Determination Of Endogenous Auxin And Expression Of MiR393b And TIR1 In Lilium During Somatic Embryogenesis

Both Lilium davidian var.unicolor and Lilium pumilum DC.Fisch.are wild lilies originating from China with relatively high ornamental and economic value.However,their bulb propagation requires a long period and is inefficient.Somatic embryogenesis has high regeneration efficiency and rapid propagation,which is of great significance for Lilium germplasm conservation,seed breeding and so on.As an important plant hormone,auxin plays an important role in plant growth and development.At present,the regulation mechanism of endogenous auxin on somatic embryogenesis is not clear.In this study,contents of endogenous auxins in both kinds of Lilium are measured during somatic embryogenesis by ultra-high-performance liquid chromatography-triple quadrupole mass spectrometry.The receptor gene T1R1 and miR393 b are screened during signal transduction of auxins based on the results of sequencing and the sequence of miR393 b mature body is cloned.Furthermore,a correlation analysis is performed on expressions of miR393 b and its target gene TIR1 by qRT-PCR,thus laying a foundation for further examining the roles of endogenous auxins in regulating embryos of lilies.The test results are mainly as follows:1.Determination of endogenous auxin contentThe auxin contents reach two peaks for both liliums during somatic embryogenesis.For Lilium pumilum DC.Fisch.,the auxin content reach the maximum peak value in the phase of endogenesis on the 15 th day and the second peak in embryogenic callus tissues on the 42 th day.For Lilium davidian var.unicolor,the auxin content reach the maximum peak value in the phase of endogenesis on the 12 th day and another peak in the phase of globular embryos on the 35 th day.This result shows that high levels of endogenous auxin are required when embryonic cells are formed and appear in a form of internal origin,and high levels of endogenous auxin also contribute to the acquisition of mature embryos.2.Clone of miR393bIn this study,miR393 b is cloned with stem-loop reverse transcription PCR(Stem-loop RT PCR)for Lilium pumilum DC.Fisch.and Lilium davidian var.unicolor.Two mature sequences of lily miR393 b are successfully obtained the,length of which is 22 nt.3.Expression analysis of miR393 b and target gene TIR1According to the results of fluorescence quantitative analysis,there is a negative correlation between miR393 b and target gene TIR1 in two lilium species.In Lilium pumilum DC.Fisch..The expression of lpu-miR393 b is the highest in the initial explant on the 0th day.and the lowest during embryogenesis of rods on the 56 th day.As a whole,it tends to decline,rise and decline.The expression of TIR1 is the highest in Phase II of embryogenic callus tissues on the 42 th day,the lowest in Phase I of embryogenic callus tissues on the 28 th day and relatively high during embryogenesis.In Lilium davidian var.unicolor the expression of lda-miR393 b is the highest when Embryogenic cells form from the outside on the 9th day.As the further development of somatic embryos begins to decline and the lowest in phase of embryogenic callus tissues on the 28 th day.The expression of TIR1 is the highest in the phase of embryogenic callus tissueson the 28 th day.The results show that miR393 b played a negative role in regulating the target gene TIR1 during the embryogenesis of Lilium,and the high level of TIR1 is beneficial to the proliferation of embryogenic cells and the maturation of somatic embryos.