| Longan (Dimocarpus longan Lour.), a species of the evergreen woody plant, belongs to Sapindaceae. Research for longan embryogensis is great significance of longan production. Plant embryo development is a complex process since there are many metabolic processes and gene regulatory networks, miRNA as a improtant factors must exist a huge role in the regulation of plant somatic embryogenesis, AGO proteins also play a important role in somatic embryogenesis, AGO family proteins related with miRNA metabolism were studied to provide new clues for reveal the molecular mechanisms regulating of miRNA, and provide a theoretical basis for the regulation of embryonic development of longan and other woody plant.. At the same time, the analysis of the study the AGO genes expression in different periods of somatic embryogenesis and different tissues by quantitative PCR technique, those can provide a basis for the miRNA metabolism research, and also give a new perspective for the regulation of plant growth and development.1 The full length genes clone of AGO1, AGO2, AGO5 and AG06 from longan embryogenic callusBy longan embryogenic callus transcriptome analysis and comparison in the GeneBank, longan embryogenic callus contains five kinds of AGO proteins that are AGO1, AGO2, AGO4, AGO5 and AGO6, where the expression of the AGO4 gene is very low and is the same as sequence homology of AGO6 gene.By using RT-PCR and RACE AGO gene family of longan embryogenic was cloned. The full-length cDNA, open reading frame (ORF), and encoding amino acid sequence of AGO1 is 3529 bp,3183 bp and 1061 aa, respectively, and are submitted to the GenBank with the name of DlAGO1 and accession number is KM001869. AGO2 has 3529 bp of full-length cDNA,3128 bp of an ORF, and encoding 976 aa. The GenBank accession number for AGO2 is KM001870 with the submitted name of DlAGO2. AGO5 has full-length cDNA of 3203 bp, an ORF is of 2919 bp, and encoding 972 amino acids, and GenBank for accession number of AGO5 is KM001871 and named as DlAGO5. The full-length cDNA, ORF, and encoding amino acid of AGO6 is 3168 bp, 2700 bp, and 900 aa, respectively. AGO6 was submitted to GenBank with the name of DlAGO6 and accession number is KF819529.2 Bioinformatics analysis of AGO1, AGO2, AGO5 and AGO6 gene of longan embryogenic callusBased on the above, the genes in longan embryogenic callus AGO gene family were analyzed about basic physical and chemical properties, phosphorylation sites, and conserved protein domains by bioinformatics software. The analysis of genetically encoded amino acids conserved domain of AGO1, AGO2, AGO5 and AGO6 show that DlAGO2 PIWI region contains DDD structure and DlAGO2, DlAGO5 and DlAGO6 contain DDH structure. Therefore, longan AGO1, AGO2, AGO5 and AGO6 proteins may have function of mRNA cleavage, and might be involved in post-transcriptional gene silencing. The predictions of phosphorylation sites show that DlAGO1, DlAGO2, DlAGO5, DlAGO6 proteins have many serine phosphorylation sites, and in the end of N and C the distribution of serine phosphorylation sites are dense while uniform distribution was found in the middle part. Having PIWI domain, AGO proteins possess RNA endonuclease activity, the serine phosphorylation of the protein-rich sites, which may be increased transcription of the RNA-binding protein with a small role in regulating the expression of post-transcriptional. DlAGO1, DlAGO2, DlAGO5, DlAGO6 family phylogenetic tree analysis, found that AGO protein longan embryogenic callus may be divided into four branches, and DlAGO1, DlAGO2, DlAGO5, DlAGO6 belong the four branches of longan embryogenic callus AGO proteins.3 Quantitative analysis of the expression of longan AGO1, AGO2, AGO5 and AGO6 genes in different stages of somatic embryogenesis and different tissues of longan treeUsing real-time quantitative technique to analysse AGO1, AGO2, AGO5 and AGO6 gene relative quantification expression at different stages of somatic embryogenesis and in different tissues of longan tree.The expression in different embryonic stages of longan is as follows. Early in the process of somatic embryogenesis, with the advancement of the process of somatic embryogenesis, AGO1 gene expression levels gradually increased but decreased in the cotyledonary stage, and in overall the highest of expression levels were detected in non-embryonic stage. The differential expression level of AG02 gene in embryonic