La3+提高拟南芥耐旱能力研究

稀土元素有着广泛的生物学效应,稀土作为微肥在我国农业广泛使用.然而,稀土对植物抗旱性影响的研究很少.以模式植物拟南芥为材料,研究了不同浓度的镧(La3+)对植物根生长以及对植物耐旱能力的影响.结果表明,10和100 μmol ·L-1的La3能显著促进根生长,提高植物耐旱能力;1μmol·L-1La3+没有明显促进根生长,但显著提高了植物耐旱能力;1和10 mmol·L-1的La3+不能促进根生长,同时还降低了植物耐旱能力.该结果提示,稀土农用过程中要重视剂量的使用,同时避免带来生态环境问题.     

蚕豆16Sr RNA基因的一级结构分析

蚕豆叶绿体DNA属无反向重复顺序类型,这种类型的16s rRNA基因的一级结构本文属首次报道。我们采用了洪国藩的非随机DNA顺序测定法,测出蚕豆叶绿体16s rRNA基因金长为1491bp。通过比较及遗传距离图的构建表明蚕豆16SrRNA基因的变化速率比具有反向重复顺序类型的烟草、油菜及玉米的要快,说明反向重复顺序具有降低反向重复顺序内的DNA变化速率的功能。同时我们的结果也支持了Kolodner等人关于反向重复顺序能重组交换进行异源修复的推测。     

拟南芥抗镧突变体筛选条件的研究

以模式植物拟南芥为材料,采用组织培养的方法,以幼苗根的向地性弯曲生长为突变指标,对拟南芥抗La突变体筛选条件进行了研究。结果表明,1/2MS La培养基不能作为抗La突变体的筛选培养基,而以MS培养基预先培养幼苗,然后将其转入纯La培养基培养的筛选方法可以确定。以5.5作为选择培养基的初始和最终pH值,在1%琼脂浓度下,达到较好的凝固效果。纯La培养基中14 mg.kg-1La3 完全抑制了拟南芥幼苗根的生长,以此浓度为最适宜筛选压力。该项研究将为拟南芥抗稀土突变体的筛选提供技术依据。     

纳米基因扩增技术及其应用

聚合酶链式反应(PCR)是20世纪80年代中期发展起来的一种应用广泛的体外DNA扩增技术,但目前该技术仍然存在着一些问题,如特异性差、灵敏度低和假阳性等.近年来,随着纳米科技的发展,一些纳米粒子如金属纳米粒子、碳纳米材料、量子点和纳米金属氧化物等被引入到PCR反应体系中,即纳米基因扩增技术(NanoPCR).该技术大幅度提高了PCR的扩增效率、选择性、灵敏度和特异性,推动了生物学技术的发展,具有非常重要的理论意义和应用价值.本文综述了近年来纳米基因扩增技术的主要研究进展、反应机理,并探讨了其应用研究.     

拟南芥和甜菜夜蛾相互作用的差异蛋白分析

以甜菜夜蛾(Spodoptera exigua)和模式植物拟南芥(Arabidopsis thanliana)作为研究体系, 应用蛋白质双向凝胶电泳(Two-dimensional gel electrophoresis, 2-DE)分析了在甜菜夜蛾取食诱导条件下拟南芥蛋白表达的差异, 从蛋白质水平揭示昆虫取食诱导条件下植物的化学防御机制. 结果发现, 在昆虫取食诱导条件下, 有28个蛋白发生显著变化, 其中17个蛋白点上调表达, 11个蛋白点下调表达. 利用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)对差异蛋白进行了鉴定, 结果发现转酮酶、S-腺苷甲硫氨酸合成酶、二氢硫辛酰胺脱氢酶和脂肪酸合成酶在植物诱导化学防御中具有重要的作用, 其中脂肪酸合成酶与茉莉酸代谢通路相关.     

人尿激酶原在CHO细胞中的基因扩增与高效表达

本文通过基因共转染和基因共扩增方法,在SV40病毒晚期启动子控制下成功地在中国仓鼠卵巢细胞二氢叶酸还原酶缺陷株(CHO-DHFR~-)中高效表达了人尿激酶原,其表达水平在5×10~(-6) mol/L氨甲喋呤存在下可达2—3μg/10~6细胞/24 h。采用PMA诱导可将表达水平提高至3.5—4μg/10~6细胞/24 h。相应的Pro-UKcDNA在细胞基因组上整合的拷贝数为200—300拷贝/细胞。Western blot分析表明,表达出来的重组Pro-UK与天然Pro-UK具有相似的分子量:S_(2444)测活法证明重组Pro-UK具有体外酰胺水解活性。     

叶绿体基因含有起源于核基因组的真核调控序列

用凝胶阻滞实验首次发现叶绿体psbA基因的上游调控序列可被核蛋白质因子特异结合,而且叶绿体中存在可与真核性质的CaMV35S启动子特异结合的蛋白质因子,认为这些实验结果表明某些叶绿体基因含有起源于核基因组的真核调控序列。     

