苏云金芽孢杆菌4.0718菌株的杀虫晶体蛋白基因分析

根据苏云金杆菌(Bacillus thuringiensis)cry1、cry2和cry3型基因的保守区分别设计了3对通用引物Un1(d)/Un1?、Un2(d)/Un2?和Un3(d)/Un3?,以Bt4.0718菌株质粒DNA为模板进行PCR扩增,通过扩增产物片段的分子量大小来确定该菌株所含有的杀虫晶体蛋白基因类型。随后根据上述3类cry基因的高变区设计特异引物再次进行PCR鉴定。结果表明:Bt4.0718菌株含有cry1Aa、cry1Ab、cry1Ac、cry1Cb、cry2Ac和新基因cry4.5等6种基因类型。这一结果为利用该菌株构建高效广谱杀虫工程菌提供了客观依据。     

对粉纹夜蛾高毒力cry9Eα基因的克隆及表达

[目的]从本实验室分离的Bt4菌株中克隆cry9Eα基因,并研究其表达和杀虫活性.[方法]以PCR-RFLP方法鉴定Bt4菌株含有cry9基因,然后以菌株Bt4的质粒为模板,利用全长引物F9EA/R9EA进行PCR扩增全长基因.[结果]将目的片段插入到表达载体pET21b,得到大肠杆菌重组表达质粒pETcrygEa.转化E.coli BL21(DE3),诱导后表达130 kDa的蛋白,再将cry9Eα7基因连接到穿梭载体pSXY422b,电激转化HD73-(cry-),得到工程菌BioHD9Ea7,提取Cry9Ea7晶体蛋白,并进行生物活性测定.生物活性测定结果显示CrygEa7蛋白对粉纹夜蛾(Trichoplusia ni)初孵幼虫具有高毒力,LC_(50)为0.044 μg/mL,而对甜菜夜蛾(Spodoptera exigua)和棉铃虫(Helicoverpa armigera)初孵幼虫未显示活性.[结论]克隆和表达了一个对粉纹夜蛾高毒力的基因cry9Eα7,并成功构建了工程菌BioHD9Ea7.     

外源基因在苏云金杆菌染色体上的定点整合及表达

[目的]研究构建稳定表达外源基因、无抗性标记基因的苏云金杆菌(Bacillus thuringiensis简称Bt)工程菌的方法.在构建Bt工程菌时,高拷贝外源质粒的转入导致Bt芽孢数量减少,芽孢形成期延滞,影响Bt菌株的杀虫活力.而且,外源质粒在Bt中的稳定性较差,外源基因容易丢失.将基因整合人染色体是一种构建遗传性状稳定、杀虫活力高的Bt工程菌的有效方法.[方法]本研究采用PCR技术,分两段扩增定位于Bt无晶体突变株XBU001染色体上的trigger factor基因片段作为同源臂,克隆入温度敏感型载体pKSV7,构建了定点整合载体pKTF12.并利用pKTF12质粒将crylAc基因定点整合入XBU001染色体上.[结果]利用载体pKTF12将crylAc定点插入triggerfactor位点,对宿主菌XBU001的正常生长没有影响.重组菌株KCTF12中的crylAc基因能够稳定遗传、表达并形成菱形晶体.与携带高拷贝外源质粒的Bt菌株HTX42相比较,KCTF12具有芽孢数量增多、芽孢形成期提前的优势.[结论]定点整合法是一种构建稳定表达外源基因、无抗性标记基因Bt工程菌的有效方法.     

苏云金芽胞杆菌cry2Ad基因的克隆及其表达产物的活性分析

苏云金芽胞杆菌(Bacillus thuringiensis,Bt)SBT2是我国新分离出的一株野生菌株.扫描电镜显示该菌株产生双锥体形晶体.琼脂糖凝胶电泳发现其质粒图谱含有5个条带.聚丙烯酰胺凝胶电泳显示此菌株产生130 kD晶体蛋白.利用PCR-RFLP法进行杀虫基因类型鉴定,发现其含有cry1Aa、cry1Da、cry1Hb、cry1Jb、cry1Ka 、cry1Ib、基因.Cry2Ad蛋白的活性至今未见研究报道,本研究克隆和测序了该基因.并对其进行了表达.生物活性测定结果表明其表达产物对舞毒蛾(Lymantria dispar)、棉铃虫(Helicoverpa armigera)、亚洲玉米螟(Ostrinia furnacalis)、小菜蛾(Plutella xylostella)有低活性;对大猿叶甲(Colaphellus bowringi)无活性.     

