Leaves are the main organ for photosynthesis and organic synthesis in cotton.Leaf shape has important effects on photosynthetic efficiency and canopy formation,thereby affecting cotton yield.Previous studies have show...Leaves are the main organ for photosynthesis and organic synthesis in cotton.Leaf shape has important effects on photosynthetic efficiency and canopy formation,thereby affecting cotton yield.Previous studies have shown that LMI1(LATE MERISTEM IDENTITY1)is the main gene regulating leaf shape.In this study,the LMI1 gene was inserted into the 35S promoter expression vector,and cotton plants overexpressing LMI1(OE)were obtained through genetic transformation.Statistical analysis of the biological traits of the T_(1) and T_(2) populations showed that compared to the wild type(WT),OE plants had significantly larger leaves,thicker stems and significantly greater dry weight.Furthermore,plant sections of the main vein and petiole showed that the numbers of cells in those tissues of OE plants were significantly greater.In addition,RNA-seq analysis revealed the differential expression of genes related to gibberellin synthesis and NAC gene family(genes containing the NAC domain)between the OE and WT plants,suggesting that LMI1 is involved in secondary wall formation and cell proliferation,which promotes stem thickening.Moreover,Gene Ontology(GO)analysis revealed enrichment in the terms of calcium ion binding,and Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis showed enrichment in the terms of fatty acid degradation,phosphatidylinositol signal transduction system,and c AMP(cyclic adenosine monophosphate)signal pathway.These results suggested that LMI1 OE plants are responsive to gibberellin hormone signals,and have altered messenger signals(c AMP,Ca^(2+))which amplify this function,to promote stronger aboveground vegetative growth.This study found the LMI1 greatly increased the vegetative growth in cotton,which is the basic requirement for higher yield.展开更多
The tumor selective over-expression of the human Hsp70 gene has been well documented in human tumors,linked to the poor prognosis,being refractory to chemo-and radio-therapies as well as the advanced stage of tumorous...The tumor selective over-expression of the human Hsp70 gene has been well documented in human tumors,linked to the poor prognosis,being refractory to chemo-and radio-therapies as well as the advanced stage of tumorous lesions in particular.However,both the nature and details of aberrations in the control of the Hsp70 expression in tumor remain enigmatic.By comparing various upstream segments of the Hsp70 gene for each''s ability to drive the luciferase reporter genes in the context of the tumor cell lines varying in their p53 status and an immortal normal liver cell line,we demonstrated in a great detail the defects in the control mechanisms at the both initiation and elongation levels of transcription being instrumental to the tumor selective profile of its expression.Our data should not only offer new insights into our understanding of the tumor specific over-expression of the human Hsp70 gene,but also paved the way for the rational utilization of the tumor selective mechanism with the Hsp70 at the central stage fortargeting the therapeutic gene expression to human tumors.展开更多
This is first report about the simultaneous over-expression of both Insulin-like growth factor (IGF- I ) and its receptor (IGF- I R) at mRNA level in human primary hepatic Cancer (PHC). In 10 PHC samples from China, I...This is first report about the simultaneous over-expression of both Insulin-like growth factor (IGF- I ) and its receptor (IGF- I R) at mRNA level in human primary hepatic Cancer (PHC). In 10 PHC samples from China, IGF-I and IGF- I R were both over-expressed, whereas only a background signal was detected in normal liver. In 5 pairs of PHC and its non- tumorous adjacent liver tissues from South Africa, IGF- I and IGF- I R were also over-expressed in PHC. mRNA expression of IGF- I in all 5 cases and IGF- I R in 4 of 5 cases were higher in cancer than non- tumorous adjacent liver tissues. These results strongly implicate that an autocrine and/ or paracrine mechanism might be Involved in formation and progression of PHC.展开更多
