【目的】克隆小黑杨(Populus×xiaohei T.S.Hwang et Liang)RAV1和RAV2基因,分析这2个基因在非生物胁迫下的表达模式,为进一步研究小黑杨基因功能提供理论依据。【方法】以抗逆性强的小黑杨根为试材,克隆2个RAV家族成员。利用生物...【目的】克隆小黑杨(Populus×xiaohei T.S.Hwang et Liang)RAV1和RAV2基因,分析这2个基因在非生物胁迫下的表达模式,为进一步研究小黑杨基因功能提供理论依据。【方法】以抗逆性强的小黑杨根为试材,克隆2个RAV家族成员。利用生物信息学方法对小黑杨2个RAV基因的理化性质、保守基序、保守结构域、系统进化进行分析,并采用半定量RT-PCR方法探讨这2个基因在小黑杨顶芽、叶、木质部、韧皮部、根、第1~14片叶、第1~11茎节中的表达情况,以及在CdCl2、NaCl、NaHCO3、PEG和ABA胁迫下的表达特性。【结果】从小黑杨中克隆出2个RAV基因,命名为PsnRAV1和PsnRAV2。PsnRAV1编码405个氨基酸,预测分子质量约为99.46 ku,等电点5.06,不存在信号肽;PsnRAV2编码407个氨基酸,预测分子质量约为99.33 ku,等电点5.06,存在信号肽;2个基因均位于细胞核上。保守结构域和保守基序分析结果表明,小黑杨RAV蛋白具有RAV家族特有的AP2和B3这2个保守蛋白结构域,基序1、2、3是构成2个结构域的主要基序。系统进化分析表明,20种植物的RAV蛋白分为3组,其中PsnRAV1和PsnRAV2与毛果杨(Populus trichocarpa)RAV同源性很高。半定量RT-PCR分析结果显示,2个PsnRAVs基因在小黑杨的顶芽、叶、木质部、韧皮部和根中均有表达;且随着叶和茎的发育,2个基因的表达量均呈现升高的趋势。在CdCl2、NaCl、NaHCO3模拟的重金属及盐胁迫下,2个基因的表达模式虽不相同,但在根部均受到诱导;在PEG模拟的干旱胁迫下,PsnRAV1和PsnRAV2在叶片和根部均有响应,其中在根部受到的诱导更为明显,尤以PsnRAV2的表达更为显著;此外,PsnRAV1和PsnRAV2也受到ABA的诱导,其在叶中的表达量在初期表现为上升,而后期下降。【结论】2个PsnRAVs基因与小黑杨的抗逆性密切相关,可能参与了小黑杨的非生物胁迫应答,其中PsnRAV2在胁迫下的作用更为显著。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
RAV1 is a novel DNA-binding protein with two distinct DNA-binding domains unique in higher plants,but its role in plant growth and development remains unknown. Using cDNA array,we found that transcription of RAV1 is d...RAV1 is a novel DNA-binding protein with two distinct DNA-binding domains unique in higher plants,but its role in plant growth and development remains unknown. Using cDNA array,we found that transcription of RAV1 is downregulated by epibrassinolide (epiBL) in Arabidopsis suspension cells. RNA gel blot analysis revealed that epiBL-regulated RAV1 transcription involves neither protein phosphorylation/dephosphorylation nor newly synthesized protein,and does not require the functional BRI1,suggesting that this regulation might be through a new BR signaling pathway.Overexpressing RAV1 in Arabidopsis results in a retardation of lateral root and rosette leaf development,and the underexpression causes an earlier flowering phenotype,implying that RAV1 may function as a negative regulatory component of growth and development.展开更多
RAV(Related to ABI3/VP1)transcription factors are unique members of the AP2-ERF superfamily with AP2 and B3 domains and play important roles in the regulation of seed germination,plant growth,and stress response.In th...RAV(Related to ABI3/VP1)transcription factors are unique members of the AP2-ERF superfamily with AP2 and B3 domains and play important roles in the regulation of seed germination,plant growth,and stress response.In the study,7 RAV genes,named HvRAVs,were identified in barley based on the available genome sequences.While five of the seven HvRAVs were located on chromosome 3,HvRAV5 and HvRAV7 were located on chromosome 1 and 4,respectively.Six of the predicted HvRAVs were intron-less,except HvRAV2,which had one intron.HvRAV proteins have shown basic,instable,and hydrophilic properties.The AP2 domain specific RAYD and WLG motifs were detected in all HvRAV proteins.Besides,B3 repression domain,R/KLFGV,is also found in the C-terminal of HvRAVs.HvRAVs were found to have stress-related cis-acting elements,including MYB,MYC,and W-BOX.HvRAV2 was predicted to have no GARE motifs,TATCCCA or TAACAA(G/A),and LTREs.Under drought conditions,the expression level of HvRAVs did not significantly change in a drought-sensitive barley genotype,whereas HvRAV5 and HvRAV7 were dramatically down-regulated in a