Expression Analyses of Soybean VOZ Transcription Factors and the Role of GmVOZ1G in Drought and Salt Stress Tolerance.

Vascular plant one-zinc-finger (VOZ) transcription factor, a plant specific one-zinc-finger-type transcriptional activator, is involved in regulating numerous biological processes such as floral induction and development, defense against pathogens, and response to multiple types of abiotic stress. Six VOZ transcription factor-encoding genes (GmVOZs) have been reported to exist in the soybean (Glycine max) genome. In spite of this, little information is currently available regarding GmVOZs. In this study, GmVOZs were cloned and characterized. GmVOZ genes encode proteins possessing transcriptional activation activity in yeast cells. GmVOZ1E, GmVOZ2B, and GmVOZ2D gene products were widely dispersed in the cytosol, while GmVOZ1G was primarily located in the nucleus. GmVOZs displayed a differential expression profile under dehydration, salt, and salicylic acid (SA) stress conditions. Among them, GmVOZ1G showed a significantly induced expression in response to all stress treatments. Overexpression of GmVOZ1G in soybean hairy roots resulted in a greater tolerance to drought and salt stress. In contrast, RNA interference (RNAi) soybean hairy roots suppressing GmVOZ1G were more sensitive to both of these stresses. Under drought treatment, soybean composite plants with an overexpression of hairy roots had higher relative water content (RWC). In response to drought and salt stress, lower malondialdehyde (MDA) accumulation and higher peroxidase (POD) and superoxide dismutase (SOD) activities were observed in soybean composite seedlings with an overexpression of hairy roots. The opposite results for each physiological parameter were obtained in RNAi lines. In conclusion, GmVOZ1G positively regulates drought and salt stress tolerance in soybean hairy roots. Our results will be valuable for the functional characterization of soybean VOZ transcription factors under abiotic stress.

The Soybean bZIP Transcription Factor Gene GmbZIP2 Confers Drought and Salt Resistances in Transgenic Plants.

Abiotic stresses, such as drought and salt, are major environmental stresses, affecting plant growth and crop productivity. Plant bZIP transcription factors (bZIPs) confer stress resistances in harsh environments and play important roles in each phase of plant growth processes. In this research, 15 soybean bZIP family members were identified from drought-induced de novo transcriptomic sequences of soybean, which were unevenly distributed across 12 soybean chromosomes. Promoter analysis showed that these 15 genes were rich in ABRE, MYB and MYC cis-acting elements which were reported to be involved in abiotic stress responses. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis indicated that 15 GmbZIP genes could be induced by drought and salt stress. GmbZIP2 was significantly upregulated under stress conditions and thus was selected for further study. Subcellular localization analysis revealed that the GmbZIP2 protein was located in the cell nucleus. qRT-PCR results show that GmbZIP2 can be induced by multiple stresses. The overexpression of GmbZIP2 in Arabidopsis and soybean hairy roots could improve plant resistance to drought and salt stresses. The result of differential expression gene analysis shows that the overexpression of GmbZIP2 in soybean hairy roots could enhance the expression of the stress responsive genes GmMYB48, GmWD40, GmDHN15, GmGST1 and GmLEA. These results indicate that soybean bZIPs played pivotal roles in plant resistance to abiotic stresses.

Comprehensive Profiling of Tubby-Like Proteins in Soybean and Roles of the GmTLP8 Gene in Abiotic Stress Responses.

Tubby-like proteins (TLPs) are transcription factors that are widely present in eukaryotes and generally participate in growth and developmental processes. Using genome databases, a total of 22 putative TLP genes were identified in the soybean genome, and unevenly distributed across 13 chromosomes. Phylogenetic analysis demonstrated that the predicted GmTLP proteins were divided into five groups (I-V). Gene structure, protein motifs, and conserved domains were analyzed to identify differences and common features among the GmTLPs. A three-dimensional protein model was built to show the typical structure of TLPs. Analysis of publicly available gene expression data showed that GmTLP genes were differentially expressed in response to abiotic stresses. Based on those data, GmTLP8 was selected to further explore the role of TLPs in soybean drought and salt stress responses. GmTLP8 overexpressors had improved tolerance to drought and salt stresses, whereas the opposite was true of GmTLP8-RNAi lines. 3,3-diaminobenzidine and nitro blue tetrazolium staining and physiological indexes also showed that overexpression of GmTLP8 enhanced the tolerance of soybean to drought and salt stresses; in addition, downstream stress-responsive genes were upregulated in response to drought and salt stresses. This study provides new insights into the function of GmTLPs in response to abiotic stresses.

