抗虫基因在转基因植株中聚合的初步研究

以质粒 pBUSCK HPT ,pCUSBK HPT和pGNA HPT作抗虫基因供体 ,优良籼型三系恢复系SH5 2 7和广亲合系W 2 0 0 8为受体 ,采用基因枪转化法 ,获得了转修饰的豇豆胰蛋白酶抑制剂基因 (sck)、修饰的Bt毒蛋白基因 (sbk)和雪花莲凝集素基因 (gna)的植株 .通过人工杂交 ,将外源基因聚合 .分子证据显示 ,作者获得了抗性基因聚合的材料 .蛋白活性测定表明 ,聚合基因的表达因载体结构相似而受到影响 ,但抗虫性却得到提高 .作者还讨论了聚合转基因在水稻育种中的作用和及其相关问题 .

聚合Cry1Ab和Cry1F基因抗虫玉米对亚洲玉米螟存活率及平均体重的影响

实验用聚合Cry1Ab和Cry1F基因抗虫玉米335YH、696YH的人工饲料饲养亚洲玉米螟幼虫,研究人工饲料中聚合基因玉米对其存活率和平均体重的影响.结果表明:在25%用量处理下,玉米螟幼虫的存活率均低于38%,平均体重均低于45 mg,说明聚合Cry1Ab和Cry1F基因抗虫玉米种子能抑制供试亚洲玉米螟幼虫的生长和存活.335YH 25%用量和50%用量的致死性差异不大,但50%用量比25%用量抑制幼虫生长的效果更好.696YH品种对幼虫的致死性和抑制生长效果受剂量影响较大.

NF-E2:a Novel Regulator of Alpha-hemoglobin Stabilizing Protein Gene Expression

Objective To investigate whether α-hemoglobin stabilizing protein (AHSP), the α-globin-specific molecular chaperone, is regulated by erythroid transcription factor NF-E2. Methods We established the stable cell line with NF-E2p45 (the larger subunit of NF-E2) short hairpin RNA to silence its expression. Western blot, real-time polymerase chain reaction, and chromatin immunoprecipitation (ChIP) analysis were performed to detect the expression of AHSP, the histone modifications at AHSP gene locus, and the binding of GATA-1 at the AHSP promoter with NF-E2p45 deficiency. ChIP was also carried out in dimethyl sulfoxide (DMSO)-induced DS19 cells and estrogen-induced G1E-ER4 cells to examine NF-E2 binding to the AHSP gene locus and its changes during cell erythroid differentiation. Finally, luciferase assay was applied in HeLa cells transfected with AHSP promoter fragments to examine AHSP promoter activity in the presence of exogenous NF-E2p45. Results We found that AHSP expression was highly dependent on NF-E2p45. NF-E2 bound to the regions across AHSP gene locus in vivo, and the transcription of AHSP was transactivated by exogenous NF-E2p45. In addition, we observed the decrease of H3K4 trimethylation and GATA-1 occupancy at the AHSP gene locus in NF-E2p45-deficient cells. Restoration of GATA-1 in G1E-ER4 cells in turn led to increased DNA binding of NF-E2p45. Conclusion NF-E2 may play an important role in AHSP gene regulation, providing new insights into the molecular mechanisms underlying the erythroid-specific expression of AHSP as well as new possibilities for β-thalassemia treatment.

Expression of Endogenous Beta Retroviruses and Hyal-2 mRNA in Immune Organs of Fetuses and Lambs

Endogenous beta retroviruses (enJSRV) are highly homologous with Jaagsiekte sheep retrovirus (exJSRV),this exogenous retrovirus is the aetiological agent of ovine pulmonary adenocarcinoma (OPA). The aim of this study was to clarify the function of enJSRV and the immunological mechanisms of its corresponding antibody, that is undetectable in JSRV-infected ovine serum. The expression of enJSRV envelope protein and Hyal-2 mRNA in immune organs and lungs of ovine fetuses and lambs were analyzed by Real-Time reverse transcription PCR and In Situ Hybridization using specific probes. In Situ Hybridization results indicated that the enJSRV envelope protein and Hyal-2 mRNA were expressed in thymus, spleen, mesenteric lymph nodes and lungs at different times, while no positive signals were detected in the negative controls. On the other hand, results from Real-Time reverse transcription PCR analysis showed that in 130d fetuses and 3d newborn lambs the enJSRV mRNA levels were much higher in organs associated with the immune system than that in lungs, especially in the thymus and spleen, but levels of Hyal-2 mRNA expression was not significantly different in all collected tissue. These results provided evidence from an immunology point of view to understand why the circulating antibodies against exJSRV are undetectable in JSRV-infected ovine, and will help to unravel the pathogenesis of JSRV-infected ovine.

