Objective: To investigate retroviral-mediated transfer and expression of human multidrug resistance (MDR) gene MDR1 in leukemic cells. Methods: Human myeloid cells, K562 and NB4, were infected by MDR retrovirus from t...Objective: To investigate retroviral-mediated transfer and expression of human multidrug resistance (MDR) gene MDR1 in leukemic cells. Methods: Human myeloid cells, K562 and NB4, were infected by MDR retrovirus from the producer PA317/HaMDR, and the resistant cells were selected with cytotoxic drug. The transfer and expression of MDR1 gene was analyzed by using polymerase chain reaction (PCR), flow cytometry (FCM) and semisolid colonies cultivation. Results: The resistant cells, K562/MDR and NB4/MDR, in which integration of the exogenous MDR1 gene was confirmed by PCR analysis, displayed a typical MDR phenotype. The expression of MDR1 transgene was detected on truncated as well as full-length transcripts. Moreover, the resistant cells were P-glycoprotein postiive at 78.0% to 98.7% analyzed with FCM. The transduction efficieny in K562 cells was studied on suspension cultures and single-cell colonies. The transduction was more efficient in coculture system (67.9%-72.5%) than in supernatant system (33.1%~46.8%), while growth factors may improve the efficiency. Conclusion: Retrovirus could allow a functional transfer and expression of MDR1 gene in human leukemia cells, and MDR1 might act as a dominant selectable gene for coexpression with the genes of interest in gene therapy.展开更多
Exogenous gene suture was used to achieve peripheral nerve anastomoses to probe into the feasibility that the sites of anastomoses of nerves directly transfer gene and thus enable gene to be expressed at the sites of...Exogenous gene suture was used to achieve peripheral nerve anastomoses to probe into the feasibility that the sites of anastomoses of nerves directly transfer gene and thus enable gene to be expressed at the sites of anastomoses under the condition that perfect nerve anastomoses are ensured. PCMVβ plasmid containing cytomegalovirus promoter (CMV promoter) and Escherichia coli (E.coli) β Galactosidase (β Gal) structural gene (lacZ gene) was conducted. A soaked medical 8 0nylon suture was used to perform epineurial repair of rabbit sciatic nerve. In the control group a suture soaked in sucrose PBS was used, while in the experimental group a suture soaked in PCMVβ plasmid solution was applied. The sites of anastomoses of nerves by stages were taken out, and β Gal histochemical staining was performed and β Gal enzyme activity was assayed with 5 bromo 4 chloro 3 indolyl β D galactoside. Results showed that the sites of anastomoses of nerves were taken out 2 days, 7 days, 14 days and 30 days respectively after the operation. The β Gal histochemical stains at the sites of anastomoses showed no indigo positive cells at different stages in the control group, whereas displayed indigo positive cells in the experimental group. In the control group, no β Gal enzyme activity was detected at different stages after operation, but in the experimental group, β Gal enzyme activity could be detected from the 3rd day to the 30th day after operation. It was concluded that by using exogenous gene suture, exogenous gene could be transferred to the sites of peripheral nerve and expressed the exogenous gene expression products with bioactivity, which provided the feasibility of using gene therapy to accelerate the recovery of nerve function.展开更多
Three rice varieties, Zhonghan 3, Shanyou 63 and Aizizhan, were used as materials in detecting differential active methyl cycle and transfer related gene expression in response to drought stress. The experiment was pe...Three rice varieties, Zhonghan 3, Shanyou 63 and Aizizhan, were used as materials in detecting differential active methyl cycle and transfer related gene expression in response to drought stress. The experiment was performed by gene chip and mRNA differential display technologies under the conditions of drought simulated with 10% PEG6000 solution. The results indicated that the methyl cycle could be activated in the leaves of Zhonghan 3 and Shanyou 63 but inhibited in the leaves of Aizizhan under drought stress. Furthermore, drought stress could induce the expression of a large number of methyltransferase genes, especially the transcription of Rubisco protein methylation related genes, which are beneficial for prevention of Rubisco protein oxidation and degradation, and drought stress could inhibit the transcription of DNA methyltransferase genes and histone methyltransferase genes. This result confirmed that the active methyl cycle and transfer related genes were involved in rice drought resistance.展开更多
Non-specific lipid transfer proteins(nsLTPs) are small, basic proteins that are characterized by an eight-cysteine motif. The biological functions of these proteins have been reported to involve plant reproduction and...Non-specific lipid transfer proteins(nsLTPs) are small, basic proteins that are characterized by an eight-cysteine motif. The biological functions of these proteins have been reported to involve plant reproduction and biotic or abiotic stress response. With the completion of the barley genome sequence, a genome-wide analysis of nsLTPs in barley(Hordeum vulgare L.)(HvLTPs) will be helpful for understanding the function of nsLTPs in plants. We performed a genome-wide analysis of the nsLTP gene family in barley and identified 70 nsLTP genes,which can be divided into five types(1, 2, C, D, and G). Each type of nsLTPs shares similar exon and intron gene structures. Expression analysis showed that barley nsLTPs have diverse expression patterns, revealing their various roles. Our results shed light on the phylogenetic relationships and potential functions of barley nsLTPs and will be useful for future studies of barley development and molecular breeding.展开更多
