高山松miR482a的鉴定及其表达分析

doi:10.14088/j.cnki.issn0439-8114.2021.17.028

湖北农业科学 ›› 2021, Vol. 60 ›› Issue (17): 135-138.doi: 10.14088/j.cnki.issn0439-8114.2021.17.028

高山松miR482a的鉴定及其表达分析

王稳利, 曾倩倩, 刘岩, 邱宗波

河南师范大学生命科学学院/河南省农业微生物生态与技术国际联合实验室,河南新乡 453007

收稿日期:2021-02-04 出版日期:2021-09-10 发布日期:2021-09-30
通讯作者: 邱宗波（1978-）,女,河南南阳人,教授,博士,主要从事植物分子生物学方面的研究,（电子信箱）qiuzongbo@126.com。
作者简介:王稳利（1993-）,女,河南周口人,在读硕士研究生,研究方向为植物发育生物学,（电话）15837329129（电子信箱）2564227677@qq.com
基金资助:
国家自然科学基金项目（31500499）; 河南省高校科技创新人才项目（16HASTIT019）

Identification and expression analysis of miR482a in Pinus densata

WANG Wen-li, ZENG Qian-qian, LIU Yan, QIU Zong-bo

College of Life Science,Henan Normal University/Henan International Joint Laboratory of Agricultural Microbial Ecology and Technology,Xinxiang 453007,Henan,China

Received:2021-02-04 Online:2021-09-10 Published:2021-09-30

摘要/Abstract

摘要： 以裸子植物高山松（Pinus densata）为试验材料,利用实验室前期构建的高山松miRNA数据库,通过生物信息学方法筛选了与生长发育相关的miR482a,并通过RLM-5′RACE 的方法验证了NBS-LRR抗性蛋白（Unigenes45569）是高山松 miR482a的靶基因。通过PCR方法克隆得到高山松miR482a前体序列是130 bp,并可形成稳定的茎环发夹结构,但其成熟序列的碱基保守性较差。系统进化显示pde-miR482a与裸子植物欧洲云杉的进化关系较近。经qRT-PCR分析表明,pde-miR482a在根中表达量最高,其次是在茎中。而靶基因Unigenes45569 在根和茎中的表达水平相对较低,暗示pde-miR482a 可以通过调控靶基因NBS-LRR抗性蛋白（Unigenes45569）而参与高山松的生长发育。

关键词: 高山松（Pinus densata）, miR482a, 靶基因, 荧光定量PCR

Abstract: Taking the gymnosperm Pinus densata as the experimental material, using the Pinus densata miRNA database constructed in the early stage of the laboratory, mir482a related to growth and development was screened by bioinformatics method, and NBS-LRR resistance protein （Unigenes45569） was verified to be the target gene of Pinus densata mir482a by RLM-5′ RACE method. The mir482a precursor sequence of Pinus densata cloned by PCR is 130 bp and can form a stable stem -loop hairpin structure, but the base conservation of its mature sequence is poor. Phylogeny showed that pde-mir482a had a close evolutionary relationship with the gymnosperm Picea europaea. qRT-PCR analysis showed that the expression of pde-mir482a was the highest in roots, followed by in stems. The expression level of target gene Unigenes45569 in roots and stems is relatively low, suggesting that pde-mir482a can participate in the growth and development of Pinus densata by regulating target gene NBS-LRR resistance protein （Unigenes45569）.

Key words: Pinus densata, miR482a, target gene, qRT-PCR

中图分类号:

Q819

王稳利, 曾倩倩, 刘岩, 邱宗波. 高山松miR482a的鉴定及其表达分析[J]. 湖北农业科学, 2021, 60(17): 135-138.

WANG Wen-li, ZENG Qian-qian, LIU Yan, QIU Zong-bo. Identification and expression analysis of miR482a in Pinus densata[J]. HUBEI AGRICULTURAL SCIENCES, 2021, 60(17): 135-138.