Establishment of an orthotopic transplantation tumor model of hepatocellular carcinoma in mice

AIM:To improve the outcome of orthotopic transplantation in a mouse model,we used an absorbable gelatin sponge(AGS) in nude mice to establish an orthotopic implantation tumor model.METHODS:MHCC-97L hepatocellular carcinoma(HCC)cells stably expressing the luciferase gene were injected into the subcutaneous region of nude mice.One week later,the ectopic tumors were harvested and transplanted into the left liver lobe of nude mice.The AGS was used to establish the nude mouse orthotopic implantation tumor model.The tumor suppressor gene,paired box gene 5(PAX5),which is a tumor suppressor in HCC,was transfected into HCC cells to validate the model.Tumor growth was measured by bioluminescence imaging technology.Semi-quantitative reverse transcription polymerase chain reaction(RT-PCR) and histopathology were used to confirm the tumorigenicity of the implanted tumor from the MHCC-97L cell line.RESULTS:We successfully developed an orthotopic transplantation tumor model in nude mice with the use of an AGS.The success rate of tumor transplantation was improved from 60% in the control group to 100% in the experimental group using AGS.The detection of fluorescent signals showed that tumors grew in all live nude mice.The mice were divided into 3 groups:AGS-,AGS+/PAX5-and AGS+/PAX5 +.Tumor size was significantly smaller in PAX5 transfected nude mice compared to control mice(P < 0.0001).These fluorescent signal results were consistent with observations made during surgery.Pathologic examination further confirmed that the tissues from the ectopic tumor were HCC.Results from RT-PCR proved that the HCC originated from MHCC-97L cells.CONCLUSION:Using an AGS is a convenient and efficient way of establishing an indirect orthotopic liver transplantation tumor model with a high success rate.

Aberrant methylation of the 3q25 tumor suppressor gene PTX3 in human esophageal squamous cell carcinoma

AIM:To identify the novel methylation-silenced gene pentraxin 3(PTX3) in esophageal squamous cell carcinoma(ESCC).METHODS:PTX3 mRNA expression was examined in six human ESCC cell lines,one human immortalized normal esophageal epithelial cell line,primary ESCC tumor tissue,and paired adjacent nontumor tissue using reverse transcription polymerase chain reaction(RTPCR).Semi-quantitative immunohistochemistry was used to examine cellular localisation and protein levels.Methylation specific PCR and bisulphite genomic sequencing were employed to investigate the methylation of the candidate gene.RESULTS:In the majority of ESCC cell lines,we found that PTX3 expression was down-regulated due to gene promoter hypermethylation,which was further confirmed by bisulphite genomic sequencing.Demethylation treatment with 5-aza-2'-deoxycytidine restored PTX3 mRNA expression in ESCC cell lines.Methylation was more common in tumor tissues(85%) than in adjacent nontumor tissues(25%)(P < 0.01).CONCLUSION:PTX3 is down-regulated through promoter hypermethylation in ESCC,and could potentially serve as a biomarker of ESCC.

