Evolutionary characteristics and the expression patterns of miR159 gene family in 'Kuerlexiangli' pear

Abstract: 【Objective】MicroRNAs (miRNAs) are short, noncoding RNAs, about 20-24 nucleotides in length, with variable sequence complementarity to longer target RNAs.Many biological processes including fruit development, senescence, metabolism, hormone response and stress responses are regulated by miRNAs.MiR159 is a conserved, ancient and multi-regulatory gene family in plants.MiR159 has been found to be involved in the plant leaf morphogenesis, flower organ development, vegetative growth, anther development, seed morphology and germination and defense response.In addition, miR159 also has been observed to participate in plant biological and abiotic stresses, such as salt stress, high temperature stress, low temperature stress and mechanical stress.Studies have showed that miR159 has a wide range of regulatory effects in plants, especially in the development of flower organs.'Kuerlexiangli' pear (Pyrus sinkiangensis Yu) is an ancient regional high-quality variety which is crisp and juicy.However, in recent years, as gradual increase of the planting area of 'Kuerlexiangli' pear, the fruit quality has gradually decreased, and the number of fruits with persistent calyx have increased, resulting in the decrease of flavor and quality of the fruits.Therefore, the study of the sequence characteristics, phylogenetic evolution, and expression patterns of the miR159 gene family is beneficial to the further exploration of the important regulatory role of miR159 in plants.What's more, it is of great significance to explore the roles of miR159 in regulating calyx persistence in' Kuerlexiangli'pear.So, we explored the evolution characteristics and expression patterns of miR159 gene family in the ovaries and sepals of the'Kuerlexiangli'pear fruits with a deciduous calyx and with a persistent calyx.【Methods】Two precursor sequence and two mature sequence of the 'Kuerlexiangli' pear miR159 family were obtained from a small RNA Illiumina high-throughput sequencing.The miR159 precursor sequences and mature sequences of Malus domestica, Arabidopsis thaliana, Carica papaya, Vitis vinifera, Citrus reticulate, Prunus persica, Cucumis melo and Populus trichocarpa were downloaded from the miRBase database (http://www.mirbase.org/index.shtml) .The precursor sequence and mature sequence multi-sequence alignment of the miR159 family were analyzed using DNAMAN (ver.6.0.3.99) software.MEGA 5.1 software was used to construct phylogenetic tree of the miR159 family in the plants including'Kuerlexiangli'pear.The construction method was Neighbor-joining (NJ) .The secondary structure prediction of the 'Kuerlexiangli' pear pre-miR159 family was performed using RNAfold (http://rna.tbi.univie.ac.at/) .WebLogo (http://weblogo.berkeley.edu/logo.cgi) was used to analyze the conservation of miR159 family in 'Kuerlexiangli' pear and some related plants.The' Kuerlexiangli'pear transcriptome database was used as a candidate sequence for the prediction of the target gene, and the target gene of the Korla Fragrant miR159 family was predicted using psRNATarget (http://plantgrn.noble.org/psRNATarget/) online software.The parameters were all default values.Quantitative real-time PCR (qRT-PCR) was performed using a CFX manager (Bio-Rad, USA) and the SYBR Green Real-time PCR Master Mix (Toyobo, Osaka, Japan) to determine the expression profiles of miR159 in the ovaries and sepals of the' Kuerlexiangli'pear fruits with a deciduous calyx and with a persistent calyx.【Results】The results show that the miR159 family of' Kuerlexiangli'pear had two precursor members (psiMIR159 a, psi-MIR159c) and two mature members (psi-miR159 a, psi-miR159 c) .RNAfold prediction results showed that the two precursors sequences of the miR159 family were able to form a typical stable stem-loop structure with a minimal folding free energies (dG) of -93.95 kal·mol-1.And the sequences of the two members were identical.Phylogenetic tree analysis showed that all members of' Kuerlexiangli'pre-miR159 gathered in different branches, and they were genetically close to Malus domestica.The Weblogo results showed that the conserved sequence of the miR159 family mature sequences was 5'-UUUGGAUUGAAGGGAGCUCUAUU-3', with higher conservation at bases 2 to 19.The mature sequence of the' Kuerlexiangli'miR159 family was less conserved, and conserved only at 5 bases.Target genes prediction of' Kuerlexiangli'miR159 family showed that their annotation of targets included heat shock 70 ku protein (HSP70) , trihelix transcription factor GT-1 (GT-1) , transcription factor bHLH91 (BHLH91) , protein argonaute 1 (AGO1) , G-type lectin S-receptor-like serine/threonine-protein kinase RLK1 (RLK1) , revealing the universality of miR159 functionality.These predicted target genes were involved in flower organ development, hormone response, apoptosis, and stress response, being closely related to organ abscission.The qRT-PCR analysis indicated that the expression of miR159 family members had similar expression pattern.There were significant differences in the expression levels between ovaries and sepals of the fruits with a deciduous calyx and those with a persistent calyx.These results suggested that the' Kuerlexiangli'pear miR159 family might be involved in the calyx persistence in' Kuerlexiangli'pear.【Conclusion】This research showed that the' Kuerlexiangli'pear miR159 family members exhibited both conservatism and diversity, and might be involved in calyx persistence in' Kuerlexiangli'pear.