不同荧光定量PCR分析方法检测大鼠脑缺血再灌注损伤中Caspase-3基因的表达研究

目的:应用实时荧光PCR检测大鼠脑缺血/再灌注损伤组织中的Caspase-3基因表达变化,并比较了相对定量与绝对定量分析方法的技术优劣.方法:相对定量实验中,以β-actin基因作为内参,采用Delta-delta Ct法计算目的基因表达变化.而在绝对定量实验中,构建了含Caspase-3基因的重组质粒,以该重组质粒作...     

草鱼MYH mRNA表达量分析中采用的内参基因稳定性比较

本研究分别以β-actin、18S rRNA和GAPDH为内参基因,采用实时荧光定量PCR对草鱼早期发育时期肌球蛋白重链(myosin heavy light,MYH)基因的mRNA表达量进行分析,并比较不同内参基因对MYH基因mRNA表达水平检测结果的准确性.研究结果表明,以β-actin和GAPDH作为内参,MYH基因mRNA表达水平完全一致,其表达量从原肠到仔鱼阶段逐次递增,仔鱼与原肠期阶段相比表达量差异显著;当采用18S rRNA作为内参时,MYH基因mRNA在发育阶段的表达量呈不稳定状态.因此,β-actin和GAPDH均可作为内参基因,用于草鱼早期发育中MYH基因mRNA的相对定量研究:而18S rRNA作为内参时,可能会对检测结果造成偏差.本研究不仅准确的揭示了草鱼MYH基因mRNA的表达特征,并且为荧光定量PCR技术在鱼类基因表达研究方面提供了有价值的参考.     

实时荧光定量PCR中内参基因的选择

实时荧光定量PCR技术是分析基因表达谱的一种常用方法,在分析中选择合适的内参基因对数据进行校正是得到可信数据的关键。以Lactobacillus helveticus H9为研究对象,应用实时荧光定量PCR技术,评价了5种常用内参基因ldh、recA、rpoB、gapdh和16S rRNA的表达稳定性,通过geNorm和NormFinder程序进行数据分析,结果表明5个候选内参基因在菌株不同的发酵时间点表达相对都较为稳定,结合两种分析得到其中最为稳定的基因是ldh,适合于用作后续实时荧光定量PCR试验中的内参基因。     

植物实时荧光定量PCR内参基因的特点及选择

实时荧光定量PCR(qRT-PCR)具有灵敏度高、特异性强、重复的动态定量范围和高通量等优点,是进行植物基因表达和转录分析最常用的技术手段之一.选择合适的内参基因是正确运用实时荧光定量PCR分析目标基因表达变化的前提.近年来,大量研究表明,内参基因的选择应取决于研究者的实验条件;随着实验条件的变化,内参基因的选择也随之变化.因此,实时荧光定量PCR结果分析的准确性在很大程度上依赖于所选择的内参基因是否适合.该文从内参基因的选择、常用内参基因的特点、新内参基因的挖掘、应用内参基因组合的优点和内参基因的稳定性评价等几方面进行综述,以期为研究者在实验中选择合适的内参基因提供参考和理论依据.     

相对定量和绝对定量-以CsSAMDC基因表达分析为例

以无芒隐子草干旱诱导的cDNA文库中获得的SAMDC序列为基础,应用实时荧光定量RT-PCR技术同时结合相对定量和绝对定量方法,以分析无芒隐子草SAMDC基因在不同组织、不同胁迫梯度下的表达为案例,详细介绍并比较相对定量和绝对定量在基因表达方面的应用.分别采集无芒隐子草幼苗自然干旱0,2,4,6,8,10d以及恢复浇水1,4d的叶片和根系材料,相对定量采用2-ΔΔCT.法;以相应基因的质粒扩增产物为标准品制作标准曲线,对定量PCR的引物浓度进行了优化,而后筛选最稳定的内参基因,以内参基因获得的标准化因子对绝对定量数据进行标准化.相对定量2-ΔΔCT法结果显示CsSAMDC基因在干旱胁迫8d后,叶片中的表达量为胁迫前的0.62±0.13倍,根中的表达量为胁迫前的5.93±0.71倍.绝对定量表明,CsSAMDC基因只在胁迫第8d和第10d的根组织中检测到表达量显著增大,胁迫第4～6d和恢复浇水后1～4d,CsSAMDC基因在叶和根组织中的表达较对照降低.相对定量和绝对定量在DsSAMDC基因表达研究中显示相对一致的趋势.     