siRNA targeting of Cdx2 inhibits growth of human gastric cancer MGC-803 cells

AIM:To investigate the effects of small interference RNA(siRNA) targeting of Cdx2 on human gastric cancer MGC-803 cells in vitro and in vivo.METHODS:The recombinant pSilencer 4.1-Cdx2 siRNA plasmids were constructed and transfected into gastric cancer MGC-803 cells in vitro.The stable transfectants were selected.The effects of Cdx2 siRNA on growth,proliferation,cell cycle,apoptosis,migration and invasiveness of human gastric cancer MGC-803 cells were evaluated and the expression of phosphatase and tensin homolog(PTEN),caspase-9 and caspase-3 was observed in vitro by reverse transcription polymerase chain reaction(RT-PCR) and Western blotting analysis.We also investigated the effect of Cdx2 siRNA on growth of MGC-803 cells in nude mice in vivo.RESULTS:Cdx2 siRNA led to inhibition of endogenous Cdx2 mRNA and protein expression as determined by RT-PCR and Western blotting analysis.Cdx2 siRNA significantly inhibited cell growth and proliferation,blocked entry into the S-phase of the cell cycle,induced cell apoptosis,and reduced the motility and invasion of MGC-803 cells.Cdx2 siRNA also increased PTEN expression,and activated caspase-9 and caspase-3 in MGC-803 cells in vitro.In addition,siRNA targeting of Cdx2 inhibited the growth of MGC-803 cells and promoted tumor cell apoptosis in vivo in nude mice tumor models.CONCLUSION:Cdx2 was involved in regulating progression of human gastric cancer cells MGC-803.Manipulation of Cdx2 expression may be a potential therapeutic strategy for gastric cancer.

RRAS:A key regulator and an important prognostic biomarker in biliary atresia

AIM:To characterize the differentially expressed gene profiles in livers from biliary atresia (BA) patients including,ascertain genes,functional categories and pathways that play a central role in the pathogenesis of BA,and identify the novel prognostic markers for BA.METHODS:Liver tissue samples from control patients,neonatal cholestasis patients,and BA patients at the age of < 60 d,60-90 d,and > 90 d were pooled for DNA microarray analysis.Bioinformatics analysis was performed using,series test cluster of gene ontology,and Pathway-Finder software.Reverse-transcription polymerase chain reaction was performed to confirm changes in selected genes.Relation between RRAS gene expression and prognosis of 40 BA patients was analyzed in a 2-year follow-up study.RESULTS:The 4 identified significant gene expression profiles could confidently separate BA liver tissue from normal and other diseased liver tissues.The included genes were mainly involved in inflammation response and reconstruction of cellular matrix.The significant pathways associated with BA were primarily involved in autoimmune response,activation of T lymphocytes and its related cytokines.The RRAS,POMC,SLC26A6 and STX3 genes were important regulatory modules in pathogenesis of BA.The expression of RRAS was negatively correlated with the elimination rate of jaundice and positively correlated with the occurrence rate of cholangitis.CONCLUSION:Autoimmune response mediated by T lymphocytes may play a vital role in the pathogenesis of BA.The RRAS gene is an important regulatory module in the pathogenesis of BA,which may serve as a novel prognostic marker for BA.

Thioredoxin and thioredoxin-interacting protein as prognostic markers for gastric cancer recurrence

AIM:To evaluate the potential of thioredoxin (TXN) and thioredoxin-interacting protein (TXNIP) expression as biomarkers for predicting gastric cancer recurrence. METHODS:TXN and TXNIP expression levels were acquired from gene expression microarray data for 65 human gastric cancer tissues. We determined whether each gene expression level was associated with cancer recurrence and investigated the relationship between the two genes. For validation, the expression levels of TXN and TXNIP were measured by quantitative real- time reverse transcription polymerase chain reaction in 68 independent stage Ⅲ gastric cancer patients. The correlation between gene expression and cancer prognosis was evaluated. Immunohistochemical staining was performed to investigate the protein expression levels of TXN and TXNIP and to characterize the expression patterns of each protein. RESULTS:TXN was a prognosis-related gene (P = 0.009), whereas TXNIP, a TXN inhibitor, demonstrated a negative correlation with TXN in the gene expression microarray data. In the 68 stage Ⅲ patients, the expression levels of both TXN and TXNIP had a statistically significant effect on recurrence-free survival (RFS, P = 0.008 and P = 0.036, respectively). The low TXN and high TXNIP expression group exhibited a better prognosis than the other groups, and the high TXN and low TXNIP expression group exhibited a poorer prognosis (P < 0.001 for RFS and P = 0.001 for overall survival). More than half of the patients in the simulta-neously high TXN and low TXNIP expression group ex- perienced a recurrence within 1 year after curative surgery, and the 5-year survival rate of the patients in this group was 29%, compared with 89% in the low TXN and high TXNIP expression group. The TXN protein was overexpressed in 65% of the gastric cancer tissues, whereas the TXNIP protein was underexpressed in 85% of the cancer cells. In a correlation analysis, TXN and TXNIP were highly correlated with many oncogenes and tumor suppressors as well as with genes related to energy, protein synthesis and autophagy. CONCLUSION:TXN and TXNIP are promising prognostic markers for gastric cancer, and performing personalized adjuvant treatment based on TXN and TXNIP expression levels would be an effective practice in the treatment of gastric cancer.

