Overexpression of B7-H3 augments anti-apoptosis of colorectal cancer cells by Jak2-STAT3

AIM:To investigate the role of the overexpression of B7-H3 in apoptosis in colorectal cancer cell lines and the underlying molecular mechanisms.METHODS:SW620 cells that highly overexpressed B7-H3(SW620-B7-H3-EGFP)and HCT8 cells stably transfected with B7-H3 sh RNA(HCT8-sh B7-H3)were previously constructed in our laboratory.Cells transfected with p IRES2-EGFP were used as negative controls(SW620-NC and HCT8-NC).Real-time PCR and western blotting analysis were used to detect the m RNA and protein expressions of the apoptosis regulator proteins Bcl-2,Bcl-xl and Bax.A cell proliferation assay was used to evaluate the survival rate and drug sensitivity of the cells.The effect of drug resistance was detected by a cell cycle assay.Active caspase-3western blotting was used to reflect the anti-apoptotic ability of cells.Western blotting was also performed to determine the expression of proteins associated with the Jak2-STAT3 signaling pathway and the apoptosis regulator proteins after the treatment with AG490,a Jak2 specific inhibitor,in B7-H3 overexpressing cells.The data were analyzed by Graph Pad Prism 6 using a non-paired t-test.RESULTS:Whether by overexpression in SW620cells or downregulation in HCT8,B7-H3 significantly affected the expression of anti-and pro-apoptotic proteins,at both the transcriptional and translational levels,compared with the negative control(P<0.05).A cell proliferation assay revealed that B7-H3overexpression increased the drug resistance of cells and resulted in a higher survival rate(P<0.05).In addition,the results of cell cycle and active caspase-3western blotting proved that B7-H3 overexpression inhibited apoptosis in colorectal cancer cell lines(P<0.05).B7-H3 overexpression improved Jak2 and STAT3phosphorylation and,in turn,increased the expression of the downstream anti-apoptotic proteins B-cell CLL/lymphoma 2(Bcl-2)and Bcl-xl,based on western blotting(P<0.05).After treating B7-H3 overexpressing cells with the Jak2-specific inhibitor AG490,the phosphorylation of Jak2 and STAT3,and the expression of Bcl-2 and Bcl-xl,decreased accordingly(P<0.05).This finding suggested that the Jak2-STAT3 pathway is involved in the mechanism mediating the anti-apoptotic ability of B7-H3.CONCLUSION:The overexpression of B7-H3 induces resistance to apoptosis in colorectal cancer cell lines by upregulating the Jak2-STAT3 signaling pathway,potentially providing new approaches to the treatment of colorectal cancer.

Overexpression of microRNA-124 promotes the neuronal differentiation of bone marrow-derived mesenchymal stem cells

microRNAs （miRNAs） play an important regulatory role in the self-renewal and differentiation of stem cells. In this study, we examined the effects of miRNA-124 （miR-124） overexpression in bone marrow-derived mesenchymal stem cells. In particular, we focused on the effect of overexpression on the differentiation of bone marrow-derived mesenchymal stem cells into neurons. First, we used GeneChip technology to analyze the expression of miRNAs in bone marrow-derived mesen- chymal stem cells, neural stem cells and neurons, miR-124 expression was substantially reduced in bone marrow-derived mesenchymal stem cells compared with the other cell types. We con- structed a lentiviral vector overexpressing miR-124 and transfected it into bone marrow-derived mesenchymal stem cells. Intracellular expression levels of the neuronal early markers [3-III tu- bulin and microtubule-associated protein-2 were significantly increased, and apoptosis induced by oxygen and glucose deprivation was reduced in transfected cells. After miR-124-transfected bone marrow-derived mesenchymal stem cells were transplanted into the injured rat spinal cord, a large number of cells positive for the neuronal marker neurofilament-200 were observed in the transplanted region. The Basso-Beattie-Bresnahan locomotion scores showed that the motor function of the hind limb of rats with spinal cord injury was substantially improved. These re- sults suggest that miR-124 plays an important role in the differentiation of bone marrow-derived mesenchymal stem cells into neurons. Our findings should facilitate the development of novel strategies for enhancing the therapeutic efficacy of bone marrow-derived mesenchymal stem cell transplantation for spinal cord injury.

