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。     

Biological soil crusts in ecological restoration: emerging research and perspectives

Drylands encompass over 40% of terrestrial ecosystems and face significant anthropogenic degradation causing a loss of ecosystem integrity, services, and deterioration of social‐ecological systems. To combat this degradation, some dryland restoration efforts have focused on the use of biological soil crusts (biocrusts): complex communities of cyanobacteria, algae, lichens, bryophytes, and other organisms living in association with the top millimeters of soil. Biocrusts are common in many ecosystems and especially drylands. They perform a suite of ecosystem functions: stabilizing soil surfaces to prevent erosion, contributing carbon through photosynthesis, fixing nitrogen, and mediating the hydrological cycle in drylands. Biocrusts have emerged as a potential tool in restoration; developing methods to implement effective biocrust restoration has the potential to return many ecosystem functions and services. Although culture‐based approaches have allowed researchers to learn about the biology, physiology, and cultivation of biocrusts, transferring this knowledge to field implementation has been more challenging. A large amount of research has amassed to improve our understanding of biocrust restoration, leaving us at an opportune time to learn from one another and to join approaches for maximum efficacy. The articles in this special issue improve the state of our current knowledge in biocrust restoration, highlighting efforts to effectively restore biocrusts through a variety of different ecosystems, across scales and utilizing a variety of lab and field methods. This collective work provides a useful resource for the scientific community as well as land managers.     

Properties of chimeric toxins with two recognition domains: interleukin 6 and transforming growth factor alpha at different locations in Pseudomonas exotoxin.

Pseudomonas exotoxin (PE) is a potent cytotoxic agent that is composed of 613 amino acids arranged into three major domains. We have previously identified two positions where ligands can successfully be placed in PE to direct it to cells with specific surface receptors. One site is at the amino terminus and the other is close to but not at the C-terminus. To examine the possibility of constructing oncotoxins with two different recognition elements that will bind to two different receptors, we have placed cDNAs encoding either transforming growth factor alpha (TGF alpha) or interleukin 6 (IL6) at the 5' end of a PE gene and also inserted a cDNA encoding TGF alpha near the 3' end of the PE gene. The plasmids encoding these chimeric toxins were expressed in Escherichia coli and the chimeric proteins purified to near homogeneity. In all the new toxins, the TGF alpha near the C-terminus was inserted after amino acid 607 of PE and followed by amino acids 604-613 so that the correct PE C-terminus (REDLK) was preserved. For each chimera, the toxin portion was either PE4E, in which the cell binding domain (domain Ia) is mutated, PE40, in which domain Ia is deleted, or PE38, in which domain Ia and part of domain Ib are deleted. These derivatives of PE do not bind to the PE receptor and allow 607, 355, or 339 amino acids, respectively, between the two ligands.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spatial and halophyte-associated microbial communities in intertidal coastal region of India

Microbial communities in intertidal coastal soils respond to a variety of environmental factors related to resources availability, habitat characteristics, and vegetation. These intertidal soils of India are dominated with Salicornia brachiata, Aeluropus lagopoides, and Suaeda maritima halophytes, which play a significant role in carbon sequestration, nutrient cycling, and improving microenvironment. However, the relative contribution of edaphic factors, halophytes, rhizosphere, and bulk sediments on microbial community composition is poorly understood in the intertidal sediments. Here, we sampled rhizosphere and bulk sediments of three dominant halophytes (Salicornia, Aeluropus, and Suaeda) from five geographical locations of intertidal region of Gujarat, India. Sediment microbial community structure was characterized using phospholipid fatty acid (PLFA) profiling. Microbial biomass was significantly influenced by the pH, electrical conductivity, organic carbon, nitrogen, and sodium and potassium concentrations. Multivariate analysis of PLFA profiles had significantly separated the sediment microbial community composition of regional sampling sites, halophytes, rhizosphere, and bulk sediments. Sediments from Suaeda plants were characterized by higher abundance of PLFA biomarkers of Gram-negative, total bacteria, and actinomycetes than other halophytes. Significantly highest abundance of Gram-positive and fungal PLFAs was observed in sediments of Aeluropus and Salicornia, respectively than in those of Suaeda. The rhizospheric sediment had significantly higher abundance of Gram-negative and fungal PLFAs biomarkers compared to bulk sediment. The results of the present study contribute to our understanding of the relative importance of different edaphic and spatial factors and halophyte vegetation on sediment microbial community of intertidal sediments of coastal ecosystem.     

Paclobutrazol Arrests Vegetative Growth and Unveils Unexpressed Yield Potential of Jatropha curcas

Although the process for making EN 14214 grade Jatropha methyl ester (biodiesel) capable of running unmodified diesel engines in neat form has been demonstrated, getting higher seed yield from Jatropha shrubs in wastelands is critical to the success of Jatropha biodiesel. But, low productivity is inherent to many Jatropha curcas germplasms and raising large-scale plantations using such untested planting material can lead to wasteful expenditures. Unreliable and poor flowering and fruiting are important factors responsible for low productivity in the species. Although much is known about growth retardants applied to field and horticultural crops, their role in improving the seed productivity of Jatropha has never been explored. Here we report for the first time that paclobutrazol could be an extremely useful chemical, whose dose and time of application, if optimized, can significantly reduce unwanted vegetative growth, with concomitant improvement in yield and seed oil content of Jatropha. In the year following application of paclobutrazol, an unexpected increase in seed yield, as high as 1127% relative to controls, was obtained from one such unproductive Jatropha germplasm. We hypothesize that low seed production in this species may be a result of excess vegetative growth caused by an unfavorable endogenous hormonal configuration which competes with growth and development of flower, fruit, or seed. This undesired physiological state can be reversed by paclobutrazol application to achieve maximum oil yield from this energy shrub that holds great promise in the future.     