苏云金芽胞杆菌Rpp39杀虫晶体蛋白基因的鉴定及cry2Aa12基因的克隆表达

[目的]本研究的目的是分析从四川生态条件下分离的苏云金芽胞杆菌Rpp39菌株的特性,从分子水平上揭示该菌株对鳞翅目高毒力的原因;进一步从中分离克隆cry2Aa基因,并对其进行初步的表达研究.[方法]本研究主要采用扫描电镜观察、PCR-RFLP鉴定法和SDS-PAGE分析法研究菌株的特性;采用PCR直接克隆法克隆cry2Aa全长基因,并亚克隆到原核表达载体pET-30a中,构建重组表达质粒pET-2Aa,再转入受体菌E.coli.BL21(DE3)中进行诱导表达;采用室内生物测定法测定表达产物对小菜蛾和水稻二化螟的毒力.[结果]经扫描电镜观察菌株Rpp39主要产生菱形、方形和圆形3种伴胞晶体;SDS-PAGE分析表明主要产生130 kDa和60 kDa左右2种蛋白;经PCR-RFLP鉴定,该菌株含有cry1Aa、cry1Ab、cry1Ac、cry1Ia和cry2Aa五类杀虫晶体蛋白基因;1种cry2Aa类杀虫晶体蛋白全长基因被克隆,序列分析显示该基因的开放阅读框(ORF)为1902 bps,编码由634个氨基酸组成的蛋白质,氨基酸序列与Cry2Aa1蛋白同源性为99.7%,被国际Bt杀虫晶体蛋白基因命名委员会命名为cry2Aa12.重组表达质粒pET-2Aa在E.coli BL21(DE3)中,经IPTG诱导能正常表达,SDS-PAGE电泳验证含有65 kDa表达蛋白.生物活性测定表明表达的包涵体蛋白对小菜蛾和二化螟具有杀虫活性,LC50分别为5.4 μg/mL和22.3μg/mL.[结论]菌株Rpp39及从中分离克隆的cry2Aa12基因来自四川生态条件,丰富了菌株及基因的资源,在资源积累方面具有重要意义.     

利用ERIC-PCR对苏云金芽胞杆菌与蜡状芽胞杆菌进行鉴定的研究

为了探索ERIC-PCR技术在苏云金芽胞杆菌和蜡状芽胞杆菌的鉴定及分型中的应用价值,本研究采用PCR方法初步检测苏云金芽胞杆菌杀虫晶体蛋白基因的组成,并对苏云金芽胞杆菌和蜡状芽胞杆菌的总DNA进行ERIC-PCR扩增,分析ERIC-PCR指纹图谱的特点并采用NTSYS2.10软件对其进行聚类。结果显示,各菌株的ERIC指纹图谱表现出不同程度的多态性,但图谱与菌株所含cry基因的类型存在一定的相关性。聚类分析结果显示,含有相同或相近cry基因类型的Bt菌株在进化树上趋向聚为一类,而不含cry基因的蜡状芽胞杆菌趋向于与不含cry基因的Bt菌株聚为一类或单独聚类。若在多种模式菌株的参考下,该方法可用于苏云金芽胞杆菌的初步鉴定和分型。     

特异性杀虫基因Bt cry3A在桑粒肩天牛幼虫两种肠道常驻内生菌中的转化和表达

[目的]将特异性杀虫毒蛋白基因Bt cry3A转入桑粒肩天牛(Apriona germari Hope,Ag)幼虫肠道常驻内生菌中,构建能在天牛幼虫肠道中定殖并表达特异性杀虫基因Bt cry3A的工程菌.[方法]以传统方法和16S rDNA分子生物学分析等方法分离、鉴定Ag幼虫肠道优势的常驻内生菌,从中筛选出适合转化的候选菌株.利用电转化技术将含有对鞘翅目昆虫具专一性毒力Bt cry3A基因的Escherichia coli-Bacillus thuringiensis穿梭表达质粒pHT305a和pHT7911分别转入Ag幼虫肠道常驻内生菌短短芽孢杆菌(Brevibacillus brevis Ag12,Ag12)和苏云金芽孢杆菌(Bacillus thuringiensis Ag13,Ag13)中.[结果]从Ag幼虫肠道共分离获得18个不同种的可培养细菌菌株,并从中选取菌株Ag12和Ag13作为出发菌株转入Bt cry3A基因.经质粒稳定性试验、转化子生长特性测试、伴胞晶体电镜检测、毒蛋白SDS-PAGE分析、工程菌定殖性分析以及生物毒力测试,结果显示cry3A基因已经成功转入Ag幼虫的常驻内生菌短短芽孢杆菌和苏云金芽孢杆菌中,并且工程菌Ag12-305a、Ag13-305a、Ag 12-7911和Ag13-7911都能在天牛幼虫肠道内稳定生长、繁殖并表达分子量约65kDa的伴孢晶体杀虫蛋白Cry3A.[结论]Bt cry3A基因已成功转入桑粒肩天牛幼虫肠道优势常驻内生菌中,获得了四株能在桑粒肩天牛幼虫肠道内定殖,并能表达目的杀虫基因Btcry3A的转基因工程菌.     

