中国农业科学 ›› 2013, Vol. 46 ›› Issue (15): 3172-3179.doi: 10.3864/j.issn.0578-1752.2013.15.011

• 园艺 • 上一篇    下一篇

mRNA差异显示法筛选‘砀山酥梨’褐皮芽变相关差异表达基因

 衡伟, 程秀云, 贾兵, 刘普, 刘莉, 叶振风, 朱立武   

  1. 安徽农业大学园艺学院果树学重点实验室,合肥230036
  • 收稿日期:2012-12-25 出版日期:2013-08-01 发布日期:2013-05-17
  • 通讯作者: 通信作者朱立武,Tel:0551-65786607;E-mail:zhuliwu@ahau.edu.cn
  • 作者简介:衡伟,E-mail:hengwei@ahau.edu.cn
  • 基金资助:

    国家现代农业产业技术体系建设专项(CARS-29-14)、国家自然科学基金项目(31101519)、高等学校省级优秀青年人才基金项目(2012SQRL060ZD)

Differential Expression Genes of ‘Dangshansuli’ Pear Russet Mutant Screened with DDRT-PCR

 HENG  Wei, CHENG  Xiu-Yun, JIA  Bing, LIU  Pu, LIU  Li, YE  Zhen-Feng, ZHU  Li-Wu   

  1. College of Horticulture, The Key Laboratory of Pomology, Anhui Agricultural University, Hefei 230036
  • Received:2012-12-25 Online:2013-08-01 Published:2013-05-17

摘要: 【目的】研究‘砀山酥梨’褐皮芽变形成机理。【方法】以‘砀山酥梨’及其褐皮芽变‘锈酥’花后110 d幼果果皮为试材,利用mRNA差异显示技术筛选相关差异表达基因,基于Gene Ontology方法、采用Blast2 Go软件进行基因功能分析,结合real-time RT-PCR技术验证差异表达基因在花后不同时期的表达水平差异。【结果】筛选出的63条目的片段中,60条质量较高、3条质量较低;差异表达基因包含α-微管蛋白、甲基转移酶、甘露糖- 6-磷酸异构酶、液泡膜焦磷酸酶和泛素等数十个基因。花后90—150 d,α-微管蛋白基因在‘锈酥’中表达量均高于‘砀山酥梨’,特别是在花后90和110 d,其表达量分别为‘砀山酥梨’的5倍和3倍。而在花后90和110 d时,Ca2+/CaM基因在‘锈酥’果皮中的表达略高于‘砀山酥梨’;在120—150 d时,Ca2+/CaM依赖的丝氨酸/苏氨酸蛋白激酶基因在‘锈酥’果皮中的表达明显低于‘砀山酥梨’。除花后130 d,甘露糖- 6-磷酸异构酶基因在‘砀山酥梨’和‘锈酥’果皮中表达差异不明显。【结论】由试验结果推测,α-微管蛋白基因在果实发育全程的增量表达,以及蛋白激酶基因在果实发育后期表达水平下调,使‘锈酥’果皮细胞壁加厚、木栓化程度加大,导致了‘锈酥’褐色果皮的形成。

关键词: 梨 , 褐皮芽变 , 果皮 , 差异基因 , 荧光定量

Abstract: 【Objective】The objective of this experiment is to study the formation mechanism of ‘Dangshansuli’ pear russet mutant. 【Method】 The peels of ‘Dangshansuli’ pear and its mutant ‘Xiusu’ on 110 d after full bloom were used to screen the differential expression genes with mRNA differential display reverse transcription-polymerase chain reaction (DDRT-PCR). 【Result】 Based on the function annotation, the key differential genes related with russet formation were confirmed with real-time PCR on 90, 110, 120, 140 and 150 d. The results showed that 63 differential genes were screened with 60 high quality and 3 low quality genes, including alpha-tubulin, N-methyltransferase, mannose-6-phosphate isomerase-like, calmodulin-binding protein kinase, vacuolar H+-PPase and so on. The expressions of α-tubulin gene in the pericarp of ‘Xiusu’ were higher than those in ‘Dangshansuli’ pear at different stages, and expecially on the 90 d and 110 d after full bloom with 5 times and 3 times, respectively. On the 90 d and 110 d after full bloom, the expression of calcium/calmodulin-dependent serine/threonine protein kinase gene in the pericarp of ‘Xiusu’ were slightly higher than those in ‘Dangshansuli’ pear, while the expressions of Ca2+/CaM gene in the pericarp of ‘Xiusu’ were distinctly lower than those in ‘Dangshansuli’ pear during 120-150 d after full bloom. The expression of mannose-6-phosphate isomerase gene in the pericarp of ‘Dangshansuli’ pear and ‘Xiusu’ showed no distinct difference except 130 d after full bloom. 【Conclusion】 These results indicated that the pericarp of russet mutant was lignified with α-tubulin accumulation regulated with the expression of calcium/calmodulin-dependent serine/threonine protein kinase gene.

Key words: Pyrus , russet mutant , fruit peel , differential gene , real-time PCR