参考文献

[1] VOINNET O.Origin, biogenesis and activity of plant microRNAs[J]. Cell, 2009, 136(4): 669-687.
[2] WAN L C, ZHANG H, LU S, et al.Transcriptome-wide identification and characterization of miRNAs from Pinus densata[J]. BMC genomics, 2012, 13(1): 132.
[3] 张俊红,张守攻,吴涛,等. 落叶松体胚发育中5个miRNA前体与成熟体的表达[J]. 植物学报,2012,47(5): 462-473.
[4] QIU Z, HE Y, ZHANG Y, et al.Characterization of miRNAs and their target genes in He-Ne laser pretreated wheat seedlings exposed to drought stress[J]. Ecotoxicology and environmental safety, 2018, 164: 611-617.
[5] 张玉娟, 王维, 陈洁, 等. 棉花中黄萎病抗性相关microRNA的挖掘及其靶基因分析[J]. 分子植物育种,2015,13(1):156-164.
[6] YANG L, MU X, LIU C, et al.Overexpression of potato miR482e enhanced plant sensitivity to Verticillium dahliae infection[J]. 植物学报(英文版), 2016, 57(12): 1078-1088.
[7] ZUO J, ZHU B, FU D, et al.Sculpting the maturation, softening and ethylene pathway:The influences of microRNAs on tomato fruits[J]. BMC genomics, 2012, 13(1): 7.
[8] LI H, DENG Y, WU T L, et al.Misexpression of miR482, miR1512, and miR1515 increases soybean nodulation[J]. Plant physiology, 2010, 153(4): 1759-1770.
[9] WANG B, MAO J F, GAO J, et al.Colonization of the Tibetan Plateau by the homoploid hybrid pine Pinus densata[J]. Molecular ecology, 2011, 20(18): 3796-3811.
[10] 孔雷, 朱向向, 王屹玮, 等. 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.
[11] LIVAK K J, SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔCt method[J]. Methods, 2001, 25(4): 402-408.
[12] 孙广鑫, 栾雨时, 崔娟娟. 番茄中与致病密切相关 miRNA 的挖掘及特性分析[J]. 遗传, 2014, 36(1): 69-76.
[13] JIANG N,MENG J,CUI J,et al.Function identification of miR482b,a negative regulator during tomato resistance to Phytophthora infestans[J]. Horticulture research, 2018, 5: 9.
[14] LU S, SUN Y H, AMERSON H, et al.MicroRNAs in loblolly pine (Pinus taeda L.) and their association with fusiform rust gall development[J]. Plant journal, 2007, 51(6): 1077-1098.
[15] ZHU Q H, FAN L, LIU Y, et al.miR482 regulation of NBS-LRR defense genes during fungal pathogen infection in cotton[J]. PLoS one, 2013, 8(12): e84390.
[16] JIANG N,CUI J,YANG G,et al.Comparative transcriptome analysis shows the defense response networks regulated by miR482b[J].Plant cell reports,2019,38(1):1-13.

高山松miR482a的鉴定及其表达分析

Identification and expression analysis of miR482a in Pinus densata

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 7

编辑推荐

Metrics

本文评价

[1]	王成, 徐石勇, 李纳, 王雅偲, 陈锐, 兰青阔, 王永, 赵新. 实时荧光定量PCR鉴定食品中绿豆源性成分[J]. 湖北农业科学, 2023, 62(9): 119-123.
[2]	张玉山, 陈洁彬, 吴亮君, 朱璇, 欧阳淑芬, 沈志华. 基于实时荧光定量PCR的番木瓜多重PCR高效检测方法[J]. 湖北农业科学, 2023, 62(9): 170-174.
[3]	刘欢, 刘格, 李锐. 樱桃病毒A实时荧光定量PCR检测技术的建立与应用[J]. 湖北农业科学, 2023, 62(7): 163-169.
[4]	李梦丹, 王美玲, 郭仰东, 张喜春. 低温胁迫下外源激素对番茄SlMYB102转录因子表达量的影响[J]. 湖北农业科学, 2021, 60(15): 136-140.
[5]	倪婧, 谢道燕, 江秀均, 杨振国, 柴建萍. 朱砂叶螨各发育阶段内参基因评估及筛选[J]. 湖北农业科学, 2021, 60(11): 141-145.
[6]	薛巨坤, 王博, 王莲萍, 杨春雨, 国会艳, 郝爱平. 与植物抗逆相关的miR-398-3p基因家族进化分析及靶基因预测[J]. 湖北农业科学, 2019, 58(14): 141-147.
[7]	许萍萍, 粟寒, 王振华, 刘中慧, 李甜甜, 李彬, 吴翠萍. 入境旅客携带苹果中粉红聚端孢菌的检测与鉴定[J]. 湖北农业科学, 2018, 57(2): 67-72.