Dickkopf3 overexpression inhibits pancreatic cancer cell growth in vitro

AIM:To elucidate the role of dickkopf3(Dkk3)in human pancreatic cancer cell growth.METHODS:Dkk3 mRNA and protein expression in human pancreatic cancer cell lines were detected by realtime reverse transcription polymerase chain reaction(realtime RTPCR),Western blotting and immunofluorescence.Methylation of the Dkk3 promoter sequence was examined by methylationspecific polymerase chain reaction(MSP)and Dkk3 mRNA expression was determined by realtime RTPCR after 5aza2'deoxycytidine(5azadC)treatment.The effects of Dkk3 on cancer cell proliferation and in vitro sensitivity to gemcitabine were investigated by CellTiter 96?AQueous One Solution Cell Proliferation Assay(MTS)after transfecting the Dkk3 expression plasmid into human pancreatic cancer cells.The expression ofβcatenin,phosphorylated extracellular signalregulated protein kinases(pERK)and extracellular signalregulated protein kinases(ERK)was also examined by realtime RTPCR and Western blotting after upregulating Dkk3 expression in human pancreatic cancer cells.RESULTS:The results show that the expression levels of both Dkk3 mRNA and protein were low in all pancreatic cancer cell lines tested.The Dkk3 promoter sequence was methylated in the MIA PaCa2 and AsPC1 cell lines,which showed reduced Dkk3 expression.These two cell lines,which initially had a methylated Dkk3 promoter,showed increased Dkk3 mRNA expression that was dependent upon the dosage and timing of the DNA demethylating agent,5azadC,treatment(P<0.05 or P<0.01).When Dkk3 expression was upregulated following the transfection of a Dkk3 expression plasmid into MIA PaCa2 cells,the ability of cells to proliferate decreased(P<0.01),and the expression ofβcatenin and pERK was downregulated(P<0.01).Sensitivity to gemcitabine was enhanced in Dkk3 expression plasmidtransfected cells.CONCLUSION:Our findings,for the first time,implicate Dkk3 as a tumor suppressor in human pancreatic cancer,through the downregulation ofβcatenin expression via the ERKmediated pathway.

Overexpression of Dickkopf-3 induces apoptosis through mitochondrial pathway in human colon cancer

AIM:To investigate the mechanisms of the biological roles of Dickkopf-3(Dkk-3) in cell invasion,survival and apoptosis in colon cancer cells.METHODS:Three human colon cancer cell lines,i.e.,HT-29,LoVo and SW480,were used.Overexpression of Dkk-3 induced by pEGFP-N1-Dkk-3-GFP plasmid in LoVo cells was performed using Lipofectamine 2000 reagent.Reverse transcription polymerase chain reaction and Western blotting were performed to determine the mRNA and protein expression levels of Dkk-3,respectively.Cell proliferation assay,cell cycle analysis,hoechst 33258 assay and Matrigel invasion assay were performed on Dkk-3 overexpressing transfectants.RESULTS:The mRNA and protein expressions of Dkk-3 in HT-29(mRNA:0.06 ± 0.02,protein:0.06 ± 0.01) and LoVo(mRNA:0.07 ± 0.02,protein:0.07 ± 0.02) cells were significantly lower than that in SW480 cells(mRNA:0.92 ± 0.04,protein:0.69 ± 0.13;all P < 0.05),and the greatest levels of invasiveness wasin LoVo cells.Dkk-3 overexpression inhibited the proliferation and invasion of LoVo cells and induced cell cycle arrest at G0/G1 phase and subsequent apoptosis,as indicated by increased chromatin condensation and fragments,upregulated Bax and cytochrome c protein,downregulated survivin and Bcl-2 protein,and the activation of caspase-3 and caspase-9.Furthermore,Dkk-3 overexpression reduced the accumulation of cytosolic fraction of β-catenin.CONCLUSION:Dkk-3 overexpression induced apoptosis in human colon cancer possibly through the mitochondrial pathway.Dkk-3 may be involved in the Wnt/β-catenin signaling pathways in colon cancer.

