【目的】探究山茶Related to ABI3 and VP1(CjRAV1)基因在开花调控中的功能及其分子机制,为四季山茶的分子育种提供理论依据。【方法】采用生物信息学分析、基因表达模式分析、转基因技术和DAP-seq等多种实验手段,系统研究CjRAV1的功能...【目的】探究山茶Related to ABI3 and VP1(CjRAV1)基因在开花调控中的功能及其分子机制,为四季山茶的分子育种提供理论依据。【方法】采用生物信息学分析、基因表达模式分析、转基因技术和DAP-seq等多种实验手段,系统研究CjRAV1的功能及其调控机制。生物信息学分析鉴定CjRAV1的基因结构、保守结构域及其系统进化关系。利用RT-qPCR技术分析CjRAV1在外源激素诱导下、不同组织以及花苞不同发育时期的表达模式。构建CjRAV1过表达转基因拟南芥植株,观察其表型变化,尤其是开花时间的改变。最后,采用DAP-seq技术筛选CjRAV1下游潜在的DNA结合位点及其调控基因,揭示CjRAV1的分子调控网络。【结果】生物信息学分析表明,CjRAV1的开放阅读框长度为1101 bp,共编码366个氨基酸,具有AP2和B3保守结构域。系统进化分析显示,山茶CjRAV1蛋白与茶树CsRAV蛋白的亲缘关系最近,表明两者可能具有相似的功能。亚细胞定位分析证实,CjRAV1转录因子定位于细胞核,提示其可能在转录调控中发挥直接作用。表达模式分析显示,CjRAV1在山茶叶中表达量最高;在花苞成熟过程中,CjRAV1的表达量总体呈现逐步下降的趋势。CjRAV1的过表达转基因拟南芥表现出晚花的表型。通过DAP-seq筛选出潜在的下游调控基因CjERF。【结论】CjRAV1过表达导致转基因拟南芥植株表现出晚花的表型,且这一功能可能与潜在的调控基因CjERF协作完成。展开更多
Xylanase 1 (Xyn1) is one of the two major representatives of the xylanase system of T. reesei; the mechanisms governing its expression were analysed throughout this study. All factors and regulatory motifs responsible...Xylanase 1 (Xyn1) is one of the two major representatives of the xylanase system of T. reesei; the mechanisms governing its expression were analysed throughout this study. All factors and regulatory motifs responsible for transcriptional regulation and the model of their interplay in induction and repression will be presented. Using in vivo foot printing analysis of xylan-induced and glucose repressed mycelia, we detected three adjacent nucleotide sequences contacted by DNA-binding proteins. Protection within the inverted repeat of the Cre1 (SYGGRG) consensus sequence on the non coding strand under repressing conditions is in perfect agreement with the previously reported Cre1 dependent glucose repression of xyn1. Constitutive protein binding could be observed to a CCAAT-box and an inverted repeat of a 5′ GGCTAA 3′ sequence. EMSA with crude extracts from induced and repressed mycelia revealed that the latter motifs are sufficient for formation of the basal transcriptional complex under all conditions. The inverted repeat of GGCTAA closely resembles the consensus sequences of the cellulase and xylanase regulators Ace1, Ace2 and, Xyr1 (encoded by xyr1, cloned and characterised in this study) EMSA with heterologously expressed components of each factor and of the T. reesei Hap2/3/5 protein complex revealed that the basal transcriptional complex is formed by Xyr1 and the Hap2/3/5. Additionally to the Cre1 mediated carbon catabolite repression a yet unknown mechanism antagonizing induction of xyn1 expression could be elucidated. Latter occurs through competition of the repressor Ace1 and Xyr1 for the GGCTAA motif. In vivo proof for the relevance of identified motifs could be given through analysis of T. reesei transformants containing correspondingly mutated versions of the xyn1 promoter fused to the A. niger goxA gene. The results indicated that the basal as well as the induction level of xyn1 gene transcription is dependent on an interaction of Xyr1 with the GGCTAA motif while formation of the CCAAT-Hap2/3/5 complex slightly reduces induction. It can be concluded that mutations impairing protein binding in vitro lead to a loss of distinct regulatory functions in xyn1 gene expression in vivo. A respective model of gene regulation will be presented.展开更多