In the present study, we illustrate the strategy and protocol required to generate rice transgenics over-expressing the 21-nt form of Osa-miR820. The miR exists in two size variants of 21-nt and 24-nt so the natural p...In the present study, we illustrate the strategy and protocol required to generate rice transgenics over-expressing the 21-nt form of Osa-miR820. The miR exists in two size variants of 21-nt and 24-nt so the natural precursor cannot be employed for the purpose of miR over-expression as the cellular machinery can process both size variants thereby masking the role of PTGS regulation. Hence, we adopted the artificial miR technology to specifically over-express the 21-nt species in the transgenics. During the course of experiments it was observed that the amiR constructs probably interfered with the regeneration of the transformed callus, necessitating protocol modifications. The results indicate the successful over-expression of the 21-nt miR species. These plants can serve as a useful source for the functional dissection of the role played by the 21-nt Osa-miR820 species. They will also be valuable in highlighting the importance for the existence of a dual mode of miR mediated target regulation.展开更多
With the availability of the whole genome sequence of Escherichia coli or Corynebacterium glutamicum, strategies for directed DNA manipulation have developed rapidly. DNA manipulation plays an important role in unders...With the availability of the whole genome sequence of Escherichia coli or Corynebacterium glutamicum, strategies for directed DNA manipulation have developed rapidly. DNA manipulation plays an important role in understanding the function of genes and in constructing novel engineering bacteria according to requirement. DNA manipulation involves modifying the autologous genes and expressing the heterogenous genes. Two alternative approaches, using electroporation linear DNA or recombinant suicide plasmid, allow a wide variety of DNA manipulation. However, the over-expression of the desired gene is generally executed via plasmid-mediation. The current review summarizes the common strategies used for genetically modifying E. coli and C. glutamicum genomes, and discusses the technical problem of multi-layered DNA manipulation. Strategies for gene over-expression via integrating into genome are proposed. This review is intended to be an accessible introduction to DNA manipulation within the bacterial genome for novices and a source of the latest experimental information for experienced investigators.展开更多
Objective:To study the influence of over-expression of three functional genes involved in GC biosynthetic pathway,GuHMGR,GuSQS1,and GuBAS on GC production.Methods:Three plant expression vectors were constructed and tr...Objective:To study the influence of over-expression of three functional genes involved in GC biosynthetic pathway,GuHMGR,GuSQS1,and GuBAS on GC production.Methods:Three plant expression vectors were constructed and transformed into Agrobacterium tumefaciens EHA105,which were used to infect Glycyrrhiza uralensis hypocotyls explants.After induction,selection,differentiation,culture,and transplantation,12,15,and 5 regenerated plants over-expressing GuHMGR,GuSQS1,and GuBAS,were obtained,respectively.Results:RT-PCR analysis showed these transgenic regenerated G.uralensis plants had 2-6 copies of GuHMGR,GuSQS1,or GuBAS.HPLC analysis showed the contents of 18α-and 18β-GC in all transgenic regenerated samples were both higher than that in the blank control.With the increase of copy numbers of GuHMGR,GuSQS1,and GuBAS,the contents of 18α-and 18β-GC were both increased in most samples.The highest 18α-and 18β-GC contents in transgenic regenerated plants were about 3.05 times and 2.80 times higher than that in the blank control,respectively.Conclusion:Over-expression of the GuHMGR,GuSQS1,and GuBAS genes enhance the accumulation of 18α-and 18β-GC in the roots and rhizomes of G.uralensis.We hope this work can lay a foundation for the molecular breeding research of G.uralensis and improving the quality of the roots and rhizomes of G.uralensis cultivars.展开更多
PtAUX1,an ortholog of Arabidopsis AtAUX1(encoding auxin influx carrier protein),has been cloned from Populus tomentosa.PtAUX1 protein is similar to Arabidopsis AtAUX1 in structure,but PtAUX1 contains an additional cAM...PtAUX1,an ortholog of Arabidopsis AtAUX1(encoding auxin influx carrier protein),has been cloned from Populus tomentosa.PtAUX1 protein is similar to Arabidopsis AtAUX1 in structure,but PtAUX1 contains an additional cAMP/cGMP-dependent protein kinase phosphorylation site.Its overexpression lines driven by 35S promoter exhibits a novel phenotype related flower development which has not been revealed in AtAUX1 mutants.Ovary-like structures each with 1～2 styles,but without a cup-like disc and a bract,can initial from regenerated transgenic bud in cluster,resembling an inflorescence.The histological dissections illuminate that the ovarylike structures have womb and transmitting tissue inner its styles,but no ovules.PtAUX1 expression pattern are ubiquitous and various in different organs,which is coincident with its candidate auxin influx,functioning in distributing auxin thorough a plant body.These results suggest that PtAUX1 or polar auxin transport has a substantial function in floral organ initiation.展开更多
