不同浓度新型分裂素PBU诱导的尾巨桉愈伤组织中miRNA396及CKX基因表达差异研究

Differential Expression of mi RNA396 and CKX Genes in Eucalyptus urophylla×E.grandis Callus Induced by Different Concentrations of Novel Mitogen PBU

尾巨桉愈伤组织的生长分化受内源激素影响,而miRNA396是一个调控植物叶片与根系生长发育的小RNA,与细胞分裂素的合成相关,CKX是负责调控细胞分裂素的氧化酶基因。为探讨miRNA396与CKX基因对尾巨桉愈伤组织生长发育的调控作用,以尾巨桉基因组为模板,进行PCR扩增及测序分析尾巨桉基因中的miRNA396序列,用不同PBU细胞分裂素浓度培养下的尾巨桉愈伤组织RNA逆转录的cDNA为模板,通过荧光定量PCR,测定不同PBU浓度处理的尾巨桉愈伤组织中miRNA396及CKX的表达差异。结果表明,相对于0.5 mg·^L(-1)PBU处理的桉树愈伤组织,1 mg·L^(-1)PBU处理的桉树愈伤组织miRNA396及CKXA、CKXB和CKXF表达量显著下调,差异达到极显著水平,CKXC、CKXD和CKXE均上调,但只有CKXC相对表达量达到极显著水平;2 mg·^L(-1)PBU处理的尾巨桉愈伤组织miRNA396A、CKXD、CKXE和CKXF表达量均下调,差异达到极显著水平,其他CKX表达量均上调,CKXA相对表达量差异达到显著水平,CKXB和CKXC相对表达量差异达到极显著水平。本研究初步确立了miRNA396和CKX基因在尾巨桉愈伤组织中的调控及表达差异,为后续进行尾巨桉miRNA调控网络的解析奠定了基础,为尾巨桉高效再生体系的建立提供了一定借鉴与参考。

Eucalyptus plays a great role in ecological environment and chemical industry.As a hybrid of Eucalyptus urophylla and E.grandis,E.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 mi RNA396 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 mi RNA396 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 mi RNA396 sequence of E.urophylla×E.grandis gene.The c DNA 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 mi RNA396 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·(-1)PBU,the expression of mi RNA396,CKXA,CKXB and CKXF in E.urophylla×E.grandis callus treated with 1 mg·(-1)PBU was significantly decreased,and the difference reached a very significant level.CKXC,CKXD and CKXE were up-regulated,but only the relative expression of CKXC reached a very significant level.The expression levels of mi RNA396A,CKXD,CKXE and CKXF in E.urophylla×E.grandis callus treated with 2 mg·(-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 CKXA,CKXB and CKXC reached a very significant level.This study preliminarily established the regulation and expression differences of mi RNA396 and CKX gene in the callus of E.urophylla×E.grandis,laid a foundation for the subsequent analysis of the mi RNA 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.