【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
Related to ABI3 and VP1(RAV)transcription factors belong to the AP2 and B3 superfamily.RAVs genes have been reported to be involved in plant growth and development regulation.This study screened three RAV genes from M...Related to ABI3 and VP1(RAV)transcription factors belong to the AP2 and B3 superfamily.RAVs genes have been reported to be involved in plant growth and development regulation.This study screened three RAV genes from Medicago truncatula and named one of them MtRAV1.The MtRAV1 overexpressing plants exhibits traits such as plant dwarfing,delayed flowering,reduced leaf and floral organs,increased branching,and reduced pods and seeds.Gene expression analysis results showed that overexpression of Mt RAV1 inhibited the expression of Flowering Locus T(MtFTa1),Suppressor of Overexpression of CO1(MtSOC1),GA3-oxidase1(MtGA3OX1),DWARF14(MtD14)and Carotenoid Cleavage Dioxygenase7(MtCCD7).To further investigate the regulation pathway involved by MtRAV1,RNA-sequencing(RNA-seq)and DNA affinity purification sequencing(DAP-seq)analysis were conducted.RNA-seq results indicated that MtRAV1 might affect plant growth and development by regulating some genes in photosynthesis,circadian rhythm and plant hormone signaling pathways,especially the auxin signaling pathway.Conjoint analysis of DAP-seq and RNA-seq revealed that Mt RAV1 might inhibit the expression of Ferredoxin(Mt Fd-l3),Light-harvesting Chlorophyll a/b Binding Protein 1(Mt Lhcb-l2)and Small Auxin Up-regulated RNA(Mt SAUR-l),which related to photosystem II and auxin signaling pathway.Summarily,MtRAV1 was preliminarily proven to be a key growth inhibitory factor in M.truncatula.展开更多
RAVE （regulator of the H ^＋ -ATPase of the vacuolar and endosomal membranes）复合物由Rav1p、Rav2p和Skp1p3（DE3）中进行表达。通过IPTG诱导，SDS-PAGE分析重组菌在诱导后表达出目的蛋白。目的蛋白经Ni-NTA凝胶纯化后再经质谱...RAVE （regulator of the H ^＋ -ATPase of the vacuolar and endosomal membranes）复合物由Rav1p、Rav2p和Skp1p3（DE3）中进行表达。通过IPTG诱导，SDS-PAGE分析重组菌在诱导后表达出目的蛋白。目的蛋白经Ni-NTA凝胶纯化后再经质谱进一步验证确定为酿酒酵母的Rav2p蛋白。目前国际上还没有有关Rav2p的结构和性质以及RAVE亚基之间相互关系的研究。重组质粒pETDuet-R2的成功构建以及Rav2p的可溶性表达为研究RAVE亚基之间的相互作用以及V-ATP酶的活性调节机理打下基础。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。
基金supported by the Key Projects in Science and Technology of Inner Mongolia,China(2021ZD0031)the China Agriculture Research System of MOF and MARA(CARS-34)+1 种基金the Performance Incentive and Guidance Special Project of Scientific Research Institution,Chongqing Science and Technology Committee,China(cstc2022jxjl80019)the Natural Science Foundation of Chongqing,China(CSTB2022NSCQ-BHX0744)。
文摘Related to ABI3 and VP1(RAV)transcription factors belong to the AP2 and B3 superfamily.RAVs genes have been reported to be involved in plant growth and development regulation.This study screened three RAV genes from Medicago truncatula and named one of them MtRAV1.The MtRAV1 overexpressing plants exhibits traits such as plant dwarfing,delayed flowering,reduced leaf and floral organs,increased branching,and reduced pods and seeds.Gene expression analysis results showed that overexpression of Mt RAV1 inhibited the expression of Flowering Locus T(MtFTa1),Suppressor of Overexpression of CO1(MtSOC1),GA3-oxidase1(MtGA3OX1),DWARF14(MtD14)and Carotenoid Cleavage Dioxygenase7(MtCCD7).To further investigate the regulation pathway involved by MtRAV1,RNA-sequencing(RNA-seq)and DNA affinity purification sequencing(DAP-seq)analysis were conducted.RNA-seq results indicated that MtRAV1 might affect plant growth and development by regulating some genes in photosynthesis,circadian rhythm and plant hormone signaling pathways,especially the auxin signaling pathway.Conjoint analysis of DAP-seq and RNA-seq revealed that Mt RAV1 might inhibit the expression of Ferredoxin(Mt Fd-l3),Light-harvesting Chlorophyll a/b Binding Protein 1(Mt Lhcb-l2)and Small Auxin Up-regulated RNA(Mt SAUR-l),which related to photosystem II and auxin signaling pathway.Summarily,MtRAV1 was preliminarily proven to be a key growth inhibitory factor in M.truncatula.
文摘RAVE （regulator of the H ^＋ -ATPase of the vacuolar and endosomal membranes）复合物由Rav1p、Rav2p和Skp1p3（DE3）中进行表达。通过IPTG诱导，SDS-PAGE分析重组菌在诱导后表达出目的蛋白。目的蛋白经Ni-NTA凝胶纯化后再经质谱进一步验证确定为酿酒酵母的Rav2p蛋白。目前国际上还没有有关Rav2p的结构和性质以及RAVE亚基之间相互关系的研究。重组质粒pETDuet-R2的成功构建以及Rav2p的可溶性表达为研究RAVE亚基之间的相互作用以及V-ATP酶的活性调节机理打下基础。
