枣树阶段转变相关microRNA家族的鉴定及其表达分析

doi:10.16420/j.issn.0513-353x.2020-0969

摘要/Abstract

摘要：

与其他果树相比,枣树具有童期短、成花快的特征。已有研究表明,多个microRNA（miRNA）家族参与植物阶段转变和开花时间调控等过程。研究枣树阶段转变相关的miRNA家族对果树童期调控具有重要意义。以枣实生后代植株不同发育阶段（节位）的当年生枝（枣吊）为材料,通过Small RNA测序,在童期、过渡期和成年期等3个时期鉴定出27个miRNA家族的58个已知miRNA和23个新miRNA,其中44个差异表达的miRNA包括32个已知miRNA和12个新miRNA;然后,从中筛选出不同时期显著差异表达的6个miRNA,即zjmiR156a-5p、zjmiR156j、zjmiR172c、zjmiR172e-3p、zjmiRnovel-16和zjmiRnovel-71,通过TargetFinder软件预测出它们的15个关键靶基因,其主要参与成花、转录调控和生物合成等过程;利用实时荧光定量PCR分析验证了12个miRNA及15个关键靶基因确实在枣树阶段转变中差异表达,并存在正调控或负调控关系,其中2个新miRNA（zjmiRnovel-16和zjmiRnovel-71）可能分别通过光周期和糖代谢途径影响阶段转变。

关键词: 枣, 阶段转变, microRNA, 靶基因, Small RNA测序

Abstract:

Current studies have shown that a number of microRNA（miRNA）families are involved in plant flowering time regulation and phase transition,etc. Therefore,it is very instructive for the phase transition regulation of fruit trees to gain insight into microRNA family associated with phase transition in jujube tree. In this study,the new shoots at different development stages（nodes）of jujube progenies were used as materials. Firstly,27 miRNA families with 58 known miRNAs and 23 novel miRNAs were identified by small RNA sequencing analysis in the three different development stages of juvenile,transition and adult period. Among them,44 miRNAs were differentially expressed,including 32 known miRNAs and 12 novel miRNAs. Afterward,six miRNAs（zjmiR156a-5p,zjmiR156j,zjmiR172c,zjmiRmir172e-3p,zjmiRnovel-16 and zjmiRnovel-71）with obviously differential expression in the three development stages were screened out. Their 15 key target genes were also predicted through TargetFinder software,and they are mainly involved in flowering,transcriptional regulation and biosynthesis. Finally,the differential expression of the above-mentioned 12 miRNAs and their 15 key target genes during phase transition were verified by qRT-PCR,and they had positive or negative regulatory relationships. Moreover,the two newly discovered miRNAs（zjmiRnovel-16 and zjmiRnovel-71）may affect the phase transition through photoperiod pathway and glucose metabolism pathway,respectively.

Key words: Ziziphus jujuba, phase transition, microRNA, target gene, small RNA sequencing

中图分类号:

S665.1

李莹, 孟宪巍, 马志航, 刘孟军, 赵锦. 枣树阶段转变相关microRNA家族的鉴定及其表达分析[J]. 园艺学报, 2022, 49(1): 23-40.

LI Ying, MENG Xianwei, MA Zhihang, LIU Mengjun, ZHAO Jin. Identification and Expression Analysis of MicroRNA Families Associated with Phase Transition in Chinese Jujube[J]. Acta Horticulturae Sinica, 2022, 49(1): 23-40.