Integrin-linked kinase overexpression promotes epithelial-mesenchymal transition via nuclear factor-κB signaling in colorectal cancer cells

AIM: To investigate the effect of integrin-linked kinase (ILK) on proliferation, metastasis, and invasion of the colorectal cancer cell line SW480.METHODS: In this study, the colorectal cancer cell line SW480 was stably transfected with ILK plasmids, and small interfering RNA (siRNA) was used to knockdown expression of nuclear factor (NF)-&#x003ba;B/p65. Methylthiazole tetrazolium (MTT) assay was performed to measure proliferation, and the wound healing migration assay and matrigel invasion assay were used to test the metastasis and invasion ability of SW480 cells. To explore the epithelial-mesenchymal transition (EMT) process, embryonic development, and the invasion and metastasis of tumors, the protein level of E-cadherin, vimentin, snail, and slug was detected by western blot. Immunofluorescence was also used to detect E-cadherin expression. Western blot was used to determine the level of phosphorylated-inhibitor of kappa B (I&#x003ba;B)a, inhibitor of gamma B (I&#x003b3;B)a, and nuclear factor kappa B (NF-&#x003ba;B) expressions and to explore the ILK signaling pathway.RESULTS: Western blot results revealed that ILK expression significantly increased when ILK was overexpressed in SW480 cells (P &#x0003c; 0.05). Proliferation, metastasis, and invasion ability were improved in the vector-ILK group compared to the vector group (P &#x0003c; 0.05). Immunofluorescence results revealed that E-cadherin fluorescence intensity decreased after ILK was overexpressed (P &#x0003c; 0.05). Western blot results revealed that the protein expression of E-cadherin was reduced, while vimentin, snail, and slug were upregulated when ILK was overexpressed in SW480 cells (P &#x0003c; 0.05). In order to determine the role of the NF-&#x003ba;B signaling pathway in ILK overexpression promoted EMT occurrence, we overexpressed ILK in SW480 cells and found that levels of NF-&#x003ba;B/p65 and cytoplasmic phosphorylated-I&#x003ba;Ba were increased and that cytoplasmic I&#x0043a;Ba levels were decreased compared to the control group (P &#x0003c; 0.05). Furthermore, NF-&#x003ba;B/p65 knockout revealed that E-cadherin was increased in the overexpressed ILK group.CONCLUSION: ILK overexpression improved the proliferation, metastasis, and invasion ability of SW480 cells, and this effect may be mediated by the NF-&#x003ba;B signaling pathway.

Overexpression of HSP27 and HSP70 is associated with decreased survival among patients with esophageal adenocarcinoma

BACKGROUND Overexpression of heat shock proteins(HSPs) is associated with several malignancies and contributes to the development, progression, and metastasis of cancer, in addition to the inhibition of cellular death. In recent years, there has been active research into using HSP inhibitors in several malignancies. Due to the poor prognosis of esophageal adenocarcinoma(EAC), it would be valuable to find new biomarkers for the development of cancer treatments.AIM To evaluate the expressions of HSP27 and HSP70 and their effect on survival in EAC.METHODS Immunohistochemical analyses and evaluations of HSP27 and HSP70 expression were performed on all available samples from 93 patients diagnosed with EACbetween 1990 and 2007 at two university hospitals. Fifteen cases with Barrett's metaplasia and 5 control cases from the same patient population were included in the analysis. HSP expression was quantitatively assessed and classified as high or low. Kaplan-Meier analyses and Cox regression models adjusting for age and sex as well as tumor site, stage, and grade were used to evaluate the effect on survival.RESULTS Tumor stage and surgical treatment were the main prognostic factors. High HSP27 expression in cancer cases was a strong negative predictive factor, with a mean survival of 23 mo compared to the 49 mo in cases with a low expression(P= 0.018). The results were similar for HSP70, with a poorer survival of 17 mo in cases with high HSP70 expression, in contrast to 40 mo(P = 0.006) in cases with a low expression. A Cox regression survival analysis was performed, adjusting for possible confounding factors, and higher HSP27 and HSP70 expressions remained an independent negative prognostic factor. The HSPs' correlation with survival was not affected by cancer treatments. When the analysis was adjusted for all factors, the odds ratios for HSP27 and HSP70 were 3.3(CI: 1.6–6.6, P =0.001) and 2.2(CI: 1.2–3.9, P = 0.02), respectively.CONCLUSION HSP27 and HSP70 overexpression is associated with poor survival in EAC, which is, to the best of our knowledge, reported for the first time.

