Analaysis of evolutionary characteristics of miR172 gene family and theirspatial and temporal expression in Dimocarpus longan Lour.

Abstract：【Objective】MicroRNAs (miRNAs) are endogenous non-coding RNAs with 20-24 nucleotides(nt) in length and play important roles in regulating growth and development, stress tolerance and hormoneresponse in plant. MiR172 was firstly obtained by miRNA sequencing in Arabidopsis thaliana, then it wasfound in rice, maize, and soybean because of its extreme conservation. MiR172 has been found to be involved in the transformation from the juvenile period to the adulthood, regulating flowering time, flower or⁃gan development in plant, and accelerating the formation of potato tubers and other growth and develop⁃ment process. In addition, miR172 also has been observed to participate in plant response to abiotic stresses, such as salt, low temperature and drought stress. Longan (Dimocarpus longan) is an important fruit treein subtropical area for its high nutritive value It is of great importance to explore the roles of miR172 inregulating growth and development in longan. Therefore, we explored the evolution characteristics ofmiR172 gene family and its expression pattern in different tissues of longan.【Methods】Firstly, five precur⁃sor sequences and one mature sequence of the longan miR172 family were downloaded from the‘Honghe⁃zi’longan embryogenic callus transcriptome database (SRA050205) and the miRNA library (SRA301402)to construct multi-sequence alignment. Secondly, the secondary structure prediction of the longan premiR172family was performed using version 3.5 Mfold online software (http://unafold.rna.albany.edu/?q=mfold). Thirdly, Mega5.1 was used to construct the phylogenetic trees of pre-miR172 in longan and somerelated plants. We downloaded the miR172 precursor sequences of Vitis vinifera, Solanum lycopersicum,Glycine max, Citrus sinensis, Arabidopsis thaliana, Malus domestica, Carica papaya, Zea mays and Populus trichocarpa from the miRBase database (http://www.mirbase.org/index.shtml) to construct phylogenetictree. Fourthly, a mature suquence miR172a from longan miRNA library was extracted to predicte its tar⁃get. The longan miR172 target gene was predicted using the longan embryogenic callus transcript dataand the psRNAtarget (http://plantgrn.noble.org/psRNAtarget//) online software. Finally, TAKARA SYBRPremix Ex TaqTM kit and Roche LightCycler 480 real-time fluorescence quantitative PCR were used to an⁃alyze the quantitative expressions of miR172 in different tissues of longan.【Results】The 5 precursor miR⁃NA sequences of the longan miR172 family were relatively conservative at both ends, while the middle re⁃gion of each member formed its specific regions. Compared with the 5 precursors and 1 mature sequencesof MiR172 family, a conserved region about 20 nucleotides was found among the 6 sequences, suggestingthat mature miR172 mainly was originated from the conservative region of the precursor sequences. Therewas (were) only 1-4 distinct base(s) in the three sequences in the overlapped area, and the mature bodydlo-miR172a completely coincided with the precursor dlo-miR172-scaffold1097 and dlo-miR172-scaf⁃fold58107, and there were some different bases among mature body dlo-miR172a and the other members,suggesting that the members of longan miR172 family might have a relatively complex evolution process,and different members might have specificity in the process of forming a mature body from the precursor.Mfold prediction results showed that all of the pre-miR172 members could form a typical stable stem loopstructures with a minimal folding free energies (dG) between -35.80 and -54.45 kal·mol-1. Phylogenetictree analysis showed that all members of longan pre-miR172 gathered in the same branch, but there was acertain distance among the 5 members of longan pre-miR172. The distance between dlo-miR172-scaf⁃fold4293 and dlo-miR172-scaffold 42790 was the closest, indicating that these two members might be relatively conservative in their evolutionary process. The distribution between the three members (dlomiR172-scaffold59107, dlo-miR172- scaffold1097 and dlo- miR172- scaffold13218) relatively dis⁃persed, indicating that the miR172 family might have diversity in the evolutionary process. The resultsshowed that the longan miR172 family members exhibited both conservatism and specificity, and theywere genetically close to Citrus sinensis and Populus trichocarpa. Target genes prediction of longanmiR172 family showed that their annotation of targets included AP2, molybdopterin cofactor, glutamyltRNAand hypothetical protein, revealing the universality of miR172a functionality. Real-time quantitative PCR revealed that longan miR172 family members had similar expression pattern in different tissues.They were highly expressed in leaf buds and in the stage of reproduction, and were lowly expressed infruits and in the stage of vegetative growth, while dlo-miR172-scaffold4293 was highly expressed inleaves. These suggested that the longan miR172 family might be involved in the formation of leaves andthe formation and development of floral organs, while dlo-miR172-scaffold4293 would not only partici⁃pate in the formation of leaves, but also play an significant role in the leaf developmental process.【Conclusion】This research showed that the longan miR172 family members exhibited both conservatism and specificity, and might be involved in the formation and development of some organs of longan.