drought-tolerant genotype.Expression of HvRAV5 was also inhibited by salinity.HvRAV7 was strongly induced by plant pathogen attack.Only HvRAV6 was induced by exogenous gibberellin application and during the germination process.Interestingly,HvRAV6 transcript was detected higher than other HvRAVs in all stress and control conditions as well as during germination.In silico analyses have shown that HvRAVs play a role in response to different abiotic and biotic stresses as well as in plant development.However,the extensive biological roles of HvRAV genes in plant development and in response to abiotic and biotic stresses need further investigation.展开更多
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。
基金supported by grants from the National Natural Science Foundation of China(No.39889003,30070074,30221002)a National Distinguished Young Scholar Award to Jia Yang LI.
文摘RAV1 is a novel DNA-binding protein with two distinct DNA-binding domains unique in higher plants,but its role in plant growth and development remains unknown. Using cDNA array,we found that transcription of RAV1 is downregulated by epibrassinolide (epiBL) in Arabidopsis suspension cells. RNA gel blot analysis revealed that epiBL-regulated RAV1 transcription involves neither protein phosphorylation/dephosphorylation nor newly synthesized protein,and does not require the functional BRI1,suggesting that this regulation might be through a new BR signaling pathway.Overexpressing RAV1 in Arabidopsis results in a retardation of lateral root and rosette leaf development,and the underexpression causes an earlier flowering phenotype,implying that RAV1 may function as a negative regulatory component of growth and development.
文摘RAV(Related to ABI3/VP1)transcription factors are unique members of the AP2-ERF superfamily with AP2 and B3 domains and play important roles in the regulation of seed germination,plant growth,and stress response.In the study,7 RAV genes,named HvRAVs,were identified in barley based on the available genome sequences.While five of the seven HvRAVs were located on chromosome 3,HvRAV5 and HvRAV7 were located on chromosome 1 and 4,respectively.Six of the predicted HvRAVs were intron-less,except HvRAV2,which had one intron.HvRAV proteins have shown basic,instable,and hydrophilic properties.The AP2 domain specific RAYD and WLG motifs were detected in all HvRAV proteins.Besides,B3 repression domain,R/KLFGV,is also found in the C-terminal of HvRAVs.HvRAVs were found to have stress-related cis-acting elements,including MYB,MYC,and W-BOX.HvRAV2 was predicted to have no GARE motifs,TATCCCA or TAACAA(G/A),and LTREs.Under drought conditions,the expression level of HvRAVs did not significantly change in a drought-sensitive barley genotype,whereas HvRAV5 and HvRAV7 were dramatically down-regulated in a drought-tolerant genotype.Expression of HvRAV5 was also inhibited by salinity.HvRAV7 was strongly induced by plant pathogen attack.Only HvRAV6 was induced by exogenous gibberellin application and during the germination process.Interestingly,HvRAV6 transcript was detected higher than other HvRAVs in all stress and control conditions as well as during germination.In silico analyses have shown that HvRAVs play a role in response to different abiotic and biotic stresses as well as in plant development.However,the extensive biological roles of HvRAV genes in plant development and in response to abiotic and biotic stresses need further investigation.