Overexpression of a Foreign Bt Gene in Cotton Affects the Low-Molecular-Weight Components in Root Exudates

Most research in the past using genetically modified crops (GM crops) has focused on the ecological safety of foreign gene (i.e., the gene flow), gene products (for example, Bt (Bacillus thuringiensis) protein), and the safety of transgenic food for humans. In this study, changes in both the species and amounts of low-molecular-weight components in cotton (Gossypium hirsutum L.) root exudates after foreign Bt gene overexpression were investigated under different nutritional conditions. Transgenic cotton containing Bt (Bt-cotton), supplemented with all the mineral nutrients, secreted more organic acids than the wild-type cotton (WT). When nitrogen was removed from the full-nutrient solution, the amount of organic acids secretion of Bt-cotton was lesser than that of WT. The roots of the transgenic cotton secreted lesser amounts of amino acids and soluble sugars than the WT roots in the full-nutrient solution. Deficiencies of P and K caused a large increase in the total amino acid and soluble sugar secretions of both Bt-cotton and WT, with larger increases observed in Bt-cotton. Because transferring the foreign Bt gene into cotton can result in alterations in the components of the root exudates, with the effect varying depending on the nutritional status, the cultivation of genetically modified crops, such as Bt-cotton, in soil environments should be more carefully assessed, and the possible effects as a result of the alterations in the root exudate components should be considered.

Overexpression of p42.3 promotes cell growth and tumorigenicity in hepatocellular carcinoma

AIM:To investigate the association of p42.3 expression with clinicopathological characteristics and the biological function of p42.3 in human hepatocellular carcinoma(HCC).METHODS:We used reverse transcription-polymerase chain reaction(RT-PCR),quantitative real-time RT-PCR and western blotting to detect p42.3 mRNA and protein expression in hepatic cell lines.We examined primary HCC samples and matched adjacent normal tissue by immunohistochemistry to investigate the correlation between p42.3 expression and clinicopathological features.HepG2 cells were transfected with a pIRES2EGFP-p42.3 expression vector to examine the function of the p42.3 gene.Transfected cells were analyzed for their viability and malignant transformation abilities by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay,colony formation assay,and tumorigenicity assay in nude mice.RESULTS:p42.3 is differentially expressed in primary HCC tumors and cell lines.Approximately 69.6%(96/138) of cells were p42.3-positive in hepatic tumor tissues,while 30.7%(35/114) were p42.3-positive in tumor-adjacent normal tissues.Clinicopathological characteristics of the HCC specimens revealed a significant correlation between p42.3 expression and tumor differentiation(P = 0.031).However,p42.3 positivity was not related to tumor tumor-node-metastasis classification,hepatitis B virus status,or hepatoma type.Regarding p42.3 overexpression in stably transfected HepG2 cells,we discovered significant enhancement of cancer cell growth and colony formation in vitro,and significantly enhanced tumorigenicity in nude mice.Western blot analysis of cell cycle proteins revealed that enhanced p42.3 levels promote upregulation of proliferating cell nuclear antigen,cyclin B1 and mitotic arrest deficient 2.CONCLUSION:p42.3 promotes tumorigenicity and tumor growth in HCC and may be a potential target for future clinical cancer therapeutics.

Effect of lentiviral vector-mediated overexpression of hypoxia-inducible factor 1 alpha delivered by pluronic F-127 hydrogel on brachial plexus avulsion in rats

