DDRT PCR PDF

Abstract Background Gene expression studies on non-model organisms require open-end strategies for transcription profiling. Gel-based analysis of cDNA fragments allows to detect alterations in gene expression for genes which have neither been sequenced yet nor are available in cDNA libraries. Both methods have been used merely as qualitative gene discovery tools so far. Results We developed procedures for the conversion of cDNA Differential Display data into quantitative transcription profiles.

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Moustafa, Email: as. Corresponding author. Received Dec 1; Accepted May In this study, Bacillus endophyticus strain SA isolated from the inner tissue of the stem of the cultivated plant Salvadora persica, Asir, Kingdom of Saudi Arabia produces an antagonistic factor. This factor has a broad spectrum of activity against Gram-positive and specifically against Staphylococcus aureus MRSA. The antagonistic factor was isolated from the bacterial culture medium and purified by thin layer chromatography technique, then analyzed by GC—MS analysis.

Identification of the producer strain was performed using the partial nucleotide sequence of 16S rRNA gene, which indicated that this strain is identical to B.

Application of differential display RT-PCR revealed that the isolate was able to up-regulate a gene with serine protease like protein. The protein is well known as antimicrobial agent and was reported to be produced by plants, animals and insects. Serine protease is also known to be produced by bacteria for purposes oth er than bacterial—bacterial antagonistic effect, which has been confirmed by this study.

Keywords: Endophytic, Bacillus endophyticus, Methicillin resistant Staphylococcus aureus, GC—MS, Differential display RT-PCR Introduction Endophytes have been defined as microorganisms bacteria or fungi which for all or part of their life cycle reside within the inner parts of plant tissues, but cause no symptoms of disease Vega et al.

They play an important role in the plant health preservation in addition to their ability to supplement their host plants with inorganic nutrients via nitrogen fixation and iron solubilization processes Porras-Soriano et al.

In addition, these microorganisms play an important role to protect their host plants from phytopathogenic fungi and bacteria Emmert and Handelsman They are able to antagonize the pathogenic microbes through the competition for space and nutrients, production of antibiotics, production of hydrolytic enzymes and by inducing plant defense mechanisms Ryan et al.

The production of antibiotics and hydrolytic enzymes is a feature of many endophytic bacilli, including Bacillus cereus Pleban et al. It has been reported that many endophytic isolates provide beneficial effects to their hosts, like preventing disease development by synthesizing novel compounds and antifungal metabolites Khan and Doty Exploring these novel compounds and metabolites may lead to the discovery of new drugs for antibiotic resistant pathogenic bacteria like methicillin resistant Staphylococcus aureus MRSA.

This study focuses on the isolation of endophytic bacteria which are able to cease and inhibit the growth and spreading of methicillin resistant S. It also tries to understand the mechanism of action using both of chromatographic and molecular levels. Materials and methods Plant materials Salvadora persica L. The sterilized cubic stem parts were then divided into two pieces using a sterilized razor blade.

The cultivated endophytic bacterial isolates were transferred to new nutrient agar NA plates for purification. The grown separate pure colonies of bacterial isolate were tested for their antagonistic activity against Klebsiella pneumoniae, Escherichia coli and methicillin resistant S. Antagonistic effect of SA isolate against S.

Both of the pathogenic strain S. After the incubation period, the S. Comparing the antagonistic effect of the isolated bacteria SA with different antibiotics To rate and determine the exact effect of the isolate SA against S. Both of sterile filter paper discs were loaded with the isolate SA and ten different antibiotic discs. Extraction of the bioactive metabolites produced by SA isolate The extraction of the active metabolites produced by the endophytic isolate SA was done as follows: the surfaces of ten NA plates were spread by S.

The collected agar pieces clear zones were assumed to contain the active materials, and were extracted using absolute ethanol. The mixture was filtered using filter papers to remove the agar medium pieces and the ethanolic filtrate was undergoing to GC—MS analysis as described previously Ezhilan and Neelamegam ; Moustafa et al. After completion, a fraction of the PCR mixture was examined using 1. Molecular detection of the antagonistic factor using differential display RT-PCR technique To investigate the upregulated or downregulated genes during the antagonistic process, both of the pathogenic and the antagonistic bacteria were grown either separately on two different nutrient agar plates or close to each other on the same plate.

The up or down regulated genes were excised from the gel and submitted for purification using a gel purification kit Qiagene, Germany and the purified PCR products were submitted for sequencing Macrogen Inc. Results and discussion Isolation and antimicrobial activity of endophytic bacteria It is well known that, the isolation of bacteria that are capable of invading the inner tissues of a certain plant is a critical process.

Plant surface sterilization is necessary to avoid the co-contamination with superficial bacteria. Using of ethanol and sodium hypochlorite at certain dilutions is recommended for complete removal of the plant surface bacteria Jan et al. Miswak stem parts were surface sterilized followed by the isolation of inner bacterial isolates on NA plates. About ten bacterial colonies with the same colony shape and characteristics were obtained. One of the colonies was chosen and transferred to a new NA plate to ensure complete purification.

The purified bacterium was checked for its ability to antagonize some human pathogenic bacteria including Klebsiella pneumoniae, Escherichia coli and S. The antagonistic effect was only observed with S.

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