猪组织中miR-103实时定量PCR分析时合适内参的确定

定量实时PCR是miRNA表达检测的主要方法之一,而利用合适内参对定量实时PCR数据进行校正处理是确保该方法分析准确性的关键.为了确定猪正常组织中miR-103定量实时PCR检测分析的合适内参,首先采用geNorm算法和扩增效率试验对miR-196、U6 snRNA和总RNA 3个候选内参进行了评价;然后以miR-103的绝对定量结果为对照,比较分析了3个候选内参的校正准确性．结果表明,miR-196的表达稳定性和扩增效率均优于U6 snRNA和总RNA;以miR-196为内参的miR-103相对定量结果同绝对定量结果具有较高的一致性,两者均显示miR-103在猪大脑中高丰度表达,在胃、小脑、小肠、心脏、肝脏和胰脏中适度表达,在肺、脾脏和腿肌中轻度表达.这些结果说明,miR-196可作为猪正常组织中miR-103定量实时PCR相对定量分析的一个合适内参.     

藜科植物藜和灰绿藜实时荧光定量PCR内参基因的选择

选择合适的内参基因是实时荧光定量PCR(qRT-PCR)研究的关键,目前对藜科耐盐(盐生)植物胁迫相关基因的表达分析中所用内参基因的报道较为有限.该研究利用GeNorm、NormFinder和BestKeeper 3个内参基因分析软件,对已选择过的β-TUBULIN、β-ACTIN、GAPDH 3个常用候选内参基因进行了比较分析,筛选出在NaCl和PEG胁迫下藜和灰绿藜中表达相对稳定的内参基因.结果表明:GeNorm、NormFinder内参软件分析在NaCl和PEG胁迫下,GAPDH是藜和灰绿藜中均共同稳定表达的内参基因,同时在藜和灰绿藜中也有各自表达较稳定的内参基因,β-ACTIN在藜中稳定表达,β-TUBULIN则在灰绿藜中稳定表达.对相同科不同种的植物内参基因表达差异进行比较,内参基因在相同科中具有相同稳定表达的内参;对相同胁迫下两种不同植物内参基因表达稳定性进行分析,内参基因的选择需根据实际的实验材料和实验条件而定.基于3个分析软件对以上3个常用内参基因的分析结果,初步确定了在藜科植物藜和灰绿藜中相对稳定的内参基因,为藜和灰绿藜胁迫相关基因的定量表达分析提供了参考依据.     

成纤维细胞生长因子21的研究进展

以无芒隐子草干旱诱导的cDNA文库中获得的SAMDC序列为基础,应用实时荧光定量RT-PCR技术同时结合相对定量和绝对定量方法,以分析无芒隐子草SAMDC基因在不同组织、不同胁迫梯度下的表达为案例,详细介绍并比较相对定量和绝对定量在基因表达方面的应用。分别采集无芒隐子草幼苗自然干旱0、2、4、6、8、10d以及恢复浇水1、4d的叶片和根系材料,相对定量采用2 -△△CT法;以相应基因的质粒扩增产物为标准品制作标准曲线,对定量PCR的引物浓度进行了优化,而后筛选最稳定的内参基因,以内参基因获得的标准化因子对绝对定量数据进行标准化。相对定量2 -△△CT法结果显示,CsSAMDC基因在干旱胁迫8d后,叶片中的表达量为胁迫前的0.62±0.13倍,根中的表达量为胁迫前的5.93±0.71倍。绝对定量表明,CsSAMDC基因只在胁迫第8d和第10d的根组织中检测到表达量显著增大,胁迫第4～6d和恢复浇水后1～4d,CsSAMDC基因在叶和根组织中的表达较对照降低。相对定量和绝对定量在CsSAMDC基因表达研究中显示相对一致的趋势。     

实时荧光定量PCR分析蒙古韭低温诱导叶片中内参基因的选择

实时荧光定量PCR (real-time fluorescence quantitative PCR,qRT-PCR)是广泛应用于基因表达分析的实验技术。在基因表达分析过程中,选择稳定表达的内参基因对实验结果的准确性非常重要。以低温诱导24 h和72 h蒙古韭(Allium mongolicum)的叶片为材料,无处理0 h叶片为对照,使用荧光定量PCR法分析了Am5S-rRNA、AmActin、AmGAPDH和AmEF1-α4个看家基因的表达情况。通过ge Norm和NormFinder程序分析,发现AmGAPDH稳定性最好,Am5S-r RNA和AmActin次之,AmEF1-α稳定性最差,因此选择AmGAPDH作为蒙古韭基因表达分析的内参基因。本研究通过qRT-PCR方法分析稳定表达的内参基因,对后续蒙古韭低温诱导基因表达分析有重要的意义。     