Salvianolate inhibits cytokine gene expression in small intestine of cirrhotic rats

AIM:To study the effect of salvianolate on expression of tumor necrosis factor (TNF)-α and interleukin (IL)-6 mRNA in small intestine of cirrhotic rats. METHODS:Cirrhosis in rats was induced using CCl4 (0.3 mL/kg). Rats were randomly divided into non-treatment group,low-dose salvianolate (12 mg/kg) treatment group,medium-dose salvianolate (24 mg/kg) treatment group,and high-dose salvianolate (48 mg/kg) treatment group,and treated for 2 wk. Another 10 healthy rats served as a normal control group. Mortality of cirrhotic rats in each group was evaluated after treatment with salvianolate. Serum samples were taken from portal vein for the detection of endotoxin. Morphological changes in tissue samples from the ileocecum were observed under a light microscope. Expression of TNF-α and IL-6 mRNA in the small intestine of rats was analyzed by real-time reverse-transcriptase polymerase chain reaction. RESULTS:The mortality of cirrhotic rats in the nontreatment group was 37.5%. No cirrhotic rat died in the high-dose salvianolate treatment group. The serum endotoxin level was significantly higher in the non-treatment group than in the salvianolate treatment and normal control groups. The intestinal mucosal and villous atrophy,necrosis and shedding of the intestinal mucosal epithelium,observed in the non-treatment group,were reversed in different salvianolate treatment groups. The TNF-α and IL-6 mRNA expression levels in small intestine were significantly lower in different salvianolate treatment groups than in the non-treatment group. CONCLUSION:Salvianolate can reduce the endotoxin level,ameliorate the injury of intestinal mucosa,and inhibit the expression of TNF-α and IL-6 mRNA in small intestine of cirrhotic rats.

QUANTIFICATION OF P4HA2 mRNA OF FIBROBLASTS WITH SYBR GREEN BASED RT-PCR FOR CORRECTING CMV INACTIVATION EFFICIENCY IN DONOR BLOOD

Objective To quantify proline 4-hydroxylase, alpha polypeptide ii （ P4HA2 ） mRNA of human embryo lung fibroblast （HELF） with SYBR green based reversed transcript PCR （RT-PCR） for correcting cytomegalovirus （CMV） inactivation or clearance efficiency in donor blood. Methods A pair of specific primers of exon 12a of P4HA2 was designed, and the related PCR-reaction system and condition were optimized. Then the recombinant plasmid containing the target fragment was constructed for making standard curve with SYBR green based real-time RT-PCR. Finally, the sensitivity, reproducibility, and specificity of this method were fully estimated. Results The sensitivity of the method was 1.5E ＋ 04 copies/mL of P4HA2 mRNA, corresponding to 10^3 fibroblasts. In addition, existence of 8. 67E ＋ 06 leukocytes could not interfere with the accurate quantification of HELF in the large dynamic range. The intra-assay variability and inter-assay variability both varied in different concentrations, being higher in low concentrations and lower in high concentrations. But all of them were below 13. 76% in variation, which showed acceptable stability of this method. Conclusion SYBR green and specific primer based real-time RT-PCR show up a good quality for quantifying HELF P4HA2 mRNA with good specificity, stability, and high sensitivity. Approximate 10 copies of P4HA2 mRNA per cell in average can be detected by the method. Therefore, this method can be used to deduct fibroblast-associated CMV for correcting CMV inactivation efficiency in leukocytes.