Brachial plexus avulsion often results in massive motor neuron death and severe functional deficits of target muscles. However, no satisfactory treatment is currently available. Hypoxia-inducible factor 1α is a critical molecule targeting several genes associated with ischemia-hypoxia damage and angiogenesis. In this study, a rat model of brachial plexus avulsion-reimplantation was established, in which C5–7 ventral nerve roots were avulsed and only the C6 root reimplanted. Different implants were immediately injected using a microsyringe into the avulsion-reimplantation site of the C6 root post-brachial plexus avulsion. Rats were randomly divided into five groups: phosphate-buffered saline, negative control of lentivirus, hypoxia-inducible factor 1α(hypoxia-inducible factor 1α overexpression lentivirus), gel(pluronic F-127 hydrogel), and gel + hypoxia-inducible factor 1α(pluronic F-127 hydrogel + hypoxia-inducible factor 1α overexpression lentivirus). The Terzis grooming test was performed to assess recovery of motor function. Scores were higher in the hypoxia-inducible factor 1α and gel +hypoxia-inducible factor 1α groups(in particular the gel + hypoxia-inducible factor 1α group) compared with the phosphate-buffered saline group. Electrophysiology, fluorogold retrograde tracing, and immunofluorescent staining were further performed to investigate neural pathway reconstruction and changes of neurons, motor endplates, and angiogenesis. Compared with the phosphate-buffered saline group, action potential latency of musculocutaneous nerves was markedly shortened in the hypoxia-inducible factor 1α and gel + hypoxia-inducible factor1α groups. Meanwhile, the number of fluorogold-positive cells and ChAT-positive neurons, neovascular area(labeled by CD31 around av ulsed sites in ipsilateral spinal cord segments), and the number of motor endplates in biceps brachii(identified by α-bungarotoxin) were all visibly increased, as well as the morphology of motor endplate in biceps brachil was clear in the hypoxia-inducible factor 1α and gel + hypoxia-inducible factor 1α groups. Taken together, delivery of hypoxia-inducible factor 1α overexpression lentiviral vectors mediated by pluronic F-127 effectively promotes spinal root regeneration and functional recovery post-brachial plexus avulsion. All animal procedures were approved by the Institutional Animal Care and Use Committee of Guangdong Medical University, China.

Overexpression of a Broccoli Defensin Gene BoDFN Enhances Downy Mildew Resistance

Plant defensins are small, basic cysteine-rich peptides that play important roles in disease resistance. A gene, designated BoDFN, was isolated from Brassica oleracea var. italica. Gene sequence has been submitted to NCBI with an accession no. of HQ436486. Complete coding sequence of BoDFN is 243 bp in length encoding 80 amino acids. Sequence comparison results showed that BoDFN shared high homology with those of crucifer plants and there were only few DNA base differences. RT-PCR results indicated an increase of gene expression in Hyaloperonospora parasitica infected leaves and revealed a significant increase at 24 and 36 h of treatment. A recombinant plasmid, named pBI121-BoDFN, was constructed and introduced into Agrobacterium tumefacien LBA4404. PCR screening for 65 regenerated plantlets, 17 positive plantlets were obtained, PCR screening results revealed that 17 out of 65 regenerated plantlets were positive. Disease resistant identification results indicated that all positive plants showed an increase in resistance to H. parasitica.

Overexpression of S-adenosylmethionine decarboxylase(SAMDC)in Xeno-pus embryos activates maternal program of apoptosis as a "fail-safe"mechanism of early embryogenesis

In Xenopus, injection of S-adenosylmethionine decarboxylase (SAMDC) mRNA into fertilized eggs or 2-cell stage embryos induces massive cell dissociation and embryo-lysis at the early gastrula stage due toactivation of the maternal program of apoptosis. We injected SAMDC mRNA into only one of the animalside blastomeres of embryos at different stages of cleavage, and examined the timing of the onset of theapoptotic reaction. In the injection at 4-and 8-cell stages, a considerable number of embryos developed intotadpoles and in the injection at 16-and 32-cell stages, all the embryos became tadpoles, although tadpolesobtained were sometimes abnormal. However, using GFP as a lineage tracer, we found that descendant cellsof the blastomere injected with SAMDC mRNA at 8-to 32-cell stages are confined within the blastocoel atthe early gastrula stage and undergo apoptotic cell death within the blastocoel, in spite of the continued development of the injected embryos. These results indicate that cells overexpressed with SAMDC undergo apoptotic cell death consistently at the early gastrula stage, irrespective of the timing of the mRNA injection.We assume that apoptosis is executed in Xenopus early gastrulae as a 'fall-safe' mechanism to eliminate physiologically-severely damaged cells to save the rest of the embryo.