柑橘黄龙病菌内参基因的筛选与评估

【背景】柑橘黄龙病是世界柑橘生产上最具毁灭性的病害之一,主要由候选韧皮部杆菌属亚洲种("Candidatus Liberibacter asiaticus",CLas)引起。CLas全基因组测序已经完成,因而该病原菌的基因表达研究和功能验证得以进行。【目的】筛选CLas内参基因并评估其不同侵染时期和在不同品种植物寄主中的表达稳定性。【方法】基于基因功能类别,利用实时荧光定量PCR技术分析23个CLas的候选内参基因相对表达情况(16Sr RNA基因作为参照基因)。结合Ct值标准差和geNorm、NormFinder、RefFinder软件,评价内参基因的表达稳定性。【结果】在感染黄龙病不同时期和不同品种的植物寄主样本中,14对引物表现出较强的特异性和稳定性。内参基因稳定性排名:ftsZgyrArpoB1ftsAsecAgapzapEgmk2rpoDsecYrpoOftsWgmk1recA,根据geNorm配对变异值Vn/n+1选择稳定性最好的ftsZ和gyrA作为内参基因作进一步评估。以ftsZ+gyrA以及16S rRNA基因分别作为内参基因检测柑橘黄龙病菌致病基因LasΔ5313的表达水平,所得的表达模式相同。【结论】柑橘黄龙病菌中涉及DNA复制和细胞分裂功能的管家基因表达较稳定,在CLas的基因表达研究中可选择ftsZ+gyrA的基因组合作为内参。本研究为后续利用实时荧光定量PCR分析CLas基因表达及研究CLas致病机理奠定基础。     

心脑血管疾病大额住院消费统计分析

通过对医院2004-2006年心脑血管疾病大额住院消费(消费大于50000元,以下简称大额消费)病例发病率高的前五种疾病的构成情况、药费、材料费消耗情况进行分析,认为加强大额病例中发病率高的病种的重点管理,是降低医疗费用的有效途径。建议制定常见病大额病种预定额付费方案和审查报销制度;采用适宜技术;控制药费,防止过度医疗,有效地遏制医疗费用的过快增长。     

Evidence that the C-terminus of OprM is involved in the assembly of the VceAB-OprM efflux pump

Although the architecture of tripartite multiple drug resistance (MDR) efflux pumps of Gram-negative bacteria has been well characterized, the means by which the components recognize each other and assemble into a functional pump remains obscure. In this study we present evidence that the C-terminal domain of the Pseudomonas aeruginosa OprM and the α-helical hairpin domain of Vibrio cholerae VceA play an important role in the recognition/specificity/recruitment step in the assembly of a functional, VceAB-OprM chimeric efflux pump. To our knowledge, this is the first evidence directly linking the C-terminal domain of an outer membrane efflux protein to its recruitment during the assembly of a tripartite efflux pump.     

两个猪苓菌株生长速度及酯酶同工酶比较研究

对不同地理分布的猪苓纯培养菌株进行了种性和酯酶同工酶的比较研究,结果表明,鸡爪苓（Z）纯培养菌株和猪屎苓（ZJ）纯培养菌株的种性有很大不同,两个纯培养菌株的酯酶同工酶酶带类型差异较大,亲缘关系较远。     

Synthesis of modified fragments of fibrinogen and their effect on the activity of proteolytic enzymes

New analogues of the Gly-Pro-Arg and Arg-Gly-Asp fragments of fibrinogen were synthesized: Gly-Pro-Arg-Pro (I), Gly-Pro-Arg-Pro-Met-OMe (II), Gly-Pro-Arg-Pro-Phe (III), Gly-Pro-Arg-Pro-Asp (IV), Gly-Pro-Arg-Pro-Glu (V), and Arg-Asn-Trp-Asp (VI). Their effect on the activity of proteases of various types was studied with the method of lysis of fibrin plates. All the peptides were found to inhibit plasmin activity (by 60–85%) and the γ-subunit of nerve growth factor (by 55–93%). Tetrapeptide (VI) proved to be an effective inhibitor of tissue activator of plasminogen and the γ-subunit of nerve growth factor (by 96 and 93%, respectively). The peptides exerted practically no effect on the activity of urokinase and moderately inhibited the activity of streptokinase [(III), IV), and (VI)], papain [(I), (II), IV), and (VI)], subtilisin [(V) and (VI)], α-chymotrypsin [(III), (V), and VI)], and Bacillus subtilis metalloprotease (VI). They inhibit trypsin [except for (I) and (III)] when applied on fibrin plates at a concentration of 1 × 10^?2 M, while, at the concentration of 1 × 10^?3 M, (I) and (II) induced an increase in proteolytic activity by 35 and 47%, respectively.     