图/表 13

参考文献 54

[1]	Allen R S, Li J Y, Stahle M I, Dubroué A, Gubler F, Millar A A. 2007. Genetic analysis reveals functional redundancy and the major target genes of the Arabidopsis miR159 Family. Proceedings of the National Academy of Sciences of the United States of America, 104 (41):16371-16376.
[2]	Ambros V. 2001. microRNAs:tiny regulators with great potential. Cell, 107 (7):823-826. doi: 10.1016/s0092-8674(01)00616-x pmid: 11779458
[3]	Arya H, Singh M B, Bhalla P L. 2018. Genomic and molecular analysis of conserved and unique features of soybean PIF4. Scientific Reports, 8 (1):12569. doi: 10.1038/s41598-018-30043-2 URL
[4]	Bartel D P. 2004. MicroRNAs:genomics,biogenesis,mechanism,and function. Cell, 116 (2):281-297. doi: 10.1016/s0092-8674(04)00045-5 pmid: 14744438
[5]	Baucher M, Moussawi J, Vandeputte O M, Monteyne D, Mol A, Pérez M D, El J M. 2013. A role for the miR396/GRF network in specification of organ type during flower development,as supported by ectopic expression of Populus trichocarpa miR396c in transgenic tobacco. Plant Biology, 15 (5):892-898. doi: 10.1111/j.1438-8677.2012.00696.x pmid: 23173976
[6]	Bhavani N, Sneha B, Anjan K B. 2017. The essential role of microRNAs in potato tuber development;a mini review. Indian Journal of Plant Physiology, 22 (4):401-410. doi: 10.1007/s40502-017-0324-x URL
[7]	Borges F, Parent J S, van E F, Wolff P, Martínez G, Köhler C, Martienssen R A. 2018. Transposon-derived small RNAs triggered by miR845 mediate genome dosage response in Arabidopsis. Nature Genetics, 50 (2):186-192. doi: 10.1038/s41588-017-0032-5 URL
[8]	Chuck G S, Tobias C, Sun Lan, Kraemer F, Li Chenlin, Dibble D, Arora R, Bragg J N, Vogel J P, Singh S, Simmons B A, Pauly M, Hake S. 2011. Overexpression of the maize Corngrass 1 microRNA prevents flowering,improves digestibility,and increases starch content of switchgrass. Proceedings of the National Academy of Sciences of the United States of America, 108 (42):17550-17555.
[9]	Damodharan S, Zhao D Z, Arazi T. 2016. A common miRNA 160-based mechanism regulates ovary patterning,floral organ abscission and lamina outgrowth in tomato. The Plant Journal, 86 (6):458-471. doi: 10.1111/tpj.13127 pmid: 26800988
[10]	Duan Zhongxin. 2012. Expression pattern and functional analysis of microRNA Peu-miR156j and Peu-miR169o from Populus euphratica[M. D. Dissertation]. Beijing: Beijing Forestry University. (in Chinese)
	段中鑫. 2012. 胡杨microRNA Peu-miR156j和Peu-miR169o表达模式分析及功能鉴定[硕士论文]. 北京: 北京林业大学.
[11]	Feng L, Xia R, Liu Y. 2019. Comprehensive characterization of miRNA and PHAS loci in the diploid strawberry(Fragaria vesca)genome. Horticultural Plant Journal, 5 (6):255-267. doi: 10.1016/j.hpj.2019.11.004
[12]	France C, François C, Christine D, Claude W. 2001. A highly specific glucosyltransferase is involved in the synthesis of crocetin glucosylesters in Crocus sativus cultured cells. Journal of Plant Physiology, 158 (5):553-560. doi: 10.1078/0176-1617-00305 URL
[13]	Friedländer M R, Mackowiak S D, Li Na, Chen Wei, Rajewsky N. 2012. miRDeep 2 accurately identifies known and hundreds of novel microRNA genes in seven animal clades. Nucleic Acids Research, 40 (1):37-52. doi: 10.1093/nar/gkr688 pmid: 21911355
[14]	Gao Y, Yang F Q, Cao X, Li C M, Wang Y, Zhao Y B, Zeng G J, Chen D M, Han Z H, Zhang X Z. 2014. Differences in gene expression and regulation during ontogenetic phase change in apple seedlings. Plant Molecular Biology Reporter, 32 (2):357-371. doi: 10.1007/s11105-013-0648-2 URL