Genome-Wide Analysis of the Catharanthus roseus RLK1-Like in Soybean and GmCrRLK1L20 Responds to Drought and Salt Stresses.

Abiotic stresses, such as drought and salinity, severely affects the growth, development and productivity of the plants. The Catharanthus roseus RLK1-like (CrRLK1L) protein kinase family is involved in several processes in the plant life cycle. However, there have been few studies addressing the functions of CrRLK1L proteins in soybean. In this study, 38 CrRLK1L genes were identified in the soybean genome (Glycine max Wm82.a2.v1). Phylogenetic analysis demonstrated that soybean CrRLK1L genes were grouped into clusters, cluster I, II, III. The chromosomal mapping demonstrated that 38 CrRLK1L genes were located in 14 of 20 soybean chromosomes. None were discovered on chromosomes 1, 4, 6, 7, 11, and 14. Gene structure analysis indicated that 73.6% soybean CrRLK1L genes were characterized by a lack of introns.15.7% soybean CrRLK1L genes only had one intron and 10.5% soybean CrRLK1L genes had more than one intron. Five genes were obtained from soybean drought- and salt-induced transcriptome databases and were found to be highly up-regulated. GmCrRLK1L20 was notably up-regulated under drought and salinity stresses, and was therefore studied further. Subcellular localization analysis revealed that the GmCrRLK1L20 protein was located in the cell membrane. The overexpression of the GmCrRLK1L20 gene in soybean hairy roots improved both drought tolerance and salt stresses and enhanced the expression of the stress-responsive genes GmMYB84, GmWRKY40, GmDREB-like, GmGST15, GmNAC29, and GmbZIP78. These results indicated that GmCrRLK1L20 could play a vital role in defending against drought and salinity stresses in soybean.

Genome-Wide Analysis of the GRAS Gene Family and Functional Identification of GmGRAS37 in Drought and Salt Tolerance.

GRAS genes, which form a plant-specific transcription factor family, play an important role in plant growth and development and stress responses. However, the functions of GRAS genes in soybean (Glycine max) remain largely unknown. Here, 117 GRAS genes distributed on 20 chromosomes were identified in the soybean genome and were classified into 11 subfamilies. Of the soybean GRAS genes, 80.34% did not have intron insertions, and 54 pairs of genes accounted for 88.52% of duplication events (61 pairs). RNA-seq analysis demonstrated that most GmGRASs were expressed in 14 different soybean tissues examined and responded to multiple abiotic stresses. Results from quantitative real-time PCR analysis of six selected GmGRASs suggested that GmGRAS37 was significantly upregulated under drought and salt stress conditions and abscisic acid and brassinosteroid treatment; therefore, this gene was selected for further study. Subcellular localization analysis revealed that the GmGRAS37 protein was located in the plasma membrane, nucleus, and cytosol. Soybean hairy roots overexpressing GmGRAS37 had improved resistance to drought and salt stresses. In addition, these roots showed increased transcript levels of several drought- and salt-related genes. The results of this study provide the basis for comprehensive analysis of GRAS genes and insight into the abiotic stress response mechanism in soybean.

Genome-Wide Identification, Evolution, and Expression of GDSL-Type Esterase/Lipase Gene Family in Soybean.

GDSL-type esterase/lipase proteins (GELPs) belong to the SGNH hydrolase superfamily and contain a conserved GDSL motif at their N-terminus. GELPs are widely distributed in nature, from microbes to plants, and play crucial roles in growth and development, stress responses and pathogen defense. However, the identification and functional analysis of GELP genes are hardly explored in soybean. This study describes the identification of 194 GELP genes in the soybean genome and their phylogenetic classification into 11 subfamilies (A-K). GmGELP genes are disproportionally distributed on 20 soybean chromosomes. Large-scale WGD/segmental duplication events contribute greatly to the expansion of the soybean GDSL gene family. The Ka/Ks ratios of more than 70% of duplicated gene pairs ranged from 0.1-0.3, indicating that most GmGELP genes were under purifying selection pressure. Gene structure analysis indicate that more than 74% of GmGELP genes are interrupted by 4 introns and composed of 5 exons in their coding regions, and closer homologous genes in the phylogenetic tree often have similar exon-intron organization. Further statistics revealed that approximately 56% of subfamily K members contain more than 4 introns, and about 28% of subfamily I members consist of less than 4 introns. For this reason, the two subfamilies were used to simulate intron gain and loss events, respectively. Furthermore, a new model of intron position distribution was established in current study to explore whether the evolution of multi-gene families resulted from the diversity of gene structure. Finally, RNA-seq data were used to investigate the expression profiles of GmGELP gene under different tissues and multiple abiotic stress treatments. Subsequently, 7 stress-responsive GmGELP genes were selected to verify their expression levels by RT-qPCR, the results were consistent with RNA-seq data. Among 7 GmGELP genes, GmGELP28 was selected for further study owing to clear responses to drought, salt and ABA treatments. Transgenic Arabidopsis thaliana and soybean plants showed drought and salt tolerant phenotype. Overexpression of GmGELP28 resulted in the changes of several physiological indicators, which allowed plants to adapt adverse conditions. In all, GmGELP28 is a potential candidate gene for improving the salinity and drought tolerance of soybean.