31株苏云金芽孢杆菌杀虫晶体蛋白基因型鉴定及表达产物研究

利用已建立的苏云金芽孢杆菌cry基因的PCRRFLP鉴定体系,鉴定了31株Bt菌株的cry基因类型,并进行了SDSPAGE分析和杀虫生物活性测定。研究表明：25株含cry1基因,表达蛋白130～150kD;其中16株含有对鞘翅目和鳞翅目害虫皆有活性的cry1I基因,其表达蛋白为81kD;15株同时含有cry1和cry2基因(13株表达蛋白约为60kD);10株含有未知待定基因;6株不含所鉴定的cry基因(其中2株有表达产物)。室内生物测定表明：cry1、cry2基因表达的菌株对鳞翅目害虫具有高杀虫活性,7株对舞毒蛾和膜翅目——杨叶蜂幼虫具有较高杀虫活性;含有cry1Aa\,cry1Ac\,cry2或cry1Ab\,cry1Ac\,cry2基因组合的菌株对棉铃虫幼虫均显示杀虫活性,其中6、12、30号菌株毒力最强。不含上述cry基因的菌株均无杀虫活性。以上结果证明,通过cry基因类型鉴定和表达产物的SDSPAGE分析可以预测菌株的杀虫活性。     

苏云金芽胞杆菌6618杀虫菌株的分离和鉴定

从我国不同地区采集118份土样,利用温度筛选法分离获得一株苏云金芽胞杆菌(Bacillus thuringiensis,简称Bt)6618,镜检观察发现该菌株能产生典型的菱形晶体,PCR分析表明其含有cry1类杀虫基因。采用SDS-PAGE和质谱分析发现该菌株主要产生130 k D原毒素,其组分由Cry1Ae和Cry1Ac原毒素组成。基因序列分析表明该菌株的cry1Ac基因为已知的cry1Ac1,而cry1Ae为新型杀虫基因。毒力生测表明该菌株对棉铃虫(Helicoverpa armigera)幼虫具有显著的杀虫效果。筛选获得的高毒力Bt菌株6618为丰富我国苏云金芽胞杆菌储备和研发新型高效生物杀虫剂提供了菌株资源。     

海南五指山热带雨林区Bt菌株的分离及其新型cry基因鉴定

五指山原始热带雨林区位于海南岛中部,是海南岛地区面积最大的热带原始雨林。我们依据不同的海拔高度对五指山原始雨林区进行了系统的取样,共采集了234份土壤样品,采用醋酸钠-高温处理的方法分离出各类产芽孢杆菌886株,并通过显微镜观察鉴定出产伴胞晶体蛋白的Bt菌株21株,其Bt菌株分离率为2.3%。为了进一步挖掘五指山丰富的Bt杀虫基因资源,还利用PCR方法结合测序技术对21株Bt分离株的cry基因进行了鉴定,鉴定结果显示cry1、cry4、cry39、cry40、cry31和cry46等基因型存在,表明五指山热带Bt菌株含有丰富cry基因资源,这将为为进一步开展Bt杀虫基因的分离克隆打下基础。     

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.     

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.     

Identification of specific binding sites for leukotriene C4 in human fetal lung

Specific leukotriene C₄ (LTC₄) binding sites were identified in membrane preparations from human fetal lung. Specific binding of [³H]-LTC₄ represented 95 percent of total binding, reached steadystate within 10 minutes and was rapidly reversible upon addition of excess unlabeled LTC₄. Binding assays were performed at 4°C under conditions which prevented metabolism of [³H]-LTC₄ (80 mM serineborate, 10 mM cysteine, 10 mM glycine). Under these conditions, greater than 95 percent of the membrane bound radioactivity, as analyzed by high performance liquid chromatography, co-eluted with the LTC₄ standard. Computer-assisted analyses of saturation binding data showed a single class of binding sites with a dissociation constant (K_d) of 26 + 6 nM and a density (B_max) of 84 ± 18 pmol/mg protein. Pharmacological specificity was demonstrated by competition studies in which specific binding of [³H]-LTC₄ was displaced by LTC₄ and its structural analogs with inhibition constants (K_j) of 10 to 30 nM, whereas LTD₄, diastereoisomers of LTD₁, LTE₄ and the end organ antagonist FPL 55712 were 150 to 700 fold less potent competitors than LTC₄. These results provide evidence for specific, reversible, saturable, high affinity binding sites for [³H]-LTC₄ in human fetal lung membranes.     

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.     