After organ transplantation, rapid repair of injured vascular endothelial cell (VEC) is a key to prevent graft chronic dysfunction besides control of immunological rejection. Many studies have confirmed that vascular ...After organ transplantation, rapid repair of injured vascular endothelial cell (VEC) is a key to prevent graft chronic dysfunction besides control of immunological rejection. Many studies have confirmed that vascular endothelial growth factor 165 (VEGF165) could accelerate the repair of VEC injury, decrease thrombosis and thrombotic occlusion, and inhibit hyperplasia of the intima. This study was designed to construct eukaryotic expression plasmid pBudCE4.1/VEGF165, and observe its effect on the prolife ration of VEC. METHODS:The VEGF165 gene cloned from human heart tissue by RT-PCR was cloned into eukaryotic expression plasmid pBudCE4.1. The recombinant expression plasmid pBudCE4.1/VEGF165 was identified by restriction enzyme (Hind III and BamH I) digestion analysis, and was sequenced. The pBudCE4.1/VEGF165 was introduced into VEC through lipofection transfection. The VEGF165 mRNA expression by Northern blot and VEGF165 protein expression was detected by immunocytochemical staining. The effect of expression protein on VEC proliferation was detected by flow cytometry. RESULTS:The RT-PCR product of the VEGF165 gene was about 576bp. Sequencing analysis revealed that the sequence of the amplified VEGF165 gene was identical with that in GenBank. Restrictive enzyme digestion analysis showed that recombinant expression plasmid pBudCE4.1/ tVEGF165 had been constructed successfully. The expression of VEGF165 at mRNA and protein levels in the transformed VSMCs had been demonstrated by Northern blot and immunocytochemical staining respectively. The expressed product of VEGF165 could notably accelerate the proliferation of VECs. CONCLUSIONS:pBudCE4.1/VEGF165 is successfully cons- tructed and is expressed in VECs. Expressed VEGF165 can accelerate the VEC proliferation. The present study has laid a foundation for potential use of VEGF165 gene transfection to prevent and treat vascular stenosis in the transplanted organ.展开更多
This investigation reports a protocol for transfer and expression of foreign chimeric genes in loblolly pine (Pinus taeda L.). Transformation was achieved by co-cultivation of mature zygotic embryos with Agrobacterium...This investigation reports a protocol for transfer and expression of foreign chimeric genes in loblolly pine (Pinus taeda L.). Transformation was achieved by co-cultivation of mature zygotic embryos with Agrobacterium tumefaciens strain LBA4404 which harbored a binary vector (pBI121) including genes for β-glucuronidase (GUS) and neomycin phosphotransferase (NPTII). Factors influencing transgene expression including seed sources of loblolly pine, concentration of bacteria, and the wounding procedures of target explants were investigated. The expression of foreign gene was confirmed by the ability of mature zygotic embryos to produce calli in the presence of kanamycin, by histochemical assays of GUS activity, by PCR analysis, and by Southern blot. The successful expression of the GUS gene in different families of loblolly pine suggests that this transformation system is probably useful for the production of the genetically modified conifers.展开更多
Objective To observe the survival and gene expression of genetically modified schwann cell (SC) with pSVPoMcat in the rat spinal cord.Methods The experimental animals were divided into three groups randomly, that is, ...Objective To observe the survival and gene expression of genetically modified schwann cell (SC) with pSVPoMcat in the rat spinal cord.Methods The experimental animals were divided into three groups randomly, that is, pSVPoMcat modified SC implantation group(Group A),highly purified SC implantation group(Group B),and the controlled group (Group C). After operation, the survival time for each group was 2, 4, 8, 12 weeks respectively, 10 rats once for each group, then the section of spinal cord grafted were checked by immunocytochemical staining of S－100 protein, myelin basic protein(MBP) and in situ hybridization.Results After operation for 2, 4. 8. 12 weeks, Group A’ S－100, MBP staining cells and ISH cells were respectively (82.3± 6.07)％ , (77.8± 12.3)％ , (67.8± 8.6)％； (81.2± 5.2)％ , (76.3± 11.8)％ , (68.4± 8.3)％； (80.7± 5.6)％ , (75.4± 11.6)％ ,(66.4± 8.2)％； (81.6± 6.2)％ , (75.8± 9.5)％ , (63.3± 8.02)％ ,(P<0.05). Among them, We can find that myelin sheath emerges in ISH positive cells Group B’S S－100 staining cells are (66.3± 6.8)％ , (40.2± 11.2)％ , (10.8± 2.6)％ , (3.2± 0.6)％ ,(P< 0.001), but no MBP staining cells and ISH cells are found. And in Group C, no positive cells are found at all.Conclusion pSVPoMcat genetically modified SC can survive for a long time and express gene in intraspinal injured and help to formation myelin of spinal cord injured (SCI) which provide a feasible way to gene care to spinal injury.展开更多