Sp1 contributes to overexpression of stanniocalcin 2 through regulation of promoter activity in colon adenocarcinoma

BACKGROUND Aberrant expression of stanniocalcin 2 (STC2) is implicated in colon adenocarcinoma (COAD). A previous study identified that STC2 functions as a tumor promoter to drive development of some cancers, but the role of its overexpression in the development of COAD remains unclear. AIM To evaluate the regulation mechanism of STC2 overexpression in COAD. METHODS The expression of STC2 in COAD was assessed by TCGA COAD database and GEO (GSE50760). Methylation level of the STC2 promoter was evaluated with beta value in UALCAN platform, and the correlation between STC2 expression and survival rate was investigated with TCGA COAD. Transcription binding site prediction was conducted by TRANSFAC and LASAGNA, and a luciferase reporter system was used to identify STC2 promoter activity in several cell lines, including HEK293T, NCM460, HT29, SW480, and HCT116. Western blotting was performed to evaluate the role of Sp1 on the expression of STC2. RESULTS The central finding of this work is that STC2 is overexpressed in COAD tissues and positively correlated with poor prognosis. Importantly, the binding site of the transcription factor Sp1 is widely located in the promoter region of STC2. A luciferase reporter system was successfully constructed to analyze the transcription activity of STC2, and knocking down the expression of Sp1 significantly inhibited the transcription activity of STC2. Furthermore, inhibition of Sp1 remarkably decreased protein levels of STC2. CONCLUSION Our data provide evidence that the transcription factor Sp1 is essential for the overexpression of STC2 in COAD through activation of promoter activity. Taken together, our finding provides new insights into the mechanism of oncogenic function of COAD by STC2.

Association of p53 codon 72 polymorphism with liver metastases of colorectal cancers positive for p53 overexpression

Objective： To evaluate the association between p53 codon 72 polymorphism （R72P） and the risk ofcolorectal liver metastases. Methods： The p53 R72P genotype was identified by polymerase chain reaction-restriction fragment length polymorphism （PCR-RFLP） method in 78 consecutive colorectal cancer patients with liver metastases and 214 age- and sex-matched cases with nonmetastatic colorectal cancer. Results： The R allele of the p53 R72P polymorphism was more frequently found in metastatic cases than in nonmetastatic cases （P=0.075）. Carriers of the 72R allele had a 2.25-fold （95% CI （confidence interval）=1.05-4.83） increased risk of liver metastases. On the stratification analysis, 72R-carrying genotype conferred a 3.46-fold （95% CI=1.02-11.72） and a 1.05-fold （95% CI=0.36-3.08） increased risk of liver metastases for p53 overexpression-positive and negative colorectal cancers, respectively. Conclusion： These results demonstrate for the first time that the 72R allele of the p53 polymorphism has an increased risk for liver metastases in colorectal cancers positive for p53 overexpression.

Comprehensive evaluation of the metabolic effects of porcine CRTC3 overexpression on subcutaneous adipocytes with metabolomic and transcriptomic analyses

Background:Meat quality is largely driven by fat deposition,which is regulated by several genes and signaling pathways.The cyclic adenosine monophosphate(cAMP)-regulated transcriptional coactivator 3(CRTC3)is a coactivator of cAMP response element binding protein(CREB)that mediates the function of protein kinase A(PKA)signaling pathway and is involved in various biological processes including lipid and energy metabolism.However,the effects of CRTC3 on the metabolome and transcriptome of porcine subcutaneous adipocytes have not been studied yet.Here,we tested whether porcine CRTC3 expression would be related to fat deposition in Heigai pigs(a local fatty breed in China)and Duroc×Landrace×Yorkshire(DLY,a lean breed)pigs in vivo.The effects of adenovirus-induced CRTC3 overexpression on the metabolomic and transcriptomic profiles of subcutaneous adipocytes were also determined in vitro by performing mass spectrometry-based metabolomics combined with RNA sequencing(RNA-seq).Results:Porcine CRTC3 expression is associated with fat deposition in vivo.In addition,CRTC3 overexpression increased lipid accumulation and the expression of mature adipocyte-related genes in cultured porcine subcutaneous adipocytes.According to the metabolomic analysis,CRTC3 overexpression induced significant changes in adipocyte lipid,amino acid and nucleotide metabolites in vitro.The RNA-seq analysis suggested that CRTC3 overexpression alters the expression of genes and pathways involved in adipogenesis,fatty acid metabolism and glycerophospholipid metabolism in vitro.Conclusions:We identified significant alterations in the metabolite composition and the expression of genes and pathways involved in lipid metabolism in CRTC3-overexpressing adipocytes.Our results suggest that CRTC3 might play an important regulatory role in lipid metabolism and thus affects lipid accumulation in porcine subcutaneous adipocytes.

