Creation of fertility-restored materials for Ogura CMS in Brassica oleracea by introducing Rfo gene from Brassica napus via an allotriploid strategy.

KEY MESSAGE: Ogura CMS fertility-restored materials, with 18 chromosomes, normal seed setting, stable fertility and closer genetic background to the parent Chinese kale, were successfully developed in B. oleracea via a triploid strategy for the first time. Ogura cytoplasmic male sterility (CMS) is the most widely used sterile type in seed production for commercial hybrids of Brassica oleracea vegetables. However, the natural Ogura CMS restorer line has not been found in B. oleracea crops. In this study, the triploid strategy was used with the aim to create euploid B. oleracea progenies with the Rfo gene. The allotriploid AAC hybrid YL2 was used as a male parent to backcross with Ogura CMS Chinese kale. After successive backcrosses, the BC2 Rfo-positive individual 16CMSF2-11 and its BC3 progenies, with 18 chromosomes, were developed, which were morphologically identical to the parent Chinese kale. Compared with F1 and BC1 plants, it showed stable fertility performance, and regular meiosis behavior and could produce seeds normally under natural pollination. The genomic composition analysis of Rfo-positive progenies by using molecular markers showed that more than 87% of the C-genome components of BC3 Rfo-progenies recovered to the parent Chinese kale, while most or all of the An-genome segments were lost in 16CMSF2-11 and its progenies. The results suggested that the genetic background of Rfo-positive individuals was closer to that of the parent Chinese kale along with backcrossing. Hereof, the Ogura CMS fertility-restored materials of Chinese kale were successfully created via triploid strategy for the first time, providing a bridge for utilizing the Ogura CMS B. oleracea germplasm in the future. Moreover, our study indicates that the triploid strategy is effective for transferring genes from B. napus into B. oleracea.

Inheritance and expression of Bt cry1Ba3 gene in progeny from transformed cabbage plants.

Stable inheritance and expression of transgene are important parameters for successful use of a transgenic crop. We previously transformed a Bt cry1Ba3 gene into cabbage inbred line CA21-3. To evaluate the stability of our Bt cabbage lineages, transgene inheritance and expression were examined in four successive generations under greenhouse conditions. In our study, T1, T2 and T3 progenies of the three independent transgenic lineages (YA-1, YA-2 and YA-3) were generated and then the inheritance and expression of cry1Ba3 were analyzed in sexually derived progeny. Segregation ratio of 2.81:1, 3.27:1 and 3.07:1 was found in T1 progeny of lineages YA-1, YA-2 and YA-3, respectively. Chi-square analysis indicated that these segregation ratios of corresponding population fit the 3:1 ratio. Segregation ratios of the transgene in T2 progeny showed either 3:1 or all expression of cry1Ba3. These data suggest that cry1Ba3 in CA21-3 can be inherited in a Mendelian manner. ELISA analysis of transgenic plants from four generations demonstrated that cry1Ba3 had been stably transmitted to the T3 progeny. Additionally, under artificial infestation conditions, the homozygous T3-YA-1-2-1 line exhibited excellent resistance to Plutella xylostella as compared with un-transformed CA21-3. All these results imply that the three cabbage lineages are genetically stable and can be used to inhibit damage on cabbage caused by P. xylostella.

Genetics and fine mapping of a purple leaf gene, BoPr, in ornamental kale (Brassica oleracea L. var. acephala).