Magnetic iron oxide nanoparticles carrying PTEN gene to reverse cisplatin-resistance of A549/CDDP cell lines

To evaluate the feasibility of using magnetic iron oxide nanoparticle as wild PTEN gene carrier for transfection in vitro to reverse cisplatin-resistance of A549/CDDP cells, A549/CDDP cells were transfected with the wild PTEN gene expression plasmid (pGFP-PTEN) by magnetic iron nanoparticle and lipo2000. The transfection efficiency was detected by fluorescence microscope and flow cytometer. The expression levels of PTEN mRNA and protein were detected by reverse transcription polymerase chain reaction (RT-PCR) and immunocytochemistry analysis. The effect of PTEN transfection on cell cycle enhances the sensitivity of A549/CDDP to cisplatin and nanoparticle-mediated transfection has a higher efficiency than that of the liposome-mediated group. The apoptosis level was up-regulated in PTEN transfection group. The magnetic iron oxide nanoparticle could be used as one of the ideal gene carriers for PTEN gene delivery in vitro. PTEN can be an effective target for reversing cisplatin-resistance in lung cancer.

Effect of Nitric Oxide on Esophageal Cancer Cell Line TE-1

Objective To investigate the radiosensitizing effect of nitric oxide(NO) combined with radiation on esophageal cancer cell line TE-1.Methods Methyl thiazolyl tetrazolium(MTT) assay was used to assess the effects of NO and radiation on TE-1 cells regarding inhibition of cell proliferation.Flow cytometry was used to examine the effect of NO and radiation on cell apoptosis and cycle.Reverse transcription polymerase chine reaction and Western blot were used to evaluete the effect of NO on mRNA and protein expression of manganese superoxide dismutase(MnSOD).Results NO inhibited the proliferation of TE-1 cells while significantly enhancing their radiosensitivity.The application of NO combined with radiation significantly increased the apoptosis rate and G2/M phase proportion of TE-1 cells,with substantial decreases in the MnSOD mRNA and protein expression levels.Conclusions NO reduces the MnSOD mRNA and protein expression levels by affecting TE-1 cell cycle,further inhibiting the apoptosis of esophageal cancer cells and enhancing the killing effect of radiation on esophageal cancer cells.

The Transcriptional Response of Arabidopsis thaliana L. Genes AtKu70, AtRAD51 and AtRadl to X-Ray Radiation

The study of influence of the fractionated and acute ionizing radiation on plants revealed that it is able to induce genomic instability. The hypothesis that transcription rate of several evolutionary conserved DNA repair genes AtKu 70, AtRAD51 and AtRadl, which keeps genome stability in cells of model plant Arabidopsis thaliana, changes differently depending on dose and mode of ionizing radiation exposure had been tested. Gel electrophoresis-based reverse transcription polymerase chain reaction （RT-PCR） method was used for quantifying mRNA transcription levels. The data demonstrated that mode and dose of irradiation affect transcription rate of the genes AtKuTO, AtRAD51 and AtRadI. The fractionated and acute X-ray irradiation may result in adaptive response through the induction of key DNA double-strand break （DSB） repair genes AtKu70 and AtRAD51, as well as in genome instability through transcriptional activation of error-prone AtRadl-mediated DNA DSB repair combined with decreased expression of AtRAD51. In plants at doses within the range of 3-9 Gy, an adaptive influence is prevailed, but at doses of 12-21 Gy an error-prone repair of double-strand DNA damage is activated. Fractionation of dose has a significant effect on the transcription of the genes AtKuTO, AtRAD51 and AtRadl only at doses of 15 Gy and 21 Gy. Acute dose of 15 Gy activates error-prone AtRadl-mediated DSB repair and repressed both AtRAD51-dependent and AtKu70-dependent repair pathways, while fractionated irradiation at the same total dose induces more accurate homologous recombination and canonical non-homologous end joining of the DNA strands. In case of A. thaliana exposed to X-rays at dose 21 Gy, the situation is going reversed because of strong induction of the all three genome caretaker genes in leaves of acutely irradiated plants in contrast to the plants under fractionated exposure.

