土壤中棉花黄萎病菌SYBR Green Ⅰ荧光RT-PCR定量检测技术研究

为实现田间土壤棉花黄萎病菌的早期检测,建立了土壤中棉花黄萎病菌的SYBR Green I荧光定量PCR检测方法。以含342bp PCR扩增产物的阳性质粒为参考,构建了标准曲线,并对该曲线的特异性、敏感性、可重复性进行了评价。结果表明,该方法具有快速、特异性强、敏感度高等特点。检测范围在3.8×103-3.8×108copies/μL之间有良好的线性关系,相关系数R2为0.996,扩增效率为101.5%,灵敏度比常规PCR方法高102倍。     

向日葵黑茎病菌的快速分子检测

从新疆采集的向日葵黑茎病罹病植株的茎秆、叶片和花盘共分离获得20个真菌分离物。经致病性测定,证明分离物XJ011和XJ111是引起该病害的病原物。采用ITS通用引物ITS1/ITS4对XJ011和XJ111菌株的rDNA-ITS区进行PCR扩增和测序,并结合形态学特征,将该菌鉴定为麦氏茎点霉Phoma macdonaldii。同时,在rDNA-ITS的多态性丰富区域设计了一对特异性引物320FOR/320REV,建立了P. macdonaldii病菌的快速分子检测体系,能特异性检测出向日葵黑茎病菌,灵敏度     

菌丝桥在日本落叶松幼苗间磷传递和植株生长中的作用

 应用四室隔网系统研究了菌丝桥在日本落叶松(Larix kaempferi)幼苗间传递磷的作用。结果表明,供体接种卷缘桩菇(Paxillus involutus)和彩色豆马勃（Pisolithus tinctorius）后,其外延菌丝可以穿过隔离层侵染受体落叶松,在供体和受体落叶松间形成了菌丝桥。供体植株接种菌根真菌后生物量明显增加,但是对受体植株没有显著的影响。菌根真菌侵染的供体和受体植株的根、地上部吸磷量均分别显著高于对照,而且供体植株根、地上部吸磷量增加的程度明显高于受体。被卷缘桩菇和彩色豆马勃侵染的受体植株体内32P的放射性强度分别是对照的10倍和6倍,两者形成菌丝桥后传递到受体植株的32P分别为供体植株体内32P的1.10%和0.22%。供体植株吸收的32P可以通过菌丝桥传递给受体,但是绝对数量十分有限,对受体植株磷营养没有产生显著的影响,但P. involutus和P. tinctorius侵染受体植株后,促进了受体落叶松对磷的吸收,这是菌丝桥形成后,真菌帮助受体植株吸收磷引起的。     

新型大肠杆菌高效表达载体pHsh的构建与应用

在现代生物学和生物技术研究中,通过基因的重组表达获得目标蛋白是一种应用最广泛的方法。因其培养简单、操作方便、遗传背景清楚、克隆表达系统成熟完善,大肠杆菌表达系统通常是人们表达重组蛋白的首选,而表达载体在重组蛋白的生产中起决定作用。pHsh及其衍生质粒是近年发展起来的新型大肠杆菌表达载体,其调控外源基因表达的原理不同于所有其他表达系统,并且具有表达水平高、成本低廉等特点。介绍大肠杆菌表达系统的组成和常用表达载体,并对由pHsh系列载体组成的Hsh表达体系的构建策略、表达调控机制及其使用方法进行综述。Hsh表达体系的建立和发展有望从一个不同的角度帮助解决基因的重组表达中常见的表达水平低、诱导剂成本高、包涵体形成等问题。     

Methylation profiling using degenerated oligonucleotide primer-PCR specific for genome-wide amplification of bisulfite-modified DNA