Studies on the energy coupling sites of photophosphorylation IV. The relation of proton fluxes to the electron transport and ATP formation associated with Photosystem II

1. By using dibromothymoquinone as the electron acceptor, it is possible to isolate functionally that segment of the chloroplast electron transport chain which includes only Photosystem II and only one of the two energy conservation sites coupled to the complete chain (Coupling Site II, observed P/e₂ = 0.3–0.4). A light-dependent, reversible proton translocation reaction is associated with the electron transport pathway: H₂O → Photosystem II → dibromothymoquinone. We have studied the characteristics of this proton uptake reaction and its relationship to the electron transport and ATP formation associated with Coupling Site II.

2. The initial phase of H⁺ uptake, analyzed by a flash-yield technique, exhibits linear kinetics (0–3 s) with no sign of transient phenomena such as the very rapid initial uptake (“pH gush”) encountered in the overall Hill reaction with methylviologen. Thus the initial rate of H⁺ uptake obtained by the flash-yield method is in good agreement with the initial rate estimated from a pH change tracing obtained under continuous illumination.

3. Dibromothymoquinone reduction, observed as O₂ evolution by a similar flash-yield technique, is also linear for at least the first 5 s, the rate of O₂ evolution agreeing well with the steady-state rate observed under continuous illumination.

4. Such measurements of the initial rates of O₂ evolution and H⁺ uptake yield an H⁺/e⁻ ratio close to 0.5 for the Photosystem II partial reaction regardless of pH from 6 to 8. (Parallel experiments for the methylviologen Hill reaction yield an H⁺/e⁻ ratio of 1.7 at pH 7.6.)

5. When dibromothymoquinone is being reduced, concurrent phosphorylation (or arsenylation) markedly lowers the extent of H⁺ uptake (by 40–60%). These data, unlike earlier data obtained using the overall Hill reaction, lend themselves to an unequivocal interpretation since phosphorylation does not alter the rate of electron transport in the Photosystem II partial reaction. ADP, P_i and hexokinase, when added individually, have no effect on proton uptake in this system.

6. The involvement of a proton uptake reaction with an H⁺/e⁻ ratio of 0.5 in the Photosystem II partial reaction H₂O → Photosystem II → dibromothymoquinone strongly suggests that at least 50% of the protons produced by the oxidation of water are released to the inside of the thylakoid, thereby leading to an internal acidification. It is pointed out that the observed efficiencies for ATP formation (P/e₂) and proton uptake (H⁺/e⁻) associated with Coupling Site II can be most easily explained by the chemiosmotic hypothesis of energy coupling.     

林带阻力系数与透风系数关系的理论分析

根据冲量定理分析了林带对气流的阻力,首次得到了林带阻力系数的估算模式（Ｃｄ＝（１．８＋０．２α）（１－α）ｓｉｎ２ω,并利用有关文献发表的资料进行了验证．文章还对来流平行林带时林带对气流的阻力进行了讨论,指出来流平行林带时林带对气流的阻力仅为来流垂直林带时林带对气流阻力的０．７—１．１％,可以不予考虑．     

