Myxobacteria Mrna Differential Expression Of The Method And The Salt-tolerant Myxococcus Hw-1 Mrna Differential Expression Analysis

The detection of differentially expressed genes is of interest in molecular analysis of biological processes including differentiation, development, and carcinogenesis. Differential display(DDRT - PCR,DD), developed by Liang and Pardee in 1992, is a powerful tool for analysing altered gene expression in eukaryotic system. Conventional DD is designed to analyze the mRNAs from eukaryotes, which have poly(A) tails. DD uses 3' anchored oligo(dT) primers for the synthesis of cDNA subsets representative of the poly(A) RNA population. In recent years DD also has been adapted to quantitative environmental stimuli on bacterial gene expression. The general strategy is based on the RNA arbitrary primed PCR (RAP-PCR) method in which one or two arbitrary primers instead of one arbitrary and one anchored oligo-dT primers arc used to reverse-transcribe and amplify the bacterial mRNAs that lack poly(A) tails.The halotolerant strain Myxococcus fulvus HW-1 (ATCC BAA-855) was isolated from a seawater sample. Unlike the normal terrestrial myxobacteria. HW-1 grew in the medium ranging from 0% to 130% seawater and the optimal growth conditions were at 0-80% seawater. The growth, morphology and development of HW-1 shifted in response to salinity. HW-1 cells from high seawater-containing medium grew independently of cell-density, while those from low-seawater media (10% or less) showed density-dependent growth. HW-1 myxospores from high seawater-containing medium had higher heat-toletance than those from low-seawater media (10% or less). The special cell behaviors of HW-1 in response to salinity were very interesting .It deserved further investigation.Conventional genetic analyses of myxobacteria were inefficient and time consuming such as transduction, electro transformation and conjugation. In the present study we first utilized mRNA differential display as a new molecular genetic analytical method of myxobacteria. RAP-PCR was used to identify and isolategenes differential expressed between HW-1 growed in no-seawater media and 50% seawater-containing medium. And the main results as following:1. Developed mRNA differential display protocol of myxobacteria. By testing and comparing, we found SV Total RNA isolation systerm (Promega) is the best protocol of RNA isolation of myxobacteria. We designed a set of 10mer arbitrary primers for high GC-contented myxobacteria. These primers were selected for a high frequency of occurrence within the coding sequences of genome of Myxococcus xanthus DK1622 by statistical analysis. Our results showed that these primers could generate anticipative bands. It also provided referenced primers sequences for other bacteria with high GC-content genome.2. Identified and isolated cDNA fragments differential expressed between HW-1 growed in no-seawater media and 50% seawater-containing medium by using of some designed arbitrary primers. Total RNA and mRNA of HW-1 were submitted to differential dispay by using sets of 10mer arbitrary primers that we designed based on the DK1622 genome sequence.Using enriched mRNA as template can decrease false positives generated by16S and 23S rRNAs from 75% to 50%.3. A further Reverse Northern blot was uesd to confirm the differential expression of those identified genes. Among the nine identified cDNA fragments, which confirmed be differential expressed in 50% seawater-containing medium, two showing homology to DK1622 genes and seven are novel genes in HW-1.