The Salmonella pathogenicity islands(SPIs) play crucial roles in the progression of Salmonella infection. In this study, we constructed an improved λ Red homologous recombination system to prepare single and triple d...The Salmonella pathogenicity islands(SPIs) play crucial roles in the progression of Salmonella infection. In this study, we constructed an improved λ Red homologous recombination system to prepare single and triple deletion mutants of 3 prominent SPIs(SPI-1, 2, and 3), aiming at the impact of deletion on morphology, carbon source metabolism, adhesion and invasion capacity, in vivo colonization, and immune efficacy in chicks. Our examination revealed that the surface of the single deletion mutants(SM6ΔSPI1, ΔSPI2, and ΔSPI3) exhibited a more rugged texture and appeared to be enveloped in a layer of transparent colloid, whereas the morphology of the triple deletion mutant(SM6ΔSPI1&2&3) remained unaltered when compared to the parent strain. The carbon metabolic spectrum of the SPI mutants underwent profound alterations, with a notable and statistically significant modification observed in 30 out of 95 carbon sources, primarily carbohydrates(17 out of 30). Furthermore, the adhesion capacity of the 4 mutants to Caco-2 cells was significantly reduced when compared to that of the parent strain. Moreover,the invasion capacity of mutants SM6ΔSPI1 and SM6ΔSPI1&2&3 exhibited a substantial decrease, while it was enhanced to varying degrees for SM6ΔSPI3 and SM6ΔSPI2. Importantly, none of the 4 mutants induced any clinical symptoms in the chicks. However, they did transiently colonize the spleen and liver. Notably, the SM6ΔSPI1&2&3mutant was rapidly cleared from both the spleen and liver within 8 days post-infection and no notable pathological changes were observed in the organs. Additionally, when challenged, the mutants immunized groups displayed a significant increase in antibody levels and alterations in the CD3+CD4+ and CD3+CD8+ subpopulations, and the levels of IL-4 and IFN-γ cytokines in the SM6ΔSPI1&2&3 immunized chicken serum surpassed those of other groups.In summary, the successful construction of the 4 SPI mutants lays the groundwork for further exploration into the pathogenic(including metabolic) mechanisms of SPIs and the development of safe and effective live attenuated Salmonella vaccines or carriers.展开更多
Nitrogen(N)is a key component in plants and their biological macromolecules,having a profound effect on developmental stages,such as germination,vegetative growth,and flowering.However,the mechanism of nitrogen-regula...Nitrogen(N)is a key component in plants and their biological macromolecules,having a profound effect on developmental stages,such as germination,vegetative growth,and flowering.However,the mechanism of nitrogen-regulated flowering time remains unclear.In this study,CmNLP7 was isolated from the chrysanthemum cultivar‘Jinba'and was characterized.CmNLP7 is a transcription factor localized in the nucleus but has no transcriptional activity.Tissue expression pattern analysis showed that CmNLP7 was mainly transcribed in leaves and roots.Knocking down CmNLP7 through the artificial-miRNA method in chrysanthemum resulted in