In recent years,zinc oxide nanoparticles(ZnO NPs)have been widely used as zinc fertilizers and pesticides.The use of ZnO NPs in this way can provide benefits to humans,but also has potential risks.ZnO NPs inevitably e...In recent years,zinc oxide nanoparticles(ZnO NPs)have been widely used as zinc fertilizers and pesticides.The use of ZnO NPs in this way can provide benefits to humans,but also has potential risks.ZnO NPs inevitably enter the environment during their production and use,which affects the ecological environment and crop growth.In order to investigate the phenotype,physiology,and gene expression of barley(Hordeum vulgare L.)seedlings under ZnO NPs stress,the barely cultivars ZJU3(P21),Golden Promise(GP)and L23 were chosen for study.Different ZnO NPs concentrations were applied to compare the physiological and biochemical indexes of the barley seedlings and the responses of six stress-related genes,when seedlings were cultured to the two-leaf stage through hydroponics.The results showed that the density of brown spots on the leaf surface increased with increasing ZnO NPs concentration.ZnO NPs stress inhibited the root growth of barley seedlings,and P21 was the most sensitive.Furthermore,ZnO NPs stress could stimulate plants to produce a large number of reactive oxygen species(ROS),resulting in an imbalance between the production and removal of ROS and membrane lipid peroxidation in plants.This imbalance inhibited the growth and development of the barley seedlings.With increasing ZnO NPs concentration,the activity of superoxide dismutase was gradually increased,the activity of catalase was progressively decreased,and the contents of malondialdehyde and proline were increased.Compared with the control,among six stress-related genes,the expression levels of five genes were downregulated and one gene was upregulated in the experimental group.This study preliminarily revealed the toxic effect of ZnO NPs on seedlings and the effect on the expression of stress-related resistance genes in different barley varieties.展开更多
Study on the transient expression of GUS gene at different growing stage of Chlorellaellipsoidea using high velocity microprojectiles, the effects of osmosis, the distance between nicroprojec-tile and target cell, bom...Study on the transient expression of GUS gene at different growing stage of Chlorellaellipsoidea using high velocity microprojectiles, the effects of osmosis, the distance between nicroprojec-tile and target cell, bombardment times, are reported in this paper. The results showed that C. ellip-soidea in exponential phase has higer level of transient expression and that treatment with osmosis can im-prove the GUS transient expression notably. The effect of distance or bombardment times was not ob-served.展开更多
Genetic engineering in filamentous N2-fixing cyanobacteria usually involves Anabaena sp.PCC 7120 and several other non-aggregating species.Mass culture and harvest of such species are more energy consuming relative to...Genetic engineering in filamentous N2-fixing cyanobacteria usually involves Anabaena sp.PCC 7120 and several other non-aggregating species.Mass culture and harvest of such species are more energy consuming relative to aggregating species.To establish a gene transfer system for aggregating species,we tested many species of Anabaena and Nostoc,and identified Nostoc muscorum FACHB244 as a species that can be genetically manipulated using the conjugative gene transfer system.To promote biodegradation of organophosphorus pollutants in aquatic environments,we introduced a plasmid containing the organophosphorus-degradation gene(opd) into Anabaena sp.PCC 7120 and Nostoc muscorum FACHB244 by conjugation.The opd gene was driven by a strong promoter,PpsbA.From both species,we obtained transgenic strains having organophosphorus-degradation activities.At 25°C,the whole-cell activities of the transgenic Anabaena and Nostoc strains were 0.163±0.001 and 0.289±0.042 unit/μg Chl a,respectively.However,most colonies resulting from the gene transfer showed no activity.PCR and DNA sequencing revealed deletions or rearrangements in the plasmid in some of the colonies.Expression of the green fluorescent protein gene from the same promoter in Anabaena sp.PCC 7120 showed similar results.These results suggest that there is the potential to promote the degradation of organophosphorus pollutants with transgenic cyanobacteria and that selection of high-expression transgenic colonies is important for genetic engineering of Anabaena and Nostoc species.For the first time,we established a gene transfer and expression system in an aggregating filamentous N2-fixing cyanobacterium.The genetic manipulation system of Nostoc muscorum FACHB244 could be utilized in the elimination of pollutants and large-scale production of valuable proteins or metabolites.展开更多
Pigeonpea [Cajanus cajan (L.) Millspaugh] is an important food legume of the semi-arid tropics (SAT) sustaining livelihood of millions of people. Stagnant and unstable yield per hectare all over the world is the chara...Pigeonpea [Cajanus cajan (L.) Millspaugh] is an important food legume of the semi-arid tropics (SAT) sustaining livelihood of millions of people. Stagnant and unstable yield per hectare all over the world is the characteristic feature of this crop. This is primarily ascribed to its susceptibility/sensitivity to a number of biotic and abiotic factors. Among biotic factors, insects such as pod borer (Helicoverpa armigera), pod fly (Melanoagromyza obtusa) and spotted borer (Maruca vitrata) substantially damage the crop and result in significant economic losses. Management of these insects by genetic means has always been considered environment friendly approach. However, genetic improvement has always been impeded by limited genetic variability in the primary gene pool of pigeonpea. Wild species present in the secondary and tertiary gene pools have been reported to carry resistance for such insects. However, transfer of resistance through conventional backcrossing has not been much successful. It calls for gene introgression through marker assisted backcrossing (MABC) or advanced backcross breeding (AB breeding). In this review, we have attempted to assess the progress made through conventional and molecular breeding and suggested the ways to move further towards genetic enhancement for insects resistance in展开更多
