5-氮胞苷与2,4-D处理对龙眼早期体胚发生及DlAGOs表达的影响

Effects of 5-azacytoside and 2,4-D Treatments on Early Somatic Embryogenesis and Expression of DlAGOs in Longan

表观遗传过程,如DNA甲基化,在调控植物的生长发育和体细胞胚胎(SE)的正常发生中起着关键作用。植物体细胞胚胎发生是一个细胞分化与器官形态建成的过程,植物体细胞胚胎发生体系可作为研究胚胎发育特别是早期胚胎发育的良好替代体系。本研究利用植物体细胞胚胎发生体系分析了DNA甲基化水平对龙眼球形胚发生的影响,并采用实时荧光定量技术检测了不同浓度5-氮胞苷(5-azac)与2,4-D处理下龙眼Argonaute基因家族(DlAGOs)的相对表达水平。结果发现:(1)当2,4-D浓度为0.1 mg/L时,对照组以及5-azac浓度为5和25μmol/L的处理组均有球形胚产生,而5-azac浓度为25μmol/L处理组中龙眼球形胚更典型,数量更多;(2)2,4-D浓度为0.5 mg/L时,只有5-azac为5μmol/L处理组中有球形胚产生;(3)DlAGOs在不同浓度5-azac和2,4-D处理下具有不同的表达模式,5-azac浓度为5μmol/L能显著诱导DlAGO4的表达。这些研究结果表明,5-azac作为甲基化抑制剂,适宜浓度的5-azac能抑制2,4-D的甲基化作用,降低龙眼胚性愈伤组织的DNA甲基化水平,促使龙眼球形胚提前发生,同时,DNA甲基化水平降低能够促进DlAGO4的表达。以上的研究结果为龙眼体胚发生过程中的DNA甲基化机制研究奠定了理论基础。

Epigenetic processes including DNA methylation play important roles in regulating plant development and normal occurrence of somatic embryogenesis(SE).Plant somatic embryogenesis is a process of cell differentiation and organ morphogenesis,and somatic embryogenesis system in plant is known as a good alternative for the study of embryonic development,especially early embryonic development.In this context an analysis was made of the impact of DNA methylation on longan early somatic embryogenesis,and the relative expression of DlAGOs after 5-azacytoside and 2,4-D treatment was detected by RT-qPCR.The results showed that when the concentration of 2,4-D was 0.1 mg·L^(-1) on the MS cultural medium,globular embryos were produced from embryonic calli of longan in the control group and the treatment group with 5-azac at the concentrations of 5 and 25μmol·L^(-1),while in the treatment group with 5-azac at a concentration of 25μmol·L^(-1),the spherical embryos of longan were more typical and higher in numbers.When the concentration of 2,4-D was 0.5 mg·L^(-1),only the treatment group with 5-azac at 5μmol·L^(-1) produced globular embryos.DlAGOs had different expression patternsμnder the 5-azac and 2,4-D treatments at different concentrations,and DlAGO4 was induced in the treatment of 5-azac at 5μmol·L^(-1).These results indicated that 5-azac as methylation inhibitor can inhibit 2,4-D methylation,reduce the DNA methylation level in embryogenic calli of longan,and promote the development of spherical embryos of longan in advance.Meanwhile,the decrease of DNA methylation level can promote the expression of DlAGO4.