early flowering under optimal nitrogen(ON)and low nitrogen(LN)conditions;whereas overexpression lines showed delayed flowering under LN conditions.Transcriptome sequencing analysis showed that the nitrate transporters NRT2.5,NPF3.1,and NPF4.6;SBP-like genes SPL7 and SPL12,and flowering integration factor FT were significantly up-regulated in the knockdown lines.Based on the KEGG pathway enrichment analysis,the differentially transcribed genes were enriched in phenylpropanoid biosynthesis and starch and sucrose metabolism pathways,which indicated their alleged function in nitrogen-regulated flowering and development in chrysanthemum.Furthermore CmPP6 as a homolog of the Arabidopsis phosphatase PP6,was verified as an interacting protein of CmNLP7 by yeast two-hybrid,BiFC,pull-down and Biacore in vitro and in vivo,and the knockdown line of CmPP6(amiR-CmPP6)flowered earlier compared to that of the wild-type chrysanthemum‘Jinba'.Collectively,these results demonstrated that CmPP6 interacts with CmNLP7 to regulate chrysanthemum flowering,and CmNLP7 could regulate flowering time in response to nitrogen,which lays a foundation for the regulation of flowering and molecular breeding of chrysanthemum through changes in nutrient signaling.展开更多
【目的】从枳[Poncirus trifoliata(L.)Raf.]中克隆SBP类[SQUAMOSA(SQUA)promoter-binding-like]转录因子基因SPL9和SPL13全长,构建SPL9和SPL13亚细胞定位表达载体验证其是否具有核定位功能,利用荧光定量PCR研究其在枳不同组织的表达特...【目的】从枳[Poncirus trifoliata(L.)Raf.]中克隆SBP类[SQUAMOSA(SQUA)promoter-binding-like]转录因子基因SPL9和SPL13全长,构建SPL9和SPL13亚细胞定位表达载体验证其是否具有核定位功能,利用荧光定量PCR研究其在枳不同组织的表达特性,初步确定SPL9和SPL13在枳生长发育过程中的作用。【方法】利用生物信息学结合RACE技术以枳花器官的cDNA为模板,克隆出SPL9和SPL13基因全长,分别命名Pt-SPL9和Pt-SPL13,大小分别是1519bp和1824bp,在GenBank的登录号分别是FJ502237和FJ502238;构建Pt-SPL9和Pt-SPL13亚细胞定位载体35S-GW-FJ502237/FJ502238-GFP,基因枪转化洋葱表皮细胞,暗培养24h后激光共聚焦显微镜下观察;利用SYBR Green I实时定量RT-PCR方法检测Pt-SPL9和Pt-SPL13在根、茎、叶、花序、花和果等不同组织中的表达。【结果】生物信息学分析表明,Pt-SPL9和Pt-SPL13的cDNA序列中都有microRNA156的识别位点,Pt-SPL9与金鱼草、拟南芥和玉米SPL9的同源性分别为48.9%、42.5%和41.7%;Pt-SPL13与拟南芥SPL13、水稻的SPL16和玉米的TGA1同源性分别为40.8%、38.1%和35.8%。Pt-SPL9和Pt-SPL13与其它植物的SBP一样有着高度保守的序列,即SBP结构域和一个双向核定位信号KRXXXRRRK。亚细胞定位结果表明,Pt-SPL9和Pt-SPL13均定位于细胞核中。SYBR Green I实时定量RT-PCR结果表明,Pt-SPL9和Pt-SPL13在各个器官均有表达,但表达量不同,Pt-SPL9在茎中的表达量最高,在花和叶中的表达量次之,在根、花芽和幼果中的表达量最低;Pt-SPL13在幼果中的表达量最高,在茎和花芽中的表达量相当,其次为叶,在花和根中的表达量很低。【结论】转录因子Pt-SPL9和Pt-SPL13均具有核定位功能,Pt-SPL9和Pt-SPL13对枳的茎和果实的发育可能有着重要作用。展开更多
实际地震波形观测表明,对于大陆结构相对简单的地壳中的地震而言,有一震相出现在P波和S波之间.一般在30～50km附近发育得较好,其能量主要集中在径向分量,而垂向分量的振幅相对径向要小,切向分量上的振幅很弱,且波形以低频为主,通常没有...实际地震波形观测表明,对于大陆结构相对简单的地壳中的地震而言,有一震相出现在P波和S波之间.一般在30～50km附近发育得较好,其能量主要集中在径向分量,而垂向分量的振幅相对径向要小,切向分量上的振幅很弱,且波形以低频为主,通常没有P波尖锐.在利用FK方法计算合成地震图的基础上,发现该震相是由S波入射到自由地表形成水平传播的P波(文献称为surface P wave,自由地表P波)或者包括S波入射到地表后形成的多次P波或其散射震相.由于该震相是由S波和P波之间耦合而形成,本文将其定义为sPL(s coupled into P)震相.理论波形研究表明,sPL相对直达P波的到时差对震中距离不敏感,而随着震源深度的增加几乎呈线性增加,因此可以很好的约束震源深度.本文以2005年江西九江地震为例,证实了sPL确定震源深度的可行性和可靠性.在观测到sPL震相的情况下,离震源50km以内的一个三分量地震台站的波形就可以帮助获得可靠的震源深度,而不需要精确的震中距离.由于sPL震相出现距离较近,对于较小(三级以上)的地震也可以应用,因此在稀疏台网布局情形下sPL对于确定中小地震深度应该具有很好的应用意义.展开更多
基金supported by the National KeyR&DProgramof China(2022YFF0710500)the National Natural Science Foundation of China(32172853 and 32373013)the Central Public-interest Scientific Institution Basal Research Fund,China(1610302022001).