Metalloproteases represent a class of extracellular proteases found in Vibrio anguillarum that can generate toxic and pathogenic eff ects in turbot(Scophthalmus maximus).The toxicological eff ect partly results from o...Metalloproteases represent a class of extracellular proteases found in Vibrio anguillarum that can generate toxic and pathogenic eff ects in turbot(Scophthalmus maximus).The toxicological eff ect partly results from oxidative damage due to the production of excessive reactive oxygen species(ROS).Catalase(CAT),superoxide dismutase(SOD),and glutathione peroxidase(GPx)are major antioxidant enzymes induced by various oxidative stresses and can scavenge peroxides generated in cells.To evaluate the eff ects of metalloprotease-induced ROS on the antioxidation defense mechanism of S.maximus head kidney cells,the cDNA of CAT gene(designated as SmCAT)was cloned and characterized.SmCAT comprises a 1584-bp coding sequence that encodes a protein containing 527 amino acids with a poly(A)tail.Bioinformatics analysis revealed an active site signature sequence,a heme-ligand signature sequence,and three catalytic amino acid residues.The deduced SmCAT amino acid sequence shares a sequence similarity of 66.1%-92.4%with those of other species.Phylogenetic analysis revealed that SmCAT is classifi ed with CAT of other fi shes.Quantitative real-time PCR analysis showed that SmCAT was extensively expressed in all tested tissues,especially in blood.The expression of SmCAT,SmMnSOD,and SmGPx were inhibited signifi cantly in head kidney cells treated with metalloprotease from 12 to 24 h.In 6 to 24 h metalloprotease-treated groups compared to that of the untreated group,it was found that the production of ROS was markedly increased,and the mitochondrial membrane potential was decreased considerably.Hoechst 33342 staining revealed the presence of apoptotic bodies when the cells were incubated with 8.0 or 40.0μg/mL metalloprotease for 12 and 24 h.Hence,the toxic eff ects of metalloprotease are associated with the down-regulation of antioxidant enzyme expression and increased ROS levels,which trigger the activation of apoptosis in the head kidney cells of turbot.Our fi ndings provide a better understanding on the mechanism of metalloprotease-induced apoptosis in fi sh.展开更多
In this study,wild eggplant germplasm No.M239,which is highly susceptible to Verticillium wilt,was used as the experimental material.The physiological and biochemical indices(SOD,PAL,MDA and soluble protein)of M239 ro...In this study,wild eggplant germplasm No.M239,which is highly susceptible to Verticillium wilt,was used as the experimental material.The physiological and biochemical indices(SOD,PAL,MDA and soluble protein)of M239 roots were measured at different times(0,12,24,36,48,60 and 72 h)post inoculation with Verticillium dahliae,and the key time points for the M239 response to Verticillium wilt infection were screened.Then,RNA-Seq technology was used to screen the differentially expressed genes(DEGs)in M239 roots at 0,12 and 48 h post-inoculation(hpi).The transcriptional results of M239 were also compared with those resistance genes from some reported wild relative Solanum species(S.sisymbriifolium and S.aculeatissimum).Then some DEGs were chosen for validation by qRT–PCR.The results showed that 12 and 48 hpi were the turning points in the changes in all physiological and biochemical indices.A total of 6,783 DEGs were identified by RNA-Seq,including 6,141 DEGs(3,046 upregulated and 3,095 downregulated)at the M_12 h vs.M_0 h,1,903 DEGs(792 upregulated and 1,111 downregulated)at M_48 h vs.M_12 h,and 1,261 DEGs that appeared simultaneously in both stages.KEGG enrichment analysis showed that there were 5 metabolic pathways enriched from DEGs,which were mostly related to primary metabolism,such as glycolysis,amino acid and ribosome biogenesis.Compared with the NCBI non-redundant protein(NR)database,one Ve2 homologous gene and 8 PR protein-related genes were screened.Transcription factor analysis showed that there were a large number of DEGs,such as MYB,AP2-EREBP,bHLH,NAC and Orphans in the two stages.Compared with the reported Verticillium wilt-resistant wild eggplant species,it was found that there were fewer genes and enriched metabolic pathways in the M239 response to Verticillium wilt infection,and it also lacked the response of some known key resistance genes.These results proved that the above resistance genes and metabolic pathways played a key role in the wild eggplant response to V.dahliae infection.展开更多
非特异性脂质转移蛋白(nsLTP,non-specific lipid transfer proteins)在植物脂质转运和分泌中发挥重要作用。本研究从薰衣草(Lavandula angustifolia)中克隆到2个II型nsLTP基因,命名为nsLTP2-1和nsLTP2-2,并对其进行功能分析。生信分析...非特异性脂质转移蛋白(nsLTP,non-specific lipid transfer proteins)在植物脂质转运和分泌中发挥重要作用。本研究从薰衣草(Lavandula angustifolia)中克隆到2个II型nsLTP基因,命名为nsLTP2-1和nsLTP2-2,并对其进行功能分析。生信分析表明,nsLTP2-1和ns LTP2-2分别编码119个和117个氨基酸,具有脂转移蛋白(LTP,lipid transfer proteins)保守结构域和8个高度保守的半胱氨酸残基;系统进化分析显示它们处于两个分支,与同科的紫苏(Perilla frutescens)相似性最高。基因表达分析显示2个基因均在花蕾中高表达,在叶片、茎和花瓣中几乎不表达,在花萼中的表达存在差异,nsLTP2-1和nsLTP2-2分别在成熟花萼和幼嫩花萼中表达量更高;2个基因在花蕾和叶片中的表达均受到强光诱导,且在花蕾中的表达均受脱落酸诱导,而叶片中nsLTP2-1和nsLTP2-2的表达分别受茉莉酸甲酯和乙烯诱导。亚细胞定位显示2个nsLTPs均定位在细胞膜和细胞壁上,可能与次生代谢物的转运有关。过表达nsLTP2-1和nsLTP2-2烟草叶片经尼罗红染色后,经485~543 nm激发光激发,叶片腺毛头部的荧光显示多于野生型,说明本研究中的nsLTPs可能在脂类的合成和转运中起重要作用。这些结果为明确薰衣草脂转移蛋白在脂类及萜类转运中的功能研究提供了参考。展开更多
基金a grant from the Public Health Bureau of Jiangsu Province (H9549).
