Molecular characteristics and expression analysis of miR397 family members during the early somatic embryogenesis in Dimocarpus longan Lour.

Abstract: 【Objective】Longan (Dimocarpus longan Lour.) is one of the important economic fruit trees in China's subtropical zone. The longan somatic embryogenesis system is a model system for the study of embryogenesis in woody fruit trees. It has been widely used in molecular biology of somatic embryogenesis. miRNA is one of the important factors to influence epigenetic growth and development of the plant. The regulation mechanism in the process of embryogenesis needs further exploration. miR397 is a kind of miRNA that is more conservative in monocotyledon and dicotyledon. Although it has been studied in other plants, the mechanism of action in the process of somatic embryogenesis in longan has not been systematically analyzed. Therefore, longan miR397 precursor molecular characterization and evolutionary analysis of mature members, and its expression analysis at different stages of early longan somatic embryogenesis and different hormone concentrations, can help to fully understand the function of longan miR397 family members in the early stage of longan somatic embryogenesis.【Methods】‘Honghezi'longan embryogenic calli were used as materials, Mfold was used to analyze the secondary structure of the longan miR397 precursor sequence, and PMTED was used to predict the target gene of miR397, MEGA 7.0 was used for the construction of phylogenetic tree of a total of 53 mature bodies.43 precursor sequence was constructed separately. The cleavage site was verified by the modified RLMRACE method. The longan embryogenic cells were cultured in MS (sucrose 20 g · L^-1, 2, 4-D 1.0 mg · L^-1) liquid medium for 1 to 12 d, and 2, 4-D and GA3 of different concentration gradients were used to treat the longan embryogenic calli, and the expression of miR397 family members at different early stages of longan somatic embryo development and different hormone concentrations were discussed.【Results】The mature sequences miR397 a and miR397 of the longan pre-miR397 could be generated on the 5-arm of the longan miR397 precursor sequence. There was only 2 bases TC difference at the 5'between them, and the precursor could form a stable stem loop. The phylogenetic tree analysis revealed that the longan pre-miR397 was closely related to peach (ppe) , cocoa (tcc) , and navel orange (csi) on the same branch. Longan miR397 and Arabidopsis (ath) , sweet orange (csi) , Grapes (vvi) were closely related, and they were located on the same conservative branch. The miR397 family members were more conservative than the precursor sequences. Target gene predictions indicated that dlo-miR397 family mainly targeted the laccase gene family and a sister chromosome binding protein (dlo＿039206.1) , miR397 a negatively regulated target gene dlo＿039206.1 in the process of somatic embryogenesis to the late stage.Longan miR397 a was down regulated under the treatments of different concentrations of 2, 4-D and GA3 hormones. The target gene sister chromatinin (dlo＿039206.1) was not significantly expressed under hormone treatments. However, the expression trend of sister chromatin (dlo＿039206.1) of target gene was not obvious under hormone treatments, which might be due to the target cleavage site outside the predicted region.【Conclusion】The targeted cleavage site was verified. miR397 a was down regulated expression under the treatments of different concentration of 2, 4-D and GA3 hormones, that might response to the auxin and cytokinin signaling.