若尔盖高原产甲烷菌数量的时空差异性

应用实时荧光定量PCR法,构建了总甲烷菌实时荧光绝对定量PCR的标准品以及标准曲线,用于总甲烷菌的定量测定,所构建的质粒DNA浓度为160mg/L,标准曲线相关性为0.992,扩增效率为98.6%。研究表明,若尔盖花湖地区4月草地生态系统和湿地生态系统甲烷菌含量相当,湿地生态系统9月甲烷菌含量高于7月,7月甲烷菌含量高于4月,在7月和9月随着深度的增加甲烷菌含量也增加,而草地生态系统的甲烷菌含量并未表现出明显的季节和深度上的规律性。     

The search for an optimal DNA, RNA, and protein detection by in situ hybridization, immunohistochemistry, and solution-based methods

Clinical trials and correlative laboratory research are increasingly reliant upon archived paraffin-embedded samples. Therefore, the proper processing of biological samples is an important step to sample preservation and for downstream analyses like the detection of a wide variety of targets including micro RNA, DNA and proteins. This paper analyzed the question whether routine fixation of cells and tissues in 10% buffered formalin is optimal for in situ and solution phase analyses by comparing this fixative to a variety of cross linking and alcohol (denaturing) fixatives. We examined the ability of nine commonly used fixative regimens to preserve cell morphology and DNA/RNA/protein quality for these applications. Epstein-Barr virus (EBV) and bovine papillomavirus (BPV)-infected tissues and cells were used as our model systems. Our evaluation showed that the optimal fixative in cell preparations for molecular hybridization techniques was "gentle" fixative with a cross-linker such as paraformaldehyde or a short incubation in 10% buffered formalin. The optimal fixatives for tissue were either paraformaldehyde or low concentration of formalin (5% of formalin). Methanol was the best of the non cross-linking fixatives for in situ hybridization and immunohistochemistry. For PCR-based detection of DNA or RNA, some denaturing fixatives like acetone and methanol as well as "gentle" cross-linking fixatives like paraformaldehyde out-performed other fixatives. Long term fixation was not proposed for DNA/RNA-based assays. The typical long-term fixation of cells and tissues in 10% buffered formalin is not optimal for combined analyses by in situ hybridization, immunohistochemistry, or--if one does not have unfixed tissues--solution phase PCR. Rather, we recommend short term less intense cross linking fixation if one wishes to use the same cells/tissue for in situ hybridization, immunohistochemistry, and solution phase PCR.     

The entomopathogenic nematode Steinernema kraussei and Metarhizium anisopliae work synergistically in controlling overwintering larvae of the black vine weevil,Otiorhynchus sulcatus,in strawberry growbags

Previously, the combination of reduced rate of entomopathogenic nematodes (EPN) and fungus caused additive or synergistic mortality to third-instar black vine weevil (BVW), Otiorhynchus sulcatus. In this study, we examined this interaction in unheated glasshouses during winter and compared a combination of commercial formulation of a cold-tolerant EPN, S. kraussei (Nemasys L?) and fungus Metarhizium anisopliae strain V275 against overwintering third-instar BVW. The combination of M. anisopliae with S. kraussei at a rate of 1×10¹⁰ conidia+250,000 nematodes/growbag resulted in additive or synergistic effects, providing 100% control of overwintering larvae.     

Susceptibility of apple clearwing moth larvae,Synanthedon myopaeformis (Lepidoptera: Sesiidae) to Beauveria bassiana and Metarhizium brunneum

Apple clearwing moth larvae, Synanthedon myopaeformis (Lepidoptera: Sesiidae) were found to be susceptible to infection by two entomopathogenic fungi: an indigenous fungus isolated from S. myopaeformis cadavers and identified as Metarhizium brunneum (Petch); and Beauveria bassiana isolate GHA. In laboratory bioassays, larvae exhibited dose related mortality after exposure to both the M. brunneum and Beauveria bassiana with 7 day LC₅₀'s of 2.9×10⁵ and 3.4×10⁵ spores/mL, respectively. Larval mortalities caused by the two isolates at 1×10⁶ spores/mL were not significantly different and 73% of the M. brunneum-treated, and 76% of the B. bassiana-treated larvae were dead 7 days post treatment, with LT₅₀'s of 5.5 and 5.1 days, respectively.     