Prostaglandin E-2(PGE(2)) is a well-known mediator of beta-cell dysfunction in both type 1 and type 2 diabetes.We recently reported that down-regulation of the Akt pathway activity is implicated in PGE(2)-induced panc...Prostaglandin E-2(PGE(2)) is a well-known mediator of beta-cell dysfunction in both type 1 and type 2 diabetes.We recently reported that down-regulation of the Akt pathway activity is implicated in PGE(2)-induced pancreatic beta-cell dysfunction.The aim of this study was to further dissect the signaling pathway of this process in pancreatic beta-cell line HIT-T15 cells and primary mouse islets.We found that PGE(2) time-dependently increased the c-Jun N-terminal kinase(JNK) pathway activity.JNK inhibition by the JNK-specific inhibitor SP600125 reversed PGE(2)-inhibited glucose-stimulated insulin secretion(GSIS).PGE(2) induced dephosphorylation of Akt and FOXO1, leading to nuclear localization and transactivation of FOXO1.Activation of FOXO1 induced nuclear exclusion but had no obvious effect on the whole-cell protein level of pancreatic and duodenal homeobox 1(PDX1).However, these effects were all attenuated by JNK inhibition.Furthermore, adenovirus-mediated overexpression of dominant-negative(DN)FOXO1 abolished whereas constitutively active(CA)-FOXO1 mimicked the effects of PGE(2) on GSIS in isolated mouse islets.In addition, we demonstrated that DN-JNK1 but not DN-JNK2 or CA-Akt abolished the PGE(2)-induced AP-1 luciferase reporter activity, whereas DN-JNK1 and CA-Akt but not DN-JNK2 reversed the effect of PGE(2) on FOXO1 transcriptional activity, and overexpression of DN-JNK1 rescued PGE(2)-impaired GSIS in mouse islets.Our results revealed that activation of the JNK is involved in PGE(2)induced beta-cell dysfunction.PGE(2)-mediated JNK1 activation, through dephosphorylation of Akt and FOXO1, leads to nuclear accumulation of FOXO1 and nucleocytoplasmic shuttling of PDX1, finally resulting in defective GSIS in pancreatic beta-cells.展开更多
文摘【目的】探究山茶Related to ABI3 and VP1(CjRAV1)基因在开花调控中的功能及其分子机制,为四季山茶的分子育种提供理论依据。【方法】采用生物信息学分析、基因表达模式分析、转基因技术和DAP-seq等多种实验手段,系统研究CjRAV1的功能及其调控机制。生物信息学分析鉴定CjRAV1的基因结构、保守结构域及其系统进化关系。利用RT-qPCR技术分析CjRAV1在外源激素诱导下、不同组织以及花苞不同发育时期的表达模式。构建CjRAV1过表达转基因拟南芥植株,观察其表型变化,尤其是开花时间的改变。最后,采用DAP-seq技术筛选CjRAV1下游潜在的DNA结合位点及其调控基因,揭示CjRAV1的分子调控网络。【结果】生物信息学分析表明,CjRAV1的开放阅读框长度为1101 bp,共编码366个氨基酸,具有AP2和B3保守结构域。系统进化分析显示,山茶CjRAV1蛋白与茶树CsRAV蛋白的亲缘关系最近,表明两者可能具有相似的功能。亚细胞定位分析证实,CjRAV1转录因子定位于细胞核,提示其可能在转录调控中发挥直接作用。表达模式分析显示,CjRAV1在山茶叶中表达量最高;在花苞成熟过程中,CjRAV1的表达量总体呈现逐步下降的趋势。CjRAV1的过表达转基因拟南芥表现出晚花的表型。通过DAP-seq筛选出潜在的下游调控基因CjERF。【结论】CjRAV1过表达导致转基因拟南芥植株表现出晚花的表型,且这一功能可能与潜在的调控基因CjERF协作完成。
文摘Xylanase 1 (Xyn1) is one of the two major representatives of the xylanase system of T. reesei; the mechanisms governing its expression were analysed throughout this study. All factors and regulatory motifs responsible for transcriptional regulation and the model of their interplay in induction and repression will be presented. Using in vivo foot printing analysis of xylan-induced and glucose repressed mycelia, we detected three adjacent nucleotide sequences contacted by DNA-binding proteins. Protection within the inverted repeat of the Cre1 (SYGGRG) consensus sequence on the non coding strand under repressing conditions is in perfect agreement with the previously reported Cre1 dependent glucose repression of