Mapping of hiv-1 Gag epitopes recognized by polyclonal antibodies using gene-fragment phage display system.

Phage display has emerged as a powerful technique for mapping epitopes recognised by monoclonal and polyclonal antibodies. We have recently developed a simple gene-fragment phage display system and have shown its utility in mapping epitope recognised by a monoclonal antibody. In the present study, we have employed this system in mapping epitopes recognised by polyclonal antibodies raised against HIV-1 capsid protein, p24 which is derived from proteolytic cleavage of Gag polyprotein. HIV-1 gag DNA was fragmented by DNase I and the fragments (50-250 bp) were cloned into gene-fragment phage display vector to construct a library of phages displaying peptides. This phage library was used for affinity selection of phages displaying epitopes recognised by rabbit anti-p24 polyclonal antibodies. Selected phages contained sequences from two discrete regions of p24, demonstrating the presence of two antigenic regions. The DNA sequences encoding these regions were also cloned and expressed as GST fusion proteins. The immunoreactivity of these epitopes as GST fusion proteins, or as phage-displayed peptides, was comparable in ELISA system using same anti-p24 polyclonal antibodies. The results indicate that the gene-fragment based phage display system can be used efficiently to identify epitopes recognised by polyclonal antibodies, and phage displayed epitopes can be directly employed in ELISA to detect antibodies.     

Activation of the c-Jun N-terminal kinase/stress-activated protein kinase pathway by overexpression of caspase-8 and its homologs.

Caspase-8 is the most proximal caspase in the caspase cascade and possesses a prodomain consisting of two homologous death effector domains (DEDs). We have discovered that caspase-8 and its homologs can physically interact with tumor necrosis factor receptor-associated factor family members and activate the c-Jun N-terminal kinase (JNK, or stress-activated protein kinase) pathway. This ability resides in the DED-containing prodomain of these proteins and is independent of their role as cell death proteases. A point mutant in the first DED of caspase-8 can block JNK activation induced by several death domain receptors. Inhibition of JNK activation blocks apoptosis mediated by caspase-10, Mach-related inducer of toxicity/cFLIP, and Fas/CD95, thereby suggesting a cooperative role of this pathway in the mediation of caspase-induced apoptosis.     

IL2-PE664Glu, a new chimeric protein cytotoxic to human-activated T lymphocytes

To produce a molecule that will kill activated T cells as well as lymphomas and leukemias expressing interleukin 2 (IL2) receptors, we have created a recombinant chimeric protein in which IL2 is attached in peptide linkage to a truncated mutant form of Pseudomonas exotoxin (PE) (Lorberboum-Galski, H., FitzGerald, D.J.P., Chandhary, V.K., Adhya, S., and Pastan, I. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 1922-1926). Although this molecule was very active on rodent cells, it had lower activity on some human cell types. A new chimeric protein termed IL2-PE664Glu has been constructed that is extremely toxic to both phytohemagglutinin blasts and mixed leukocyte reaction blasts prepared from monkey and human lymphocytes. The chimeric gene encoding this protein was constructed by fusing a cDNA clone for human interleukin 2 to the 5' end of a mutated cDNA encoding a full-length PE molecule. Four amino acids in domain I of PE were changed thus decreasing its nonspecific toxicity. IL2-PE664Glu is a much more active cytotoxic molecule for primate and human-activated T cells than IL2-PE40 which is a chimeric protein that was found to be an effective immunosuppressive agent in rodent models. Our results indicate that IL2-PE664Glu should be evaluated as an immunosuppressive agent for the treatment of human immune disorders in which activated T cells expressing the IL2 receptor are prominent.     

Cloning and expression of the H chain V region of antibody OVB3 that reacts with human ovarian cancer

Hybridoma OVB3 produces an antibody (IgG2b kappa) that reacts with an Ag present on the surface of almost all human ovarian carcinomas. An EcoRI fragment of genomic DNA containing the rearranged H chain V region of monoclonal OVB3 was cloned from a lambda gtWES library and then sub-cloned into a pGEM4 vector. To show that the cloned DNA sequence did encode the V region of a functional antibody, the DNA fragment was inserted into plasmid pSV-VNP gamma SNase in place of VNp to produce pSV-VOVB3 gamma SNase. This plasmid was then transfected into variant OVB3 hybridoma cells, which no longer produced the H chain of antibody OVB3, and functional antibody was secreted by the recipient cells. The recombinant chimeric antibody bound to ovarian cancer cells and contained Staphylococcus aureus nuclease activity, proving that a functional V region gene had been cloned.