文摘Objective: To investigate retroviral-mediated transfer and expression of human multidrug resistance (MDR) gene MDR1 in leukemic cells. Methods: Human myeloid cells, K562 and NB4, were infected by MDR retrovirus from the producer PA317/HaMDR, and the resistant cells were selected with cytotoxic drug. The transfer and expression of MDR1 gene was analyzed by using polymerase chain reaction (PCR), flow cytometry (FCM) and semisolid colonies cultivation. Results: The resistant cells, K562/MDR and NB4/MDR, in which integration of the exogenous MDR1 gene was confirmed by PCR analysis, displayed a typical MDR phenotype. The expression of MDR1 transgene was detected on truncated as well as full-length transcripts. Moreover, the resistant cells were P-glycoprotein postiive at 78.0% to 98.7% analyzed with FCM. The transduction efficieny in K562 cells was studied on suspension cultures and single-cell colonies. The transduction was more efficient in coculture system (67.9%-72.5%) than in supernatant system (33.1%~46.8%), while growth factors may improve the efficiency. Conclusion: Retrovirus could allow a functional transfer and expression of MDR1 gene in human leukemia cells, and MDR1 might act as a dominant selectable gene for coexpression with the genes of interest in gene therapy.
基金a grant from the ScientificCommittee of Hebei Province(No.982 76 110 1)
文摘Exogenous gene suture was used to achieve peripheral nerve anastomoses to probe into the feasibility that the sites of anastomoses of nerves directly transfer gene and thus enable gene to be expressed at the sites of anastomoses under the condition that perfect nerve anastomoses are ensured. PCMVβ plasmid containing cytomegalovirus promoter (CMV promoter) and Escherichia coli (E.coli) β Galactosidase (β Gal) structural gene (lacZ gene) was conducted. A soaked medical 8 0nylon suture was used to perform epineurial repair of rabbit sciatic nerve. In the control group a suture soaked in sucrose PBS was used, while in the experimental group a suture soaked in PCMVβ plasmid solution was applied. The sites of anastomoses of nerves by stages were taken out, and β Gal histochemical staining was performed and β Gal enzyme activity was assayed with 5 bromo 4 chloro 3 indolyl β D galactoside. Results showed that the sites of anastomoses of nerves were taken out 2 days, 7 days, 14 days and 30 days respectively after the operation. The β Gal histochemical stains at the sites of anastomoses showed no indigo positive cells at different stages in the control group, whereas displayed indigo positive cells in the experimental group. In the control group, no β Gal enzyme activity was detected at different stages after operation, but in the experimental group, β Gal enzyme activity could be detected from the 3rd day to the 30th day after operation. It was concluded that by using exogenous gene suture, exogenous gene could be transferred to the sites of peripheral nerve and expressed the exogenous gene expression products with bioactivity, which provided the feasibility of using gene therapy to accelerate the recovery of nerve function.
基金supported by the Open Research Fund Program of Jiangsu Key Laboratory of Crop Cultivation and Physiology,China (Grant No.0273880036)
文摘Three rice varieties, Zhonghan 3, Shanyou 63 and Aizizhan, were used as materials in detecting differential active methyl cycle and transfer related gene expression in response to drought stress. The experiment was performed by gene chip and mRNA differential display technologies under the conditions of drought simulated with 10% PEG6000 solution. The results indicated that the methyl cycle could be activated in the leaves of Zhonghan 3 and Shanyou 63 but inhibited in the leaves of Aizizhan under drought stress. Furthermore, drought stress could induce the expression of a large number of methyltransferase genes, especially the transcription of Rubisco protein methylation related genes, which are beneficial for prevention of Rubisco protein oxidation and degradation, and drought stress could inhibit the transcription of DNA methyltransferase genes and histone methyltransferase genes. This result confirmed that the active methyl cycle and transfer related genes were involved in rice drought resistance.
基金supported by funds from the National Key Research and Development Program of China (2016YFD0100903)
文摘Non-specific lipid transfer proteins(nsLTPs) are small, basic proteins that are characterized by an eight-cysteine motif. The biological functions of these proteins have been reported to involve plant reproduction and biotic or abiotic stress response. With the completion of the barley genome sequence, a genome-wide analysis of nsLTPs in barley(Hordeum vulgare L.)(HvLTPs) will be helpful for understanding the function of nsLTPs in plants. We performed a genome-wide analysis of the nsLTP gene family in barley and identified 70 nsLTP genes,which can be divided into five types(1, 2, C, D, and G). Each type of nsLTPs shares similar exon and intron gene structures. Expression analysis showed that barley nsLTPs have diverse expression patterns, revealing their various roles. Our results shed light on the phylogenetic relationships and potential functions of barley nsLTPs and will be useful for future studies of barley development and molecular breeding.