The first branchial arch malformation(FBAM) is a rare congenital defect associated with anomalous development of the first and second branchial arches.Cause of FBAM still remains unknown,and is thought in most cases t...The first branchial arch malformation(FBAM) is a rare congenital defect associated with anomalous development of the first and second branchial arches.Cause of FBAM still remains unknown,and is thought in most cases to be multifactorial,involving both genetic and enviromental factors.Dlx2 as a member of the Dlx homeobox gene family,plays a crucial role in the development of the first branchial arch.The tissues regulated mainly by Dlx2 are coincident with the tissues mainly involved in FBAM.Dlx2 over-expression generated by electroporation transfection can disturb the migration and differentiation of cranial neural crest cells(CNCCs),which migrate to the branchial arches and in turn give rise to much of the facial skeleton and connective tissues.Furthermore,Dlx2 over-expression can be found in the first branchial arch spontaneous mutant mice.So we hypothesize that Dlx2 over-expression mutation causes FBAM due to an increase in cell-cell adhesion and inhibiting the migration of CNCC to the first branchial arch in the early stage,or migrating to an incorrect position and can't differentiate into normal tissues.What an exact role of Dlx2 over-expression in FBAM remains to be investigated and Dlx2 over-expression transgenic mouse will be a nice model for further research in FBAM.展开更多
Background Many researchers studied the possibility of using stem cells as gene therapeutic vector. But few related reports on the adipose tissue-derived stem cells （ADSCs） are available. Therefore we intended to co...Background Many researchers studied the possibility of using stem cells as gene therapeutic vector. But few related reports on the adipose tissue-derived stem cells （ADSCs） are available. Therefore we intended to construct a lentiviral VEGF165 expression vector and then infect the ADSCs to produce therapeutic seed cells.Methods EHS1001-68950485313912 clone was mutated by PCR method to produce consensus fragment of VEGF165 transcript （NM_001025368）. Lentivirus was enveloped with pGC-FU, pHelper 1.0 and pHelper 2.0 plasmids in 293T cells.And then the ADSCs （multiplicity of infection=20） were transfected with the vectors after titer determination. Stable expression of VEGF165 in ADSCs was confirmed by immunofluorescence staining, enzyme-linked immunosorbent assay （ELISA） and Western blotting analysis.Results DNA sequencing and 293T transfection verified VEGF165 was linked to the GFP fused vector. The virus titer is up to 2x10a determined by quantitative PCR. VEGF165 transduced cells could show green fluorescence confirmed by immunofluorescence staining （almost 95%）. ELISA analyses could detect out the density of VEGF was 850.86-1202.13pg/ml （mean （923.00±31.22） pg/ml） in the supernatant of VEGF16s-transduced cells but not detected in the GFP-transduced cells （P 〈0.001） and the Western blotting analyses also confirmed VEGF165 expression in VEGF165-transduced cells.Conclusions The VEGF165 over-expression ADSCs were obtained and may be used as a cell therapeutic tool and may be applied for vascular regeneration, especially in the treatment of erectile dysfunction.展开更多
Objsective: Glycyrrhizia uralensis, one of the most widely-used traditional Chinese medicines, is mainly cropped in China. However, many cultivars are less in glycyrrhizic acid than Chinese Pharmacopoeia requires. In ...Objsective: Glycyrrhizia uralensis, one of the most widely-used traditional Chinese medicines, is mainly cropped in China. However, many cultivars are less in glycyrrhizic acid than Chinese Pharmacopoeia requires. In this paper, we improved glycyrrhizic acid by regulating β-amyrin synthase gene(GuBAS).Methods: Tobacco root-specific promoter TobRB7 and Gu BAS c DNA were obtained and combined with linearized pCAMBIA1305.1 to construct root-specific plant expression vector which was later transformed into Agrobacterium rhizogenes ACCC10060 by electrotransformation. The cotyledons and hypocotyls of G.uralensis were infected by the recombinant A. rhizogenes ACCC10060 to induce hairy roots. The GA content was quantified by HPLC.Results: The PCR and sequencing results both showed that three transgenic hairy root lines were obtained. The copy number of Gu BAS in these transgenic hairy roots was intended by q RT-PCR to be 3, 7,and 4. GA was detected by HPLC, and the results showed that GA was present in the three transgenic hairy roots, while absent in wild hairy roots.Conclusion: Over-expressing Gu BAS root-specifically in hairy roots of G. uralensis enhanced GA accumulation.展开更多
