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中国农学通报

中国农学通报 ›› 2015, Vol. 31 ›› Issue (7): 125-130.doi: 10.11924/j.issn.1000-6850.casb14110072

所属专题： 生物技术

• 林学 园艺 园林 • 上一篇    下一篇

基于实时荧光定量PCR技术的蝴蝶兰PPO和PAL基因表达分析

赵和文,崔金腾,周田田   

  1. 北京农学院 园林学院 北京 102206,北京农学院 园林学院 北京 102206,北京农学院 园林学院 北京 102206
  • 收稿日期:2014-11-12 修回日期:2014-11-27 接受日期:2014-12-01 出版日期:2015-04-03 发布日期:2015-04-03
  • 通讯作者: 赵和文
  • 基金资助:
    北京市属高等学校创新团队建设与教师职业发展计划项目

Phalaenopsis aphrodite Rchb. F. PPO and PAL Expression Based on Real-time PCR Technology

赵和文,崔金腾 and 周田田   

  • Received:2014-11-12 Revised:2014-11-27 Accepted:2014-12-01 Online:2015-04-03 Published:2015-04-03

摘要： 为研究蝴蝶兰在组织培养过程中的褐化机制，通过观察与褐化相关基因PPO和PAL在组织培养过程中不同时期的表达量，判断上述两基因对褐化的影响。采用实时荧光定量的方法，设计特异性引物，获得PPO基因与PAL基因的目的片段，再以实时荧光定量方式，检验上述2个基因在蝴蝶兰组培苗中的表达量。结果表明，获得的PPO基因目的片段与蝴蝶兰x五唇兰相似性达到97%，蝴蝶兰PPO基因目的片段序列与蝴蝶兰x朵丽兰相似性达到98%。通过对蝴蝶兰组培苗培养不同阶段中PPO基因及PAL基因的表达差异研究，发现：在对组培苗叶片切割后PPO基因的表达量迅速增加，随培养时间的推移，表达量经过缓慢的降低后逐渐增高，在10天到12天阶段表达量急剧增加，在12天达到最大量，随后降低，后期表达量浮动不大；PAL基因分别在1天，8天，12天表达量急剧增加，12天的表达量最大，随后，总体表达量有降低趋势。两个基因在第12d表达量都达到最高，说明此时是褐化发生的关键时期，若此时期对其进行有效预防措施，将会减少褐化现象。

关键词: 茶叶, 茶叶, 花青素, 分离, 稳定性

Abstract: For researching browning of tissue cultured-seeding of Phalaenopsis aphrodite Rchb. F. it has been judged the influence of PPO gene and PAL gene by observing the expression of tissue culture time differently of Phalaenopsis aphrodite Rchb. F.. We obtain the target sequences of PPO gene and PAL gene by designing specific primers and test the expression of the above mentioned genes by real-time PCR. In our study , the similarities between Phalaenopsis aphrodite Rchb. F. PPO and Phalaenopsis aphrodite Rchb. F.× pulcherrima PPO are up to 97% and the similarities between Phalaenopsis aphrodite Rchb. F.t × Doritis PAL are up to 98%.We find that after slicing the leave of tissue cultured-seeding, the expression of PPO dramatically increases, and as time goes by, the expression first slowly descends and then increases. The expression dramatically increases between 10d and 12d and then is up to the maximum, at last the expression is lower and not change much; The expression of PAL gene rapidly increases, respectively 1d,8d and 12d ,and the expression is the maximum at 12d.At last, the expression is down. The expression receive maximum at 12d both PPO and PAL, which has been illustrated that it is the critical period for browning. Only taking precaution at that time , can it increase browning.