茶树miR164a及其靶基因的鉴定与表达分析

doi:10.13305/j.cnki.jts.2018.06.001

摘要/Abstract

摘要： 植物miR164通过负调控靶基因NAC转录因子（NAC transcription factors）参与植物生长发育调控和逆境胁迫等复杂的生理过程。利用茶树microRNA高通量测序和降解组测序,对茶树miR164a及其靶基因NAC1进行了克隆、序列分析与鉴定,并对其在叶片发育、高温和低温胁迫中表达模式进行了研究。茶树miR164a前体序列长度为126βbp,成熟序列21βbp。CsNAC1 ORF框长度912βbp,编码303个氨基酸,在CsNAC1蛋白的N端有典型NAM结构域,C端有Csn-miR164a识别位点。RLM-5´RACE验证CsNAC1为茶树miR164a的靶基因。荧光定量表达分析表明,Csn-miR164a在芽中表达量最高,第7叶中最低。在高温和低温胁迫下,Csn-miR164a都下调表达,而CsNAC1表达水平上升,CsNAC1与Csn-miR164a呈现负相关。研究表明茶树miR164a及其靶基因CsNAC1可能与茶树生长及抗逆相关。

关键词: 茶树, 靶基因, 温度胁迫, miR164a, NAC

Abstract: Plant miR164 is involved in complex physiological processes such as plant growth and development regulation and stress responses by negatively regulating the target gene NAC transcription factors. The identification, cloning and sequence analysis of miR164a and its target gene NAC1 in tea plant were carried out by high-throughput sequencing of microRNA and degradome analysis. The expression patterns of Csn-miR164a and its target gene CsNAC1 during leaf development and under high or low temperature stresses were also studied. The length of Csn-miR164a precursor is 126βbp, and the mature sequence is 21βbp. The CsNAC1 ORF is 912βbp, encoding 303 amino acids. There are a typical NAM domain at the N terminus of CsNAC1 protein and a Csn-miR164a recognition site at the C-terminus. Quantitative real-time PCR analysis showed that the expression of Csn-miR164a was the highest in the bud and the lowest in the seventh leaves. Under high and low temperature treatments, the expression of Csn-miR164a was down regulated, while the expression level of CsNAC1 increased. The expression of Csn-miR164a was negatively correlatied with the expression of the target gene CsNAC1. The results revealed that Csn-miR164a and its target gene CsNAC1 might be closely related to the growth and stress responses in tea plant.

Key words: Camellia sinensis, target gene, temperature stress, miR164a, NAC

中图分类号:

S571.1
Q52

孔雷, 朱向向, 王屹玮, 谢小芳, 江昌俊, 李叶云. 茶树miR164a及其靶基因的鉴定与表达分析[J]. 茶叶科学, 2018, 38(6): 547-558. doi: 10.13305/j.cnki.jts.2018.06.001.

KONG Lei, ZHU Xiangxiang, WANG Yiwei, XIE Xiaofang, JIANG Changjun, LI Yeyun. Identification and Expression Analysis of Tea Plant (Camellia sinesis) miR164a and Its Target Gene[J]. Journal of Tea Science, 2018, 38(6): 547-558. doi: 10.13305/j.cnki.jts.2018.06.001.