文摘After organ transplantation, rapid repair of injured vascular endothelial cell (VEC) is a key to prevent graft chronic dysfunction besides control of immunological rejection. Many studies have confirmed that vascular endothelial growth factor 165 (VEGF165) could accelerate the repair of VEC injury, decrease thrombosis and thrombotic occlusion, and inhibit hyperplasia of the intima. This study was designed to construct eukaryotic expression plasmid pBudCE4.1/VEGF165, and observe its effect on the prolife ration of VEC. METHODS:The VEGF165 gene cloned from human heart tissue by RT-PCR was cloned into eukaryotic expression plasmid pBudCE4.1. The recombinant expression plasmid pBudCE4.1/VEGF165 was identified by restriction enzyme (Hind III and BamH I) digestion analysis, and was sequenced. The pBudCE4.1/VEGF165 was introduced into VEC through lipofection transfection. The VEGF165 mRNA expression by Northern blot and VEGF165 protein expression was detected by immunocytochemical staining. The effect of expression protein on VEC proliferation was detected by flow cytometry. RESULTS:The RT-PCR product of the VEGF165 gene was about 576bp. Sequencing analysis revealed that the sequence of the amplified VEGF165 gene was identical with that in GenBank. Restrictive enzyme digestion analysis showed that recombinant expression plasmid pBudCE4.1/ tVEGF165 had been constructed successfully. The expression of VEGF165 at mRNA and protein levels in the transformed VSMCs had been demonstrated by Northern blot and immunocytochemical staining respectively. The expressed product of VEGF165 could notably accelerate the proliferation of VECs. CONCLUSIONS:pBudCE4.1/VEGF165 is successfully cons- tructed and is expressed in VECs. Expressed VEGF165 can accelerate the VEC proliferation. The present study has laid a foundation for potential use of VEGF165 gene transfection to prevent and treat vascular stenosis in the transplanted organ.
文摘This investigation reports a protocol for transfer and expression of foreign chimeric genes in loblolly pine (Pinus taeda L.). Transformation was achieved by co-cultivation of mature zygotic embryos with Agrobacterium tumefaciens strain LBA4404 which harbored a binary vector (pBI121) including genes for β-glucuronidase (GUS) and neomycin phosphotransferase (NPTII). Factors influencing transgene expression including seed sources of loblolly pine, concentration of bacteria, and the wounding procedures of target explants were investigated. The expression of foreign gene was confirmed by the ability of mature zygotic embryos to produce calli in the presence of kanamycin, by histochemical assays of GUS activity, by PCR analysis, and by Southern blot. The successful expression of the GUS gene in different families of loblolly pine suggests that this transformation system is probably useful for the production of the genetically modified conifers.
文摘Objective To observe the survival and gene expression of genetically modified schwann cell (SC) with pSVPoMcat in the rat spinal cord.Methods The experimental animals were divided into three groups randomly, that is, pSVPoMcat modified SC implantation group(Group A),highly purified SC implantation group(Group B),and the controlled group (Group C). After operation, the survival time for each group was 2, 4, 8, 12 weeks respectively, 10 rats once for each group, then the section of spinal cord grafted were checked by immunocytochemical staining of S－100 protein, myelin basic protein(MBP) and in situ hybridization.Results After operation for 2, 4. 8. 12 weeks, Group A’ S－100, MBP staining cells and ISH cells were respectively (82.3± 6.07)％ , (77.8± 12.3)％ , (67.8± 8.6)％； (81.2± 5.2)％ , (76.3± 11.8)％ , (68.4± 8.3)％； (80.7± 5.6)％ , (75.4± 11.6)％ ,(66.4± 8.2)％； (81.6± 6.2)％ , (75.8± 9.5)％ , (63.3± 8.02)％ ,(P<0.05). Among them, We can find that myelin sheath emerges in ISH positive cells Group B’S S－100 staining cells are (66.3± 6.8)％ , (40.2± 11.2)％ , (10.8± 2.6)％ , (3.2± 0.6)％ ,(P< 0.001), but no MBP staining cells and ISH cells are found. And in Group C, no positive cells are found at all.Conclusion pSVPoMcat genetically modified SC can survive for a long time and express gene in intraspinal injured and help to formation myelin of spinal cord injured (SCI) which provide a feasible way to gene care to spinal injury.
基金This research was supported by the New Talent College Student Plan in Zhejiang Province(2020R4270332020R427034)the National Undergraduate Training Programs for Innovation and Entrepreneurship(202010346043).