文摘The Salmonella pathogenicity islands(SPIs) play crucial roles in the progression of Salmonella infection. In this study, we constructed an improved λ Red homologous recombination system to prepare single and triple deletion mutants of 3 prominent SPIs(SPI-1, 2, and 3), aiming at the impact of deletion on morphology, carbon source metabolism, adhesion and invasion capacity, in vivo colonization, and immune efficacy in chicks. Our examination revealed that the surface of the single deletion mutants(SM6ΔSPI1, ΔSPI2, and ΔSPI3) exhibited a more rugged texture and appeared to be enveloped in a layer of transparent colloid, whereas the morphology of the triple deletion mutant(SM6ΔSPI1&2&3) remained unaltered when compared to the parent strain. The carbon metabolic spectrum of the SPI mutants underwent profound alterations, with a notable and statistically significant modification observed in 30 out of 95 carbon sources, primarily carbohydrates(17 out of 30). Furthermore, the adhesion capacity of the 4 mutants to Caco-2 cells was significantly reduced when compared to that of the parent strain. Moreover,the invasion capacity of mutants SM6ΔSPI1 and SM6ΔSPI1&2&3 exhibited a substantial decrease, while it was enhanced to varying degrees for SM6ΔSPI3 and SM6ΔSPI2. Importantly, none of the 4 mutants induced any clinical symptoms in the chicks. However, they did transiently colonize the spleen and liver. Notably, the SM6ΔSPI1&2&3mutant was rapidly cleared from both the spleen and liver within 8 days post-infection and no notable pathological changes were observed in the organs. Additionally, when challenged, the mutants immunized groups displayed a significant increase in antibody levels and alterations in the CD3+CD4+ and CD3+CD8+ subpopulations, and the levels of IL-4 and IFN-γ cytokines in the SM6ΔSPI1&2&3 immunized chicken serum surpassed those of other groups.In summary, the successful construction of the 4 SPI mutants lays the groundwork for further exploration into the pathogenic(including metabolic) mechanisms of SPIs and the development of safe and effective live attenuated Salmonella vaccines or carriers.
基金supported by grants from the National Natural Science Foundation of China(Grant No.31930100)a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions。
文摘Nitrogen(N)is a key component in plants and their biological macromolecules,having a profound effect on developmental stages,such as germination,vegetative growth,and flowering.However,the mechanism of nitrogen-regulated flowering time remains unclear.In this study,CmNLP7 was isolated from the chrysanthemum cultivar‘Jinba'and was characterized.CmNLP7 is a transcription factor localized in the nucleus but has no transcriptional activity.Tissue expression pattern analysis showed that CmNLP7 was mainly transcribed in leaves and roots.Knocking down CmNLP7 through the artificial-miRNA method in chrysanthemum resulted in early flowering under optimal nitrogen(ON)and low nitrogen(LN)conditions;whereas overexpression lines showed delayed flowering under LN conditions.Transcriptome sequencing analysis showed that the nitrate transporters NRT2.5,NPF3.1,and NPF4.6;SBP-like genes SPL7 and SPL12,and flowering integration factor FT were significantly up-regulated in the knockdown lines.Based on the KEGG pathway enrichment analysis,the differentially transcribed genes were enriched in phenylpropanoid biosynthesis and starch and sucrose metabolism pathways,which indicated their alleged function in nitrogen-regulated flowering and development in chrysanthemum.Furthermore CmPP6 as a homolog of the Arabidopsis phosphatase PP6,was verified as an interacting protein of CmNLP7 by yeast two-hybrid,BiFC,pull-down and Biacore in vitro and in vivo,and the knockdown line of CmPP6(amiR-CmPP6)flowered earlier compared to that of the wild-type chrysanthemum‘Jinba'.Collectively,these results demonstrated that CmPP6 interacts with CmNLP7 to regulate chrysanthemum flowering,and CmNLP7 could regulate flowering time in response to nitrogen,which lays a foundation for the regulation of flowering and molecular breeding of chrysanthemum through changes in nutrient signaling.