参考文献 38

[1]	Bartel D P.MicroRNAs: genomics, biogenesis, mechanism, and function[J]. Cell, 2004, 116(2): 281-297.
[2]	谢小芳. 茶树miRNA靶基因的鉴定及在低温胁迫下的表达分析[D]. 合肥: 安徽农业大学, 2016.
[3]	牟桂萍, 纪春艳, 许东林, 等. 植物miR164家族研究进展[J]. 生命科学, 2013, 25(5): 532-538.
[4]	王浩然, 李爽爽, 乐丽娜, 等. miR164a及其靶基因PeNAC1相互作用研究[J]. 南京林业大学学报(自然科学版), 2016, 40(5): 29-33.
[5]	Guo H S, Xie Q, Fei J F, et al.MicroRNA directs mRNA cleavage of the transcription factor NAC1 to downregulate auxin signals for arabidopsis lateral root development[J]. Plant Cell, 2005, 17(5): 1376-1386.
[6]	Válóczi A, Várallyay É, Kauppinen S, et al.Spatio-temporal accumulation of microRNAs is highly coordinated in developing plant tissues[J]. Plant Journal, 2006, 47(1): 140-151.
[7]	Lu S, Sun YH, Shi R, et al.Novel and mechanical stress-responsive MicroRNAs in Populus trichocarpa that are absent from Arabidopsis[J]. Plant Cell, 2005, 17(8): 2186-2203.
[8]	孙宗艳. 盐/干旱胁迫下甜菜幼苗中miR160/164及其靶基因的表达与分析[D]. 哈尔滨: 哈尔滨工业大学, 2017.
[9]	彭辉, 于兴旺, 成慧颖, 等. 植物NAC转录因子家族研究概况[J]. 植物学报, 2010, 45(2): 236-248.
[10]	孙利军, 李大勇, 张慧娟, 等. NAC转录因子在植物抗病和抗非生物胁迫反应中的作用[J]. 遗传, 2012, 34(8): 993-1002.
[11]	Olsen A N, Ernst H A, Leggio L L, et al.NAC transcription factors: structurally distinct, functionally diverse[J]. Trends in Plant Science, 2005, 10(2): 79-87.
[12]	Nakashima K, Takasaki H, Mizoi J, et al.NAC transcription factors in plant abiotic stress responses[J]. Biochimica et Biophysica Acta, 2012, 1819(2): 97-103.
[13]	Fang Y, Xie K, Xiong L.Conserved miR164-targeted NAC genes negatively regulate drought resistance in rice[J]. Journal of Experimental Botany, 2014, 65(8): 2119-2135.
[14]	Puranik S, Sahu PP, Srivastava PS, et al.NAC proteins: regulation and role in stress tolerance[J]. Trends in plant science, 2012, 17(6): 369-381.
[15]	Fujita M, Fujita Y, Maruyama K, et al.A dehydration-induced NAC protein, RD26, is involved in a novel ABA-dependent stress-signaling pathway[J]. The Plant Journal, 2004, 39(6): 863-876.
[16]	Yoo S Y, Kim Y, Kim S Y, et al.Control of flowering time and cold response by a NAC-domain protein in Arabidopsis[J]. Plos One, 2007, 2(7): e642. DOI: 10.1371/ journal.pone.0000642.
[17]	Honghong Hu, Mingqiu Dai, Jialing Yao, et al.Overexpressing a NAM, ATAF, and CUC (NAC) transcription factor enhances drought resistance and salt tolerance in rice[J]. Proceedings of the National Academy of Sciences of the United States of America, 2006, 103(35): 12987-12992.
[18]	Laufs P, Peaucelle A, Morin H, et al.MicroRNA regulation of the CUC genes is required for boundary size control in Arabidopsis meristems[J]. Development, 2004, 131(17): 4311-4322.
[19]	Kim J H, Woo H R, Kim J, et al.Trifurcate feed-forward regulation of age-dependent cell death involving miR164 in Arabidopsis[J]. Science, 2009, 323(5917): 1053-1057.
