土壤中多氯联苯的生物有效性及其影响机制研究

多氯联苯(PCBs)属于环境中广泛存在的持久性有机污染物(POPs),威胁生态系统安全与人体健康。为评价典型土壤中PCBs的生态风险,揭示其生物有效性以及影响生物有效性的分子机制将具有重要科学意义。本文以赤子爱胜蚓(Eisenia fetida)为土壤模式生物,模拟土壤中6种典型PCBs化合物(PCB-18、PCB-20、PCB-28、PCB-101、PCB-105、PCB-114)的富集动力学过程,并分析了蚯蚓吸收累积对土壤中PCBs总量的影响。结果表明,对于PCB-18、PCB-20和PCB-28,蚯蚓富集15天能够达到动力学过程的近平衡状态,而PCB-101、PCB-105和PCB-114则在30天达到近平衡状态,这可能是由于分子体积增大致使PCBs自由穿越生物膜并为生物体有机脂相富集的过程受到一定程度的限制,吸收速率变慢。PCBs生物有效性与典型分子性质(α、Kow)之间高度线性相关,从微观层面上揭示了影响生物有效性的分子机制,进一步表明PCBs分子体积、分子极化率及其疏水作用是影响生物有效性的主要因子,为典型土壤中PCBs污染物的生态风险预测与评价提供科学依据。     

高pH对植物生长发育的影响及其分子生物学研究进展

盐碱是影响作物产量的主要非生物胁迫之一。pH恒稳态是植物正常生长和发育的必要条件,也是植物细胞生命过程中的重要信号,在许多信号转导中起重要的调控作用。但植物对高pH逆境响应和信号转导的分子机理研究尚未得到充分重视。本文对盐碱地高pH因素对植物生长发育的影响及相关分子生物学研究进展进行了综述,并提出了今后植物在盐碱复杂胁迫条件下遗传机理的研究策略。     

盐碱胁迫对果树的危害及其分子机理研究进展

盐碱胁迫是影响果树生长发育最严重的限制因子之一.果树受盐碱胁迫后根系生长受阻、叶片变小、发育迟缓,造成植株矮化、早衰甚至死亡.从细胞膜透性、POD、SOD等生理特性方面分析了果树对盐碱复合胁迫的响应机制,多角度分析了果树抗盐碱的分子机理.     

烯效唑干拌种对小麦种子萌发过程中DNA合成的影响

以小麦品种绵阳 2 6为材料 ,利用3 H TdR掺入法 ,研究了烯效唑干拌种对小麦种子萌发过程中DNA合成的影响。结果表明 :烯效唑处理后 ,小麦种子萌发早期DNA合成加快 ,当浓度为 2 0mg kg时 ,DNA合成速率最快 ;同时 ,在小麦种子萌发早期烯效唑处理促进3 H 胸苷掺入可能反映了烯效唑有助于萌发中DNA修复和DNA复制 ,为种子萌发奠定了较好的基础。     

硅调节植物抗病性的机理:进展与展望

【目的】硅素营养增强作物对病虫害的防御能力已得到充分证实,但其作用机理至今仍然没有明确。本文对国内外有关硅素营养与作物病害发展的相互关系及相关机理的最新研究进展进行了归纳总结,为通过植物营养调节技术来提高作物病害防御能力的研究提供理论支撑。【内容】土壤有效硅包括土壤溶液中的单硅酸和易转化为单硅酸的盐类,土壤中有效硅含量一般在50～260 mg/kg。硅虽然不是植物生长发育的必需矿质营养元素,但是硅在减轻植物多种生物和非生物胁迫以及提高植物对病菌的抵抗能力等方面起着重要作用。施硅可以显著地抑制水稻稻瘟病、 纹枯病、 白叶枯病、 胡麻叶斑病,小麦、黄瓜、番茄等植物白粉病等多种病害的发生。关于硅调节植物抗病性的机理,首先提出了机械或物理屏障假设,认为施硅促进了细胞硅化作用的增强,细胞壁角质-硅双层以及表皮细胞乳突的增强,对病菌的入侵起到了物理防御作用。但随着研究的深入,发现物理屏障并非唯一机制,而后提出硅积极参与了生物化学防御过程,发现硅可以诱导感病植物产生酚醛类、黄酮类等抗毒素物质,以及施硅可以提高植物中几丁质酶、过氧化物酶、多酚氧化酶的活性、苯丙氨酸解氨酶等感病植物中病程相关蛋白酶的活性,从而通过化学防御过程提高植物对病害的抵抗能力。随着现代分子技术的发展,从基因组、转录组水平对其防御机制进行了阐明。研究认为硅通过主动的上调感病植物防卫基因及病程相关蛋白基因的表达,以应对病菌侵染。硅诱导植物产生乙烯、茉莉酸、活性氧等系列信号,使植物处于预激活化状态,从而减轻生物胁迫,但是硅在调节植物胁迫信号转导方面的机制还需要深入的研究。【结论】在缺硅土壤中施用硅肥,可以增强作物对病害的抵抗能力,从而大量降低杀菌剂的使用。关于硅调节植物抗病性机理,不能单一归因于某一方面,物理屏障防御机制与生物化学防御过程兼在。硅可能与关键的植物胁迫信号系统相互作用,而最终诱导产生对病原菌的抵抗, 但是这方面的确切机制还不是很清楚,是今后的研究重点。     

