Highly efficient Agrobacterium rhizogenes-mediated transformation for functional analysis in woodland strawberry.

BACKGROUND: The diploid woodland strawberry (Fragaria vesca) is an excellent model plant for investigating economically significant traits and several genetic resources within the Rosaceae family. Agrobacterium rhizogenes-mediated hairy root transformation is an alternative for exploring gene functions, especially the genes specifically expressed in roots. However, the hairy root transformation has not been established in strawberry. RESULTS: Here, we described an efficient and rapid hairy root transgenic system for strawberry using A. rhizogenes. Strain of A. rhizogenes MSU440 or C58C1 was the most suitable for hairy root transformation. The transformation efficiency was highest when tissues contained hypocotyls as explants. The optimal procedure involves A. rhizogenes at an optical density (OD600) of 0.7 for 10 min and co-cultivation duration for four days, achieving a transgenic efficiency of up to 71.43%. An auxin responsive promoter DR5ver2 carrying an enhanced green fluorescent protein (eGFP) marker was transformed by A. rhizogenes MSU440, thereby generating transgenic hairy roots capable of high eGFP expression in root tip and meristem of strawberry where auxin accumulated. Finally, this system was applied for functional analysis using jGCaMP7c, which could sense calcium signals. A significant upsurge in eGFP expression in the transgenic hairy roots was displayed after adding calcium chloride. The results suggested that this approach was feasible for studying specific promoters and could be a tool to analyze gene functions in the roots of strawberries. CONCLUSION: We established a rapid and efficient hairy root transformation in strawberry by optimizing parameters, which was adequate for promoter analysis and functional characterization of candidate genes in strawberry and other rosaceous plants.

RrTTG1 promotes fruit prickle development through an MBW complex in Rosa roxburghii.

Fruit prickles are widely distributed on the pericarp and exhibit polymorphic traits at different developmental stages. Although they are multicellular appendages that are well-known for helping plants defend against biotic and abiotic stresses, their origination and molecular mechanism are still less known. Here, we studied the origination and molecular mechanism of fruit prickles in Rosa roxburghii. Using morphological and histological observations, we found that the fruit prickle primordium of R. roxburghii originated from the ground meristem that underwent cell division to form flagelliform prickles, continued to enlarge, and finally lignified to form mature fruit prickles. We amplified a homolog of candidate gene TRANSPARENT TESTA GLABRA1 (TTG1) from R. roxburghii, named RrTTG1. RrTTG1 harbored four conserved WD-repeat domains and was exclusively nuclear-localized. Using qRT-PCR and in situ hybridization, we found that RrTTG1 was constitutively expressed and highly expressed during the initiation and cell expansion phases of fruit prickles. Ectopic expression analysis in Arabidopsis proved that RrTTG1 substantially enhanced the number of trichome and pigmentation production and inhibited root hair formation. Besides, RrTTG1 complemented the phenotypes of the ttg1 mutant in Arabidopsis, thus indicating that RrTTG1 played pleiotropic roles akin to AtTTG1. We demonstrated that the RrTTG1 only interacted with RrEGL3, a homolog of ENHANCER OF GLABRA3 (EGL3), via yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays. Briefly, RrTTG1 might positively regulate the initiation of fruit prickle primordium and cell enlargement by forming the RrTTG1-RrEGL3-RrGL1 complex in R. roxburghii. Therefore, our results help characterize the RrTTG1 in R. roxburghii and also elucidate the establishment of the prickles regulatory system in the Rosaceae plants.

Two NF-κB subunits are associated with antimicrobial immunity in Hyriopsis cumingii.