[15]	Gou Yanli, Zhang Le, Guo Huan, Ma Hongping, Bao Aike. 2020. Research progress on the AP2/ERF transcription factor in plants. Pratacultural Science, 37 (6):1150-1159. (in Chinese)
	苟艳丽, 张乐, 郭欢, 马红萍, 包爱科. 2020. 植物AP2/ERF类转录因子研究进展. 草业科学, 37 (6):1150-1159.
[16]	Gu Jie. 2008. Localization of regulator of G protein signaling(RGS)Protein and its functional analyses in glucose signaling in Arabidopsis thaliana[M. D. Dissertation]. Yangzhou: Yangzhou University. (in Chinese)
	顾杰. 2008. 拟南芥G蛋白信号转导调节蛋白(AtRGS1蛋白)的定位及在葡萄糖信号转导中的功能研究[硕士论文]. 扬州: 扬州大学.
[17]	Hu Z W, Shen X P, Xiang X, Cao J S. 2019. Evolution of MIR159/ 319 genes in Brassica campestris and their function in pollen development. Plant Molecular Biology, 101 (6):537-550. doi: 10.1007/s11103-019-00920-z URL
[18]	Huang Jingmiao. 2018. Prediction and analysis of cis-acting elements and transcription factors of related MdMIR156s during vegetative phase change in apple[M. D. Dissertation]. Beijing: China Agriculture University. (in Chinese)
	黄晶淼. 2018. 苹果阶段转变相关MdMIR156s顺式元件及转录因子预测与分析[硕士论文]. 北京: 中国农业大学.
[19]	José M F Z, Adrián V, Marco T, Isabel M, María I P, Ignacio R S, Antonio L, Detlef W, Juan A G, Javier P A. 2007. Target mimicry provides a new mechanism for regulation of microRNA activity. Nature Genetics, 39 (8):1033-1037. doi: 10.1038/ng2079 URL
[20]	Ketting R F. 2010. MicroRNA biogenesis and function. An overview. Advances in Experimental Medicine and Biology, 700:1-14. pmid: 21627025
[21]	Kim Wanhui, Ahn H J, Chiou T J, Ahn J H. 2011. The role of the miR399-PHO 2 module in the regulation of flowering time in response to different ambient temperatures in Arabidopsis thaliana. Molecules and Cells, 32 (1):83-88. doi: 10.1007/s10059-011-1043-1 pmid: 21533549
[22]	Kunz H H, Häusler R E, Fettke J, Herbst K, Niewiadomski P, Gierth M, Bell K, Steup M, Flügge U I, Schneider A. 2010. The role of plastidial glucose-6-phosphate/phosphate translocators in vegetative tissues of Arabidopsis thaliana mutants impaired in starch biosynthesis. Plant Biology, 12:115-128. doi: 10.1111/j.1438-8677.2010.00349.x URL
[23]	Li Chenjing, Niu Jianxin, Pei Maosong, Cao Fujun, Quan Shaowen. 2016. Cloning and identification of novel miRNA genes related to calyx persistence in Korla Fragrant Pear. Acta Horticulturae Sinica, 43 (9):1803-1815. (in Chinese)
	李陈静, 牛建新, 裴茂松, 曹福军, 全绍文. 2016. 库尔勒香梨9个新miRNA克隆鉴定. 园艺学报, 43(9):1803-1815.
[24]	Li Zongmei. 2013. Identification and characterization of a novel hydroxycinnamoyl transferase from the Physcomitrella patens and the regulation of P. patens HCT[M. D. Dissertation]. Wuhan;Huazhong Agricultural University. (in Chinese)
	李宗梅. 2013. 小立碗藓羟基肉桂酰酰基转移酶基因功能及调控研究初探[硕士论文]. 武汉: 华中农业大学.
[25]	Liu M J, Wang J R, Wang L L, Liu P, Zhao J, Zhao Z H, Yao S R, Stănică Florin, Liu Z G, Wang L X, Ao C W, Dai L, Li X S, Zhao X, Jia C X. 2020. The historical and current research progress on jujube-a superfruit for the future. Horticulture Research, 7 (1):1683-1698.
[26]	Liu M J, Zhao J, Cai Q L, Liu G C, Wang J R, Zhao Z H, Liu P, Dai L, Yan G J, Wang W J, Li X S, Chen Y, Sun Y D, Liu Z G, Lin M J, Xiao J, Chen Y Y, Li X F, Wu B, Ma Y, Jian J B, Yang W, Yuan Z, Sun X C, Wei Y L, Yu L L, Zhang C, Liao S G, He R J, Guang X M, Wang Z, Zhang Y Y, Luo L H. 2014. The complex jujube genome provides insights into fruit tree biology. Nature Communications, 5 (1):1309-1324.