stages was detected in the overall tend and the relative expression levels look like M-shaped curve. The two vertices of M-shaped curve were at the embryonic callus and globular embryo stages In non-embryonic stage, incomplete compact pro-embryogenic callus, and cotyledonary stage relatively low expression were detected. The gene expression of AGO5 is significantly different in different developmental stages of somatic embryogenesis. The overall trend showed a gradual decrease expression with advance the process of somatic embryogenesis, non-embryonic stage higher than other embryonic stage; AGO6 gene expression in different stages of somatic embryogenesis in a significant difference in the overall trend showed a gradual decrease, with the advancement of the process of somatic embryogenesis, AGO6 gene expression decreased, but not embryonic callus tissue relatively much lower.The expression of AGO genes in different tissues of longan was also detected. AGO gene expression in different tissues of longan has significant difference. The gene expression levels of AGO1 were relatively low in mature leaves, floral buds, small fruit, large fruit, peel and seeds while the highest relative expression was detected in the pulp. This result suggested that AGO1 may be related to the formation of pulp. Also in the flower and roots, the relative expression was high, so AGO1 gene might interact with the miRNA which may have relationships with flower and root development. The expression of AGO2 gene in the root, mature leaves, floral buds, flower, small fruit, large fruit, pulp, and seeds are relatively low while high expression was found in the peel, presumably related to the development of peel by interactions of miRNA with AGO2. AGO5 expression in peel was significantly higher than other parts of the tissues as in AG02 gene. AGO5 may interact with AG02 gene. Very low expression level of AGO5 was detected in small fruit as well as expression level in large fruit was not high. But high levels of expression was detected in the peel, presumably it has an important role in the development of peel in late ripening process. AG06 gene expressed the highest level in flower and pulp while in mature leaves, young fruit and mature fruit the expression was relatively low. In roots and floral buds the relative expression higher, the relative expression of small fruit and seeds such as old leaves, large fruit.4 The subcellular localization of longan embryogenic callus AGO1, AGO2, AGO5 and AGO6 gene family analysisThis study used the subcellular localization of GFP as a reporter gene, constructed longan embryogenic callus AGO1, AGO2, AGO5 and AGO6 protein subcellular localization carrier. Using Agrobacterium transient expression, transiently expressed in onion epidermal cells, observe AGO1, AGO2, AGO5, AGO6 subcellular localization fluorescence signal, the result show:AGO1 mainly concentrated in the nucleus and microbodies, AGO2 mainly concentrated in the nucleus and cytoplasm, AGO5 and AGO6 focused on the cell membrane and nuclear membrane. Longan embryogenic callus AGO proteins are all localized in the nucleus, the nucleus is the main place organisms and genetic material of ribosome biogenesis, and has important functions for the cell cycle and cell senescence. Subcellular localization of AGO proteins, helps to further explore gene function.In summary, by longan embryogenic callus AGO1, AGO2, AGO5 and AGO6 gene sequences and bioinformatics analysis,it is discovered that longan embryogenic callus AGO gene family sequences, a kind of conservative basic protein, are highly conserved, and longan AGO proteins can be divided into four branches similar to rice.By the expressive difference of AGO], AGO2,AGO5 and AGO6 genes in the longan somatic embryogenesis, they may be related to the rate of cell division, and the expressive difference of in Sijimi different tissues suggest that they are probably involved in a specific organ morphogenesis.Subcellular localization show that AGO1, AGO2, AGO5 and AGO6 are localized in the nucleus. The nucleus is the main site of the genetic material of an organism and ribosome biogenesis, and has important functions in cell cycle and cell senescence.The study of AGO protein cloning, bioinformatics analysis, expression analysis and subcellular localization contribute to further explore the gene the function. |
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