DNA分子标记技术及其在小麦育种及遗传研究中的应用

本文介绍了几种常用的DNA分子标记,如RFLP、RAPD、AFLP、SSR、STS、SNP等,并简要综述了分子标记技术在小麦遗传育种研究中的应用现状,包括基因标记与定位、遗传图谱构建、外源染色体鉴定与标记、种质资源鉴定和辅助育种等。     

水稻对UV-B辐射增强的生理响应及其分子机制研究

本文以水稻为研究对象,从细胞、个体和群体水平系统分析了不同水稻品种对UV-B辐射增强差异响应的遗传生理与防卫机制。试从农业生态系统角度,结合作者近期研究成果,系统分析了近年来国内外的研究重点及其成就。已有研究认为水稻对UV-B辐射增强的生理响应存在明显的种间差异,通常认为起源于靠近赤道附近的低纬度地区的籼稻品种比高纬度地区的粳稻品种更抗(耐)UV-B辐射污染,但许多研究结果不支持这一假说,即在籼粳稻品种中均存在明显对UV-B辐射增强呈不同抗性的种质资源。进一步研究结果表明水稻对UV-B辐射的响应差异是可遗传的数量性状。QTL定位分析结果发现多数抗UV-B辐射相关性状的加性QTL主要集中在第1、2、3、6染色体上,并检测到一些加性QTL还存在加性×加性上位性及其与环境的显著互作效应。作者还深入分析了水稻抗UV-B辐射增强的分子生理与调控机制,提出适当增加植物的硅营养,可以有效提高其抗逆性。最后,作者提出从农田生态系统角度研究和评价UV-B辐射增强所带来的生态风险及其影响是今后研究的重点,强调应重视研究田间条件下UV-B辐射增强及其与其他生态环境因子的互作对作物生长发育的综合影响,在此基础上,探索建立作物遗传改良与栽培调控的减灾防灾技术,为应对全球环境变化,制订相关防护策略提供理论依据和技术支撑。     

复合材料对砷污染土壤稳定效果及其影响因素的研究

添加Fe S、电石渣、菌渣复配材料稳定处理砷污染土壤,采用国标硫酸-硝酸法(SNP)和美国毒性浸出程序(TCLP)检测处理前后污染土壤砷的浸出浓度,依据浸出浓度评价稳定效果,并探讨土壤含水量、土壤pH、竞争性离子、反应时间、污染初始浓度对砷稳定效果的影响。结果表明:(1)SNP法和TCLP法浸提土壤,砷的浸出浓度为污染原土>对照处理>>稳定处理,稳定处理有效降低砷的浸出毒性。(2)砷的稳定效果影响因素研究表明,土壤含水率以30%为宜;土壤pH于2.20~9.85时砷的稳定效果良好,且pH=6.05时最佳,pH高于9.85至12.01时稳定效果减弱;竞争性离子对土壤砷稳定效果的抑制作用表现为PO_4~(3-)垌SO_4~(2-)≈NO_3~->Cl~-;土壤砷的稳定效率随反应时间先上升后平缓,于15 d后趋于稳定并持续至120 d;土壤中砷的污染初始浓度为506 mg kg~(-1)、833 mg kg~(-1)、2951 mg kg~(-1)和5290 mg kg~(-1),砷的稳定效率分别为92.36%、90.53%、55.57%和47.54%,稳定效率随着污染初始浓度升高而降低,稳定药剂适用于一定污染浓度范围。     

