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Bulletin of Botanical Research ›› 2022, Vol. 42 ›› Issue (5): 840-847.doi: 10.7525/j.issn.1673-5102.2022.05.015

• Molecular biology • Previous Articles     Next Articles

Differential Expression of miRNA396 and CKX Genes in Eucalyptus urophylla × E. grandis Callus Induced by Different Concentrations of Novel Mitogen PBU

Yamei LIU1,2, Yupeng WU2, Limei LI2, Min SU1,2, Zhu YU2, Lejun OUYANG1,2()   

  1. 1.College of Life and Geographic Sciences,the Key Laboratory of Ecology and Biological Resources in Yarkand Oasis at Colleges & Universities under the Department of Education of Xinjiang Uygur Autonomous Region,Kashi University,Kashi 844000
    2.College of Biological and Food Engineering,Guangdong University of Petrochemical Technology,Maoming 525000
  • Received:2021-11-01 Online:2022-09-20 Published:2022-09-15
  • Contact: Lejun OUYANG E-mail:ouyanglejun@gdupt.edu.cn
  • About author:LIU Yamei(1996—),female,master degree candidate,major in plant genetic engineering.
  • Supported by:
    National Natural Science Foundation of China(32071780);Guangdong Natural Science Foundation of China(2019A1515010709);Guangdong Science and Technology Program(mmkj2020035);Guangdong Provincial Department of Education Key Project(2018KZDXM047);Guangdong Rural Revitalization special fund project(2021S0074);Autonomous Region graduate innovation project in 2021(XJ2021G313)


Eucalyptus plays a great role in ecological environment and chemical industry. As a hybrid of Eucalyptus urophylla and E. grandisE.urophylla × E.grandis has good heterosis and is more synthetical than other Eucalyptus species. In order to meet the requirements of cultivating superior Eucalyptus cultivars with different uses by genetic engineering technology, it is of great significance to establish an efficient in vitro regeneration system of E.urophylla × E.grandis and study its callus differentiation mechanism. The growth and differentiation of E.urophylla × E.grandis callus is affected by its endogenous phytohormones and miRNA396 is a small RNA regulating the growth and development of plant leaves and roots, which is related to the biosynthesis of cytokinin. CKX is responsible for regulating cytokinin oxidase gene. In order to explore the regulatory effects of miRNA396 and CKX genes on the growth and development of E.urophylla × E.grandis callus, in this study, the genome of E.urophylla × E.grandis was used as a template for PCR amplification and sequencing to analyse the miRNA396 sequence of E. urophylla × E.grandis gene. The cDNA reverse transcribed by RNA from E.urophylla × E.grandis callus cultured on media with different PBU cytokinin concentrations was used as a template by fluorescence quantitative PCR and the expression differences of miRNA396 and CKX in E.urophylla × E.grandis callus treated with different PBU concentrations were determined. The results showed that compared with the E.urophylla × E.grandis callus treated with 0.5 mg·L-1 PBU, the expression of miRNA396, CKXACKXB and CKXF in E.urophylla × E.grandis callus treated with 1 mg·L-1 PBU was significantly decreased, and the difference reached a very significant level. CKXCCKXD and CKXE were up-regulated, but only the relative expression of CKXC reached a very significant level. The expression levels of miRNA396A, CKXDCKXE and CKXF in E.urophylla × E.grandis callus treated with 2 mg·L-1 PBU were down-regulated, and the difference reached a very significant level. The rest of gene expression levels were up-regulated. The relative expression levels of CKXACKXB and CKXC reached a very significant level. This study preliminarily established the regulation and expression differences of miRNA396 and CKX gene in the callus of E.urophylla × E.grandis, laid a foundation for the subsequent analysis of the miRNA regulation network of E.urophylla × E.grandis, and provided a certain reference for the establishment of an efficient regeneration system of E.urophylla × E.grandis.

Key words: Eucalyptus urophylla × E.grandis, miRNA396, CKX, PBU

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