Complete genome sequence of a novel lytic bacteriophage isolated from Ralstonia solanacearum.

A lytic podophage RSPI1 was isolated from tobacco field soil collected in Fujian Province, South China using host bacterium Ralstonia solanacearum Tb15-14. Whole genome sequencing of this phage was performed using the high-throughput Ion Torrent PGM Sequencer. The complete genome of RSPI1 was 43,211 bp in length with a mean DNA G + C content of 61.5%. A total of 48 open reading frames were identified with lengths ranging from 132 bp to 5,061 bp, of which, 11, 12 and 25 were identified as functional, structural and unknown genes, respectively. A BLAST analysis revealed that this phage genome had a query cover of 78-79% and a highest identity of 84% with four podophages that infect Burkholderia pseudomallei. Two neighbor-joining phylogenetic trees were constructed using phage DNA polymerase I and tail fiber protein sequences and showed that this phage is closely related to Burkholderia phage Bp-AMP1, and also related to several phages that infect Ralstonia solanacearum. These findings indicate that RSPI1 is a novel phage that infects the notorious plant pathogen Ralstonia solanacearum.

The Diversity and Geographic Distribution of Cultivable Bacillus-Like Bacteria Across Black Soils of Northeast China.

Bacillus-like species are gram-positive bacteria that are ubiquitous in soils. Many of Bacillus-like bacteria are demonstrated as beneficial microbes widely used in industry and agriculture. However, the knowledge related to their diversity and distribution patterns in soils is still rudimentary. In this study, we developed a combined research method of using culture-dependent and high-throughput sequencing to investigate the composition and diversity of cultivable Bacillus-like bacterial communities across 26 soil samples obtained from the black soil zone in northeast China. Nearly all bacterial 16S rDNA sequences were classified into the order Bacillales. Fifteen genera were detected, with Bacillus, Paenibacillus, and Brevibacillus being the three most abundant genera. Although more than 2,000 OTUs were obtained across all samples, 33 OTUs were confirmed as the abundant species with a relative abundance over 5% in at least one sample. Pairwise analysis showed that the diversity of Bacillus-like bacterial communities were significantly and positively correlated with soil total carbon contents and soil sampling latitudes, which suggests that a latitudinal gradient diversity of Bacillus-like bacterial communities exists in the black soil zone. The principal coordinates analysis revealed that the Bacillus-like bacterial communities were remarkably affected by soil sampling latitudes and soil total carbon content. In general, this study demonstrated that a distinct biogeographic distribution pattern of cultivable Bacillus-like bacterial communities existed in the black soil zone, which emphasizes that the strategy of local isolation and application of beneficial Bacillus-like strains is rather important in black soil agriculture development.

Functional characterization of a potassium transporter gene NrHAK1 in Nicotiana rustica.

The purpose of this study is to investigate the function of a novel potassium transporter gene (NrHAK1) isolated from Nicotiana rustica roots using yeast complement and real-time PCR technique. The complementary DNA (cDNA) of NrHAK1, 2 488 bp long, contains an open reading frame (ORF) of 2 334 bp encoding a protein of 777 amino acids (87.6 kDa) with 12 predicted transmembrane domains. The NrHAK1 protein shows a high sequence similarity to those of high-affinity potassium transporters in Mesembryanthemum, Phytolacca acinosa, Arabidopsis thaliana, and so on. We found that the NrHAK1 gene could complement the yeast-mutant defect in K+ uptake. Among several tissues surveyed, the expression level of NrHAK1 was most abundant in the root tip and was up-regulated when exposed to potassium starvation. Moreover, the transcript accumulation was significantly reduced by adding 5 mmol/L NH4+ to the solution. These results suggest that NrHAK1 plays an important role in potassium absorption in N. rustica.

Directed evolution of beta-galactosidase from Escherichia coli into beta-glucuronidase.

In vitro directed evolution through DNA shuffling is a powerful molecular tool for creation of new biological phenotypes. E. coli beta-galactosidase and beta-glucuronidase are widely used, and their biological function, catalytic mechanism, and molecular structures are well characterized. We applied an in vitro directed evolution strategy through DNA shuffling and obtained five mutants named YG6764, YG6768, YG6769, YG6770 and YG6771 after two rounds of DNA shuffling and screening, which exhibited more beta-glucuronidase activity than wild-type beta-galactosidase. These variants had mutations at fourteen nucleic acid sites, resulting in changes in ten amino acids: S193N, T266A, Q267R, V411A, D448G, G466A, L527I, M543I, Q626R and Q951R. We expressed and purified those mutant proteins. Compared to the wild-type protein, five mutant proteins exhibited high beta-glucuronidase activity. The comparison of molecular models of the mutated and wildtype enzymes revealed the relationship between protein function and structural modification.

[Expression of TMV coat protein gene RNAi in transgenic tobacco plants confer immunity to tobacco mosaic virus infection].

RNAi technique has been proved as a powerful tool for plant breeding. In this paper, the coat protein of tobacco mosaic virus (TMV) was used for constructing the RNAi interference vector. The tobacco varieties K326 and Longjiang 911 were transformed via Agrobacterium tumefaciens-mediated transformation, and transgenic plants were generated. The expression analysis with real-time PCR indicated that TMV RNA had been degraded varied in different transgenic lines. Field assay revealed that 83% and 90 % transgenic plants showed immunity resistance to TMV in K326 and Longjiang 911 respectively.

Isolation and characterization of a novel cDNA encoding ERF/AP2-type transcription factor OsAP25 from Oryza sativa L.

Using a yeast one-hybrid method, a transcription factor, OsAP25, which interacts specifically with a GCC box was isolated from rice. The OsAP25 protein contained a conserved ethylene-responsive element binding factor (ERF) domain which shared identity with other reported ERF domains. Phylogenetic analysis showed that OsAP25 could be categorized into class III ERF of the previously characterized ERF proteins on an evolutionary relationship. The semi-quantitative RT-PCR analysis revealed that OsAP25 gene was constitutively expressed in leaves, roots, growing points, flower, bolting stage and grain filling stage. In addition, OsAP25 gene was induced by NaCl, cold, drought, abscisic acid and exogenous ethylene.