文摘In recent years,zinc oxide nanoparticles(ZnO NPs)have been widely used as zinc fertilizers and pesticides.The use of ZnO NPs in this way can provide benefits to humans,but also has potential risks.ZnO NPs inevitably enter the environment during their production and use,which affects the ecological environment and crop growth.In order to investigate the phenotype,physiology,and gene expression of barley(Hordeum vulgare L.)seedlings under ZnO NPs stress,the barely cultivars ZJU3(P21),Golden Promise(GP)and L23 were chosen for study.Different ZnO NPs concentrations were applied to compare the physiological and biochemical indexes of the barley seedlings and the responses of six stress-related genes,when seedlings were cultured to the two-leaf stage through hydroponics.The results showed that the density of brown spots on the leaf surface increased with increasing ZnO NPs concentration.ZnO NPs stress inhibited the root growth of barley seedlings,and P21 was the most sensitive.Furthermore,ZnO NPs stress could stimulate plants to produce a large number of reactive oxygen species(ROS),resulting in an imbalance between the production and removal of ROS and membrane lipid peroxidation in plants.This imbalance inhibited the growth and development of the barley seedlings.With increasing ZnO NPs concentration,the activity of superoxide dismutase was gradually increased,the activity of catalase was progressively decreased,and the contents of malondialdehyde and proline were increased.Compared with the control,among six stress-related genes,the expression levels of five genes were downregulated and one gene was upregulated in the experimental group.This study preliminarily revealed the toxic effect of ZnO NPs on seedlings and the effect on the expression of stress-related resistance genes in different barley varieties.
文摘Study on the transient expression of GUS gene at different growing stage of Chlorellaellipsoidea using high velocity microprojectiles, the effects of osmosis, the distance between nicroprojec-tile and target cell, bombardment times, are reported in this paper. The results showed that C. ellip-soidea in exponential phase has higer level of transient expression and that treatment with osmosis can im-prove the GUS transient expression notably. The effect of distance or bombardment times was not ob-served.
基金Supported by the National High Technology Research and Development Program of China (863 Program) (No. 2002AA601170)the National Natural Science Foundation of China (No. 30623001)
文摘Genetic engineering in filamentous N2-fixing cyanobacteria usually involves Anabaena sp.PCC 7120 and several other non-aggregating species.Mass culture and harvest of such species are more energy consuming relative to aggregating species.To establish a gene transfer system for aggregating species,we tested many species of Anabaena and Nostoc,and identified Nostoc muscorum FACHB244 as a species that can be genetically manipulated using the conjugative gene transfer system.To promote biodegradation of organophosphorus pollutants in aquatic environments,we introduced a plasmid containing the organophosphorus-degradation gene(opd) into Anabaena sp.PCC 7120 and Nostoc muscorum FACHB244 by conjugation.The opd gene was driven by a strong promoter,PpsbA.From both species,we obtained transgenic strains having organophosphorus-degradation activities.At 25°C,the whole-cell activities of the transgenic Anabaena and Nostoc strains were 0.163±0.001 and 0.289±0.042 unit/μg Chl a,respectively.However,most colonies resulting from the gene transfer showed no activity.PCR and DNA sequencing revealed deletions or rearrangements in the plasmid in some of the colonies.Expression of the green fluorescent protein gene from the same promoter in Anabaena sp.PCC 7120 showed similar results.These results suggest that there is the potential to promote the degradation of organophosphorus pollutants with transgenic cyanobacteria and that selection of high-expression transgenic colonies is important for genetic engineering of Anabaena and Nostoc species.For the first time,we established a gene transfer and expression system in an aggregating filamentous N2-fixing cyanobacterium.The genetic manipulation system of Nostoc muscorum FACHB244 could be utilized in the elimination of pollutants and large-scale production of valuable proteins or metabolites.
文摘Pigeonpea [Cajanus cajan (L.) Millspaugh] is an important food legume of the semi-arid tropics (SAT) sustaining livelihood of millions of people. Stagnant and unstable yield per hectare all over the world is the characteristic feature of this crop. This is primarily ascribed to its susceptibility/sensitivity to a number of biotic and abiotic factors. Among biotic factors, insects such as pod borer (Helicoverpa armigera), pod fly (Melanoagromyza obtusa) and spotted borer (Maruca vitrata) substantially damage the crop and result in significant economic losses. Management of these insects by genetic means has always been considered environment friendly approach. However, genetic improvement has always been impeded by limited genetic variability in the primary gene pool of pigeonpea. Wild species present in the secondary and tertiary gene pools have been reported to carry resistance for such insects. However, transfer of resistance through conventional backcrossing has not been much successful. It calls for gene introgression through marker assisted backcrossing (MABC) or advanced backcross breeding (AB breeding). In this review, we have attempted to assess the progress made through conventional and molecular breeding and suggested the ways to move further towards genetic enhancement for insects resistance in
基金Supported by the National Natural Science Foundation of China(No.31502187)the Natural Science Foundation of Hebei Province(No.C2018407049)+1 种基金the Hebei Provincial Department of Science and Technology(Nos.20286701Z,20567621H)the Talent Engineering Training Funding Project of Hebei Province(No.A201901057)。
文摘Metalloproteases represent a class of extracellular proteases found in Vibrio anguillarum that can generate toxic and pathogenic eff ects in turbot(Scophthalmus maximus).The toxicological eff ect partly results from oxidative damage due to the production of excessive reactive oxygen species(ROS).Catalase(CAT),superoxide dismutase(SOD),and glutathione peroxidase(GPx)are major antioxidant enzymes induced by various oxidative stresses and can scavenge peroxides generated in cells.To evaluate the eff ects of metalloprotease-induced ROS on the antioxidation defense mechanism of S.maximus head kidney cells,the cDNA of CAT gene(designated as SmCAT)was cloned and characterized.SmCAT comprises a 1584-bp coding sequence that encodes a protein containing 527 amino acids with a poly(A)tail.Bioinformatics analysis revealed an active site signature sequence,a heme-ligand signature sequence,and three catalytic amino acid residues.The deduced SmCAT amino acid sequence shares a sequence similarity of 66.1%-92.4%with those of other species.Phylogenetic analysis revealed that SmCAT is classifi ed with CAT of other fi shes.Quantitative real-time PCR analysis showed that SmCAT was extensively expressed in all tested tissues,especially in blood.The expression of SmCAT,SmMnSOD,and SmGPx were inhibited signifi cantly in head kidney cells treated with metalloprotease from 12 to 24 h.In 6 to 24 h metalloprotease-treated groups compared to that of the untreated group,it was found that the production of ROS was markedly increased,and the mitochondrial membrane potential was decreased considerably.Hoechst 33342 staining revealed the presence of apoptotic bodies when the cells were incubated with 8.0 or 40.0μg/mL metalloprotease for 12 and 24 h.Hence,the toxic eff ects of metalloprotease are associated with the down-regulation of antioxidant enzyme expression and increased ROS levels,which trigger the activation of apoptosis in the head kidney cells of turbot.Our fi ndings provide a better understanding on the mechanism of metalloprotease-induced apoptosis in fi sh.