Photoinactivation of Photosystem II in leaves

Photoinactivation of Photosystem II (PS II), the light-induced loss of ability to evolve oxygen, inevitably occurs under any light environment in nature, counteracted by repair. Under certain conditions, the extent of photoinactivation of PS II depends on the photon exposure (light dosage, x), rather than the irradiance or duration of illumination per se, thus obeying the law of reciprocity of irradiance and duration of illumination, namely, that equal photon exposure produces an equal effect. If the probability of photoinactivation (p) of PS II is directly proportional to an increment in photon exposure (p = kΔx, where k is the probability per unit photon exposure), it can be deduced that the number of active PS II complexes decreases exponentially as a function of photon exposure: N = N_oexp(−kx). Further, since a photon exposure is usually achieved by varying the illumination time (t) at constant irradiance (I), N = N_oexp(−kI t), i.e., N decreases exponentially with time, with a rate coefficient of photoinactivation kI, where the product kI is obviously directly proportional to I. Given that N = N_oexp(−kx), the quantum yield of photoinactivation of PS II can be defined as −dN/dx = kN, which varies with the number of active PS II complexes remaining. Typically, the quantum yield of photoinactivation of PS II is ca. 0.1μmol PS II per mol photons at low photon exposure when repair is inhibited. That is, when about 10⁷ photons have been received by leaf tissue, one PS II complex is inactivated. Some species such as grapevine have a much lower quantum yield of photoinactivation of PS II, even at a chilling temperature. Examination of the longer-term time course of photoinactivation of PS II in capsicum leaves reveals that the decrease in N deviates from a single-exponential decay when the majority of the PS II complexes are inactivated in the absence of repair. This can be attributed to the formation of strong quenchers in severely-photoinactivated PS II complexes, able to dissipate excitation energy efficiently and to protect the remaining active neighbours against damage by light.     

2种嵩草属植物形态特征对生境干旱化的响应

该研究于西藏自治区东南部的东达山高山草甸沿生境干旱化梯度设置10个样地,采用线性回归方法分析优势种高山嵩草(Kobresia pygmaea)和矮生嵩草(K.humilis)叶片数量与其它构件数值(分株数量、茎基直径、根系数量和根系长度)之间的关系,并采用线性回归斜率测度分株功效、叶片萌生能力、根系分生功效和根系伸长功效,以探讨嵩草的分株能力、茎基生长和根系生长对生境干旱化过程的响应机制。结果显示:(1)2种嵩草植物的叶片数量与4种构件数值均为显著线性正相关关系。(2)随着生境干旱化程度增加,高山嵩草分株数量增加,矮生嵩草分株受干旱抑制程度高,分株数量呈下降趋势;2种嵩草分株功效下降,即单株叶片数量因干旱化程度增加而减少。(3)2种嵩草的茎基直径、叶片萌生能力随着生境干旱化程度增加而下降;高山嵩草叶片萌生能力的变化与生境干旱化梯度一致,具有连续性;矮生嵩草叶片萌生能力对生境干旱化的适应性弱,干旱到一定程度发生骤降。(4)随着生境干旱化,高山嵩草的根系数量和长度均增加,矮生嵩草根系长度增加,但数量却无规律变化;2种嵩草的根系分生功效和根系伸长功效均下降。研究表明:嵩草属植物分布的最适宜生境为表面稍有积水的沼生生境。为适应生境干旱化,高山嵩草降低叶片萌生率以减少蒸腾作用,增加分株数量以增强对空间的占有能力,并增加根系数量和长度来提高对土壤水分的吸收能力,因此对干旱有较强适应性且分布范围广;矮生嵩草只通过降低叶片萌生率,增加根系长度响应干旱化生境,其分布范围较窄。     

The detection of salivary enzymes of phytophagous Hemiptera: a compilation of methods

The role of Hemipteran saliva and salivary enzymes is central to an understanding of the etiology of damage that these insects cause to plants. The dilute nature of the salivary secretions and the minute quantities in which they are often obtainable have made analysis and detection of salivary components very difficult. Such investigations in this laboratory have led us to formalise the techniques involved and we believe that the compilation of these methods presented herein may be useful to other research workers in this area. Methods are described for acid and alkaline phosphatase, esterase, /S-glucosidase, carbohydrases, invertase, amylase, proteinase, pectinase, cellulase, catalase, peroxidase, catechol oxidase, superoxide dismutase and ascorbic oxidase.     

民勤绿洲边缘地下水位变化对植物种群生态位的影响

在民勤绿洲边缘,利用空间区域不同地下水位(湖区8～12m,泉山区15～17m,坝区20～23m)和时间序列(1984～1992年)民勤沙井子地区地下水位下降(7.45～11.65m)梯度,研究地下水位下降对荒漠植物种群生态位的影响。结果表明:空间区域地下水位下降,植物种群生态位宽度均减小,种群退化;时间序列地下水位下降,白刺种群在扩展,其它植物种群在退化。白刺种群生态位宽度在民勤绿洲边缘荒漠植物群落中最大,是该区的建群种。由于白刺种群在地下水位7.45～11.65m范围内扩展,民勤绿洲生态环境治理中地下水位达到该范围是一个主要目标。