The rate-limiting enzyme in the mevalonic acid(MVA)pathway which can lead to triterpenoid saponin glycyrrhizic acid(GA)is 3-hydroxy-3-methylglutaryl-CoA reductase(HMGR).In order to reveal the effect of copy number var...The rate-limiting enzyme in the mevalonic acid(MVA)pathway which can lead to triterpenoid saponin glycyrrhizic acid(GA)is 3-hydroxy-3-methylglutaryl-CoA reductase(HMGR).In order to reveal the effect of copy number variation in the HMGR gene on the MVA pathway,the HMGR gene from Glycyrrhiza uralensis Fisch.(GuHMGR)was cloned and over-expressed in Pichia pastoris GS115.Six recombinant P.pastoris strains containing different copy numbers of the GuHMGR gene were obtained and the content of ergosterol was analyzed by HPLC.The results showed that all the recombinant P.pastoris strains contained more ergosterol than the negative control and the strains with 8 and 44 copies contained significantly more ergosterol than the other strains.However,as the copy number increased,the content of ergosterol showed an increasing–decreasing–increasing pattern.This study provides a rationale for increasing the content of GA through over-expressing the GuHMGR gene in cultivars of G.uralensis.展开更多
Porcine reproductive and respiratory syndrome virus （PRRSV） is a major pathogen that causes reproductive failure and respiratory disease in pigs, resulting in devastating economic losses worldwide. Porcine alveolar ...Porcine reproductive and respiratory syndrome virus （PRRSV） is a major pathogen that causes reproductive failure and respiratory disease in pigs, resulting in devastating economic losses worldwide. Porcine alveolar macrophages （PAMs） are the primary target cells of PRRSV , and the putative receptors, including CD163, CD169, and CD151, play key roles during infection . However, the understanding of PRRSV infection and pathogenesis is limited by its narrow host range. Pig is the unparalleled animal susceptible to PRRSV, but not well-suited for the study of long-term chronic infection or immune response in vivo because of their long breed- ing cvcle, size, high cost, and lack of biological materials.展开更多
基金supported by the National Natural Science Foundation of China(5201101621)。
文摘Leaves are the main organ for photosynthesis and organic synthesis in cotton.Leaf shape has important effects on photosynthetic efficiency and canopy formation,thereby affecting cotton yield.Previous studies have shown that LMI1(LATE MERISTEM IDENTITY1)is the main gene regulating leaf shape.In this study,the LMI1 gene was inserted into the 35S promoter expression vector,and cotton plants overexpressing LMI1(OE)were obtained through genetic transformation.Statistical analysis of the biological traits of the T_(1) and T_(2) populations showed that compared to the wild type(WT),OE plants had significantly larger leaves,thicker stems and significantly greater dry weight.Furthermore,plant sections of the main vein and petiole showed that the numbers of cells in those tissues of OE plants were significantly greater.In addition,RNA-seq analysis revealed the differential expression of genes related to gibberellin synthesis and NAC gene family(genes containing the NAC domain)between the OE and WT plants,suggesting that LMI1 is involved in secondary wall formation and cell proliferation,which promotes stem thickening.Moreover,Gene Ontology(GO)analysis revealed enrichment in the terms of calcium ion binding,and Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis showed enrichment in the terms of fatty acid degradation,phosphatidylinositol signal transduction system,and c AMP(cyclic adenosine monophosphate)signal pathway.These results suggested that LMI1 OE plants are responsive to gibberellin hormone signals,and have altered messenger signals(c AMP,Ca^(2+))which amplify this function,to promote stronger aboveground vegetative growth.This study found the LMI1 greatly increased the vegetative growth in cotton,which is the basic requirement for higher yield.