基金supported by the National Natural Science Foundation of China(31960594)the Yunnan Fundamental Research Projects(202201AT070074,2019FB059)the Yunnan Technical Innovation Talent Training Project(202205AD160029).
文摘In this study,wild eggplant germplasm No.M239,which is highly susceptible to Verticillium wilt,was used as the experimental material.The physiological and biochemical indices(SOD,PAL,MDA and soluble protein)of M239 roots were measured at different times(0,12,24,36,48,60 and 72 h)post inoculation with Verticillium dahliae,and the key time points for the M239 response to Verticillium wilt infection were screened.Then,RNA-Seq technology was used to screen the differentially expressed genes(DEGs)in M239 roots at 0,12 and 48 h post-inoculation(hpi).The transcriptional results of M239 were also compared with those resistance genes from some reported wild relative Solanum species(S.sisymbriifolium and S.aculeatissimum).Then some DEGs were chosen for validation by qRT–PCR.The results showed that 12 and 48 hpi were the turning points in the changes in all physiological and biochemical indices.A total of 6,783 DEGs were identified by RNA-Seq,including 6,141 DEGs(3,046 upregulated and 3,095 downregulated)at the M_12 h vs.M_0 h,1,903 DEGs(792 upregulated and 1,111 downregulated)at M_48 h vs.M_12 h,and 1,261 DEGs that appeared simultaneously in both stages.KEGG enrichment analysis showed that there were 5 metabolic pathways enriched from DEGs,which were mostly related to primary metabolism,such as glycolysis,amino acid and ribosome biogenesis.Compared with the NCBI non-redundant protein(NR)database,one Ve2 homologous gene and 8 PR protein-related genes were screened.Transcription factor analysis showed that there were a large number of DEGs,such as MYB,AP2-EREBP,bHLH,NAC and Orphans in the two stages.Compared with the reported Verticillium wilt-resistant wild eggplant species,it was found that there were fewer genes and enriched metabolic pathways in the M239 response to Verticillium wilt infection,and it also lacked the response of some known key resistance genes.These results proved that the above resistance genes and metabolic pathways played a key role in the wild eggplant response to V.dahliae infection.
文摘非特异性脂质转移蛋白(nsLTP,non-specific lipid transfer proteins)在植物脂质转运和分泌中发挥重要作用。本研究从薰衣草(Lavandula angustifolia)中克隆到2个II型nsLTP基因,命名为nsLTP2-1和nsLTP2-2,并对其进行功能分析。生信分析表明,nsLTP2-1和ns LTP2-2分别编码119个和117个氨基酸,具有脂转移蛋白(LTP,lipid transfer proteins)保守结构域和8个高度保守的半胱氨酸残基;系统进化分析显示它们处于两个分支,与同科的紫苏(Perilla frutescens)相似性最高。基因表达分析显示2个基因均在花蕾中高表达,在叶片、茎和花瓣中几乎不表达,在花萼中的表达存在差异,nsLTP2-1和nsLTP2-2分别在成熟花萼和幼嫩花萼中表达量更高;2个基因在花蕾和叶片中的表达均受到强光诱导,且在花蕾中的表达均受脱落酸诱导,而叶片中nsLTP2-1和nsLTP2-2的表达分别受茉莉酸甲酯和乙烯诱导。亚细胞定位显示2个nsLTPs均定位在细胞膜和细胞壁上,可能与次生代谢物的转运有关。过表达nsLTP2-1和nsLTP2-2烟草叶片经尼罗红染色后,经485~543 nm激发光激发,叶片腺毛头部的荧光显示多于野生型,说明本研究中的nsLTPs可能在脂类的合成和转运中起重要作用。这些结果为明确薰衣草脂转移蛋白在脂类及萜类转运中的功能研究提供了参考。