[27]	Liu Weihua, Lin Yuling, Lin Zhengchun, Ni Shanshan, Lai Zhongxiong. 2018. Analysis of evolution and molecular characteristics of miR172 family members in plants. Chinese Journal of Tropical Crops, 39 (3):525-533. (in Chinese)
	刘炜婳, 林玉玲, 林争春, 倪珊珊, 赖钟雄. 2018. 植物miR172家族成员进化与分子特性分析. 热带作物学报, 39 (3):525-533.
[28]	Luo Hongyu, Yang Jiangwei, Feng Ya, Zhang Huanhuan, Liu Shengyan, Zhang Ning, Si Huaijun. 2021. The effect of Stu-miR156 silencing by STTM technology on potato lateral root development. Acta Horticulturae Sinica, 48 (3):531-538. (in Chinese)
	罗红玉, 杨江伟, 冯亚, 张欢欢, 刘升燕, 张宁, 司怀军. 2021. STTM 技术沉默马铃薯Stu-miR156 对其侧根发育的影响. 园艺学报, 48 (3):531-538.
[29]	Ma J Y, Zhao P, Liu S B, Yang Q, Guo H H. 2020. The Control of developmental phase transitions by microRNAs and their targets in seed plants. International Journal of Molecular Sciences, 21 (6):1971. doi: 10.3390/ijms21061971 URL
[30]	Ma Li, Zhou Li, Xu Hang, Quan Shaowen, Yang Jieping, Niu LJianxini. 2019. Evolutionary characteristics and the expression patterns of miR159 gene family in‘Kuerlexiangli’pear. Journal of Fruit Science, 36 (1):1-10. (in Chinese)
	马丽, 周丽, 徐航, 全绍文, 杨洁萍, 牛建新. 2019. ‘库尔勒香梨’miR159家族成员进化特性及表达分析. 果树学报, 36 (1):1-10.
[31]	Martin R C, Asahina M, Liu Popu, Kristof J R, Coppersmith J L, Pluskota W E, Bassel G W, Goloviznina N A, Nguyen T T, Martínez A C, Arun Kumar M B, Pupel P, Nonogaki H. 2010. The microRNA156 and microRNA 172 gene regulation cascades at post-germinative stages in Arabidopsis. Seed Science Research, 20 (2):79-87. doi: 10.1017/S0960258510000085 URL
[32]	Meng J, Yang J, Peng M D, Liu X L, He H B. 2020a. Genome-wide characterization,evolution,and expression analysis of the Leucine-Rich Repeat Receptor-Like Protein Kinase(LRR-RLK)gene family in Medicago truncatula. Life, 10 (176):176. doi: 10.3390/life10090176 URL
[33]	Meng X W, Li Y, Yuan Y, Zhang Y, Li H T, Zhao J, Liu M J. 2020b. The regulatory pathways of distinct flowering characteristics in Chinese jujube. Horticulture Research, 7 (1):13-19. doi: 10.1038/s41438-019-0236-1 URL
[34]	Mi S J, Cai T, Hu Y G, Chen Y M, Hodges E, Ni F R, Wu L, Li S, Zhou H Y, Long C Z, Chen S, Hannon G J, Qi Y J. 2008. Sorting of small RNAs into Arabidopsis argonaute complexes is directed by the 5′ terminal nucleotide. Cell, 133 (1):116-127. doi: 10.1016/j.cell.2008.02.034 URL
[35]	Milo J A, Hajime S. 2003. Regulation of flowering time and floral organ identity by a MicroRNA and Its APETALA2-Like target Genes. The Plant Cell, 15:2730-2741 doi: 10.1105/tpc.016238 URL
[36]	Rubenach A J S, Hecht V, Vander Schoor J K, Liew L C, Aubert G, Burstin J, Weller J L. 2017. EARLY FLOWERING 3 redundancy fine-tunes photoperiod sensitivity. Plant Physiology, 173 (4):2253-2264. doi: 10.1104/pp.16.01738 pmid: 28202598
[37]	Schmid M, Uhlenhaut N H, Godard F, Demar M, Bressan R, Weigel D, Lohmann J U. 2003. Dissection of floral induction pathways using global expression analysis. Development, 130 (24):6001-6012. doi: 10.1242/dev.00842 URL
[38]	Shuai Minmin. 2018. Advances of GIGANTEA and CONSTANS,the key genes of flowering in photoperiod pathway[M. D. Dissertation]. Lin’an:Zhejiang A & F University. (in Chinese)
	帅敏敏. 2018. 光周期途径成花关键基因GIGANTEA和CONSTANS的进化机制[硕士论文]. 临安: 浙江农林大学.
[39]	Shuai Minmin, Zhang Qixiang, Huang Youjun. 2019. Evolution of the flowering time gene CONSTANS in a photoperiod pathway. Journal of Zhejiang A & F University, 36 (1):7-13. (in Chinese)
	帅敏敏, 张启香, 黄有军. 2019. 光周期途径成花关键基因CONSTANS的进化机制. 浙江农林大学学报, 36 (1):7-13.