文摘The tumor selective over-expression of the human Hsp70 gene has been well documented in human tumors,linked to the poor prognosis,being refractory to chemo-and radio-therapies as well as the advanced stage of tumorous lesions in particular.However,both the nature and details of aberrations in the control of the Hsp70 expression in tumor remain enigmatic.By comparing various upstream segments of the Hsp70 gene for each''s ability to drive the luciferase reporter genes in the context of the tumor cell lines varying in their p53 status and an immortal normal liver cell line,we demonstrated in a great detail the defects in the control mechanisms at the both initiation and elongation levels of transcription being instrumental to the tumor selective profile of its expression.Our data should not only offer new insights into our understanding of the tumor specific over-expression of the human Hsp70 gene,but also paved the way for the rational utilization of the tumor selective mechanism with the Hsp70 at the central stage fortargeting the therapeutic gene expression to human tumors.
文摘This is first report about the simultaneous over-expression of both Insulin-like growth factor (IGF- I ) and its receptor (IGF- I R) at mRNA level in human primary hepatic Cancer (PHC). In 10 PHC samples from China, IGF-I and IGF- I R were both over-expressed, whereas only a background signal was detected in normal liver. In 5 pairs of PHC and its non- tumorous adjacent liver tissues from South Africa, IGF- I and IGF- I R were also over-expressed in PHC. mRNA expression of IGF- I in all 5 cases and IGF- I R in 4 of 5 cases were higher in cancer than non- tumorous adjacent liver tissues. These results strongly implicate that an autocrine and/ or paracrine mechanism might be Involved in formation and progression of PHC.
文摘In the present study, we illustrate the strategy and protocol required to generate rice transgenics over-expressing the 21-nt form of Osa-miR820. The miR exists in two size variants of 21-nt and 24-nt so the natural precursor cannot be employed for the purpose of miR over-expression as the cellular machinery can process both size variants thereby masking the role of PTGS regulation. Hence, we adopted the artificial miR technology to specifically over-express the 21-nt species in the transgenics. During the course of experiments it was observed that the amiR constructs probably interfered with the regeneration of the transformed callus, necessitating protocol modifications. The results indicate the successful over-expression of the 21-nt miR species. These plants can serve as a useful source for the functional dissection of the role played by the 21-nt Osa-miR820 species. They will also be valuable in highlighting the importance for the existence of a dual mode of miR mediated target regulation.
基金supported by the Natural Science Foundation of Jiangsu Province(No.BK20150149)the Fundamental Research Funds for the Central Universities(No.JUSRP51504)the Youth Foundation of Jiangnan University(No.JUSRP115A19),China
文摘With the availability of the whole genome sequence of Escherichia coli or Corynebacterium glutamicum, strategies for directed DNA manipulation have developed rapidly. DNA manipulation plays an important role in understanding the function of genes and in constructing novel engineering bacteria according to requirement. DNA manipulation involves modifying the autologous genes and expressing the heterogenous genes. Two alternative approaches, using electroporation linear DNA or recombinant suicide plasmid, allow a wide variety of DNA manipulation. However, the over-expression of the desired gene is generally executed via plasmid-mediation. The current review summarizes the common strategies used for genetically modifying E. coli and C. glutamicum genomes, and discusses the technical problem of multi-layered DNA manipulation. Strategies for gene over-expression via integrating into genome are proposed. This review is intended to be an accessible introduction to DNA manipulation within the bacterial genome for novices and a source of the latest experimental information for experienced investigators.