The NF-κB pathway activated by bacteria and viruses produces a series of antimicrobial peptides that participate in the innate immune response. In this study, two NF-κB subunits were cloned and identified from Hyriopsis cumingii (named Hcp65 and Hcp105) using RT-PCR and RACE. The predicted Hcp65 protein possessed a N-terminal Rel homology domain (RHD) and an Ig-like/plexins/transcription factors domain (IPT); the Hcp105 contained an RHD, an IPT domain, 6 ankyrin (ANK) domain and a death domain. Quantitative reverse transcription PCR (qRT-PCR) showed that Hcp65 and Hcp105 were constitutively expressed in the detected tissues, and were significantly up-regulated in hemocytes, hepatopancreas and gill of mussels challenged with lipopolysaccharide (LPS), peptidoglycan (PGN) and polyinosinic-polycytidylic acid (poly I: C). The dsRNA-mediated silencing of Hcp65 and Hcp105 caused significant reduction of immune genes such as lysozyme (HcLyso), theromacin (Hcther), whey acid protein (HcWAP), LPS-binding protein/bactericidal permeability protein (HcLBP/BPI) 1 and 2. In addition, subcellular localization experiments showed that Hcp65 and Hcp105 proteins were expressed in both the nucleus and cytoplasm of HEK-293T cells, and Hcp50 proteins (mature peptide of Hcp105) were mainly localized in the nucleus. The recombinant Hcp65 and Hcp50 protein could form homodimer and heterodimer and bind κB site in vitro. These results provide useful information for understanding the role of NF-κB in mollusks.

The effect of hormonal levels and oxidative stress on bisphenol A and soy isoflavone reproductive toxicity in murine offspring.

Previous studies have suggested that human exposure to bisphenol A (BPA) and soy isoflavones (SIFs) can occur during pregnancy. The combination of these chemicals is hypothesized to have a toxic impact on the fetus. While BPA is an industrial chemical used widely in the manufacture of polycarbonate plastics and epoxy resins, SIFs are naturally occurring estrogen­like phytoestrogens. To determine the impact of the combination of BPA and SIFs on fetal development, the body weight, organ weight, anogenital distance and histopathological changes in the testes of F1 offspring were assessed in mice. Hormonal effects were determined by measuring serum levels of estrogen receptor (ESR), follicle­stimulating hormone (FSH), luteinizing hormone (LH) and testosterone (T). Additionally, mitochondrial DNA copy numbers, and the serum levels of malondialdehyde and superoxide dismutase, were determined to evaluate alterations in oxidative stress and potential toxicity. Exposure to BPA increased the body weight of the pups and reduced the ratio of anogenital distance to body weight, as well as testes weight. Moreover, BPA exposure also induced testicular lesions. The seminiferous tubules of testis were denatured in varying degrees and the lumen wall structure was disordered. The levels of ESR in all offspring and the T levels in male offspring significantly increased, compared with controls. Co­exposure to BPA and SIFs exacerbated these changes in body weight, testicular lesions and hormonal levels, relative to BPA exposure alone. Additionally, oxidative damage was only induced by high­dose BPA. Collectively, these findings suggested that BPA and SIFs could have synergistic effect on the reproductive system, which could be mediated by the regulation of ESR expression and testosterone release.

Deaths of colon neuroendocrine tumors are associated with increasing metastatic lymph nodes and lymph node ratio.

BACKGROUND: Colon neuroendocrine tumors (NETs) are uncommon. Currently, the impact of the number of metastatic lymph nodes (LNs) and lymph node ratio (LNR) on survival has been well investigated in other colon malignancies, but both remain nebulous for patients with colon NETs. METHODS: Surgically resected patients with histologically proven nonmetastatic colon NETs were queried from the Surveillance, Epidemiology, and End Results database between 1988 and 2011. Patients with lymph nodes involved were investigated and categorized into four LNs-based classifications (≤4, >4-10, >10-13, and >13) or three LNR-based subgroups (≤0.51, >0.51-0.71, and >0.71) according to the threshold, determined by Harrell's C statistic. Univariate and multivariate survival analyses were performed by log-rank test and Cox stepwise regression analysis, respectively. RESULTS: Eight hundred fifty-one patients met the inclusion criteria. Among them, higher LNR and LNs classification are associated with a worse prognosis. The 10-year NETs-specific survival rate was 78.3% (74.2-82.6%), 61.3% (52.4-71.7%), 40.8% (20.7-80.7%) for patients in the ≤4, >4-10, and 10-13 LNs groups, respectively. When patients were classified with LNR, the observed 10-year NETs-specific survival rate was 79.9% (74.8-85.5%) for ≤0.51, 57.4% (43.8-75.2%) for >0.51-0.71, and 40.0% (31.0-51.5%) for >0.71. In stratified analysis, higher LNs and LNR groups have worse prognosis only in patients with advanced T stage (T3-T4). Regarding stage migration, the LNR-based system did not show superiority to LNs-based classification. CONCLUSIONS: Current TNM staging classification could be improved by considering the count of metastatic nodes and LNR instead of a simple record of lymph node status (N1 or N0) for colon NETs.