xyn1. Constitutive protein binding could be observed to a CCAAT-box and an inverted repeat of a 5′ GGCTAA 3′ sequence. EMSA with crude extracts from induced and repressed mycelia revealed that the latter motifs are sufficient for formation of the basal transcriptional complex under all conditions. The inverted repeat of GGCTAA closely resembles the consensus sequences of the cellulase and xylanase regulators Ace1, Ace2 and, Xyr1 (encoded by xyr1, cloned and characterised in this study) EMSA with heterologously expressed components of each factor and of the T. reesei Hap2/3/5 protein complex revealed that the basal transcriptional complex is formed by Xyr1 and the Hap2/3/5. Additionally to the Cre1 mediated carbon catabolite repression a yet unknown mechanism antagonizing induction of xyn1 expression could be elucidated. Latter occurs through competition of the repressor Ace1 and Xyr1 for the GGCTAA motif. In vivo proof for the relevance of identified motifs could be given through analysis of T. reesei transformants containing correspondingly mutated versions of the xyn1 promoter fused to the A. niger goxA gene. The results indicated that the basal as well as the induction level of xyn1 gene transcription is dependent on an interaction of Xyr1 with the GGCTAA motif while formation of the CCAAT-Hap2/3/5 complex slightly reduces induction. It can be concluded that mutations impairing protein binding in vitro lead to a loss of distinct regulatory functions in xyn1 gene expression in vivo. A respective model of gene regulation will be presented.
文摘Prostaglandin E-2(PGE(2)) is a well-known mediator of beta-cell dysfunction in both type 1 and type 2 diabetes.We recently reported that down-regulation of the Akt pathway activity is implicated in PGE(2)-induced pancreatic beta-cell dysfunction.The aim of this study was to further dissect the signaling pathway of this process in pancreatic beta-cell line HIT-T15 cells and primary mouse islets.We found that PGE(2) time-dependently increased the c-Jun N-terminal kinase(JNK) pathway activity.JNK inhibition by the JNK-specific inhibitor SP600125 reversed PGE(2)-inhibited glucose-stimulated insulin secretion(GSIS).PGE(2) induced dephosphorylation of Akt and FOXO1, leading to nuclear localization and transactivation of FOXO1.Activation of FOXO1 induced nuclear exclusion but had no obvious effect on the whole-cell protein level of pancreatic and duodenal homeobox 1(PDX1).However, these effects were all attenuated by JNK inhibition.Furthermore, adenovirus-mediated overexpression of dominant-negative(DN)FOXO1 abolished whereas constitutively active(CA)-FOXO1 mimicked the effects of PGE(2) on GSIS in isolated mouse islets.In addition, we demonstrated that DN-JNK1 but not DN-JNK2 or CA-Akt abolished the PGE(2)-induced AP-1 luciferase reporter activity, whereas DN-JNK1 and CA-Akt but not DN-JNK2 reversed the effect of PGE(2) on FOXO1 transcriptional activity, and overexpression of DN-JNK1 rescued PGE(2)-impaired GSIS in mouse islets.Our results revealed that activation of the JNK is involved in PGE(2)induced beta-cell dysfunction.PGE(2)-mediated JNK1 activation, through dephosphorylation of Akt and FOXO1, leads to nuclear accumulation of FOXO1 and nucleocytoplasmic shuttling of PDX1, finally resulting in defective GSIS in pancreatic beta-cells.
