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Bacillus strain WYCQ02 was obtained from soil samples by high-temperature screening. A 1,327-bp DNA fragment containing a 1,152-bp long open reading frame named phyC-WYCQ02 was amplified from the genomic DNA of strain WYCQ02 by PCR. The ORF encoded a polypeptide of 383 amino acid residues with a putative signal peptide of 26 amino acids. The 1,089-bp fragment encoding the mature peptide of neutral phytase and a 6 × histidine tag was cloned into the plasmid pPIC9K. The expression vector, pPIC9K-phyC, was linearized and transformed in Pichia pastoris. The molecular weight of phytase was estimated to be approximately 53 kDa by electrophoresis. The optimal temperature of the purified phytase was 55°C and the optimal pH value was between 7.0 and 8.0. After incubation at 70°C for 10 and 30 min, the relative activity was still over 80 and 62%, and over 70% of enzyme activity remained in the pH range of 5.0-10.0. There was no significant difference in enzymatic activity after incubation for 30 or 60 min in buffer with different pH values, therefore the purified phytase had some acid and alkali resistance. The phytase gene and the engineered yeast strain may have value in industrial applications. Copyright © 2013 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Sclerotinia sclerotiorum (Ss) is a devastating fungal pathogen that causes Sclerotinia stem rot in rapeseed (Brassica napus), and is also detrimental to mulberry and many other crops. A wild mulberry germplasm, Morus laevigata, showed high resistance to Ss, but the molecular basis for the resistance is largely unknown. Here, the transcriptome response characteristics of M. laevigata to Ss infection were revealed by RNA-seq. A total of 833 differentially expressed genes (DEGs) were detected after the Ss inoculation in the leaf of M. laevigata. After the GO terms and KEGG pathways enrichment analyses, 42 resistance-related genes were selected as core candidates from the upregulated DEGs. Their expression patterns were detected in the roots, stems, leaves, flowers, and fruits of M. laevigata. Most of them (30/42) were specifically or mainly expressed in flowers, which was consistent with the fact that Ss mainly infects plants through floral organs, and indicated that Ss-resistance genes could be induced by pathogen inoculation on ectopic organs. After the Ss inoculation, these candidate genes were also induced in the two susceptible varieties of mulberry, but the responses of most of them were much slower with lower extents. Based on the expression patterns and functional annotation of the 42 candidate genes, we cloned the full-length gDNA and cDNA sequences of the Ss-inducible chitinase gene set (MlChi family). Phylogenetic tree construction, protein interaction network prediction, and gene expression analysis revealed their special roles in response to Ss infection. In prokaryotic expression, their protein products were all in the form of an inclusion body. Our results will help in the understanding of the molecular basis of Ss-resistance in M. laevigata, and the isolated MlChi genes are candidates for the improvement in plant Ss-resistance via biotechnology. [ABSTRACT FROM AUTHOR]