[20]	Kaur A, Gupta O P, Meena N L, et al.Comparative temporal expression analysis of microRNAs and their target genes in contrasting wheat genotypes during osmotic stress[J]. Applied Biochemistry & Biotechnology, 2017, 181(2): 613-626.
[21]	Lu X, Dun H, Lian C, et al.The role of peu-miR164 and its target PeNAC genes in response to abiotic stress in Populus euphratica[J]. Plant Physiology & Biochemistry Ppb, 2017, 115: 418-438.
[22]	王永鑫, 刘志薇, 吴致君, 等. 茶树中2个NAC转录因子基因的克隆及温度胁迫的响应[J]. 西北植物学报, 2015, 35(11): 2148-2156.
[23]	Zhang Y, Zhu X, Chen X, et al. Identification and characterization of cold-responsive microRNAs in tea plant (Camellia sinensis) andtheir targets using high-throughput sequencing and degradome analysis [J]. Bmc Plant Biology, 2014, 14(1): 271. https://doi.org/10.1186/s12870-014-0271-x.
[24]	Zheng C, Zhao L, Wang Y, et al. Integrated RNA-Seq and sRNA-Seq analysis identifies chilling and freezing responsive key molecular players and pathways in tea plant (Camellia sinensis) [J]. Plos One, 2015, 10(4): e0125031. https://doi.org/10.1371/journal.pone.0125031.
[25]	刘亚芹, 田坤红, 孙琪璐, 等. 茶树miR156a靶基因SPL6和SPL9的克隆及表达分析[J]. 茶叶科学, 2017, 37(6): 551-564.
[26]	顾冕, 孟大千, 徐国华. 烟草microRNA827及其靶基因的鉴定与分析[J]. 南京农业大学学报, 2016, 39(6): 965-972.
[27]	Fang Y, Xie K, Xiong L.Conserved miR164-targeted NAC genes negatively regulate drought resistance in rice[J]. Journal of Experimental Botany, 2014, 65(8): 2119-2135.
[28]	Varkonyi-Gasic E, Hellens R P. qRT-PCR of small RNAs[J]. Methods in Molecular Biology, 2010, 631:109-122.
[29]	谢小芳, 添先凤, 江昌俊, 等. 茶树低温胁迫下microRNA实时定量PCR内参基因的筛选[J]. 茶叶科学, 2015, 35(6): 596-604.
[30]	Hao X, Horvath D P, Chao W S, et al.Identification and evaluation of reliable reference genes for quantitative real-time pcr analysis in tea plant (Camellia sinensis (L.) O. Kuntze)[J]. International Journal of Molecular Sciences, 2014, 15(12):22155-22172.
[31]	Zhang B H, Pan X P, Cox S B, et al.Evidence that miRNAs are different from other RNAs[J]. Cellular & Molecular Life Sciences Cmls, 2006, 63(2): 246-254.
[32]	Wang Y X, Liu Z W, Wu Z J, et al.Transcriptome-wide identification and expression analysis of the NAC gene family in tea plant [Camellia sinensis (L.) O. Kuntze][J]. Plos One, 2016, 11(11): e0166727. DOI: 10.1371/journal.pone.0166727.
[33]	吴骏, 张俊红, 黄蒙慧, 等. 光皮桦miR164及其靶基因NAC1在低氮胁迫中的表达分析[J]. 遗传, 2016, 38(2): 155-162.
[34]	Guleria P, Yadav S K.Identification of miR414 and expression analysis of conserved miRNAs from stevia rebaudiana[J]. Genomics, Proteomics & Bioinformatics, 2011, 9(6): 211-217.
[35]	王丽丽, 赵韩生, 孙化雨, 等. 胁迫条件下毛竹miR164b及其靶基因PeNAC1表达研究[J]. 林业科学研究, 2015, 28(5): 605-611.
[36]	罗中钦. 大豆逆境胁迫相关microRNA的发掘与验证[D]. 北京: 中国农业科学院, 2012.
[37]	孙润泽, 侯琦, 章文乐, 等. 甜杨低温响应microRNAs的克隆与分析[J]. 基因组学与应用生物学, 2011, 30(2): 204-211.
[38]	Gupta O P, Meena N L, Sharma I, et al.Differential regulation of microRNAs in response to osmotic, salt and cold stresses in wheat[J]. Molecular Biology Reports, 2014, 41(7): 4623-4629.