用~3H-TdR研究混合重金属对鲫鱼DNA合成的影响

应用3 H 胸腺嘧啶核苷 ( 3 H TdR)示踪法研究混合重金属对鲫鱼 (Carassiusaura tus)遗传毒性的影响。结果表明 ,最佳取样时间为注射3 H TdR后 6h。鱼体各组织对混合重金属的毒性反应呈现双向效应 ,即低浓度时 ,表现出损伤、修复作用 ;高浓度时 ,DNA合成受到抑制。鱼卵、鱼鳃对重金属最敏感 ,损伤浓度为 0 0 773mg L ;而大脑对混合重金属耐受性最强。DNA损伤修复作用和DNA合成作用都存在剂量效应 ,随着暴露时间的延长 ,毒性作用增大。鱼体各组织对重金属的毒性反应可由DNA损伤修复变成合成抑制。经过 72h暴露后 ,3 H TdR在肝脏、鱼鳃、鱼卵中掺入量约低于对照的 5 0 %。     

一种可用于PCR扩增的直接提取土壤细菌DNA的方法

本文以澳大利亚桉树林和松树林的土壤为例 ,采用Napp提取液和SDS直接溶解土壤细菌 ,并配合温浴 -玻璃珠震荡、苯酚 -氯仿萃取和异丙醇提取以及纯化DNA等步骤 ,直接从土壤样品中提取了土壤细菌DNA。所得DNA完全适用于酶解和PCR扩增的要求。该方法高效简单 ,费用低 ,在土壤微生物研究中具有重要的应用价值     

转基因水稻DNA样品浓度以及存放条件对PCR定性检测的影响

通过在不同模板DNA浓度(0.2～16 ng/μL)、不同转基因含量(0.05%～50%W/W)以及不同模板存放温度(22℃、4℃和-20℃)和存放时间(1、2、3周和1,3个月)条件下进行转基因水稻外源成分的定性PCR检测,发现当模板浓度在0.4 ng/μL以上时所有引物均能扩增出目标片段,对于转基因含量为1.0%和0.1%的混合样品,能稳定检测出转基因成分所需的DNA最低浓度分别为1和2～4 ng/μL.模板DNA的不同温度存放条件对检测没有明显影响,长时间存放后PCR扩增产物条带亮度有所减弱.     

一种适于PCR扩增的植物基因组快速提取新方法

在传统的碱裂解法提取基因组的基础上建立了一种新的基因组提取方法,这种方法对传统碱裂解制备基因组的方法进行优化,并且调整了中和剂的用量,引入异丙醇沉淀步骤对基因组进行纯化.实验证明这种方法扩大了碱裂解法制备基因组的适用范围,可直接从植物种子中快速提取出用于PCR(polymerasechain reaction)模板的基因组,对于600 bp以下的目的基因的检测效果显著,可以发现待测组分含量大于1%的样品.整个基因组提取时间控制在21 min,大大缩短了基因组提取时间,并且避免了酚/仿等对人体有害药品的使用.     

PCR Amplification of Wheat Sequences from DNA Extracted During Milling and Baking

DNA‐based analyses are highly sensitive and specific. Because processing steps can have profound effects on the proteins and DNA present in foods, this project examined the effects of breadmaking on wheat DNA size and polymerase chain reaction (PCR)‐based detection of sequences. DNA was extracted from wheat kernels, milling fractions, and flour, and from samples taken at various steps during and after the baking process. Kernels contained primarily high molecular weight DNA (>12,000 base pairs [bp]), whereas flour DNA exhibited a broad range of molecular weights from >12,000 bp to <300 bp. A marked reduction in DNA yield and size occurred after the first 5 min of baking. PCR successfully amplified products of both high and low copy number genes, even from DNA extracted from bread loaves five days after baking. However, successful amplification required that the maximum product size be no more than the average molecular weight of the DNA recovered from the source. The data also demonstrate that PCR can be used to detect the presence of yeast (Saccharomyces cerevisiae), a minor ingredient.     

生物质炭对盆栽黑麦草生长的影响及机理

通过盆栽试验,采用实时定量PCR和微孔板荧光法,分别研究了生物质炭添加对太湖地区农田土壤黑麦草生长、微生物群落丰度和酶活性的影响。结果表明:生物质炭添加量为4%(炭/土质量比)处理显著提高了土壤p H、有机碳、全氮、碳氮比、速效钾含量及黑麦草生物量;提高了土壤细菌、古菌和固氮菌nif H基因拷贝数,而对真菌无影响;提高了β-葡萄糖苷酶、纤维二糖水解酶、木糖苷酶、β-N-乙酰氨基葡萄糖苷酶和酸性磷酸酶的活性。微生物丰度(除真菌外)与多数土壤酶活性(除亮氨酸氨基肽酶)均成显著正相关。因此,生物质炭可增加土壤矿质养分,提高主要微生物类群和功能菌的丰度及土壤碳、氮和磷转化酶活性,这可能是施用生物质炭提升农田土壤养分转化功能和生产力的主要原因。     

