光皮桦miR164及其靶基因NAC1在低氮胁迫中的表达分析

doi:10.16288/j.yczz.15-231

遗传 ›› 2016, Vol. 38 ›› Issue (2): 155-162.doi: 10.16288/j.yczz.15-231

光皮桦miR164及其靶基因NAC1在低氮胁迫中的表达分析

吴骏,张俊红,黄蒙慧,朱敏慧,童再康

浙江农林大学,亚热带森林培育国家重点实验室培育基地,临安 311300

收稿日期:2015-05-26 修回日期:2015-07-28 出版日期:2016-02-20 发布日期:2015-12-30
通讯作者: 童再康,教授,研究方向：林木遗传育种。E-mail: zaikangtong@163.com
作者简介:吴骏,在读硕士研究生,专业方向：林木分子生物学。E-mail: wujunjames@126.com张俊红,副教授,研究方向：植物逆境生理与分子生物学。E-mail: zjh6719036@163.com吴骏和张俊红为共同第一作者。
基金资助:
国家自然科学基金项目(编号：31300566)和浙江省竹木新品种选育重大科技专项(编号：2012C12908-8)资助[Supported by the National Natural Science Foundation of China (No; 31300566) and Zhejiang Province Science and Technology Support Program (No; 2012C12908-8)]

Expression analysis of miR164 and its target gene NAC1 in response to low nitrate availability in Betula luminifera

Jun Wu, Junhong Zhang, Menghui Huang, Minhui Zhu, Zaikang Tong

Nurturing Station for the State Key Laboratory of Subtropical Silviculture, Zhejiang Agriculture and Forestry University, Lin’an 311300, China

Received:2015-05-26 Revised:2015-07-28 Online:2016-02-20 Published:2015-12-30

摘要/Abstract

摘要： 氮是植物生长发育所必需的大量营养元素,植物缺氮后严重影响地上部分生物量的积累,因此,揭示植物如何抵抗或适应低氮胁迫的分子机制具有重要意义。杨树(Populus tremula × P. alba)NAC1(NAM, ATAF, CUC 1)基因位于调控网络上游,在低氮环境下调控下游关键基因的表达,进而调控根系生长以抵抗低氮胁迫。本文以光皮桦(Betula luminifera)G49-3无性系组培苗为材料,探讨了miR164及其靶基因NAC1对低氮胁迫的响应。通过RACE技术克隆了光皮桦NAC1基因(GenBank登录号：KT900889),全长1497 bp,编码358个氨基酸,N端具有高度保守的NAM结构域;运用5′-RACE验证了NAC1为miR164靶基因,切割位点在第10和11位碱基之间;采用qRT-PCR分析miR164与靶基因NAC1在低氮胁迫时的表达模式,发现miR164表达在根中的低氮处理前期(4 d)受到抑制,而后升高,而茎叶中表达模式与根不同;靶基因NAC1与miR164表达水平呈负相关,且在恢复实验组(重新添加全营养液)中,根中miR164表达上升,NAC1显示出相应的表达变化,暗示miR164及其靶基因NAC1可能在低氮胁迫响应中发挥调控功能。本研究结果有助于揭示miR164对NAC1在低氮胁迫响应中转录后水平的分子调控机制,为进一步研究miR164-NAC1在低氮胁迫响应中的功能提供有价值的信息。

关键词: 光皮桦, 低氮胁迫, miR164, 靶基因, 表达分析

Abstract: Nitrogen, an essential macronutrient for the growth and development of plants, affects above- ground biomass accumulation dramatically. Thus, it is very important to reveal the molecular mechanisms of how plants resist or adapt to low nitrogen availability. The NAC1(NAM, ATAF, CUC 1) gene, located in the upstream regulatory network, has been reported to resist low nitrogen by regulating expression of key downstream genes and thus root growth in (Populus tremula × alba).In this study, we detected the responses of miR164 and its target gene NAC1 under nitrate-starvation condition using the Betula luminifera somaclones G49-3 as material. The NAC1 gene which contains 1497 bp sequence, encodes 358 amino acids and contains a highly conserved NAM domain at N terminal was cloned by the RACE method. The NAC1 was then validated to be the target gene of miR164 via 5′-RACE, and the cleavage site was between the 10^th and 11^th base. The expression patterns of miR164 and its target gene NAC1 were further detected under nitrate-starvation condition through qRT-PCR analysis. The results showed that miR164 expression was repressed by nitrate-starvation at the beginning of the treatment (4 d) and then ascended. However, the expression pattern of miR164 in roots was different from that in shoots and leaves. Moreover, the expression levels of target gene NAC1 and miR164 were negatively correlated. The expression level of miR164 in root was increased while that of NAC1 was decreased under Re treatment, which indicated that miR164 and its target gene NAC1 play a regulatory role in response to low nitrate availability. The findings of our study may help elucidate the molecular mechanisms by which miR164 regulates target gene NAC1 at post-transcriptional level, and provide valuable information for further study of the regulatory roles of miR164-NAC1 under nitrate-starvation condition.

Key words: Betula luminifera, nitrate-starvation, miR164, target gene, expression analysis

吴骏,张俊红,黄蒙慧,朱敏慧,童再康. 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.

Jun Wu, Junhong Zhang, Menghui Huang, Minhui Zhu, Zaikang Tong. Expression analysis of miR164 and its target gene NAC1 in response to low nitrate availability in Betula luminifera[J]. HEREDITAS(Beijing), 2016, 38(2): 155-162.

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光皮桦miR164及其靶基因NAC1在低氮胁迫中的表达分析

Expression analysis of miR164 and its target gene NAC1 in response to low nitrate availability in Betula luminifera

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