龙眼成花转变与成花逆转的差异蛋白质组学研究
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摘要
本研究以龙眼正常成花和成花逆转的花芽为研究对象,运用蛋白质组学方法和技术比较龙眼正常成花和成花逆转花芽的蛋白质组变化,应用双向电泳对花芽蛋白进行分离,从2-D凝胶上均检测到分离的蛋白质点约1000个,应用PDQuest软件对凝胶图谱进行分析,获得65个表达量差异在2倍以上的差异蛋白,其中28个蛋白在龙眼成花逆转花芽中上调表达, 37个蛋白下调表达。MALDI-TOF-TOF/MS串联质谱分析和数据库检索鉴定了其中41个差异表达蛋白,鉴定率为63%。鉴定得到的蛋白中,与龙眼成花逆转关系密切的物质和能量代谢相关蛋白,转录和翻译相关的蛋白,次生代谢相关蛋白,调控相关蛋白,抗逆相关蛋白和细胞骨架蛋白的生物学功能都得到了讨论。通过对这些差异蛋白在成花过程中的功能分析,表明这些蛋白在龙眼成花逆转过程中的差异表达可能影响了花芽的正常发育,进而导致了龙眼成花逆转。应用RT-PCR和RACE方法克隆其中4个与龙眼成花逆转关系密切的差异蛋白的基因:获得了上调表达的调控因子14-3-3蛋白(登录号FJ479618),细胞骨架α-微管蛋白(登录号FJ479617),转醛醇酶(登录号FJ472991)和下调表达的花青素合成酶(登录号FJ479616)的完整开放阅读框,4个基因的全长cDNA都分别在大肠杆菌中表达,获得相应外源蛋白,其中14-3-3蛋白和α-微管蛋白经过Western blotting验证确认。半定量RT-PCR结果显示,ANS和TAL在转录和翻译水平上具有同样表达差异,TUB和14-3-3在转录水平上无明显差异。
Proteomics method was used to compare and identify the differential proteins of normal flowering and floral reversion buds in longan (Dimocarpus longan Lour.). over 1000 protein spots were detected on 2 DE gels, PDQuest was applied on gel pattern analysis, 65 differentially expressed proteins were detected. Among these proteins, 28 were up-regulated and 37 were down-regulated on floral reversion buds. 41 differential proteins were identified by MALDI-TOF-TOF/MS and protein database searching, the identification rate was 63%. Biological function analysis showed these identificated proteins related with substance and energy metabolism, transcription and translation, secondary metabolism, regulation, stress tolerance and cytoskeleton. Open reading frame(ORF) of up-regulated protein regulation factor 14-3-3(GenBank Accession No. FJ479618), cytoskeleton alpha-tubulin(No. FJ479617), transaldolase(No. FJ472991) and down-regulated anthocyanidin synthase(No. FJ479616)were cloned with RACE method. All 4 genes were expressed in E.coli, and western blotting confirmed 14-3-3 and alpha-tubulin. Semi-Quantitative RT-PCR result showed ANS and TAL each has identical regulation on transcription and translation level, whileα-tublin and 14-3-3 each has minor expression differential on transcription level. The changes in expression level of these proteins might cause flower buds developing abnormally and lead to flowering reversion in longan.The results has an important significance in revealing the molecular mechanism of flowering reversion in longan.
Proteomics method was used to compare and identify the differential proteins of normal flowering and floral reversion buds in longan (Dimocarpus longan Lour.). over 1000 protein spots were detected on 2 DE gels, PDQuest was applied on gel pattern analysis, 65 differentially expressed proteins were detected. Among these proteins, 28 were up-regulated and 37 were down-regulated on floral reversion buds. 41 differential proteins were identified by MALDI-TOF-TOF/MS and protein database searching, the identification rate was 63%. Biological function analysis showed these identificated proteins related with substance and energy metabolism, transcription and translation, secondary metabolism, regulation, stress tolerance and cytoskeleton. Open reading frame(ORF) of up-regulated protein regulation factor 14-3-3(GenBank Accession No. FJ479618), cytoskeleton alpha-tubulin(No. FJ479617), transaldolase(No. FJ472991) and down-regulated anthocyanidin synthase(No. FJ479616)were cloned with RACE method. All 4 genes were expressed in E.coli, and western blotting confirmed 14-3-3 and alpha-tubulin. Semi-Quantitative RT-PCR result showed ANS and TAL each has identical regulation on transcription and translation level, whileα-tublin and 14-3-3 each has minor expression differential on transcription level. The changes in expression level of these proteins might cause flower buds developing abnormally and lead to flowering reversion in longan.The results has an important significance in revealing the molecular mechanism of flowering reversion in longan.
引文
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