A highly sensitive biotin-based probe for small RNA northern blot and its application in dissecting miRNA function in pepper.

Small RNAs play important roles in regulation of plant development and response to various stresses. Northern blot is an important technique in small RNA research. Isotope- and biotin- (or digoxigenin) labeled probes are frequently used in small RNA northern blot. However, isotope-based probe is limited by strict environmental regulation and availability in many places in the world while biotin-based probe is usually suffered from low sensitivity. In this study, we developed a T4 DNA polymerase-based method for incorporation of a cluster of 33 biotin-labeled C in small RNA probe (T4BC33 probe). T4BC33 probe reaches similar sensitivity as 32P-labeled probe in dot blot and small RNA northern blot experiments. Addition of locked nucleic acids in T4BC33 probe further enhanced its sensitivity in detecting low-abundance miRNAs. With newly developed northern blot method, expression of miR6027 and miR6149 family members was validated. Northern blot analysis also confirmed the successful application of virus-based miRNA silencing in pepper, knocking down accumulation of Can-miR6027a and Can-miR6149L. Importantly, further analysis showed that knocking-down Can-miR6027a led to upregulation of a nucleotide binding-leucine rich repeat domain protein coding gene (CaRLb1) and increased immunity against Phytophthora capsici in pepper leaves. Our study provided a highly sensitive and convenient method for sRNA research and identified new targets for genetic improvement of pepper immunity against P. capsici.

Genomic insights into biased allele loss and increased gene numbers after genome duplication in autotetraploid Cyclocarya paliurus.

BACKGROUND: Autopolyploidy is a valuable model for studying whole-genome duplication (WGD) without hybridization, yet little is known about the genomic structural and functional changes that occur in autopolyploids after WGD. Cyclocarya paliurus (Juglandaceae) is a natural diploid-autotetraploid species. We generated an allele-aware autotetraploid genome, a chimeric chromosome-level diploid genome, and whole-genome resequencing data for 106 autotetraploid individuals at an average depth of 60 × per individual, along with 12 diploid individuals at an average depth of 90 × per individual. RESULTS: Autotetraploid C. paliurus had 64 chromosomes clustered into 16 homologous groups, and the majority of homologous chromosomes demonstrated similar chromosome length, gene numbers, and expression. The regions of synteny, structural variation and nonalignment to the diploid genome accounted for 81.3%, 8.8% and 9.9% of the autotetraploid genome, respectively. Our analyses identified 20,626 genes (69.18%) with four alleles and 9191 genes (30.82%) with one, two, or three alleles, suggesting post-polyploid allelic loss. Genes with allelic loss were found to occur more often in proximity to or within structural variations and exhibited a marked overlap with transposable elements. Additionally, such genes showed a reduced tendency to interact with other genes. We also found 102 genes with more than four copies in the autotetraploid genome, and their expression levels were significantly higher than their diploid counterparts. These genes were enriched in enzymes involved in stress response and plant defense, potentially contributing to the evolutionary success of autotetraploids. Our population genomic analyses suggested a single origin of autotetraploids and recent divergence (~ 0.57 Mya) from diploids, with minimal interploidy admixture. CONCLUSIONS: Our results indicate the potential for genomic and functional reorganization, which may contribute to evolutionary success in autotetraploid C. paliurus.

A Genome-Wide Analysis of StTGA Genes Reveals the Critical Role in Enhanced Bacterial Wilt Tolerance in Potato During Ralstonia solanacearum Infection.

TGA is one of the members of TGACG sequence-specific binding protein family, which plays a crucial role in the regulated course of hormone synthesis as a stress-responsive transcription factor (TF). Little is known, however, about its implication in response to bacterial wilt disease in potato (Solanum tuberosum) caused by Ralstonia solanacearum. Here, we performed an in silico identification and analysis of the members of the TGA family based on the whole genome data of potato. In total, 42 StTGAs were predicted to be distributed on four chromosomes in potato genome. Phylogenetic analysis showed that the proteins of StTGAs could be divided into six sub-families. We found that many of these genes have more than one exon according to the conserved motif and gene structure analysis. The heat map inferred that StTGAs are generally expressed in different tissues which are at different stages of development. Genomic collinear analysis showed that there are homologous relationships among potato, tomato, pepper, Arabidopsis, and tobacco TGA genes. Cis-element in silico analysis predicted that there may be many cis-acting elements related to abiotic and biotic stress upstream of StTGA promoter including plant hormone response elements. A representative member StTGA39 was selected to investigate the potential function of the StTGA genes for further analysis. Quantitative real-time polymerase chain reaction (qRT-PCR) assays indicated that the expression of the StTGAs was significantly induced by R. solanacearum infection and upregulated by exogenous salicylic acid (SA), abscisic acid (ABA), gibberellin 3 (GA3), and methyl jasmonate (MeJA). The results of yeast one-hybrid (Y1H) assay showed that StTGA39 regulates S. tuberosum BRI1-associated receptor kinase 1 (StBAK1) expression. Thus, our study provides a theoretical basis for further research of the molecular mechanism of the StTGA gene of potato tolerance to bacterial wilt.

