mRNA差异显示法筛选‘砀山酥梨’褐皮芽变相关差异表达基因  

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

作　　者：衡伟[1] 程秀云[1] 贾兵[1] 刘普[1] 刘莉[1] 叶振风[1] 朱立武[1] 

机构地区：[1]安徽农业大学园艺学院果树学重点实验室,合肥230036

出　　处：《中国农业科学》2013年第15期3172-3179,共8页Scientia Agricultura Sinica

基　　金：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)

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Blast2Go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。【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.

关 键 词：梨 褐皮芽变 果皮 差异基因 荧光定量 

分 类 号：S661.2[农业科学—果树学]