摘要
【目的】了解‘库尔勒香梨’miR159家族成员的进化特征及其在萼片脱落与宿存中的作用。【方法】以‘库尔勒香梨’Small RNA高通量测序中获得的miR159家族前体序列和成熟体序列为基础进行系统发育树构建、前体和成熟体的序列比对、前体二级结构预测、WebLogo保守性分析、靶基因预测和表达量分析。【结果】‘库尔勒香梨’miR159家族存在2个前体成员(psi-MIR159a、psi-MIR159c)和2个成熟体成员(psi-miR159a、psi-miR159c)。2个前体成员的序列均能形成典型稳定的茎环结构,且序列完全一致,它们的最小折叠自由能(dG)都为-93.95 kal·mol^(-1)。系统发育进化树显示,‘库尔勒香梨’和苹果MIR159家族亲缘关系很近。WebLogo分析表明,miR159家族成熟体的保守性很高,其保守序列为5'-UUUGGAUUGAAGGGAGCUCUAUU-3',但‘库尔勒香梨’的碱基保守性较低。靶基因预测显示,‘库尔勒香梨’miR159基因家族靶基因为转录因子bHLH91、BES1/BZR1同源蛋白4、热激70 ku蛋白质、Trihelix转录因子GT-1、G型凝集素S-受体类丝氨酸/苏氨酸蛋白激酶RLK1等。实时荧光定量PCR(qPCR)显示,‘库尔勒香梨’miR159家族的2个成员在初花期、盛花期和末花期3个时期萼片脱落组和宿存组的表达量有显著差异。【结论】‘库尔勒香梨’miR159家族在进化过程中既有保守性又有多样性,可能在萼片脱落过程中起作用。
[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.
作者
马丽
周丽
徐航
全绍文
杨洁萍
牛建新
MA Li;ZHOU Li;XU Hang;QUAN Shaowen;YANG Jieping;NIU Jianxin(Department of Horticulture,College of Agriculture,Shihezi University/Xinjiang Production and Construction Corps Key Laboratory of Special Fruits and Vegetables Cultivation Physiology and Germplasm Resources Utilization,Shihezi 832003 ,Xinjiang ,China)
出处
《果树学报》
CAS
CSCD
北大核心
2019年第1期1-10,共10页
Journal of Fruit Science
基金
国家自然科学基金(31360474)
高等学校博士学科点专项科研基金博导类联合资助课题(2013651810002)