蚕豆16S rRNA基因前导顺序的一级结构分析

通过测定蚕豆16S rRNA基因前导顺序的一级结构并和其它物种相应顺序的比较发现:(1)在蚕豆16S rRNA基因上游不存在F1和X蛋白基因;(2)蚕豆的F3的保守区域更接近于保守顺序;(3)蚕豆的F3的—10区3’端后更富含A-T核苷酸对;(4)油菜、烟草、玉米和菠菜的16s rRNA基因5’端上游能形成一个稳定的基环结构,而蚕豆则不能。我们由此认为蚕豆的rDNA启动子比油菜的要强,增加转录是一个担负起两个rDNA拷贝功能的原因之一。     

螺旋通道微流控PCR芯片连续自动扩增DNA片段的研究

研制了由内向外流动的螺旋通道微流控PCR玻璃芯片,减少了PCR反应液在微通道中流动时的分散和阻力;讨论了扩增循环数和进样速度对长片段基因扩增的影响,在26min内成功扩增了质量浓度仅为10ng/mL的6012bpλ-DNA;通过将小孔径石英毛细管作为顺序注射(SI)系统的连接管路,使其死体积降到0.30μL.实现了微升级样品的自动换样、连续PCR扩增和微通道洗涤等功能.样品间无交叉污染.每小时可扩增500bpλ-DNA试样7个.扩增产物片段大小和荧光强度的相对标准偏差分别为0.4%和6.7%.     

Tb3+离子发光探针研究Ca2+,La3+和Al3+与拟南芥钙调素的竞争结合

利用Tb3 离子的发光, 研究了Tb3 与拟南芥钙调素 (CaM)结合的荧光光谱及荧光滴定曲线特点, 然后利用Tb3 4*CaM系统在221 nm直接激发和280 nm敏化激发的光谱变化研究了Ca2 , La3 和Al3 与拟南芥钙调素 (CaM) 的竞争结合作用. 结果表明: Tb3 , La3 与钙调素的竞争结合能力强于Ca2 , 而Tb3 的竞争结合力又大于La3 , Ca2 与钙调素的结合力远大于Al3 . 竞争实验的结果从分子水平上揭示了Tb3 , La3 和Al3 等金属离子生物效应可能的分子机制.     

Immobilisation of quantum dots by bio-orthogonal PCR amplification and labelling for direct gene detection and quantitation

A sensitive and versatile detection scheme based on quantum dot immobilisation on a solid support through bio-orthogonal PCR amplification and labelling has been developed for detection and quantification of gene targets in complex DNA mixtures.     

Ionic liquids promote PCR amplification of DNA

A bicyclic imidazolium ionic liquid (4d), [b-4C-im][Br], was found to be highly effective not only for promoting PCR of GC-rich DNA by minimizing non-specific amplification, but also for facilitating PCR of normal-GC DNA under mild conditions.     

Quantitative evaluation of bias in PCR amplification and next-generation sequencing derived from metabarcoding samples

Unbiased identification of organisms by PCR reactions using universal primers followed by DNA sequencing assumes positive amplification. We used six universal loci spanning 48 plant species and quantified the bias at each step of the identification process from end point PCR to next-generation sequencing. End point amplification was significantly different for single loci and between species. Quantitative PCR revealed that Cq threshold for various loci, even within a single DNA extraction, showed 2,000-fold differences in DNA quantity after amplification. Next-generation sequencing (NGS) experiments in nine species showed significant biases towards species and specific loci using adaptor-specific primers. NGS sequencing bias may be predicted to some extent by the Cq values of qPCR amplification.     

A proteomic analysis of organelles from Arabidopsis thaliana

We introduce the use of Arabidopsis thaliana callus culture as a system for proteomic analysis of plant organelles using liquid-grown callus. This callus is relatively homogeneous, reproducible and cytoplasmically rich, and provides organelles in sufficient quantities for proteomic studies. A database was generated of mitochondrial, endoplasmic reticulum (ER), Golgi/prevacuolar compartment and plasma membrane (PM) markers using two-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (2-D SDS-PAGE) and peptide sequencing or mass spectrometric methods. The major callus membrane-associated proteins were characterised as being integral or peripheral by Triton X-114 phase partitioning. The database was used to define specific proteins at the Arabidopsis callus plasma membrane. This database of organelle proteins provides the basis for future characterisation of the expression and localisation of novel plant proteins.     