[40]	Spanudakis E, Jackson S. 2014. The role of microRNAs in the control of flowering time. Journal of experimental botany, 65 (2):365-380. doi: 10.1093/jxb/ert453 pmid: 24474808
[41]	Teng Yunlong, Li Chunmin, Zhang Xinzhong, Chen Dongmei, Zeng Guangjuan, Zhao Yongbo, Dong Wenxuan. 2009. SDS-PAGE analysis of phase-transition-related proteins in buds of apple trees. Journal of Fruit Science, 26 (3):375-378. (in Chinese)
	滕云龙, 李春敏, 张新忠, 陈东玫, 曾广娟, 赵永波, 董文轩. 2009. 苹果实生树嫩芽阶段转变相关蛋白质的SDS-PAGE分析. 果树学报, 26 (3):375-378.
[42]	Wang L K, Feng Z X, Wang X, Wang X W, Zhang X G. 2010. DEGseq;an R package for identifying differentially expressed genes from RNA-seq data. Bioinformatics, 26 (1):136-138. doi: 10.1093/bioinformatics/btp612 URL
[43]	Wen M, Shen Y, Shi S H, Tang T. 2012. miREvo;an integrative microRNA evolutionary analysis platform for next-generation sequencing experiments. BMC Bioinformatics 13 (1):140. doi: 10.1186/1471-2105-13-140 URL
[44]	Wu G, Park M Y, Conway S R, Wang J W, Weigel D, Poethig R S. 2009. The sequential action of miR156 and miR172 regulates developmental timing in Arabidopsis. Cell, 138 (4):750-759. doi: 10.1016/j.cell.2009.06.031 URL
[45]	Xu Zihan, Hu Fengrong. 2020. Research progress of miR172 in plant development and regulation. Biotechnology Bulletin, 36 (8):173-184. (in Chinese) doi: 10.13560/j.cnki.biotech.bull.1985.2020-0181
	徐子涵, 胡凤荣. 2020. miR172参与植物发育调控的研究进展. 生物技术通报, 36 (8):173-184. doi: 10.13560/j.cnki.biotech.bull.1985.2020-0181
[46]	Yang L L, Xu M L, Koo Y, He J,Poethig R Scott. 2013. Sugar promotes vegetative phase change in Arabidopsis thaliana by repressing the expression of MIR156A and MIR156C. eLife, 2:e00260. doi: 10.7554/eLife.00260 URL
[47]	Yu S, Cao L, Zhou C M, Zhang T Q, Lian H, Sun Y, Wu J Q, Huang J R, Wang G D, Wang J W. 2013. Sugar is an endogenous cue for juvenile-to-adult phase transition in plants. eLife, 2:17.
[48]	Yu S, Lian H, Wang J W. 2015. Plant developmental transitions;the role of microRNAs and sugars. Current Opinion in Plant Biology, 27:1-7. doi: 10.1016/j.pbi.2015.05.009 URL
[49]	Zhang C, Xian Z Q, Huang W, Li Z G. 2015. Evidence for the biological function of miR403 in tomato development. Scientia Horticulturae, 197:619-626. doi: 10.1016/j.scienta.2015.10.027 URL
[50]	Zhao Guomiao. 2019. Studies on the miRNA regulation mechanism of heteromorphic leaf development in Populus euphratica[Ph. D. Dissertation]. Beijing: Beijing Forestry University. (in Chinese)
	赵国淼. 2019. 胡杨异形叶发育的miRNA调控机制研究[博士论文]. 北京: 北京林业大学.
[51]	Zhao Hang. 2020. Molecular mechanism of Arabidopsis NF-YA 8 in regulating juvenile-to-adult transition and flowering time[Ph. D. Dissertation]. Tai'an: Shandong Agricultural University. (in Chinese)
	赵航. 2020. 拟南芥NF-YA8调控幼年向成年阶段转型及开花时间的分子机理研究[博士论文]. 泰安: 山东农业大学.
[52]	Zhao Jianguo, Cui Jiawen, Jin Biao. 2015. Research advances of developmental changes of juvenile to adult transition in woody plants. Plant Physiology Journal, 51 (11):1765-1774. (in Chinese)
	赵建国, 崔佳雯, 金飚. 2015. 树木幼年向成年转变的发育调控机制研究进展. 植物生理学报, 51 (11);1765-1774.
[53]	Zheng C F, Ye M X, Sang M G, Wu R L. 2019a. A regulatory network for miR156-SPL module in Arabidopsis thaliana. International Journal of Molecular Sciences, 20 (24):6166. doi: 10.3390/ijms20246166 URL
[54]	Zheng G H, Wei W, Li Y P, Kan L J, Wang F X, Zhang X, Li F, Liu Z C, Kang C Y. 2019b. Conserved and novel roles of miR164-CUC 2 regulatory module in specifying leaf and floral organ morphology in strawberry. New Phytologist, 224 (1):480-492. doi: 10.1111/nph.v224.1 URL