BACKGROUND: Due to its variegated and colorful leaves, ornamental kale (Brassica oleracea L. var. acephala) has become a popular ornamental plant. In this study, we report the fine mapping and analysis of a candidate purple leaf gene using a backcross population and an F2 population derived from two parental lines: W1827 (with white leaves) and P1835 (with purple leaves). RESULTS: Genetic analysis indicated that the purple leaf trait is controlled by a single dominant gene, which we named BoPr. Using markers developed based on the reference genome '02-12', the BoPr gene was preliminarily mapped to a 280-kb interval of chromosome C09, with flanking markers M17 and BoID4714 at genetic distances of 4.3 cM and 1.5 cM, respectively. The recombination rate within this interval is almost 12 times higher than the usual level, which could be caused by assembly error for reference genome '02-12' at this interval. Primers were designed based on 'TO1000', another B. oleracea reference genome. Among the newly designed InDel markers, BRID485 and BRID490 were found to be the closest to BoPr, flanking the gene at genetic distances of 0.1 cM and 0.2 cM, respectively; the interval between the two markers is 44.8 kb (reference genome 'TO1000'). Seven annotated genes are located within the 44.8 kb genomic region, of which only Bo9g058630 shows high homology to AT5G42800 (dihydroflavonol reductase), which was identified as a candidate gene for BoPr. Blast analysis revealed that this 44.8 kb interval is located on an unanchored scaffold (Scaffold000035_P2) of '02-12', confirming the existence of assembly error at the interval between M17 and BoID4714 for reference genome '02-12'. CONCLUSIONS: This study identified a candidate gene for BoPr and lays a foundation for the cloning and functional analysis of this gene.

Recessive male sterility in cabbage (Brassica oleracea var. capitata) caused by loss of function of BoCYP704B1 due to the insertion of a LTR-retrotransposon.

KEY MESSAGE: The LTR-retrotransposon insertion in BoCYP704B1 is proved to be the primary cause of the male sterility in cabbage. Effective allele-specific markers were developed for marker-assisted selection of male sterile gene. 83121A is a spontaneous male sterile mutant identified from cabbage. Genetic analysis indicated that male sterility is controlled by a single recessive gene. Pollen wall formation in the 83121A mutant was severely defective, with a lack of sporopollenin or exine. To understand the mechanisms of male sterility in 83121A, transcription analysis using RNA-Seq was carried out in the buds of the male sterile line 83121A and the male fertile line 83121B, which are near-isogenic lines differing only in the fertility trait. Via expression analysis of differentially expressed genes involved in pollen exine development before the bicellular pollen stage, BoCYP704B1 was identified as a candidate gene, which was approximately downregulated 30-fold in 83121A. BoCYP704B1 is a member of the evolutionarily conserved CYP704B family, which is essential for sporopollenin formation. The BoCYP704B1 transcript is specifically detected in the developing anthers of wild-type cabbage. Further sequence analysis revealed that a 5424-bp long terminal repeat-retrotransposon (LTR-RT) was inserted into the first exon of BoCYP704B1 in 83121A, which is not found in wild-type plants. The insertion of LTR-RT not only reduced the expression of BoCYP704B1 but also altered structure of protein encoded by BoCYP704B1. Moreover, linkage analysis showed that the homozygotic mutational BoCYP704B1 always cosegregated with male sterility. These data suggest that the LTR-RT insertion in BoCYP704B1 hinders sporopollenin formation in 83121A leading to male sterility. The allele-specific markers developed in this study were effective for marker-assisted selection of the male sterile gene.

Development of a novel allele-specific Rfo marker and creation of Ogura CMS fertility-restored interspecific hybrids in Brassica oleracea.

KEY MESSAGE: A novel allele-specific Rfo marker was developed and proved to be effective for MAS of Rfo gene in B. oleracea background and six Ogu-CMS fertility-restored interspecific hybrids were created for the first time. Ogura cytoplasmic male sterility (Ogu-CMS) has been extensively used for Brassica oleracea hybrid production. However, because of maternal inheritance, all the hybrids produced by CMS lines are male sterile and cannot be self-pollinated, which prohibits germplasm maintenance and innovation. This problem can be overcome by using the Ogu-CMS restorer line, but restorer material is absent in B. oleracea crops. Here, Rfo, a fertility-restored gene of Ogu-CMS, was transferred from rapeseed restorer lines into a Chinese kale Ogu-CMS line using interspecific hybridization combined with embryo rescue. Nine interspecific, triploid plant progenies were identified at morphological and ploidy level, with phenotypes intermediate between those of rapeseed and Chinese kale. Because the Rfo marker (Hu et al., Mol Breeding 22:663-674, 2008) cannot distinguish the Rfo and its homologies under a B. oleracea background, a novel allele-specific Rfo marker was developed based on the BLAST analysis of highly homologous Rfo sequences in B. oleracea. Screening using the novel Rfo marker found that six interspecific hybrids carrying Rfo were also fertile, although fertility varied during different flowering periods. Furthermore, BC1 offsprings with the Rfo gene were selected with the allele-specific Rfo marker and showed restored fertility. These results indicated that the novel allele-specific marker could be used for the MAS of Rfo gene in B. oleracea, and this study lays the foundation for the development of Ogu-CMS restorer material in cabbage and its related other subspecies.