文摘Objective:To study the influence of over-expression of three functional genes involved in GC biosynthetic pathway,GuHMGR,GuSQS1,and GuBAS on GC production.Methods:Three plant expression vectors were constructed and transformed into Agrobacterium tumefaciens EHA105,which were used to infect Glycyrrhiza uralensis hypocotyls explants.After induction,selection,differentiation,culture,and transplantation,12,15,and 5 regenerated plants over-expressing GuHMGR,GuSQS1,and GuBAS,were obtained,respectively.Results:RT-PCR analysis showed these transgenic regenerated G.uralensis plants had 2-6 copies of GuHMGR,GuSQS1,or GuBAS.HPLC analysis showed the contents of 18α-and 18β-GC in all transgenic regenerated samples were both higher than that in the blank control.With the increase of copy numbers of GuHMGR,GuSQS1,and GuBAS,the contents of 18α-and 18β-GC were both increased in most samples.The highest 18α-and 18β-GC contents in transgenic regenerated plants were about 3.05 times and 2.80 times higher than that in the blank control,respectively.Conclusion:Over-expression of the GuHMGR,GuSQS1,and GuBAS genes enhance the accumulation of 18α-and 18β-GC in the roots and rhizomes of G.uralensis.We hope this work can lay a foundation for the molecular breeding research of G.uralensis and improving the quality of the roots and rhizomes of G.uralensis cultivars.
基金ShanDong Provincial Education Department Supported Item(no.J07YF08)
文摘PtAUX1,an ortholog of Arabidopsis AtAUX1(encoding auxin influx carrier protein),has been cloned from Populus tomentosa.PtAUX1 protein is similar to Arabidopsis AtAUX1 in structure,but PtAUX1 contains an additional cAMP/cGMP-dependent protein kinase phosphorylation site.Its overexpression lines driven by 35S promoter exhibits a novel phenotype related flower development which has not been revealed in AtAUX1 mutants.Ovary-like structures each with 1～2 styles,but without a cup-like disc and a bract,can initial from regenerated transgenic bud in cluster,resembling an inflorescence.The histological dissections illuminate that the ovarylike structures have womb and transmitting tissue inner its styles,but no ovules.PtAUX1 expression pattern are ubiquitous and various in different organs,which is coincident with its candidate auxin influx,functioning in distributing auxin thorough a plant body.These results suggest that PtAUX1 or polar auxin transport has a substantial function in floral organ initiation.
文摘The first branchial arch malformation(FBAM) is a rare congenital defect associated with anomalous development of the first and second branchial arches.Cause of FBAM still remains unknown,and is thought in most cases to be multifactorial,involving both genetic and enviromental factors.Dlx2 as a member of the Dlx homeobox gene family,plays a crucial role in the development of the first branchial arch.The tissues regulated mainly by Dlx2 are coincident with the tissues mainly involved in FBAM.Dlx2 over-expression generated by electroporation transfection can disturb the migration and differentiation of cranial neural crest cells(CNCCs),which migrate to the branchial arches and in turn give rise to much of the facial skeleton and connective tissues.Furthermore,Dlx2 over-expression can be found in the first branchial arch spontaneous mutant mice.So we hypothesize that Dlx2 over-expression mutation causes FBAM due to an increase in cell-cell adhesion and inhibiting the migration of CNCC to the first branchial arch in the early stage,or migrating to an incorrect position and can't differentiate into normal tissues.What an exact role of Dlx2 over-expression in FBAM remains to be investigated and Dlx2 over-expression transgenic mouse will be a nice model for further research in FBAM.
