Analysis of a suppressive subtractive hybridization library of Alternaria alternata resistant to 2-propenyl isothiocyanate

Background Isothiocyanates (ITCs) are natural products obtained from plants of the Brassicas family. They represent an environmentally friendly alternative for the control of phytopathogenic fungi. However, as it has been observed with synthetic fungicides, the possibility of inducing ITC-resistant strains is a major concern. It is, therefore, essential to understanding the molecular mechanisms of fungal resistance to ITCs. We analyzed a subtractive library containing 180 clones of an Alternaria alternata strain resistant to 2-propenyl ITC (2-pITC). After their sequencing, 141 expressed sequence tags (ESTs) were identified using the BlastX algorithm. The sequence assembly was carried out using CAP3 software; the functional annotation and metabolic pathways identification were performed using the Blast2GO program. Results The bioinformatics analysis revealed 124 reads with similarities to proteins involved in transcriptional control, defense and stress pathways, cell wall integrity maintenance, detoxification, organization and cytoskeleton destabilization; exocytosis, transport, DNA damage control, ribosome maintenance, and RNA processing. In addition, transcripts corresponding to enzymes as oxidoreductases, transferases, hydrolases, lyases, and ligases, were detected. Degradation pathways for styrene, aminobenzoate, and toluene were induced, as well as the biosynthesis of phenylpropanoid and several types of N-glycan. Conclusions The fungal response showed that natural compounds could induce tolerance/resistance mechanisms in organisms in the same manner as synthetic chemical products. The response of A. alternata to the toxicity of 2-pITC is a sophisticated phenomenon including the induction of signaling cascades targeting a broad set of cellular processes. Whole-transcriptome approaches are needed to elucidate completely the fungal response to 2-pITC.

Construction of a virulence-related gene library of Streptococcus mutans by suppression subtractive hybridization / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To construct a suppression subtractive library of virulence-related genes from c serotype Streptococcus mutans (S. mutans), and lay foundations for screening the virulent genes.</p><p><b>METHODS</b>After being isolated from virulent and avirulent strain of S. mutans respectively, the intact and high-pure genomic DNA was digested with three appropriate four-base-cutting restriction endonueleases to produce fragments of optimal length. The digested DNA of the virulent strain ligated with adaptor was used as tester DNA, and that of the avirulent strain as driver DNA. Then the suppression subtractive hybridization was carried out, and the efficiency of ligation and subtraction detected respectively. The subtracted fragments were inserted into vector pCR2. 1 using T/A cloning kit, and transformed into E. coli TOP10F' competent cells. Those white colonies were selected to construct the suppression subtractive library.</p><p><b>RESULTS</b>Alu I chosen from three restriction endonucleases was verified to be suitable for preparing restriction fragments from S. mutans genomic DNA. Through electrophoresis of Alu I -digested DNA, a smear ranged from 0.1 to 2.0 kb was observed. The ligation efficiency of tester DNA with adaptor was at least higher than 25 percent. The subtraction efficiency of suppression subtractive hybridization confirmed the success in enrichment of differential genes between virulent and avirulent strain of S. mutans. In the subtracted group, the appearance time of the 23S rRNA gene both in tester and driver DNA was later than that in the unsubtracted group by six cycles. It suggested that suppression subtractive hybridization happened indeed. After the subtracted fragments were cloned, 96 colonies were picked up for constructing the suppression subtractive library of virulence-related genes of S. mutans.</p><p><b>CONCLUSION</b>Suppression subtractive hybridization allows rapid and easy construction of virulence-related gene library of S. mutans.</p>