The molecular mechanisms of Yang Wei Kang Liu powder on anticancer and reducing chemotherapy side-effect in combination with chemotherapy

Objective:We studied the molecular mechanisms of Yang Wei Kang Liu Power(YWKL,traditional Chinese medicine for nourishing stomach and anticancer) on anticancer and reducing chemotherapy side-effect in combination with chemotherapy.Methods:615 pre-cancer mouse model of YWKL for 10 days and CTX 1 time,semi-quantitative reverse transcription-polymerase chain reaction(RT-PCR) to detect bone marrow granulocyte-macrophage colony-stimulating factor(GM-CSF) gene and cancer proto-oncogene Bcl-2,c-myc expression.Results:YWKL in combination with chemotherapy could obviously promoted the expression of GM-CSF gene and inhibited the expression of Bcl-2 and c-myc oncogenes of FC 615 mice.Conclusion:The molecular mechanisms of anticancer and reducing chemotherapy side-effect of YWKL in combination with chemotherapy are to promote the expression of GM-CSF gene and inhibit the expression of Bcl-2 and c-myc oncogenes.

Cloning and functional analysis of a novel ascorbate peroxidase(APX) gene from Anthurium andraeanum

An 888-bp ful-length ascorbate peroxidase （APX） complementary DNA （cDNA） gene was cloned from Anthurium andraeanum, and designated as AnAPX. It contains a 110-bp 5′-noncoding region, a 28-bp 3′-noncoding region, and a 750-bp open reading frame （ORF）. This protein is hydrophilic with an aliphatic index of 81.64 and its structure consisting ofα-helixes,β-turns, and random coils. The AnAPX protein showed 93%, 87%, 87%, 87%, and 86% similarities to the APX homologs from Zantedeschia aethiopica, Vitis pseudoreticulata, Gossypium hirsutum, Elaeis guineensis, and Zea mays, respectively. AnAPX gene transcript was measured non-significantly in roots, stems, leaves, spathes, and spadices by real-time polymerase chain reaction （RT-PCR） analysis. Interestingly, this gene expression was remarkably up-regulated in response to a cold stress under 6 °C, implying that AnAPX might play an important role in A. andraeanum tolerance to cold stress. To confirm this function we overexpressed AnAPX in tobacco plants by transformation with an AnAPX expression construct driven by CaMV 35S promoter. The transformed tobacco seedlings under 4 °C showed less electrolyte leakage （EL） and malondialdehyde （MDA） content than the control. The content of MDA was correlated with chilling tolerance in these transgenic plants. These results show that AnAPX can prevent the chilling challenged plant from cellmembrane damage and ultimately enhance the plant cold tolerance.

Transient expression of organophosphorus hydrolase to enhance the degrading activity of tomato fruit on coumaphos

We constructed an expression cassette of the organophosphorus pesticide degrading （opal） gene under the control of the E8 promoter. Then opd was transformed into tomato fruit using an agroinfiltration transient expression system. β-Glucuronidase （GUS） staining, reverse transcription-polymerase chain reaction （RT-PCR）, wavelength scanning, and fluorescent reaction were performed to examine the expression of the opd gene and the hydrolysis activity on coumaphos of organo- phosphorus hydrolase （OPH） in tomato fruit. The results show that the agroinfiltrated tomato fruit-expressed OPH had the maximum hydrolysis activity of about 11.59 Uhng total soluble protein. These results will allow us to focus on breeding transgenic plants that could not only enhance the degrading capability of fruit and but also hold no negative effects on pest control when spraying organophosphorus pesticides onto the seedlings in fields.

A new method for improving the accuracy of miRNA detection with NaYF4：Yb,Er upconversion nanoparticles

MicroRNAs(miRNAs)are small noncoding RNAs,which play a central role in gene expression regulation and have been considered as excellent biomarker candidates for clinical diagnosis and prognosis.So far,many miRNAs detection methods require polymerase chain reaction(PCR)amplification following reverse transcription of miRNAs.These processes are complicated and time-consuming.In this work,we have developed a simpler method for miRNA detection based on base stacking hybridization happening on the surface of NaYF_4:Yb,Er upconversion nanoparticles.In this method,the fluorescence of NaYF_4:Yb,Er upconversion nanoparticles were functionalized as a reference standard,which can improve the accuracy of miRNA detection.On the basis of these findings,we suggest this novel approach for miRNA detection could be applied as an accurate and specific technique for miRNAs detection.