Endotoxin molecule lipopolysaccharide-induced zebrafish inflammation model: a novel screening method for anti-inflammatory drugs.

Lipopolysaccharide (LPS), an endotoxin molecule, has been used to induce inflammatory responses. In this study, LPS was used to establish an in vivo inflammation model in zebrafish for drug screening. We present an experimental method that conveniently and rapidly assesses the anti-inflammatory properties of drugs. The yolks of 3-day post-fertilization (dpf) larvae were injected with 0.5 mg/mL LPS to induce fatal inflammation. After LPS stimulation, macrophages were tracked by NR and SB staining and neutrophil migration was observed using the MPO:GFP line. Larval mortality was used as the primary end-point. Expression levels of key cytokines involved in the inflammatory response including IL-1ß, IL-6, and TNF-α, were measured using quantitative reverse transcription polymerase chain reaction (RT-PCR). Macrophages and neutrophils were both recruited to the LPS-injected site during the inflammatory response. Mortality was increased by LPS in a dose-dependent manner within 48 h. Analyses of IL-1ß, IL-6, and TNF-α expression levels revealed the upregulation of the inflammatory response in the LPS-injected larvae. Further, the anti-inflammatory activity of chlorogenic acid (CA) was evaluated in this zebrafish model to screen for anti-inflammatory drugs. A preliminary result showed that CA revealed a similar effect as the corticosteroid dexamethasone (DEX), which was used as a positive control, by inhibiting macrophage and neutrophil recruitment to the LPS site and improving survival. Our results suggest that this zebrafish screening model could be applied to study inflammation-mediated diseases. Moreover, the Traditional Chinese Medicine CA displays potential anti-inflammatory activity.

[Progress of researches on acupuncture-moxibustion promoting wound repair].

Wound is a common surgical disease characterized by skin defect or functional limitation. Studies have found that acupuncture-moxibustion plays an important role in wound healing. In this paper, we reviewed the mechanisms of acupuncture-moxibustion in promoting wound repair. Outcomes display that acupuncture-moxibustion has an action in promoting wound restoration by improving wound flow perfusion, promoting angiogenesis, increasing the number of fibroblasts and regulating collagen synthesis. In addition, acupuncture can effectively promote wound healing by controlling the release of inflammatory cytokines, up-regulating the expression of growth factors such as vascular endothelial growth factor and transforming growth factor-ß1, and affecting phosphatidylinositol-3-kinase/protein kinase B, mitogen-activated protein kinase and advanced glycation end products/receptor for AGEs signaling pathways. Based on the above studies, it is highly recommended that future studies should pay more attention to the multi-mechanism coordinated regulation target center, and the therapeutic means and dose-effect relationship of acupuncture-moxibustion in tissue repair.

Scutellarin benzyl ester partially secured the ischemic injury by its anti-apoptosis mechanism in cardiomyocytes of neonatal rats.