文摘【目的】从枳[Poncirus trifoliata(L.)Raf.]中克隆SBP类[SQUAMOSA(SQUA)promoter-binding-like]转录因子基因SPL9和SPL13全长,构建SPL9和SPL13亚细胞定位表达载体验证其是否具有核定位功能,利用荧光定量PCR研究其在枳不同组织的表达特性,初步确定SPL9和SPL13在枳生长发育过程中的作用。【方法】利用生物信息学结合RACE技术以枳花器官的cDNA为模板,克隆出SPL9和SPL13基因全长,分别命名Pt-SPL9和Pt-SPL13,大小分别是1519bp和1824bp,在GenBank的登录号分别是FJ502237和FJ502238;构建Pt-SPL9和Pt-SPL13亚细胞定位载体35S-GW-FJ502237/FJ502238-GFP,基因枪转化洋葱表皮细胞,暗培养24h后激光共聚焦显微镜下观察;利用SYBR Green I实时定量RT-PCR方法检测Pt-SPL9和Pt-SPL13在根、茎、叶、花序、花和果等不同组织中的表达。【结果】生物信息学分析表明,Pt-SPL9和Pt-SPL13的cDNA序列中都有microRNA156的识别位点,Pt-SPL9与金鱼草、拟南芥和玉米SPL9的同源性分别为48.9%、42.5%和41.7%;Pt-SPL13与拟南芥SPL13、水稻的SPL16和玉米的TGA1同源性分别为40.8%、38.1%和35.8%。Pt-SPL9和Pt-SPL13与其它植物的SBP一样有着高度保守的序列,即SBP结构域和一个双向核定位信号KRXXXRRRK。亚细胞定位结果表明,Pt-SPL9和Pt-SPL13均定位于细胞核中。SYBR Green I实时定量RT-PCR结果表明,Pt-SPL9和Pt-SPL13在各个器官均有表达,但表达量不同,Pt-SPL9在茎中的表达量最高,在花和叶中的表达量次之,在根、花芽和幼果中的表达量最低;Pt-SPL13在幼果中的表达量最高,在茎和花芽中的表达量相当,其次为叶,在花和根中的表达量很低。【结论】转录因子Pt-SPL9和Pt-SPL13均具有核定位功能,Pt-SPL9和Pt-SPL13对枳的茎和果实的发育可能有着重要作用。
文摘实际地震波形观测表明,对于大陆结构相对简单的地壳中的地震而言,有一震相出现在P波和S波之间.一般在30～50km附近发育得较好,其能量主要集中在径向分量,而垂向分量的振幅相对径向要小,切向分量上的振幅很弱,且波形以低频为主,通常没有P波尖锐.在利用FK方法计算合成地震图的基础上,发现该震相是由S波入射到自由地表形成水平传播的P波(文献称为surface P wave,自由地表P波)或者包括S波入射到地表后形成的多次P波或其散射震相.由于该震相是由S波和P波之间耦合而形成,本文将其定义为sPL(s coupled into P)震相.理论波形研究表明,sPL相对直达P波的到时差对震中距离不敏感,而随着震源深度的增加几乎呈线性增加,因此可以很好的约束震源深度.本文以2005年江西九江地震为例,证实了sPL确定震源深度的可行性和可靠性.在观测到sPL震相的情况下,离震源50km以内的一个三分量地震台站的波形就可以帮助获得可靠的震源深度,而不需要精确的震中距离.由于sPL震相出现距离较近,对于较小(三级以上)的地震也可以应用,因此在稀疏台网布局情形下sPL对于确定中小地震深度应该具有很好的应用意义.