miRNA名称 miRNA name	引物序列（5′-3′） Primer sequence	miRNA名称 miRNA name	引物序列（5′-3′） Primer sequence
zjmiR156a-5p	TGGCTGGGTGACAGAAGAGAGT	zjmiRnovel-16	GGTTAGTGATCCTCCGGAAGATC
zjmiR156j	GGCTGGGTGACAGAAGAGAGA	zjmiRnovel-71	TTGAAGTGTTTGGGGGAACC
zjmiR172c	TGAGAATCTTGATGATGCTGCAG	zjmiRnovel-35	GGCTGCCTCTTGTCTTTCATG
zjmiR172e-3p	TGGAATCTTGATGATGCTGCAT	zjmiRnovel-17	TGCATTTGCACCTGCACCT
zjmiR160a-5p	TGCCTGGCTCCCTGTATGC	zjmiR845a	TCGGCTCTGATACCAATTGATG
zjmiR157a-5p	CGGTTGACAGAAGATAGAGAGCAC	U6	GGGACATCCGATAAAATTG
zjmiR396b-5p	TGGTTCCACAGCTTTCTTGAACTT	U6	CCAATTTTATCGGATGTCC

miRNA名称 miRNA name	引物序列（5′-3′） Primer sequence	miRNA名称 miRNA name	引物序列（5′-3′） Primer sequence
zjmiR156a-5p	TGGCTGGGTGACAGAAGAGAGT	zjmiRnovel-16	GGTTAGTGATCCTCCGGAAGATC
zjmiR156j	GGCTGGGTGACAGAAGAGAGA	zjmiRnovel-71	TTGAAGTGTTTGGGGGAACC
zjmiR172c	TGAGAATCTTGATGATGCTGCAG	zjmiRnovel-35	GGCTGCCTCTTGTCTTTCATG
zjmiR172e-3p	TGGAATCTTGATGATGCTGCAT	zjmiRnovel-17	TGCATTTGCACCTGCACCT
zjmiR160a-5p	TGCCTGGCTCCCTGTATGC	zjmiR845a	TCGGCTCTGATACCAATTGATG
zjmiR157a-5p	CGGTTGACAGAAGATAGAGAGCAC	U6	GGGACATCCGATAAAATTG
zjmiR396b-5p	TGGTTCCACAGCTTTCTTGAACTT	U6	CCAATTTTATCGGATGTCC

