vitamin E with methionine (doses: 10 mg NaF/kg of body mass/24 h, 3 mg vitamin E per 10 μl per rat for 24 h, 2 mg methionine per rat for 24 h). The influence of administered sodium fluoride and antioxidants upon the antioxidative system in kidney was examined by analyzing the concentration of malondialdehyde (MDA) and the activity of the most important antioxidative enzymes (SOD, total and both its isoenzymes, GPX, GST, GR, and CAT). The studies carried out confirmed the disadvantageous effect of the administered dose of NaF upon the antixodiative system in rats (increase in the concentration MDA, decrease activity of all antioxidative enzymes). The administration of vitamin E increased the activity of studied enzymes with the exception of glutathione reductase GR; it also reduced the procesess of lipid peroxidation. It has been found that combined doses of vitamin E and methionine were most effective in inhibiting lipid peroxidation processes. The results confirmed the antioxidative properties of methionine.     

Reorientation of Microfibrils and Microtubules at the Outer Epidermal Wall of Maize Coleoptiles During Auxin-Mediated Growth

Auxin-mediated elongation growth of isolated subapical coleoptile segments of maize (Zea mays L.) is controlled by the extensibility of the outer cell wall of the outer epidermis (Kutschera et al., 1987). Here we investigate the hypothesis that auxin controls the extensibility of this wall by changing the orientation of newly deposited microfibrils through a corresponding change in the orientation of cortical microtubules. On the basis of electron micrographs it is shown that cessation of growth after removal of the endogenous source of auxin is correlated with a relative increase of longitudinally orientated microfibrils and microtubules at the inner wall surface. Conversely, reinduction of growth by exogenous auxin is correlated with a relative increase of transversely orientated microfibrils and microtubules at the inner wall surface. These changes can be detected 30–60 min after the removal and addition of auxin, respectively. The functional significance of directional changes of newly desposited wall microfibrils for the control of elongation growth is discussed.     

Gamma-glutamylcysteine protects ergothioneine-deficient Mycobacterium tuberculosis mutants against oxidative and nitrosative stress

Mycobacterium tuberculosis (M.tb.), the causative agent of tuberculosis (TB), cannot synthesize GSH, but synthesizes two major low molecular weight thiols namely mycothiol (MSH) and ergothioneine (ERG). Gamma-glutamylcysteine (GGC), an intermediate in GSH synthesis, has been implicated in the protection of lactic acid bacteria from oxidative stress in the absence of GSH. In mycobacteria, GGC is an intermediate in ERG biosynthesis, and its formation is catalysed by EgtA (GshA). GGC is subsequently used by EgtB in the formation of hercynine-sulphoxide-GGC. In this study, M.tb. mutants harbouring unmarked, in-frame deletions in each of the fives genes involved in ERG biosynthesis (egtA, egtB, egtC, egtD and egtE) or a marked deletion of the mshA gene (required for MSH biosynthesis) were generated. Liquid chromatography tandem mass spectrometry analyses (LC-MS) revealed that the production of GGC was elevated in the MSH-deficient and the ERG-deficient mutants. The ERG-deficient ΔegtB mutant which accumulated GGC was more resistant to oxidative and nitrosative stress than the ERG-deficient, GGC-deficient ΔegtA mutant. This implicates GGC in the detoxification of reactive oxygen and nitrogen species in M.tb.     

In vitro studies of the rabbit immune system. IV. Differential mitogen responses of isolated T and B cells.

The mitogenic responses of separated rabbit lymphocyte populations functionally analogous to mouse T and B cells have been tested in vitro. Purified T cells were prepared by passage over nylon wool (NW) and purified B cells prepared by treatment with antithymocyte serum and complement (ATS + C). ATS + C kills 70% of peripheral blood lymphocytes (PBL's) and 50% of the spleen cells while passage over NW yields 40% of the applied PBL's and 5–23% of the applied spleen cells. NW-purified T cells from the spleen or PBL's respond fully to concanavalin A (Con A) but have a reduced response to phytohemaglutinin (PHA) and little or no response to goat anti-rabbit immunoglobulin (anti-Ig). PBL's that survive ATS + C (B cells) are stimulated by anti-Ig but not by Con A or PHA. B cells purified from spleen do not respond to Con A or PHA but will respond to anti-Ig under appropriate conditions. A full spleen B-cell response to anti-Ig required removal of Ig produced by the cultures that blocked anti-Ig stimulation. It is concluded that, for rabbit lymphocytes, Con A and PHA are primarily T-cell mitogens and that anti-Ig is primarily a B-cell mitogen. However, the mitogen response of unfractionated PBL or spleen cell populations indicates an overlap in reactivity. This could be due to cells sharing T and B properties, alteration of cell populations by the fractionation procedures used, or recruitment of one population in the presence of a mitogenic response of the other population.     

八种脑-肠肽侧脑室内注射对大鼠基础胃酸分泌的影响

用乌拉坦麻醉大鼠作急性实验,采用连续灌流胃并收集流出液的方法,观察向侧脑室内注射微量脑-肠肽对大鼠基础胃酸分泌的影响。实验结果如下:(1)雨蛙肽、八肽胆囊收缩素、促甲状腺素释放激素及四肽胃泌素均使总酸排出量增加;(2)生长抑素、胰多肽、P 物质、胰高血糖素则使总酸排出量减少;(3)上述肽类用侧脑室注射的剂量作肌肉注射,除四肽胃泌素也产生明显的刺激胃酸分泌作用外,对胃酸分泌均无明显影响。以上结果提示,脑内的一些肽类可能以神经递质或调制物的方式,参与中枢对胃酸分泌的调节。     

A chromium-tolerant plant growing in Cr-contaminated land

In this paper, a kind of Typhaceae plant species, Typha angustifolia L, with a high tolerance to Cr is described. Experiments were carried out to examine its ability to tolerant Cr and its physiological response. The results showed that there was no difference in growth, plant height, and biomass response to external Cr (VI) between the plants exposed to 100 microM Cr (VI) and control (0 microM), while increasing Cr levels to 200-800 microM induced a significant decrease in plant height and biomass, but no significant injury was detected, even for the plants exposed to 800 microM Cr. Chromium induced significant increases in superoxide dismutase (SOD) and peroxidase (POD) activities. Meanwhile, a significantly positive correlation was found between Cr and Mn or Cu in leaves and roots, respectively. The Cr tolerance of the plant appeared to be associated with the enhancement of SOD and POD activities and the improvement in uptake and translocation of the essential microelements.