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园艺学报 ›› 2019, Vol. 46 ›› Issue (10): 1907-1918.doi: 10.16420/j.issn.0513-353x.2018-0845

• 研究论文 • 上一篇    下一篇

核桃不定根发生阶段内参基因筛选与关键基因表达分析

宋晓波1,常英英1,刘 昊1,2,徐慧敏3,裴 东1,*   

  1. 1林木遗传育种国家重点实验室,国家林业和草原局林木培育重点实验室,中国林业科学研究院林业研究所,北京100091;2天津市林业果树研究所,天津 300112;3河南省洛宁县林业局,河南洛阳 471700
  • 出版日期:2019-10-25 发布日期:2019-10-25
  • 基金资助:
    中央级公益性科研院所基本科研业务费专项(CAFYBB2017ZA004-2);国家自然科学基金项目(31672126)

Reference Gene Selection and Genes Expression Analysis During Adventitious Root Formation in Walnut

SONG Xiaobo1,CHANG Yingying1,LIU Hao1,2,XU Huimin3,and PEI Dong1,*   

  1. 1State Key Laboratory of Tree Genetics and Breeding,Key Laboratory of Tree Breeding and Cultivativation of State Forestry Administration,Research Institute of Forestry Chinese Academy of Forestry,Beijing 100091,China;2Tianjin Research Institute of Forestry and Pomology,Tianjin 300112,China;3Forestry Bureau of Luoning County,Luoyang,Henan 471700,China
  • Online:2019-10-25 Published:2019-10-25

摘要: 通过荧光定量PCR技术和geNorm、NormFinder软件对8个候选内参基因(18S、ACT、ACT2、TUB、H2B、UBI、EF-2α和UBQ)在核桃(Juglans reiga)不定根发生过程中的表达稳定性进行评估。利用筛选出的最佳内参基因ACT2进行校正,对核桃不定根发生过程中的12个重要功能基因(YUC4、PIN6、AUX/IAA8、SAUR24、WOX11、WOX5、LBD16、WRKY14、SHR、SCL、CYCA和CYCD)的表达水平进行定量分析。结果显示,YUC4在扦插过程中持续显著下调表达,其余11个基因均在不同时期显著上调表达。结合基因功能推测:PIN6等基因介导的生长素极性运输对于不定根发生初期所需的高浓度IAA富集具有重要作用;SHR、WOX11、LBD16、WRKY14和SCL主要参与形成层细胞向根原基原始细胞的转化过程,SAUR24、WOX5、CYCA和CYCD主要参与根原基原始细胞向根原基细胞转变的调控;同时,细胞周期蛋白相关基因(CYCA、CYCD)可以作为插穗中根原基原始细胞向根原基细胞转变的标志基因。

关键词: 核桃, 不定根, 内参基因筛选, 表达分析

Abstract: The expression stability of eight candidate internal reference genes(18S,ACT,ACT2,TUB,H2B,UBI,EF-2α and UBQ)during adventitious root development was evaluated by real-time PCR and geNorm and NormFinder software. Using the best selected internal reference genes for calibration,12 important functional genes(YUC4,PIN6,AUX/IAA8,SAUR24,WOX11,WOX5,LBD16,WRKY14,SHR,SCL,CYCA and CYCD)were found,and the level of expression was quantified during the adventitious root formation of walnut. The results of stability analysis of internal reference gene expression showed that ACT2 gene could be used as the best reference gene in qRT-PCR study of walnut adventitious root formation. ACT2 was used as reference to quantitatively analyze the expression levels of 12 genes in adventitious root formation. The results showed:the YUC gene was down-regulated significantly during the cutting process,and the other 11 genes were significantly up-regulated during different periods of adventitious root development. It is speculated that PIN6 and other gene-mediated auxin polar transport plays an important role in the high concentration of IAA enrichment required in the early stage of adventitious root formation. The SHR,WOX11,LBD16,WRKY14 and SCL genes are mainly involved in the transformation of the stratified cells into the root primordial cells. The SAUR24,WOX5,CYCA and CYCD genes are mainly involved in the regulation of root primordial cells to root primordial cells. At the same time,cyclin-related genes(CYCA,CYCD)can serve as marker genes for the transformation of root primordial cells into root primordial cells.

Key words: walnut, adventitious root, reference gene selection, expression analysis

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