miRNA名称 miRNA name	靶基因 Gene	引物名称 Primer	上游引物（5′-3′） Forward primer	下游引物（5′-3′） Reversed primer
zjmiR156a-5p	LOC107411088	GE1	TCACTCTAAACGACCCGCAC	GAAGGCACGGAGGATATCGG
	LOC107411586	TC4M	GTCACAGAGCCCGACATTCA	TTCCAGTTGGCAGGGTTGTT
	LOC107420411	GPT2	TTCGTCTGGTGGGTTGTAGC	GAAGGTGCCGAGGATAGCAA
zjmiR156j	LOC107414106	ERF105-like	ACGTCGTCGGAGAGTAAGGA	AGGTGACAAAGGAGGCACAC
zjmiR156j	LOC107425063	SPL	GCAATGCTGACTTGACCGAC	TACGACGCTCATTGTGTCCA
zjmiR172c	LOC107411289	AP2c	AGAGCCTATGATCGAGCTGC	TCCCATTCGAGCTTCCCATC
zjmiR172e-3p	LOC107413613	AP2e	TTAGGTGGGTTCGACACTGC	CCGACGAAGTATCAGCACGA
zjmiRnovel-16	LOC107416596	PIF4-like	TAGCAGGGTGTCCAGCAATG	TCTGATTGCCTCCGCCTTTT
	LOC107416597	PIF4	GAGTCAACGGTCGGGTTCAA	ATTGGTGCCATCCCACTTCC
	LOC107415240	ELF 3	TGCTGAAGGAAGGGATGCTC	ATTGTGCGGTACAACCCTGA
	LOC107420622	CO-LIKE7-like	AGAGGCAAGCGTGTTGAGAT	GAACCGGCCCTTCATACGAG
zjmiRnovel-71	LOC107422007	cro-GT1	GCTACCGTGTTCGGCATCTA	CCATGAACGTGGGAAGGTCA
	LOC107422008	cro-GT2	CGGACCGTTGATTCAGTCCA	GTGGCTTTGGTAACACGCAG
	LOC107418233	NFYA7	AAGCTGGAGTTCCTTTGCCA	CCGCTTTTGCACGAGATTGT
	LOC107412400	RLK1	GCGCAACAAAGTGGAACCTT	GCATTTGTGCACAGGAGAGC