Amplification facilitators and multiplex PCR: Tools to overcome PCR-inhibition in DNA-gut-content analysis of soil-living invertebrates

Polymerase chain reaction (PCR) can be used to detect prey within the gut contents of predators and allows specific trophic interactions to be studied among soil-dwelling invertebrates which cannot be examined by other approaches. PCR-inhibitory substances, however, are commonly found in DNA prepared from soil organisms or from biological material contaminated with soil. This can lead to false-negative results and the risk of not detecting trophic connections or of underestimating predation rates in field studies. In the present study, we developed mitochondrial DNA markers to detect Amphimallon solstitiale (Coleoptera: Scarabaeidae) in the gut contents of invertebrate predators. Larvae of A. solstitiale can cause serious damage in grasslands, field crops, and forests by feeding on roots. Adequate methodologies to study predation on these pests are lacking, and their invertebrate predator guild is, therefore, barely known. To test the new molecular markers for prey detection, larvae and eggs of A. solstitiale were fed to Poecilus versicolor larvae (Coleoptera: Carabidae), which are abundant below-ground predators in grassland ecosystems. Unfortunately, even when specific DNA extraction and purification methods were used, DNA extracts from predators were of poor quality and not amplifiable by PCR; this yielded false-negative results and a dramatically lower prey-detection rate. We overcame PCR-inhibition by applying ?1.28 μg μl⁻¹ bovine serum albumin to the PCR reaction mix. This enabled us to detect A. solstitiale DNA within fed carabid larvae up to 48 and 40 h post-feeding for 127 and 463 bp sized DNA fragments, respectively. When single A. solstitiale eggs were consumed by the carabid larvae, predation could be verified in 100% of the predators within the first 8 h of digestion; some carabid larvae even tested positive 32 h after feeding. Moreover, by multiplexing primers targeting both prey and predator, we were able to simultaneously screen for prey consumption and check for co-purified PCR inhibitors. Sensitivity in prey detection was not reduced compared to singleplex PCR. We recommend the multiplex approach because it considerably reduces time and costs compared to singleplex assays. We also show that multiplex PCR not only detects specific prey, but also can identify the predator itself. This allows the identification of taxa which are difficult or not identifiable based on morphological characters, such as soil-dwelling predatory beetle larvae.     

用复合PCR方法检测6种转基因玉米中外源DNA的特异性

利用本实验室研制的植物DNA提取试剂盒，从玉米（Zea mays）种子中提取符合PCR检测要求的DNA。根据不同转基因玉米中重组DNA的结构，分别设计引物，可对Btl1、Event176、T25、MON810、GA21和NK603等6种转基因玉米进行特异性PCR检测。在此基础上建立了针对6种转基因玉米的特异性DNA片段和玉米内参照基因（zein）的复合PCR检测方法，能同时对7对引物进行扩增，通过产物的长度差异对不同的转基因玉米进行辨别，实现了在同一个PCR反应管中同时对6种不同转基因玉米的特异性检测。     

环介导等温扩增技术快速诊断猪肺炎支原体

利用环介导等温扩增（loop-mediated isothermal amplification,LAMP）方法建立了猪肺炎支原体（Mycoplasma hyopneumoinae）的快速检测方法。该方法以猪肺炎支原体保守性基因16SrRNA为靶序列设计了6条特异引物,在63℃等温条件下,30min即可完成反应。结果显示,LAMP技术优于PCR技术。在敏感性实验中,LAMP方法的能检测出10个包含靶基因片段的重组质粒,敏感性高于PCR方法10倍;特异性实验中,LAMP方法和PCR方法均显示出了高度的特异性;在对127个临床鼻拭子样本的检测中,LAMP方法检测结果是所有样本都为阳性,而PCR检测出了116份阳性。证明本研究所建立的方法,对临床样本的检测的敏感度高于常规的PCR法。LAMP方法具有操作简便、快速、高效、敏感、特异、经济的特点,在研究机构以及基层实验室、现场监测的广泛使用具有一定的潜力。     

An improved method for purifying genomic DNA from forest leaf litters and soil suitable for PCR