[Advanced biological studies on the cytoplasmic male sterility and its fertility restoration in plants].

It was reviewed here that the major advances of biological research on the cytoplasmic male sterility (cms) and its fertility restoration in plants in the past decade. It focused on the identification of the cms- associated regions in the plant mitochondrial genome and their expressing profile, the possible mechanisms of cms and of fertility restoration, the genetics and localization of the restorer genes and their molecular characteristics. Sequencing the mitochondrial genome in Arabidopsis thaliana and rice were completed and their bio-information and subsequent researching advances would greatly impelled the similar work in other plants.

MEK1 and MEK2 differentially regulate human insulin- and insulin glargine-induced human bladder cancer T24 cell proliferation / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Increased risk of bladder cancer has been reported in diabetic patients. This study was to investigate the roles of mitogen-activated protein kinase kinase (MEK) 1 and 2 in the regulation of human insulin- and insulin glargine-induced proliferation of human bladder cancer T24 cells.</p><p><b>METHODS</b>In the absence or presence of a selective inhibitor for MEK1 (PD98059) or a specific siRNA for MEK2 (siMEK2), with or without addition of insulin or glargine, T24 cell proliferation was evaluated by cell counting kit (CCK)-8 assay. Protein expression of MEK2, phosphorylation of ERK1/2 and Akt was analyzed by Western blotting.</p><p><b>RESULTS</b>T24 cell proliferation was promoted by PD98059 at 5 - 20 µmol/L, inhibited by siMEK2 at 25 - 100 nmol/L. PD98059 and siMEK2 remarkably reduced phosphorylated ERK1/2. Insulin- and glargine-induced T24 cell proliferation was enhanced by PD98059, suppressed while not blocked by siMEK2. Insulin- and glargine-induced ERK1/2 activation was blocked by PD98059 or siMEK2 treatment, whereas activation of Akt was not affected.</p><p><b>CONCLUSION</b>MEK1 inhibits while MEK2 contributes to normal and human insulin- and insulin glargine-induced human bladder cancer T24 cell proliferation.</p>

Comparison of the surgical stress between endoscopic thyroidectomy via anterior chest approach and conventional thyroidectomy / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To evaluate the difference in surgical stress between gasless endoscopic thyroidectomy through anterior chest approach and conventional thyroidectomy.</p><p><b>METHODS</b>The patients with thyroid nodules who would undergo thyroidectomy between November 2006 and February 2008 in Department of Otorhinolaryngology Head and Neck Surgery, Second Affiliated Hospital of Sun Yat-sen University, were randomly divided into gasless endoscopic thyroidectomy group or conventional thyroidectomy group, with 25 cases and 22 cases respectively. Before and after surgery, white blood cell count (WBC), serum C-reactive protein (CRP) and interleukin-6 (IL-6) were measured to assess the surgical stress response.</p><p><b>RESULTS</b>At 12 h, 24 h and 48 h after surgery, no significant difference was found between the two groups in WBC (t = -0.172, 1.774 and 2.039 respectively, P > 0.05), serum CRP (t = -0.927, -1.701 and -1.813, P > 0.05) and IL-6 (t = 0.098, -2.019 and -1.121, P > 0.05).</p><p><b>CONCLUSION</b>The stress response of gasless endoscopic thyroidectomy is similar with that of conventional thyroidectomy.</p>