Cloning and characteristic of MMP1 gene from Hyriopsis cumingii and collagen hydrolytic activity of its recombinant protein.

Matrix metalloproteinases (MMPs) play an essential role in a variety of biological processes including wound healing, inflammation, cell invasion, angiogenesis and immune defense. In this study, a putative MMP1 cDNA was cloned and characterized from Hyriopsis cumingii (designated as HcMMP1). The cDNA was 1822 bp in length and encoded a putative protein of 510 amino acids, with a predicted molecular mass of 58.28 kDa and an isoelectric point (pI) of 9.27. HcMMP1 contained all prototype MMPs family signatures, such as signal peptide, prodomain, catalytic center, hinge region, and hemopexin like domain. Quantitative real time-PCR (qRT-PCR) revealed that in mussels HcMMP1 mRNA was expressed in all tissues tested, and the transcriptional expression levels were significantly up-regulated in hepatopancreas and hemocytes after Aeromonas hydrophila, peptidoglycan stimulations and in mantle after wounding. Moreover, the recombination HcMMP1 protein, successfully expressed in Escherichia coli, was purified by affinity chromatography with the concentration of final yield at 0.3 mg/mL. The recombinase had an essentially hydrolytic activity toward rat type I collagen, mouse II and IV collagen after renaturation.

Identification and characterization of two distinct sigma-class glutathione-S-transferase from freshwater bivalve Cristaria plicata.

Glutathione-S-transferases (GSTs) are multifunctional phase II detoxification enzymes that catalyze the attachment of electrophilic substrates to glutathione, and play an important role in protecting organisms against the toxicity of reactive oxygen species. In this study, two distinct sigma-class GST (CpGSTσ1 and 2) cDNA sequences were cloned from freshwater bivalve Cristaria plicata. The full length cDNA of CpGSTσ1 and 2 was 826 bp and 1609 bp, which encoded 213 and 248 amino acid residues, respectively. Their transcripts were expressed in all detected tissues and the highest expression level was in hepatopancreas from C. plicata. The expression level of CpGSTσ1 and 2 in hepatopancreas and hemocytes showed a significantly increased trend after bacterial challenge. The recombinant CpGSTσ1 was successfully expressed as a soluble form in Escherichia coli DE3. The specific activity of recombinase toward CDNB was 46.965 ±â€¯0.082 µmol/min/mg, and its optimum temperature and pH was 37 °C and 9.0, respectively. The recombinant of CpGSTσ1 could bear 6 M urea and 8% SDS, when the concentration of urea was 8 M and its activity was only below 20%. The results might provide a better perspective on the mechanisms of resistance to bacterial infection in molluscs.

Molecular characterization of two distinct Smads gene and their roles in the response to bacteria change and wound healing from Hyriopsis cumingii.

The proteins of Smad family are critical components of the TGF-ß superfamily signal pathway. In this paper, we cloned two intracellular mediators of TGF-ß signaling, Smad3 and Smad5, from the pearl mussel Hyriopsis cumingii. The full length cDNA of HcSmad3 and HcSmad5 were 2052 bp and 1908 bp and encoded two polypeptides of 418 and 461amino acid residues, respectively. The deduced amino acid of HcSmad3 and HcSmad5 possessed two putative conserved domains, MH1 and MH2, a conserved phosphorylation motif SSXS at the carboxyl-terminal. The two Smad genes were detected muscle, mantle, hepatopancreas and gill, but with a very low level in heamocytes. The transcripts of Smad3 and Smad5 were up-regulated in hemocytes and hepatopancreas after A. hydrophila and PGN stimulation. However, the expression of Smad3 and Smad5 were only up-regulated in hepatopancreas after A. hydrophila stimulation. The transcripts of Smad3 and Smad5 had a slight change in hepatopancreas after PGN stimulation. The transcripts of HcSmad3 showed very little increase and HcSmad5 mRNA significantly up-regulated after wounding.