DNA methylation is one of the essential epigenetic processes that play a role in regulating gene expression. Aberrant methylation of CpG-rich promoter regions has been associated with many forms of human cancers. The current method for determining the methylation status relies mainly on bisulfite treatment of genomic DNA, followed by methylation-specific PCR (MSP). The difficulty in acquiring a methylation profiling often is limited by the amount of genomic DNA that can be recovered from a given sample, whereas complex procedures of bisulfite treatment further compromise the effective template for PCR analysis. To circumvent these obstacles, we developed degenerated oligonucleotide primer (DOP)-PCR to enable amplification of bisulfite-modified genomic DNA at a genome-wide scale. A DOP pair was specially designed as follows: first 3' DOP, CTCGAGCTGHHHHHAACTAC, where H is a mixture of base consisting of 50% A, 25% T, and 25% C; and second 5' DOP, CTCGAGCTGDDDDDGTTTAG, where D is a mixture of base consisting of 50% T, 25% G, and 25% A. Our results showed that bisulfite-modified DNAs from a cell line, cord blood cells, or cells obtained by laser capture microdissection were amplified by up to 1000-fold using this method. Subsequent MSP analysis using these amplified DNAs on nine randomly selected cancer-related genes revealed that the methylation status of these genes remained identical to that derived from the original unamplified template.     

Microsatellite Instability in Chicken Lymphoma Induced by Gallid Herpesvirus 2

Microsatellite instability (MSI) has been found in a range of human tumors, and little is known of the links between MSI and herpesvirus. In order to investigate the relationship between MSI and Gallid herpesvirus 2 (GaHV-2)-induced lymphoma, fifteen Marek’s disease (MD) lymphomas were analyzed through using 46 microsatellite markers, which were amplified by PCR from DNA specimens of lymphoma and normal muscular tissues from the same chicken. PCR products were evaluated by denaturing polyacrylamide gel electrophoresis for MSI analysis. MSI was proved in all lymphomas, at least in one locus. Thirty of the 46 microsatellite markers had microsatellite alterations. These results suggested that GaHV-2-induced lymphoma in chickens is related to MSI, and this is the first report to demonstrate that MSI is associated with the GaHV-2 induced lymphoma in chicken.     

乙氧基磷酸酯类有机磷农药单克隆抗体的制备与鉴定

制备针对乙氧基磷酸酯类有机磷农药的单克隆抗体,以此为基础建立该类农药的快速免疫筛选检测方法．以二乙基磷酸乙酸为通用结构半抗原,分别使之与牛血清白蛋白和鸡卵清蛋白共价偶联,合成免疫原和包被原并对其进行结构鉴定．偶联成功后的免疫原用于免疫Balb/c小鼠．将免疫成功小鼠的脾细胞与小鼠SP2/0骨髓瘤细胞融合,筛选能稳定分泌抗乙氧基磷酸酯类有机磷农药单克隆抗体的杂交瘤细胞株．获得的小鼠腹水用辛酸-硫酸铵法纯化,所得纯化抗体以琼脂双扩散法鉴定其免疫球蛋白类型,间接竞争ELISA方法测定其对半抗原的灵敏度、特异性和亲和性．结果表明,该抗体分泌IgG1亚类的单克隆抗体,且与二乙基磷酸乙酸的亲和性较高(1.4×10⁷ L/mol),所得抗体对毒死蜱、对硫磷、丙溴磷、氧化乐果、除线磷、二嗪农、溴硫磷、辛硫磷、喹硫磷、三唑磷等农药有特异性反应．该检测技术可用于上述农药的快速定性或定量检测．     

Landscape and evolutionary dynamics of Helitron transposons in plant genomes as well as construction of online database HelDB

Helitron transposons play an important role in host genome evolution due to their ability to capture genes and regulatory elements. In this study, we developed a pipeline to identify and annotate Helitrons systematically from 358 plant and 178 animal high-quality genomes. All these data were organized into HelDB, a database where Helitrons can be explored with a user-friendly Web interface and related software. Based on these data, further analysis showed that the number or the cumulative length of Helitrons is positively correlated with genome size. Helitrons had experienced two expansion periods in plants, with the first occurring 20–30 Ma and peaking at approximately 24 Ma. The second expansion occurred in the last 4 million years. The expansions might be due to stimulation of paleogeographic environment. Detailed investigation of gene capture by Helitrons in Brassicaceae and Solanaceae plants showed that the captured genes showed diverse functions. Interestingly, metal ion binding function was enriched in these captured genes in most species. This phenomenon might be due to the need for binding of divalent metal ions to the Rep domain required for Helitron transposition. This study improves our knowledge of the landscape and evolution of Helitron transposons in plants and paves a way for further functional studies of this kind of transposable element.