大豆C3HC4型RING锌指蛋白基因GmRZFP1克隆与表达分析

锌指蛋白在调节植物防卫基因表达和抗性反应上起关键作用。目前,对大豆中C3HC4型RING锌指蛋白基因的研究不多。本研究利用核蛋白筛选系统(NTT)筛选大豆(铁丰8号)干旱处理5h的cDNA文库,获得一个RING锌指蛋白基因。该基因全长927bp,编码308个氨基酸,含有C3HC4-type RING锌指结构域,命名为GmRZFP1。系统进化树分析显示,Gm-RZFP1属于C3HC4-type锌指亚家族。Real-time PCR结果表明,GmRZFP1基因受干旱、高盐、高温、低温、乙烯和ABA等胁迫诱导表达,表明该蛋白涉及多种胁迫相关的信号传导途径。亚细胞定位结果表明,163hGFP-GmRZFP1融合蛋白定位于细胞核中。本研究结果有助于研究该类基因在大豆逆境应答反应中的作用,阐明大豆抗逆分子机制。     

Preimplantation genetic diagnosis of β-thalassemia using real-time polymerase chain reaction with fluorescence resonance energy transfer hybridization probes

Preimplantation genetic diagnosis (PGD) is employed increasingly to allow transfer of embryos to the uterus in assisted reproduction procedures. There are three stages of biopsy: polar bodies, one or two blastomeres from the cleavage-stage embryos, and trophectoderm cells (∼5 cells) from the blastocyst-stage embryos. Validation of polymerase chain reaction (PCR)-based assays are challenging because only limited genetic material can be obtained for PGD. In the current study, we modified a valid single-cell PCR protocol for PGD using real-time PCR assay with fluorescence resonance energy transfer (FRET) hybridization probes followed by melting curve analysis. We optimized and clinically applied the protocol, permitting molecular genetic analysis to amplify a specific region on the beta-globin (HBB) gene for a couple, carriers of two mutations: c.-78A>G and c.52A>T. Among a total of eight embryos obtained after ovarian stimulation, a single blastomere per embryo at the six- to eight-cell stage was biopsied. This PGD method showed that four embryos were unaffected, two embryos were selected for transfer, and one pregnancy was achieved. Finally, a healthy male baby was delivered at 38 weeks’ gestation. The results obtained using the new method, FRET hybridization probes, were compared with findings using an existing method, primer extension minisequencing.     

Normalizing genes for real-time polymerase chain reaction in epithelial and nonepithelial cells of mouse small intestine

Gene expression studies in intestinal epithelial and stromal cells are a common tool for investigating the mechanisms by which the homeostasis of the small intestine is regulated under normal and pathological conditions. Quantitative real-time PCR (qPCR) is a sensitive and highly reproducible method of gene expression analysis, with expression levels quantified by normalization against reference genes in most cases. However, the lack of suitable reference genes for epithelial cells with different differentiation states and nonepithelial tissue cells has limited the application of qPCR in gene expression studies of small intestinal samples. In this study, 13 housekeeping genes, ACTB, B2M, GAPDH, GUSB, HPRT1, HMBS, HSP90AB1, RPL13A, RPS29, RPLP0,PPIA, TBP, and TUBA1, were analyzed to determine their applicability for isolated crypt cells, villus cells, deepithelialized mucosa, and whole mucosa of the mouse small intestine. Using geNorm and NormFinder software, GUSB and TBP were identified as the most stably expressed genes, whereas the expressions of the commonly used reference genes GAPDH, B2M, and ACTB, and ribosomal protein genes RPL13A, RPS29, and RPLP0 were relatively unstable. Thus, this study demonstrates that GUSB and TBP are the optimal reference genes for the normalization of gene expression in the mouse small intestine.     

Gene inactivation mediated by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> in the filamentous fungi <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis>

The list of fungal species with known complete genome and/or expressed sequence tag collections is extending rapidly during the last couple of years. Postgenomic gene function assignment is an obvious follow-up and depends on methodologies to test gene function in vivo. One of such methods is the generation of null mutants via homologous recombination at the wild–type loci by using inactivation cassettes. In this paper, the ability of Agrobacterium tumefaciens to genetically transform filamentous fungi was exploited to drive homologous recombination at the trp1 locus of the enthomopathogenic fungus Metarhizium anisopliae. The trp1 disruptants exhibited a clearly distinguishable phenotype from wild-type cells and were recovered with high efficiency of homologous recombination (22%). The complementation of such mutants with the wild-type gene generates only transformants with homologous integration.