Construction and selection of siRNA expression cassettes targeting human telomerase reverse transcriptase gene in vitro / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To determine whether the human telomerase reverse transcriptase (hTERT) gene silencing could be effectively induced by PCR-derived siRNA expression cassettes (SEC) transfected by the fifth generation polyamidoamine dendrimer (G5 PAMAM-D) in Tca8113 cells.</p><p><b>METHODS</b>Four SEC were rationally designed and constructed based on a two-step PCR reaction. The SEC were then transferred into Tca8113 cells using G5 PAMAM-D, and hTERT expression was investigated by real-time fluorescence-quantitative reverse transcriptase-PCR and western blot analysis.</p><p><b>RESULTS</b>The RNA interference effects of the SEC targeted for varying hTERT mRNA positions showed a significant disparity. Among them, SEC-A revealed the most potent inhibitory effects (above 95% of reduction), followed by SEC-D and SEC-C, and SEC-B had no effect on hTERT expression (P > 0.05). That the endogenous hTERT gene silencing induced by G5 PAMAM dendrimer-mediated SEC-A was highly sequence-specific, and multiple transfection as well as properties of the vectors were routinely attributable to the specific suppression.</p><p><b>CONCLUSIONS</b>Specific inhibition of endogenous hTERT expression by use of a PCR-based short hairpin siRNA technique and dendrimer transfer system may serve as a novel strategy for treatment of tongue cancers expressing hTERT in vitro.</p>

Estrogen receptor alpha gene polymorphism associated with type 2 diabetes mellitus and the serum lipid concentration in Chinese women in Guangzhou / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Estrogen might play an important role in type 2 diabetes mellitus pathogenesis. A number of polymorphisms have been reported in the estrogen receptor alpha (ERalpha) gene (also named ESR1), including the XbaI and PvuII restriction enzyme polymorphisms of ESR1, which may be involved in disease pathogenesis. The aim of this study was to determine whether ERX gene polymorphisms are associated with type 2 diabetes mellitus and serum lipid level.</p><p><b>METHODS</b>Two hundred and ninety-nine patients with type 2 diabetes mellitus were compared with three hundred and forty-one health controls of Guangzhou in China, both were male and postmenopausal female residents at 51 - 70 years. ESR1 genotyping was performed using polymerase chain reaction (PCR) and PvuII and XbaI restriction fragment length polymorphism (PCR-RFLP) analysis.</p><p><b>RESULTS</b>ESR1 allelic frequencies of P, p and X, x alleles were 0.408, 0.592; 0.360, 0.640 in the type 2 diabetes mellitus group and 0.318, 0.682; 0.328, 0.672 in the control group, respectively. In case-control study, there was significant difference in PvuII, but not XbaI, allele frequency between the type 2 diabetes mellitus and control groups (P = 0.001 and P = 0.122). When the group was separated into men and women, the difference was significant in women (P < 0.001) but not in men (P = 0.854) with the PvuII genotype, and the effect of PvuII variant on the development of type 2 diabetes mellitus was improved with aging. In addition, PvuII genotype was associated with blood glucose [fasting blood glucose (FBG), postprandial blood glucose (PBG)] and serum lipid [total cholesterol (TC) and low density lipoprotein (LDL)-c] concentration in healthy women.</p><p><b>CONCLUSIONS</b>PvuII polymorphism of ESR1 increases susceptibility to type 2 diabetes mellitus in Chinese Guangzhou women. ESR1 variants may also impact serum lipid metabolism, which might provide a mechanism connecting ESR1 to type 2 diabetes.</p>

Determination of ofloxacin in human fallopian tube, uterus and serum by high performance liquid chromatography / 药学学报

<p><b>AIM</b>To establish a method for determineation of the concentration of ofloxacin in human fallopian tube, uterus and serum.</p><p><b>METHODS</b>The separation was performed on a Spherisob C18 column (Hypersil, 250 mm x 4.6 mm ID, 5 microns) with a mobile phase of acetonitrile-0.01 moL.L-1 potassium dihydrogen phosphate-0.5 mol.L-1 tetrabutylammonium bromide (9:91:4, pH 2.5). The flow rate was 1.0 mL.min-1 and detection was at 294 nm. The samples were homogenated or ground to powder after freezing with liquid nitrogen. 1% triton-100 and certain volume of ethylacetate-isopropanol (10:1) were added, shaken and centrifuged. Then the entire organic layer was transferred to a tube and vacuum dried. The residue was reconstituted in the mobile phase for HPLC.</p><p><b>RESULTS</b>There was a linear relationship between the peak area ratio and the ofloxacin concentration over the range of 0.2-8.0 micrograms.mL-1. The limits of detection was 40 ng.mL-1. Using this method to determine the ofloxacin concentrations in relevant organs as well as in the plasma of patients of the Department of Gynecology, and achieved satisfactary results.</p><p><b>CONCLUSION</b>The method can be applied to assay the ofloxacin concentration in human tissues. Ofloxacin was well distributed in woman fallopian tube, uterus and serum after single oral administration.</p>