Different rates of pollen and seed gene flow cause branch-length and geographic cytonuclear discordance within Asian butternuts.

Topological cytonuclear discordance is commonly observed in plant phylogenetic and phylogeographic studies, yet few studies have attempted to detect two other forms of cytonuclear discordance (branch length and geographical) and to uncover the causes of the discordance. We used the whole nuclear and chloroplast genome data from 80 individual Asian butternuts to reveal the pattern and processes of cytonuclear discordance. Our findings indicate that the chloroplast genome had substantially deeper divergence (branch-length discordance) and a steeper cline in the contact zone (geographic discordance) compared with the nuclear genome. After various hypothesis have been tested, the results suggest that incomplete lineage sorting, positive selection and cytonuclear incompatibility are probably insufficient to explain this pattern. However, isolation-by-distance analysis and gene flow estimation point to a much higher level of gene flow by pollen compared with by seeds, which may have slowed down lineage divergence and mediated wider contact for nuclear genome compared with the chloroplast genome. Altogether, this study highlights a critical role of sex-biased dispersal in causing discordance between the nuclear and plastid genome of Asian butternuts. Given its ubiquity among plants, asymmetric gene flow should be given a high priority in future studies of cytonuclear discordance.

StMBF1c positively regulates disease resistance to Ralstonia solanacearum via it's primary and secondary upregulation combining expression of StTPS5 and resistance marker genes in potato.

Multiprotein bridging factor 1 (MBF1) is a transcription coactivator that has a general defense response to pathogens. However, the regulatory mechanisms of MBF1 resistance bacterial wilt remain largely unknown. Here, the role of StMBF1c in potato resistance to Ralstonia solanacearum infection was characterized. qRT-PCR assays indicated that StMBF1c could was elicited by SA, MJ and ABA and the time-course expression pattern of the StMBF1c gene induced by R. solanacearum was found to be twice significant upregulated expression during the early and middle stages of bacterial wilt. Combined with the co-expression analysis of disease-resistant marker genes, gain-of-function and loss-of-function assays demonstrated that StMBF1c was associated with defence priming. Overexpression or silencing the MBF1c could enhance plants resistance or sensitivity to R. solanacearum through inducing or reducing NPR and PR genes related to SA signal pathway. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) experiment results confirmed the interaction of StMBF1c with StTPS5 which played a key role in ABA signal pathway in potato. It is speculated that by combining StTPS5 and resistance marker genes, StMBF1c is activated twice to participate in potato bacterial wilt resistance, in which EPI, PTI involved.

NAC transcription factor involves in regulating bacterial wilt resistance in potato.

Bacterial wilt (BW) is a serious disease that affects potato (Solanum tuberosum L.) production. Although resistance to this disease has been reported, the underlying mechanism is unknown. In this study, we identified a NAC family transcription factor (StNACb4) from potato and characterised its structure, function, expression, its localisation at the tissue and its role in BW resistance. To this end, the transgenic Nicotiana benthamiana Domin lines were generated in which the expression of NACb4 was constitutively upregulated or suppressed using RNAi. Different tobacco mutants were stained after inoculating with Ralstonia solanacearum to observe the cell death and callose deposition. The results indicated that StNACb4 could be upregulated under the induction of R. solanacearum, and salicylic acid, abscisic acid and methyl jasmonate could also induce the expression of StNACb4. Tissue localisation analysis indicated that its expression was tissue specific, and it was mainly in the phloem of the vascular system of stems and leaves. NbNACb4 gene silencing can enhance the sensitivity of tobacco to R. solanacearum; on the contrary, StNACb4 gene overexpression can enhance the tolerance of tobacco to R. solanacearum. Meanwhile, StNACb4 gene overexpression can induce cell death and callose deposition in tobacco. The upregulated expression of StNACb4 can also activate the StPR10 gene expression. Our results provide important new insights into the regulatory mechanisms of bacterial wilt resistance in potato.