文摘Background Many researchers studied the possibility of using stem cells as gene therapeutic vector. But few related reports on the adipose tissue-derived stem cells （ADSCs） are available. Therefore we intended to construct a lentiviral VEGF165 expression vector and then infect the ADSCs to produce therapeutic seed cells.Methods EHS1001-68950485313912 clone was mutated by PCR method to produce consensus fragment of VEGF165 transcript （NM_001025368）. Lentivirus was enveloped with pGC-FU, pHelper 1.0 and pHelper 2.0 plasmids in 293T cells.And then the ADSCs （multiplicity of infection=20） were transfected with the vectors after titer determination. Stable expression of VEGF165 in ADSCs was confirmed by immunofluorescence staining, enzyme-linked immunosorbent assay （ELISA） and Western blotting analysis.Results DNA sequencing and 293T transfection verified VEGF165 was linked to the GFP fused vector. The virus titer is up to 2x10a determined by quantitative PCR. VEGF165 transduced cells could show green fluorescence confirmed by immunofluorescence staining （almost 95%）. ELISA analyses could detect out the density of VEGF was 850.86-1202.13pg/ml （mean （923.00±31.22） pg/ml） in the supernatant of VEGF16s-transduced cells but not detected in the GFP-transduced cells （P 〈0.001） and the Western blotting analyses also confirmed VEGF165 expression in VEGF165-transduced cells.Conclusions The VEGF165 over-expression ADSCs were obtained and may be used as a cell therapeutic tool and may be applied for vascular regeneration, especially in the treatment of erectile dysfunction.
基金supported by the National Natural Science Foundation of China (81503181)
文摘Objsective: Glycyrrhizia uralensis, one of the most widely-used traditional Chinese medicines, is mainly cropped in China. However, many cultivars are less in glycyrrhizic acid than Chinese Pharmacopoeia requires. In this paper, we improved glycyrrhizic acid by regulating β-amyrin synthase gene(GuBAS).Methods: Tobacco root-specific promoter TobRB7 and Gu BAS c DNA were obtained and combined with linearized pCAMBIA1305.1 to construct root-specific plant expression vector which was later transformed into Agrobacterium rhizogenes ACCC10060 by electrotransformation. The cotyledons and hypocotyls of G.uralensis were infected by the recombinant A. rhizogenes ACCC10060 to induce hairy roots. The GA content was quantified by HPLC.Results: The PCR and sequencing results both showed that three transgenic hairy root lines were obtained. The copy number of Gu BAS in these transgenic hairy roots was intended by q RT-PCR to be 3, 7,and 4. GA was detected by HPLC, and the results showed that GA was present in the three transgenic hairy roots, while absent in wild hairy roots.Conclusion: Over-expressing Gu BAS root-specifically in hairy roots of G. uralensis enhanced GA accumulation.
基金This work was supported by the National Natural Science foundation of China(81072988).
文摘The rate-limiting enzyme in the mevalonic acid(MVA)pathway which can lead to triterpenoid saponin glycyrrhizic acid(GA)is 3-hydroxy-3-methylglutaryl-CoA reductase(HMGR).In order to reveal the effect of copy number variation in the HMGR gene on the MVA pathway,the HMGR gene from Glycyrrhiza uralensis Fisch.(GuHMGR)was cloned and over-expressed in Pichia pastoris GS115.Six recombinant P.pastoris strains containing different copy numbers of the GuHMGR gene were obtained and the content of ergosterol was analyzed by HPLC.The results showed that all the recombinant P.pastoris strains contained more ergosterol than the negative control and the strains with 8 and 44 copies contained significantly more ergosterol than the other strains.However,as the copy number increased,the content of ergosterol showed an increasing–decreasing–increasing pattern.This study provides a rationale for increasing the content of GA through over-expressing the GuHMGR gene in cultivars of G.uralensis.
基金supported in part by the National Key Research and Development Program of China (2017YFA0104401)the National Natural Science Foundation of China (31422037 and 31571522)
文摘Porcine reproductive and respiratory syndrome virus （PRRSV） is a major pathogen that causes reproductive failure and respiratory disease in pigs, resulting in devastating economic losses worldwide. Porcine alveolar macrophages （PAMs） are the primary target cells of PRRSV , and the putative receptors, including CD163, CD169, and CD151, play key roles during infection . However, the understanding of PRRSV infection and pathogenesis is limited by its narrow host range. Pig is the unparalleled animal susceptible to PRRSV, but not well-suited for the study of long-term chronic infection or immune response in vivo because of their long breed- ing cvcle, size, high cost, and lack of biological materials.