OBJECTIVE: To investigate the protective effects of scutellarin benzyl ester on neonatal rats' cardiomyocytes injured by ischemia and its anti-apoptosis mechanism. METHODS: The cardiomyocytes in primary culture were prepared from ventricular tissue of 1- to 3-day-old Sprague-Dawley rats and the cells in good condition were assigned to five groups: control group, ischemic model group and three scutellarin benzyl ester groups (doses of 100, 50 and 25µmol/L, respectively). The model of ischemic injury was established in the primary culture of cardiomyocytes under glucose-free anoxic condition. After ischemia for 6 h, the metabolic ability of the cells was detected by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) assay and the activity of lactate dehydrogenase (LDH) in the media was measured by biochemistry approaches. The nuclear damage was revealed by Hoechst-propidium iodide staining. The percentage of apoptotic cells was monitored by flow cytometry. The expression levels of cytochrome c and caspase-3 mRNAs and proteins were determined by reverse transcription-polymerase chain reaction and Western blotting, respectively. RESULTS: After exposure to ischemic condition, the cell viability of the model group was degraded compared with that of the control group (P<0.01) and scutellarin benzyl ester (high and medium doses) could attenuate the loss of cell viability induced by ischemia (P<0.01 and P<0.05). In addition, each dose of scutellarin benzyl ester could significantly reduce the release of LDH from cardiomyocytes injured by ischemia (P<0.01). In morphology, the injured nuclei presented significant changes such as condensation of chromatin, and shrinkage and fragmentation of nuclei, which could be attenuated remarkably by pretreatment with scutellarin benzyl ester. Furthermore, scutellarin benzyl ester could significantly decrease the percentage of apoptosis induced by ischemia (P<0.01) and inhibit the increased expression levels of cytochrome c and caspase-3 mRNAs and proteins (<0.01). CONCLUSION: Scutellarin benzyl ester has a remarkable protective effect against myocardial ischemic injury and the protective mechanism may associate with its anti-apoptosis effect by inhibiting cytochrome c release and caspase-3 activation.

Synthesis of a virus electrode for measurement of prostate specific membrane antigen.

Though relatively unexploited in biosensor applications, phage display technology can provide versatile recognition scaffolds for detection of cancer markers and other analytes. This chapter details protocols for covalent attachment of viruses directly to electrodes for reagent-free detection of analytes in real-time. Customization of binding specificity leverages selections with large phage display libraries prior to covalent attachment of the selected virus to the electrode. The methods described here utilize electrochemical impedance spectroscopy (EIS) to detect molecular recognition between M13 phage bound to a Au electrode and the following analytes: prostate specific membrane antigen (PSMA), positive and negative control antibodies (p-Ab and n-Ab, respectively). Because of a thick layer built on the Au electrode, the real impedance (Zre) increases reliably with S/N ratios upon noncovalent binding to PSMA (approximately 14) and p-Ab (approximately 20).

Direct electrical transduction of antibody binding to a covalent virus layer using electrochemical impedance.

Electrochemical impedance spectroscopy is used to detect the binding of a 148.2 kDa antibody to a "covalent virus layer" (CVL) immobilized on a gold electrode. The CVL consisted of M13 phage particles covalently anchored to a 3 mm diameter gold disk electrode. The ability of the CVL to distinguish this antibody ("p-Ab") from a second, nonbinding antibody ("n-Ab") was evaluated as a function of the frequency and phase of the measured current relative to the applied voltage. The binding of p-Ab to the CVL was correlated with a change in the resistance, reducing it at low frequency (1-40 Hz) while increasing it at high frequency (2-140 kHz). The capacitance of the CVL was virtually uncorrelated with p-Ab binding. At both low and high frequency, the electrode resistance was linearly dependent on the p-Ab concentration from 20 to 266 nM but noise compromised the reproducibility of the p-Ab measurement at frequencies below 40 Hz. A "signal-to-noise" ratio for antibody detection was computed based upon the ratio between the measured resistance change upon p-Ab binding and the standard deviation of this change obtained from multiple measurements. In spite of the fact that the impedance change upon p-Ab binding in the low frequency domain was more than 100 times larger than that measured at high frequency, the S/N ratio at high frequency was higher and virtually independent of frequency from 4 to 140 kHz. Attempts to release p-Ab from the CVL using 0.05 M HCl, as previously described for mass-based detection, caused a loss of sensitivity that may be associated with a transition of these phage particles within the CVL from a linear to a coiled conformation at low pH.

Genome-wide identification of small RNAs in Bifidobacterium animalis subsp. lactis KLDS 2.0603 and their regulation role in the adaption to gastrointestinal environment.