Overexpression of ARAhPR10, a Member of the PR10 Family, Decreases Levels of <i>Aspergillus flavus</i>Infection in Peanut Seeds

Peanut (Arachis hypogaea L.) is one of the most susceptible host crops to Aspergillus flavus invasion and subsequent aflatoxin contamination. In this report, a new member of PR10 family putative resistant gene (designated as ARAhPR10, No. EU661964.1) encoding a PR10 protein was isolated and characterized. Analysis of qRT-PCR showed that the expression of ARAhPR10 was induced by pre-harvested A. flavus infection, but no significant difference was observed between resistant genotype “GT-C20” and susceptible genotype “Yueyou 7”. Seven transgenic peanut lines expressing the ARAhPR10 gene under the control of 35S promoter were obtained using the Agrobacterium tumefaciens-mediated method. Real time RT-PCR results showed that the expression level of the ARAhPR10 was significantly higher and the A. flavus infection and aflatoxin content were significantly lower in seeds of transgenic lines than that of the wild type. A significant negative correlation between ARAhPR10 expression at transcript level and seeds aflatoxin production was observed. Combining the previous results, it is suggested that ARAhPR10 expression play an important role in peanut host resistance to A. flavus infection and aflatoxin producing.

Overexpression of StCYS1 gene enhances tolerance to salt stress in the transgenic potato(Solanum tuberosum L.)plant

Salt stress seriously restricts the growth and yield of potatoes.Plant cystatins are vital players in biotic stress and development,however,their roles in salt stress resistance remain elusive.Here,we report that StCYS1 positively regulates salt tolerance in potato plants.An in vitro biochemical test demonstrated that StCYS1 is a bona fide cystatin.Overexpression of StCYS1 in both Escherichia coli and potato plants significantly increased their resistance to high salinity.Further analysis revealed that the transgenic plants accumulated more proline and chlorophyll under salt stress conditions.Moreover,the transgenic plants displayed higher H2O2 scavenging capability and cell membrane integrity compared with wild-type potato.These results demonstrate that StCYS1 is closely correlated with salt stress and its overaccumulation can substantially enhance salt stress resistance.

Overexpression of Brassica napus cytosolic fructose-1,6-bisphosphatase and sedoheptulose-1,7-bisphosphatase genes significantly enhanced tobacco growth and biomass

Elevated activities of cytosolic fructose-1,6-bisphosphatase(cyFBPase) and sedoheptulose-1,7-bisphosphatase(SBPase)are associated with higher yields in plants. In this study, the expression levels of the cyFBPase and SBPase genes were increased by overexpressing rape(Brassica napus) cDNA in tobacco(Nicotiana tabacum) plants. The transgenic plants coexpressing cy FBPase and SBPase(TpFS), or expressing single cy FBPase(TpF) or SBPase(TpS) had 1.77-, 1.55-, 1.23-fold cyFBPase and 1.45-, 1.12-, 1.36-fold SBPase activities as compared to the wild-type(WT), respectively. Photosynthesis rates of TpF, TpS and TpFS increased 4, 20 and 25% compared with WT plants. The SBPase and cyFBPase positively regulated each other and functioned synergistically in transgenic tobacco plants. In addition, the sucrose contents of the three transgenic plants were higher than that of WT plants. The starch accumulation of the TpFS and TpS plants was improved by 53 and 37%, but slightly decreased in TpF plants. Moreover, the transgenic tobacco plants harbouring SBPase and/or cyFBPase genes showed improvements in their growth, biomass, dry weight, plant height, stem diameter, leaf size,flower number, and pod weight. In conclusion, co-expression of SBPase and cyFBPase may pave a new way for improving crop yield in agricultural applications.