miRNA名称 miRNA name	靶基因 Gene	引物名称 Primer	上游引物（5′-3′） Forward primer	下游引物（5′-3′） Reversed primer
zjmiR156a-5p	LOC107411088	GE1	TCACTCTAAACGACCCGCAC	GAAGGCACGGAGGATATCGG
	LOC107411586	TC4M	GTCACAGAGCCCGACATTCA	TTCCAGTTGGCAGGGTTGTT
	LOC107420411	GPT2	TTCGTCTGGTGGGTTGTAGC	GAAGGTGCCGAGGATAGCAA
zjmiR156j	LOC107414106	ERF105-like	ACGTCGTCGGAGAGTAAGGA	AGGTGACAAAGGAGGCACAC
zjmiR156j	LOC107425063	SPL	GCAATGCTGACTTGACCGAC	TACGACGCTCATTGTGTCCA
zjmiR172c	LOC107411289	AP2c	AGAGCCTATGATCGAGCTGC	TCCCATTCGAGCTTCCCATC
zjmiR172e-3p	LOC107413613	AP2e	TTAGGTGGGTTCGACACTGC	CCGACGAAGTATCAGCACGA
zjmiRnovel-16	LOC107416596	PIF4-like	TAGCAGGGTGTCCAGCAATG	TCTGATTGCCTCCGCCTTTT
	LOC107416597	PIF4	GAGTCAACGGTCGGGTTCAA	ATTGGTGCCATCCCACTTCC
	LOC107415240	ELF 3	TGCTGAAGGAAGGGATGCTC	ATTGTGCGGTACAACCCTGA
	LOC107420622	CO-LIKE7-like	AGAGGCAAGCGTGTTGAGAT	GAACCGGCCCTTCATACGAG
zjmiRnovel-71	LOC107422007	cro-GT1	GCTACCGTGTTCGGCATCTA	CCATGAACGTGGGAAGGTCA
	LOC107422008	cro-GT2	CGGACCGTTGATTCAGTCCA	GTGGCTTTGGTAACACGCAG
	LOC107418233	NFYA7	AAGCTGGAGTTCCTTTGCCA	CCGCTTTTGCACGAGATTGT
	LOC107412400	RLK1	GCGCAACAAAGTGGAACCTT	GCATTTGTGCACAGGAGAGC

样本类型 Sample type	童期Juvenile period		过渡期Transition period		成年期Adult period
样本类型 Sample type	种类（%） Category	数量（%） Number	种类（%） category	数量（%） Number	种类（%） category	数量（%） Number
已知miRNA Known miRNA	121（0.03）	22 367（0.33）	152（0.04）	36 906（0.57）	167（0.04）	34 796（0.35）
rRNA	118 138（31.30）	5 535 824（82.23）	112 531（32.56）	5 295 448（81.29）	134 295（28.46）	8 073 860（80.84）
tRNA	4 740（1.26）	181 112（2.69）	4 537（1.31）	152 992（2.35）	5 847（1.24）	340 580（3.41）
snRNA	965（0.26）	4 027（0.06）	1 007（0.29）	4 148（0.06）	1 328（0.28）	7 269（0.07）
snoRNA	576（0.15）	1 674（0.02）	487（0.14）	1 550（0.02）	699（0.15）	2 483（0.02）
重复序列 Repeat	14 323（3.80）	37 164（0.55）	13 090（3.79）	33 685（0.52）	17 230（3.65）	48 302（0.48）
NAT	69 876（18.51）	340 047（5.05）	62 771（18.16）	433 170（6.65）	89 355（18.94）	579 299（5.80）
新miRNA Novel miRNA	61（0.02）	4 221（0.06）	67（0.02）	8 903（0.14）	78（0.02）	9 116（0.09）
exon：+	25 350（6.72）	52 607（0.78）	22 878（6.62）	54 968（0.84）	38 512（8.16）	86 381（0.86）
exon：-	2 263（0.60）	12 125（0.18）	2 841（0.82）	20 279（0.31）	3 489（0.74）	24 477（0.25）
intron：+	6 317（1.67）	48 369（0.72）	5 486（1.59）	34 062（0.52）	8 825（1.87）	79 221（0.79）
intron：-	3 386（0.90）	11 592（0.17）	2 934（0.85）	9 272（0.14）	4 064（0.86）	18 475（0.18）
其他Other	131 297（34.79）	481 402（7.15）	116 873（33.81）	429 158（6.59）	167 937（35.59）	683 147（6.84）
总和Total	377 413	6 732 531	345 654	6 514 541	471 826	9 987 406