OBJECTIVE: Bifidobacteria are one of the predominant bacterial species in the human gastrointestinal tract (GIT) and play a vital role in the host's health by acting as probiotics. However, how they regulate themselves to adapt to GIT of their host remains unknown. METHODS: Eighteen bifidobacterial strains were used to analyze their adaptive capacities towards simulated GIT environment. The strain with highest survival rate and adhesion ability was selected for comparative genome as well as transcriptomic analysis. RESULTS: The Bifidobacterium animalis subsp. lactis KLDS 2.0603 strain was demonstrated to have the highest survival rate and adhesion ability in simulated GIT treatments. The comparative genome analysis revealed that the KLDS 2.0603 has most similar whole genome sequence compared with BB-12 strain. Eleven intergenic sRNAs were identified after genomes prediction and transcriptomic analysis of KLDS 2.0603. Transcriptomic analysis also showed that genes (mainly sRNAs targeted genes) and sRNAs were differentially expressed in different stress conditions, suggesting that sRNAs might play a crucial role in regulating genes involved in the stress resistance of this strain towards environmental changes. CONCLUSIONS: This study first provided deep and comprehensive insights into the regulation of KLDS 2.0603 strain at transcription and post-transcription level towards environmental.

[Epidemiological characteristics on the clustering nature of pandemic (H1N1) 2009 in China].

OBJECTIVE: To study the epidemiological characteristics on the clustering nature of pandemic (H1N1) 2009 in China. METHODS: Time and place distribution of pandemic (H1N1) 2009 on the nature of clustering through data from Public Health Emergency Management Information System were described. RESULTS: As of August 10, 2010, 2773 pandemic (H1N1) 2009 clusters, a total of 77 363 cases (including 20 deaths) were reported in the mainland of China. The most reported number of clusters was from schools and kindergartens with the total number of 2498 (accounted for 90.08% of the total number). Middle schools appeared the have the most clusters (1223, accounting for 48.96%). The number of clusters reported in the southern provinces (cities) accounted for 77.03% of the total, and was more than that in the northern provinces (cities). Two reported peaks in the southern provinces (cities) were in June and November, 2009, respectively. There was only one reported peak in the northern provinces in September, 2009. CONCLUSION: Time and place distribution characteristics on the clusters of pandemic (H1N1) 2009 were similar to the seasonal influenza, but the beginning of winter peak was much earlier and intensity of reporting was much higher on the clusters of pandemic (H1N1) 2009 than that of seasonal influenza.

Covalent virus layer for mass-based biosensing.

M13 virus particles were covalently attached to a planar gold-coated quartz crystal microbalance (QCM) through reaction with a self-assembled monolayer of N-hydroxysuccinimide thioctic ester, followed by incorporation of the blocking agent bovine serum albumin. This immobilization chemistry produced a phage multilayer having a coverage equivalent to approximately 6.5 close-packed monolayers of the virus. The properties of this "covalent virus surface" or CVS for the mass-based detection of a 148.2 kDa antibody were then evaluated in a phosphate buffer using a flow injection analysis system. The mass of the CVS increased with exposure to an antibody (p-Ab) known to bind the phage particles with high affinity. Bound p-Ab was removed by washing with 0.5 M HCl thereby regenerating the sensor surface. A calibration plot for p-Ab binding was constructed by repetitively exposing the surface to p-Ab at concentrations between 6.6 and 200 nM and HCl rinsing after each exposure. The mass-concentration relationship was linear with a sensitivity of 0.018 microg/(cm2 nM) and a limit of detection of 7 nM or 1.3 pmol. The CVS could be saturated with high doses of p-Ab enabling the determination that an average of approximately 140 binding sites are available per M13 phage particle. Exposure of the CVS to a second, nonbinding antibody (n-Ab) did not cause a measurable mass change. These results demonstrate that the covalent virus layer is a rugged, selective, and sensitive means for carrying out mass-based biodetection.

Virus electrodes for universal biodetection.

A dense virus layer, readily tailored for recognition of essentially any biomarker, was covalently attached to a gold electrode surface through a self-assembled monolayer. The resistance of this "virus electrode", Z(Re), measured in the frequency range from 2 to 500 kHz in a salt-based pH 7.2 buffer, increased when the phage particles selectively bound either an antibody or prostate-specific membrane antigen (PSMA), a biomarker for prostate cancer. In contrast to prior results, we show the capacitive impedence of the virus electrode, Z(Im), is both a noisier and a less sensitive indicator of this binding compared to Z(Re). The specificity of antibody and PSMA binding, and the absence of nonspecific binding to the virus electrode, was confirmed using quartz crystal microbalance gravimetry.