Overexpression of BnKCS1-1,BnKCS1-2,and BnCER1-2 promotes cuticular wax production and increases drought tolerance in Brassica napus

Higher amounts of cuticular wax in plants have been associated with improved plant stress tolerance and increased potential for industrial use.In this study,orthologs of KCS1 and CER1 in Arabidopsis,designated BnKCS1-1,BnKCS1-2,and BnCER1-2,were isolated from Brassica napus.Transcription of BnKCS1-1 and BnKCS1-2 in B.napus were induced by abscisic acid(ABA)and drought treatment,while transcription of BnCER1-2 was induced only by drought treatment.All three gene transcripts decreased significantly when plants were treated with methyl jasmonate(MeJA)or subjected to cold stress.Overexpression of BnKCS1-1,BnKCS1-2,and BnCER1-2 under the control of the CaMV35S promoter led to a significant increase in cuticular wax on transgenic B.napus leaves.BnKCS1-1 and BnKCS1-2 overexpression led to similar differences from non-transformed plants,with significantly higher levels of aldehydes(C29 and C30),alkanes(C28,C29,and C31)and secondary alcohols(C28 and C29),and a significantly lower level of C29 ketone.Overexpression of BnCER1-2 led to an increase in alkanes(C27,C28,C29,and C31),a decrease in secondary alcohols(C28 and C29),and insignificant changes in other wax components.Scanning electron microscopy revealed that overexpression of BnKCS1-1,BnKCS1-2,and BnCER1-2 in B.napus resulted in a higher density of wax crystals on the leaf surface than observed in non-transformed plants.Transgenic plants showed a reduced rate of water loss and increased drought tolerance compared to non-transformed plants.These results suggest that BnKCS1-1,BnKCS1-2,and BnCER1-2 gene products can modify the cuticular wax of B.napus.Changing cuticular waxes using transgenic approaches is a new strategy for genetic improvement of plant drought tolerance and provides an opportunity for development of B.napus as a surface-wax crop.

Overexpression of the MADS-box gene SIMBP21 alters leaf morphology and affects reproductive development in tomato

Fruit yield is the most important horticultural trait of tomato.SIMBP21,a SEPALLATA subclass MADS-box gene has been reported to have functions in regulating pedicel abscission zone identity and development and controlling sepal size in tomato.However,we generated transgenic tomato plants which overexpress SIMBP21 and found the transformants displayed curly leaves,abnormally shaped flowers with twisted and opened stamens,reduced yield parameters,and small and light seeds.Our studies on the gain-of-function phenotype and gene expression level showed that its novel aspects played important roles in determining leaf morphology,flower and inflorescence architecture,and seed size,as well as the fruit yield.Overexpression of SIMBP21 in tomato resulted in curly leaves with fewer leaflets due to the regulation of the critical leaf polarity genes that cause an imbalance between the midvein adaxial-abaxial cell growth.Defects in the architecture of flowers and inflorescences resulted in reduced fruit set.Furthermore,we demonstrated that SIMBP21 plays its role through inhibiting the expression of the genes involved in the determination of seed development in tomato and SIMBP21 protein can interact with other MADS-box protein(SIAGL11,TAGL1 and SIMBP3)to control seed size.Thus,these results suggest that overexpression of SIMBP21 causes multiple types of damage to plant growth and development,especially fruit yield,in tomato.

Overexpression of tyrosine phosphorylated proteins in reproductive tissues of polycystic ovary syndrome rats induced by letrozole

Objective:To identify the alteration of tyrosine phosphorylated protein expression in rats with polycystic ovary syndrome(PCOS).Methods:Sixteen female Sprague-Dawley rats were divided into the control and letrozole-induced PCOS groups.The oestrus cycle of rats was performed by vaginal smear.Sex hormones and morphology of the ovary,oviduct,and uterus were observed.Expressions and intensity of androgen receptor and tyrosine phosphorylated proteins of reproductive organs were investigated by Western blot.Results:Various polycysts and increased androgen receptor expression were present in the ovary of the PCOS group.The levels of follicle-stimulating hormone and testosteone were significantly higher in the PCOS group while progesterone and estradiol levels were significantly decreased as compared with the control group(P<0.05).Only the size of uterus in the PCOS group was significantly smaller than the control group.However,the density of collagen fibers observed in PCOS uterus was greater than the control group.Moreover,tyrosine phosphorylated proteins were significantly overexpressed in ovary(52,42,and 28 kDa),oviduct(72,56,42,and 28 kDa),and uterus(53 and 42 kDa)of the PCOS group compared to the control group.Conclusions:Presence of tyrosine phosphorylated proteins in the ovary,oviduct and uterus suggests that overexpression of tyrosine phosphorylated proteins may be involved in potential mechanism of female infertility especially in PCOS.