果树学报2017，Vol.34Issue(11)：1385-1393,9.DOI:10.13925/j.cnki.gsxb.20170218
龙眼miR172家族成员进化特性及其时空表达分析
Analaysis of evolutionary characteristics of miR172 gene family and their spatial and temporal expression in Dimocarpus longan Lour.
摘要
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 hormone response in plant.MiR172 was firstly obtained by miRNA sequencing in Arabidopsis thaliana,then it was found 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 organ development in plant,and accelerating the formation of potato tubers and other growth and development 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 tree in subtropical area for its high nutritive value It is of great importance to explore the roles of miR 172 in regulating growth and development in longan.Therefore,we explored the evolution characteristics of miR 172 gene family and its expression pattern in different tissues of longan.[Methods] Firstly,five precursor sequences and one mature sequence of the longan miR 172 family were downloaded from the ‘Honghezi’ longan embryogenic callus transcriptome database (SRA050205) and the miRNA library (SRA301402) to construct multi-sequence alignment.Secondly,the secondary structure prediction of the longan premiR172 family 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-miR 172 in longan and some related 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 phylogenetic tree.Fourthly,a mature suquence miR172a from longan miRNA library was extracted to predicte its target.The longan miR172 target gene was predicted using the longan embryogenic callus transcript data and the psRNAtarget (http://plantgrn.noble.org/psRNAtarget//) online software.Finally,TAKARA SYBR Premix Ex TaqTM kit and Roche LightCycler 480 real-time fluorescence quantitative PCR were used to analyze the quantitative expressions of miR 172 in different tissues of longan.[Results] The 5 precursor miRNA sequences of the longan miR172 family were relatively conservative at both ends,while the middle region of each member formed its specific regions.Compared with the 5 precursors and 1 mature sequences of MiR172 family,a conserved region about 20 nucleotides was found among the 6 sequences,suggesting that mature miR172 mainly was originated from the conservative region of the precursor sequences.There was (were) only 1-4 distinct base(s) in the three sequences in the overlapped area,and the mature body dlo-miR172a completely coincided with the precursor dlo-miR172-scaffold1097 and dlo-miR172-scaffold58107,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 loop structures with a minimal folding free energies (dG) between-35.80 and-54.45 kal· mol-1.Phylogenetic tree analysis showed that all members of longan pre-miR 172 gathered in the same branch,but there was a certain distance among the 5 members of longan pre-miR172.The distance between dlo-miR172-scaffold4293 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 dispersed,indicating that the miR172 family might have diversity in the evolutionary process.The results showed that the longan miR172 family members exhibited both conservatism and specificity,and they were genetically close to Citrus sinensis and Populus trichocarpa.Target genes prediction of longan miR172 family showed that their annotation of targets included AP2,molybdopterin cofactor,glutamyltRNA and 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 in fruits and in the stage of vegetative growth,while dlo-miR172-scaffold4293 was highly expressed in leaves.These suggested that the longan miR172 family might be involved in the formation of leaves and the formation and development of floral organs,while dlo-miR172-scaffold4293 would not only participate 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关键词
龙眼/miR172家族/进化特性/实时荧光定量PCRKey words
Longan/MiR172 family/Evolutionary characteristics/Real-time quantitative PCR分类
农业科技引用本文复制引用
张舒婷,林玉玲,朱晨,王培育,陈旭,陈晓慧,白玉,张梓浩,陈裕坤,赖钟雄..龙眼miR172家族成员进化特性及其时空表达分析[J].果树学报,2017,34(11):1385-1393,9.基金项目
福建省自然科学基金杰出青年项目(2015J06004) （2015J06004）
国家自然科学基金(31672127,31572088) （31672127,31572088）
福建省重大专项(2015NZ0002-1) （2015NZ0002-1）
福建省新世纪人才项目(20151104) （20151104）
福建农林大学“杰出青年科研人才”培养专项基金(xjq201405) （xjq201405）