Absorption and fluorescence spectroscopic characterization of cryptochrome 3 from Arabidopsis thaliana

The blue light photoreceptor cryptochrome 3 (cry3) from Arabidopsis thaliana was characterized at room temperature in vitro in aqueous solution by optical absorption and emission spectroscopic studies. The protein non-covalently binds the chromophores flavin adenine dinucleotide (FAD) and N5,N10-methenyl-5,6,7,8-tetrahydrofolate (MTHF). In the dark-adapted state of cry3, the bound FAD is present in the oxidized form (FAD(ox), ca. 38.5%), in the semiquinone form (FADH., ca. 5%), and in the fully reduced neutral form (FAD(red)H2) or fully reduced anionic form (FAD(red)H-, ca. 55%). Some amount of FAD (ca. 1.5%) in the oxidized state remains unbound probably caused by chromophore release and/or denaturation. F?rster-type energy transfer from MTHF to FAD(ox) is observed. Photo-excitation reversibly modifies the protein conformation causing a slight rise of the MTHF absorption strength and an increase of the MTHF fluorescence efficiency (efficient protein conformation photo-cycle). Additionally there occurs reversible reduction of bound FAD(ox) to FAD(red)H2 (or FAD(red)H-, FAD(ox) photo-cycle of moderate efficiency), reversible reduction of FADH. to FAD(red)H2 (or FAD(red)H-, FADH. photo-cycle of high efficiency), and modification of re-oxidable FAD(red)H2 (or FAD(red)H-) to permanent FAD(red)H2 (or FAD(red)H-) with low quantum efficiency. Photo-excitation of MTHF causes the reversible formation of a MTHF species (MTHF', MTHF photo-cycle, moderate quantum efficiency) with slow recovery to the initial dark state, and also the formation of an irreversible photoproduct (MTHF').     

Molecular beacon-based fluorescence biosensor for the detection of gene fragment and PCR amplification products related to chronic myelogenous leukemia

A novel fluorescence method has been established for the determination of gene fragment and PCR amplification products related to chronic myelogenous leukemia (CML). A molecular beacon (MB) which comprises a DNA loop section, a pair of fluorophore (tetramethoxyl rhodamine, TAMRA), and a quencher (4-(2-methyl-on-amino-azobenzene) benzoate, DABCYL) was designed. The loop sequence of MB was designed according to the DNA sequence relating to CML (type b3a2) which contained a single-stranded oligonucleotide. Before hybridization, the fluorescence from the TAMRA had been quenched by the DABCYL. After hybridization with the complementary DNA, the quencher will become far away from the TAMRA, and the fluorescence intensity detected will increase. Changes in the fluorescence intensity have a linear relationship with the concentration of complementary DNA (C) in the range of 4.0 × 10⁻⁹–3.2 × 10⁻⁸ mol/L, with a correlation coefficient of 0.9973; the detection limit was 6.0 × 10⁻¹⁰ mol/L (S/N = 3). The developed method has high selectivity, which can be used to discriminate single-base mismatch sequence. The method has been applied to detect the short-stranded CML DNA fragment (278 bp) with high sensitivity. This approach is a promising method for the detection of CML in real samples for medical diagnostics.     

Centrifugal microfluidic system for primary amplification and secondary real-time PCR

Pre-amplification is a basis for numerous polymerase chain reaction (PCR) protocols but bears severe contamination risks due to handling of high-copy DNA samples. Therefore we developed a self-contained centrifugal microfluidic system comprising pre-stored reagents; it enables pre-amplification of specific DNA sequences prior to automated aliquoting and real-time PCR in a modified commercial thermocycler.     

A quantitative model of error accumulation during PCR amplification

The amplification of target DNA by the polymerase chain reaction (PCR) produces copies which may contain errors. Two sources of errors are associated with the PCR process: (1) editing errors that occur during DNA polymerase-catalyzed enzymatic copying and (2) errors due to DNA thermal damage. In this study a quantitative model of error frequencies is proposed and the role of reaction conditions is investigated. The errors which are ascribed to the polymerase depend on the efficiency of its editing function as well as the reaction conditions; specifically the temperature and the dNTP pool composition. Thermally induced errors stem mostly from three sources: A+G depurination, oxidative damage of guanine to 8-oxoG and cytosine deamination to uracil. The post-PCR modifications of sequences are primarily due to exposure of nucleic acids to elevated temperatures, especially if the DNA is in a single-stranded form. The proposed quantitative model predicts the accumulation of errors over the course of a PCR cycle. Thermal damage contributes significantly to the total errors; therefore consideration must be given to thermal management of the PCR process.     

Fast extraction, amplification and analysis of genes from human blood

In order to shorten the time spent on the sample preparation for gene analysis, a novel method was proposed through the combination of fast DNA extraction and purification by Generation capture disk, amplification by capillary polymerase chain reaction, and confirmation of amplification products by microchip electrophoresis. With this method, 3 microL blood was enough to obtain adequate target fragments in human genes. Under the optimal conditions in each step, the sample preparation for eight fragments in beta-globin gene and four fragments in ras gene could be finished within 20 min. Since all the experiments were performed on commercial instruments, this method showed a wide range of applicability. In addition, other advantages such as fast speed and low consumption of samples were demonstrated. All these merits proved that such a combination method was of great potential for the clinical diagnostics.     

Detection of bond formations by DNA-programmed chemical reactions and PCR amplification

A system capable of performing both DNA-templated chemical reactions and detection of bond formations is reported. Photocleavable DNA templates direct reactions. Products from bond-forming events re-ligate original templates, amplifiable by PCR, therefore distinguishing bond formation from background. This system provides a novel approach for discovering potential new chemical reactions.