Background, aim and scope  An improving knowledge of bacterial community within natural environments including forest soils and leaf litters requires extraction of nucleic acids directly from environmental samples since molecular approaches provide less biased access to a larger portion of uncultivable microorganisms. However, when DNA was extracted successfully from these samples, it might still have been difficult to apply it as a template for polymerase chain reaction (PCR) amplifications due to the effect of PCR inhibitors. Various compounds from plant tissues including polysaccharides, phenolic compounds and especially humic acids can inhibit PCR amplification. Some of these inhibitors could inhibit PCR amplification by chelating the Mg²⁺ (cofactor for Taq polymerase), or by binding to target DNA, and PCR amplification would consequently be interfered with. Therefore, eliminating the effects of these PCR inhibitors is one of the most important steps for PCR-based molecular techniques. Four different methods were assessed in this study to purify the genomic DNA extracted from F, L layer leaf litters and forest soil in an exotic pine plantation of southeast Queensland, Australia. Materials and methods  Three samples including two leaf litters and one forest soil were collected with a core (25 × 40 cm) from a 22-year-old slash pine plantation in southeast Queensland, Australia. The DNA fragments were extracted directly using the Ultra Clean™ Mega Prep Soil DNA kit (Mo Bio Labs, Solana Beach, CA). Then, four different purification methods were applied and compared to purify the DNA for PCR amplification, which include PVPP, Sephadex ^TM spin column, low-melting agarose gel and a new modified gel purification method. The purified DNA from these four purification methods was detected by agarose gel electrophoresis, and the purity and usefulness of DNA samples were ultimately determined by successful PCR amplifications. Results and discussion  The DNA was extracted from each sample using the Ultra Clean™ Mega Prep Soil DNA kit, and the DNA eluents were dark in colour and sometimes formed compact aggregates. Subsequently, PCR amplification from such samples failed, although a series of dilutions had been made from neat to 1:10³. The DNA purification step could not, therefore, be avoided. It was observed that both the colour of eluent and the DNA concentration decreased gradually after elution. Considering the difficulties of removing PCR inhibitors and the possibility of high DNA losses, 50–200 μl of sample DNA was used for purification. Four DNA purification methods (the PVPP spin column, Sephadex™ spin column, low-melting agarose gel and the modified gel purification method) were applied and compared on leaf litter and soil samples. The DNA purified by the modified gel purification method provided the best PCR products for 16S rRNA gene amplification, but the other methods, PVPP, Sephadex™ spin column and low-melting agarose gel, produced very weak or no products. Thus, in this study, DNA fragments which were purified by the modified gel purification method were amplified efficiently. This may be attributed to running the low-melting agrose gel for a longer time, which could remove substantial humic substances and also some other compounds from the samples and, thus, prevent them from being involved in PCR amplification. Conclusions  A new modified gel purification method which can improve DNA purification and PCR amplification of environmental DNA is first introduced in this study. Comparing PVPP, Sephadex ™ spin column, low-melting agarose gel and modified gel purification method for the effect of DNA purification, the modified gel purification method is more successful in removing the PCR amplification inhibitors and obtaining the highly purified PCR amplifiable high-molecular-weight DNA. The method described here is cheap, fast and easy to operate. It suggests in this study that the method containing less and easier following steps should be widely used to relieve the heavy working load of molecular-biological researchers. Recommendations and perspectives  This study introduces a new modified DNA purification method, and it is found that this modified gel purification method is effective in removing the PCR inhibitors and obtains highly purified DNA from leaf litters for PCR amplification. The modified gel purification method may have wider applications, although it was only assessed on leaf litter and soil samples. The effect of the modified gel purification method on the DNA purification would need to be further investigated on a variety of samples which suffered from PCR inhibitors, such as clinical samples, plant tissues and environmental samples.     

PCR扩增法检测江苏省5大淡水湖泊产毒微囊藻的空间分布

微囊藻毒素（Microcystin,MC）的产生受微囊藻毒素合成酶基因簇（microcystin biosynthesis gene,mcy）调控,常用PCR扩增mcy基因检测产毒微囊藻。采集江苏省5大淡水湖泊——太湖、滆湖、高宝-邵伯湖、洪泽湖和骆马湖的水样,测定水体营养盐浓度和叶绿素a浓度,根据叶绿素a浓度计算5个湖泊的富营养化指数（Trophic state index,TSI）,同时应用单一和多重PCR扩增mcy基因。结果表明,太湖和滆湖处于富营养和超富营养化水平,洪泽湖和骆马湖处于中营养和富营养化水平,高宝-邵伯湖处于寡营养水平。太湖、滆湖、洪泽湖和骆马湖的所有水样均检出mcy基因,4个湖泊水体受到微囊藻毒素的潜在威胁,高宝-邵伯湖没有检测出mcy基因的存在,尚未受微囊藻毒素的污染。