多马胺能药物对鲇鱼促性腺激素（GtH）分泌活动的影响

以珠江流域鲇鱼（silurus asotus)为实验材料,研究了多巴胺（DA）能药物（DA及其D－2型受体拮抗物 ,DOM）对鲇鱼促性腺激素（GtH)释放的影响,结果表明,在性腺发育的各个时期,单独注射DOM（5ug/g)均不能显著提高鲇鱼血液基础GtH水平,当DOM与LHRH－A联合注射时能显著增强LHRH－A刺激GtH释放的作用;DA只能抑制GnRH诱导的GtH释放,对基础GtH释放无抑制作用,这种生殖内分泌调节方式与鲇形目的革胡子鲇（Clarias gariepinus)和大鳍Hu（Mystus macropterus)相似,而与鲤形目的鲁科（Cyrpindiae)鱼类不同。     

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.     

Interactions of an antitumor platinum compound with deoxyribonucleic acid, histones, L-amino acids, poly-L-amino acids, nucleosides and nucleotides

Interaction of cis-dichloro(dipyridine)platinum(II) (cis-PPC) with calf thymus DNA, calf thymus histone, l-amino acids, poly-l-amino acids, nucleosides, and nucleotides has been evaluated by equilibrium dialysis technics. At least a 28 % decrease in the association of cis-PPC with DNA occurs when the platinum compound is pre-incubated with l-amino acids. The greatest decrease in association is seen upon pre-incubation of the platinum compound with the free amino acids. Glut, Asp, Lys, Arg, and CySH, before the addition of a sack containing a solution of DNA. The low level of association between DNA and the amino acids tends to rule out competition between cis-PPC and amino acids for DNA association sites. cis-PPC was repelled from sacks containing positively charged poly-l-Lys, poly-l-Arg, and calf thymus histone; however, in the presence of poly-l-Glut and poly-l-Asp, cis-PPC associated with these negatively charged polymers to a considerable degree. Enhanced chloride dissociation from cis-PPC was observed in the presence of all of the amino acids and the nucleotides GMP, CMP, UMP, and TMP, but not in the presence of AMP or the nucleosides rG and dG. In the presence of calf thymus histone, the association of cis-PPC with calf thymus DNA was reduced by more than 50% at histone/DNA ratios of 0.8–1.0.These data suggest that cis-PPC or cis-Pt(II) may associate with electron-rich areas of not only nucleic acids and proteins but also with body pools of free nucleotides and amino acids. The presence of positively charged histones shielding DNA strands in vivo suggests that the most probable point of platinum-DNA association would be at de-repressed areas of DNA which are undergoing RNA synthesis. The aquated form of the platinum complex may also associate with acidic proteins which appear to be involved in the positive control of RNA synthesis and, as a result, this interaction may be of pharmacological significance.     

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.     

Biofortification of crops with seven mineral elements often lacking in human diets – iron, zinc, copper, calcium, magnesium, selenium and iodine

The diets of over two-thirds of the world's population lack one or more essential mineral elements. This can be remedied through dietary diversification, mineral supplementation, food fortification, or increasing the concentrations and/or bioavailability of mineral elements in produce (biofortification). This article reviews aspects of soil science, plant physiology and genetics underpinning crop biofortification strategies, as well as agronomic and genetic approaches currently taken to biofortify food crops with the mineral elements most commonly lacking in human diets: iron (Fe), zinc (Zn), copper (Cu), calcium (Ca), magnesium (Mg), iodine (I) and selenium (Se). Two complementary approaches have been successfully adopted to increase the concentrations of bioavailable mineral elements in food crops. First, agronomic approaches optimizing the application of mineral fertilizers and/or improving the solubilization and mobilization of mineral elements in the soil have been implemented. Secondly, crops have been developed with: increased abilities to acquire mineral elements and accumulate them in edible tissues; increased concentrations of 'promoter' substances, such as ascorbate, β-carotene and cysteine-rich polypeptides which stimulate the absorption of essential mineral elements by the gut; and reduced concentrations of 'antinutrients', such as oxalate, polyphenolics or phytate, which interfere with their absorption. These approaches are addressing mineral malnutrition in humans globally.     

Invited Review Free Radicals in Foods

During the last decade increasing attention has been given to the role of free radicals in biological oxidations. The subject has been of increasing interest to both the food scientist and the physiologist. Free radical scavengers in the form of both indigenous and added antioxidants are necessary for the successful preservation of food; free radicals are increasingly being implicated in the onset of, among others, ischaemic heart disease and for protection against these diseases it is suggested that the dietary intake of the antioxidant vitamins should be increased especially for diets high in polyunsaturated fats. ^1,2 Convenience and snack foods which absorb substantial amounts of frying oils are being increasingly consumed. Since poly-unsaturated fatty acids are particularly susceptible to oxidation by free radicals during the storage, cooking and frying of foods, the potential risk of exposure to lipid degradation products' is likely to have increased. In foods the non-enzymic and lipoxygenase catalysed oxidation of polyunsaturated fatty acids, β-carotene and vitamin A can result in the loss of essential nutrients and the development of off-flavours.