中国农业科学 ›› 2021, Vol. 54 ›› Issue (22): 4709-4727.doi: 10.3864/j.issn.0578-1752.2021.22.001

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

小麦TaNAC基因基于可变剪切和microRNA的转录后调控分析

吕士凯(),马小龙,张敏,邓平川,陈春环,张宏(),刘新伦(),吉万全()   

  1. 西北农林科技大学农学院/旱区作物逆境生物学国家重点实验室,陕西杨凌 712100
  • 收稿日期:2021-02-10 接受日期:2021-04-07 出版日期:2021-11-16 发布日期:2021-11-19
  • 通讯作者: 张宏,刘新伦,吉万全
  • 作者简介:吕士凯,E-mail: lvshikaiyd@163.com
  • 基金资助:
    陕西省重点研发计划(2019DLNY04-06)

Post-transcriptional Regulation of TaNAC Genes by Alternative Splicing and MicroRNA in Common Wheat (Triticum aestivum L.)

LÜ ShiKai(),MA XiaoLong,ZHANG Min,DENG PingChuan,CHEN ChunHuan,ZHANG Hong(),LIU XinLun(),JI WanQuan()   

  1. College of Agronomy, Northwest A&F University/State Key Laboratory of Crop Stress Biology for Arid Areas, Yangling 712100, Shaanxi
  • Received:2021-02-10 Accepted:2021-04-07 Online:2021-11-16 Published:2021-11-19
  • Contact: Hong ZHANG,XinLun LIU,WanQuan JI

摘要:

【目的】以条锈菌和白粉菌胁迫的普通小麦(Triticum aestivum L.)为研究对象,分析由可变剪切(alternative splicing,AS)形成的TaNAC结构变异转录本,同时分析TaNAC基因的microRNA调控位点,为进一步解析TaNAC基因通过转录后调控参与小麦响应真菌胁迫奠定基础。【方法】普通小麦兼抗种质N9134在被白粉菌和条锈菌分别侵染后,各8个时间点取样并混合,然后从混合样本中克隆得到大量TaNAC转录本。参考中国春小麦基因组注释信息(IWGSC RefSeq v1.1)进行比对,选择由可变剪切形成的TaNAC序列结构变异转录本,分析它们的序列结构特征。利用生物信息学软件和在线工具,对这些TaNAC结构变异转录本编码产物的功能结构域、高级结构、理化性质、亚细胞定位等特征和变异情况进行比对分析。同时,利用洋葱表皮细胞瞬时表达系统验证其中1对TaNAC结构变异转录本的亚细胞定位预测结果,并选取5组TaNAC基因的可变剪切序列结构变异转录本进行酵母转录自激活试验,研究序列结构变异对TaNAC基因转录调控活性的影响。此外,利用miRBase数据库收录的小麦中已报道的miRNAs和TaNAC基因,进行靶基因预测分析,建立小麦TaNAC家族成员与miRNAs的靶向关系。【结果】以条锈菌和白粉菌侵染的普通小麦兼抗种质N9134为材料,克隆得到的TaNAC转录本中的35条序列结构变异转录本由13个TaNAC基因可变剪切形成。通过分析发现,同一TaNAC基因由可变剪切形成的不同结构变异转录本的核酸序列结构存在差异,而且其对应编码产物的功能结构域、高级结构、理化性质和亚细胞定位等方面均会存在差异,同时也会表现出不同的转录调控活性;不同TaNAC基因的可变剪切方式存在差异,而且它们的结构变异转录本及其编码产物在结构特征、理化性质和转录调控活性等方面也均呈现出多样性的特征。通过分析TaNAC基因与其在编码序列区域的靶标tae-miRNAs,发现具有可变剪切转录本的TaNAC基因与tae-miRNA的结合位点均在其非可变剪切区域。【结论】TaNAC基因可以通过可变剪切这种转录后调控方式参与小麦对真菌胁迫的响应;同时发现,调控TaNAC基因的tae-miRNA可以独立于可变剪切这种转录后调控方式行使功能。

关键词: 小麦, TaNAC转录因子, microRNA, 可变剪切, 转录后调控, 胁迫响应

Abstract:

【Objective】 In the present study, the common wheat (Triticum aestivum L.) was exposed to the stress of stripe rust and powdery mildew. Then the cloned TaNAC structural variation transcripts formed by alternative splicing were analyzed. And putative information of TaNAC genes regulated by miRNAs was annotated. It would shed light on the study of TaNAC genes in response to fungal stress of wheat at the post-transcriptional level. 【Method】 After common wheat resistance germplasm N9134 being infected stripe rust and powdery mildew respectively, the leaves were sampled at eight time points. Then a large number of TaNAC transcripts were cloned from the mixed sample pool. Referring to the wheat genome annotation of Chinese Spring (IWGSC RefSeqv1.1), the sequence structure characteristics of TaNAC structural variation transcripts formed by alternative cutting were revealed. Using bioinformatics software and online tools, the coding products derived from these TaNAC transcripts were compared and analyzed, including the functional domain, advanced structure, physical and chemical properties, subcellular localization and other characteristics. And then, one pair of TaNAC structural variation transcripts were selected to further verify the predicted subcellular localizations by onion epidermis transient expression system. Meanwhile, five groups of TaNAC transcripts were conducted transcriptional self-activation experiments in yeast. It was aimed to analyze the effects of structural variation, which caused by alternative splicing on transcriptional regulation activity. Additionally, using the miRBase database, the targeting relationship between TaNAC genes and tae-miRNAs was forecasted and established in wheat. 【Result】 In this study, 35 TaNAC structural variation transcripts were formed by alternative splicing form 13 TaNAC genes, and they all were cloned from common wheat N9134 after infecting by stripe rust and powdery mildew. After analyzing, it was found that there were differences in the nucleic acid sequence structure of different structural variation transcripts from the same TaNAC gene, as well as in the functional domain, advanced structure, physicochemical properties and subcellular localization of their corresponding coding products. And they might be with different transcriptional regulatory activities. Moreover, different TaNAC genes could be with different patterns of alternative splicing, and the coded products of the structural variation transcripts from different TaNAC genes showed diversity in characteristics of structure, physical and chemical properties, transcriptional regulatory activity and so on. By analysis of TaNAC genes and their target tae-miRNAs, which is in the coding region, the result showed that the binding sites of tae-miRNAs were all in the non-alternative splicing region. 【Conclusion】 In conclusion, TaNAC genes might be involved in the response of wheat to fungal stress through the post-transcriptional regulation of alternative splicing. And the tae-miRNAs targeted to TaNAC genes could function post-transcriptional regulation independently of alternative splicing.

Key words: wheat, TaNAC transcription factor, microRNA, alternative splicing, post-transcriptional regulation, stress responses