摘要
【目的】柑橘自然芽变材料是柑橘育种的重要来源。‘明柳甜橘’(Citrus reticuiata Blancocv.Mingliutianju,MP)是从‘春甜橘’(C.reticuiata Blanco cv.Chuntianju,CP)中选育出的自然芽变品种。分析‘春甜橘’与其自然芽变‘明柳甜橘’果皮差异蛋白质,探讨导致两品种形态差异的原因。【方法】以花后12周和23周的‘明柳甜橘’和‘春甜橘’果皮为材料,采用双向电泳技术(Two-dimensional gel electrophoresis,2-DE)分离获得野生型与突变型差异表达蛋白点,利用MALDI-TOF-MS质谱鉴定技术分析相关差异蛋白质,并进行功能注释和生物信息学分析。【结果】将提取的果皮总蛋白质经双向电泳后,分别采用硝酸银和考马斯亮蓝染色法进行蛋白质凝胶染色,均获得了清晰的2-DE电泳图。2-DE图谱经Image Master 2D软件分析表明,硝酸银染色法能检测到约1 200个蛋白点,考马斯亮蓝染色法能检测到约500个蛋白质点,从中各选取20个变化丰度2倍以上且重复性好的蛋白点进行MALDI-TOF-MS质谱鉴定和数据库检索分析,共有33个蛋白质有功能注释,其中17个蛋白质在‘明柳甜橘’果皮中表达上调,16个表达下调。按照33个差异蛋白质的功能,可将其分为11类,它们分别参与糖类/能量代谢、胁迫/防御反应、核酸代谢、氨基酸代谢、转录、氮代谢、脂肪酸代谢、蛋白修饰与降解以及未知类。利用KOBAS(KEGG Orthology-Based Annotation System)在线功能分析平台对33个差异表达蛋白质进行信号通路分析,其中18个差异蛋白质有KO注释,共涉及31条信号通路,按照P值大小排列,类黄酮生物合成过程位居第一,说明该信号通路最有可能参与春甜橘芽变形成过程。【结论】综合分析‘春甜橘’和‘明柳甜橘’基因水平及蛋白质水平的差异,推测可能是由参与类黄酮生物合成过程的差异表达基因尿苷二磷酸葡萄糖基转移酶基因和差异表达蛋白咖啡酰�
【Objective】 Bud mutation of citrus is one of the most important breeding technique for new cultivars. However, little is known about the mechanisms of citrus bud mutation, particularly at the molecular level. Mingliutianju（C. reticuiata Blanco cv. Mingliutianju, MP） is a new cultivar selected from Chuntianju（C. reticuiata Blanco cv. Chuntianju, CP） bud mutation. To identify the differentially expressed proteins in peels of Chuntianju（C. reticuiata Blanco cv. Chuntianju, CP） and its bud mutant Mingliutianju（C. reticuiata Blanco cv. Mingliutianju, MP） and to clarify the possible mechanism of this phenotype bud mutation. 【Method】Two-dimensional gel electrophoresis（2-DE） combined with the mass spectrometry（MS） technology were applied to separate the differentially expressed proteins（DEPs） between the MP and CP using peels at 12 weeks and 23 weeks after flowering. Meanwhile, the identified differentially expressed proteins（DEPs） with fold-change of more than 2 were analyzed using MALDI-TOF-MS and Bioinformatics. 【Result】 Clear 2-DE gel maps of peels of both CP and MP were successfully obtained. In total, more than 1200 protein spots and 500 protein spots were detected by silver stained（SS） 2-DE gels and by Coomassie brilliant blue stained（CS） 2-DE gels respectively. Forty DEP spots（20 identified from SS 2-DE gels and 20 from CS 2-DE gels） were further analyzed through MALDI-TOF-MS. Totally, 33 annotated DEPs, including 17 up-regulated and 16 down-regulated DEPs, were identified in MP. Gene ontology analysis revealed that these 33 DEPs could be divided into 11 categories including carbohydrate/energy metabolism, stress/defense response, nucleotide metabolism, amino acid metabolism, transcription, nitrogen metabolism, lipid metabolism, unclassified, protein modification and degradation and unknown pathways. Moreover, by using the web-based platform KOBAS（KEGG Ontology-Based Annotation System）, 18 of the 33 DEPs were successfully mapped to 31 KEGG pathways, amon
出处
《中国农业科学》
CAS
CSCD
北大核心
2015年第24期4965-4978,共14页
Scientia Agricultura Sinica
基金
国家科技支撑计划(2013BAD92B02)
国家现代农业(柑橘)产业技术体系建设专项(CARS-27)
广东省科技计划(2012NL010
2015B090906026)