[In silico cloning, expression and bioinformatics analysis of StZnT11 in Solanum tuberosum].

Solanum tuberosum Zinc transporter 11 (StZnT11) is very important for maintaining zinc homeostasis in cells. The study on the expression of StZnT11 under abiotic stress and biotic stress laid a foundation for verifying the role of potato StZnT11 in the process of biotic stress of Ralstonia solanacearum species complex. According to the designated EST sequence, the homology of the original sequence was analyzed by using the Blast tool in NCBI, and a homologous object sequence with the highest similarity, coverage and e expectation value was selected. StZnT11 gene is obtained by Silico Cloning. The sequence and coding amino acid composition, physicochemical properties, molecular evolution, phosphorylation site and advanced structure of Solanum tuberosum StZnT11 gene were analyzed by bioinformatics method. The results showed that the cDNA gene is 1 300 bp in length, encoding a protein containing 348 amino acid residues, including 23 phosphorylation sites, one signal peptide and nine transmembrane regions, and is a hydrophobic protein located the plasma membrane. Through amino acid sequence alignment, StZnT11 protein has a high homology with zinc transporter from tobacco, tomato, pepper and other plants. The results of real-time fluorescence quantitative polymerase chain reaction showed that, StZnT11 is up-regulated by different concentrations of exogenous plant hormone abscisic acid (ABA). Tissue localization showed that StZnT11 was mainly expressed in specific tissues (phloem and leaf vascular bundles of stem vascular system). These results provide a theoretical basis for further experimental cloning and functional verification of the gene.

[The influence of elective course of emergency treatment for medical students on the cultivation of first aid knowledge and skill of cardio-pulmonary resuscitation].

OBJECTIVE: To investigate the influence of elective course of emergency treatment for medical students on the cultivation of first aid knowledge and skills of cardio-pulmonary resuscitation. METHODS: Senior students major in medicine of our university were randomly divided into observation group and contrast group with 30 students in each group according to whether an elective course of emergency treatment was given or not. All of them then received a test of first aid knowledge and cardio-pulmonary resuscitation skills. RESULTS: The theoretical exam scores in observation group and contrast group were respectively 78.5+/-9.1 and 46.7+/-15.6. The scores in observation group were significantly higher than that in contrast group (P < 0.01). Cardio-pulmonary resuscitation skills scores in observation group and contrast group were respectively 7.32+/-0.83 and 6.63+/-0.91. The scores in observation group were significantly higher than that in contrast group (P < 0.01). The number of failure for closed cardiac massage in 60 times in observation group and contrast group was respectively 5.06+/-0.58 and 5.77+/-0.63. The number of mouth to mouth artificial respiration in 4 times in observation group and contrast group was 0.92+/-0.16 and 1.10+/-0.17, respectively. There were notable differences in the number of failure in resuscitation maneuvers between two groups (both P < 0.01), observation group being obviously poorer than contrast group. CONCLUSION: An elective course of emergency treatment given to medical students plays an important role in the cultivation of first aid knowledge and skills in cardio-pulmonary resuscitation. It is therefore necessary that emergency medicine is included as a required course in medical college.

[Effects of rosiglitazone on inflammatory reaction and insulin resistance in obese patients with newly diagnosed type 2 diabetes].

OBJECTIVE: To study the effect of rosiglitazone on serum high-sensitivity C-reactive protein (hs-CRP), interleukin-1beta (IL-1beta), IL-6, tumor necrosis factor-alpha (TNF-alpha) and insulin resistance in obese patients with newly diagnosed type 2 diabetes. METHODS: This study involved 118 patients with newly diagnosed type 2 diabetes and obesity, who were randomly assigned into two groups for a 12-week treatment with rosiglitazone (4 mg/day, group A) or sulfonylureas (group B). Serum hs-CRP, IL-1beta, IL-6, TNF-alpha, fasting plasma glucose (FPG) and fasting insulin (FINS) were measured before and after the treatment. Insulin resistance index was calculated according to the HOMA Model. RESULTS: In group A, rosiglitazone treatment resulted in significantly reduced serum hs-CRP, IL-1beta, IL-6, TNF-alpha, FPG and insulin resistance index (P<0.01). No difference in FPG was found between the two groups after the treatment (P>0.05), but serum hs-CRP, IL-1beta, IL-6, TNF-alpha and insulin resistance index were significantly lower in group A than in group B (P<0.05). CONCLUSION: Rosiglitazone can decrease FPG, reduce the inflammation reaction and improve insulin resistance in obese patients with type 2 diabetes.