Analyses of MiRNA Functions in Maize Using a Newly Developed ZMBJ-CMV-2bN81-STTM Vector
Analyses of MiRNA Functions in Maize Using a Newly Developed ZMBJ-CMV-2bN81-STTM Vector
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使用新开发的 ZMBJ-CMV-2b(N81)-STTM 载体分析玉米中的 miRNA 功能
DOI：
10.3389/fpls.2019.01277
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发表时间：
2019-10-17
影响因子：
5.6
通讯作者：
Zhou, Tao
中科院分区：
文献类型：
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作者：
Liu, Xuedong;Liu, Sijia;Zhou, Tao
内源性microRNAs （miRNAs）在植物发育和对各种生物或非生物胁迫的反应中起着关键作用。到目前为止，已经鉴定了500多个玉米mirna。然而，由于缺乏快速可靠的工具，这些已鉴定的mirna的功能在很大程度上仍然未知。我们先前报道了一种基于黄瓜花叶病毒株ZMBJ （ZMBJ- cmv）的基因沉默载体，用于快速高效的玉米系基因功能研究，具有重要的农学意义。由于ZMBJ-CMV在玉米中引起的疾病症状非常轻微，但基因沉默性很强，因此我们决定进一步修饰该载体以抑制玉米中miRNA的表达。新开发的ZMBJ-CMV-2b(N81)-STTM载体表达一个短串联靶模拟物（STTM），其中包含两个由短间隔序列分隔的靶模拟物序列。结果表明，ZMBJ-CMV-2b(N81)-STTM可用于研究本烟和玉米幼苗中miRNA的功能。基于ZMBJ-CMV-2b(N81)- sttm的下调Nbe-miR165/166或Nbe-miR159诱导特异性和强mirna隔离表型，并增加其预测靶基因的表达。对于玉米，基于ZMBJ-CMV-2b(N81)-STTM的zma-miR167或zma-miR482的下调分别导致侧根生长减少和植株发育迟缓表型。在这两种情况下，zma-miR167-或zma-miR482的靶基因都显著增加。因此，我们认为基于ZMBJ-CMV的VbMS系统是高通量研究玉米miRNA功能的有用工具。
Endogenous microRNAs (miRNAs) play pivotal roles in plant development and responses to various biotic or abiotic stresses. Up to now, more than 500 maize miRNAs have been identified. However, functions of these identified miRNAs remained largely unknown due mainly to the lack of rapid and reliable tools. We previously reported a cucumber mosaic virus strain ZMBJ (ZMBJ-CMV)-based gene silencing vector for rapid and efficient gene function studies in maize lines with agronomical importance. Because ZMBJ-CMV induces very mild disease symptoms but strong gene silencing in maize, we decided to further modify this vector to suppress miRNA expressions in maize. The newly developed ZMBJ-CMV-2b(N81)-STTM vector expresses a short tandem target mimic (STTM) containing two target-mimic sequences separated by a short spacer sequence. Our results showed that ZMBJ-CMV-2b(N81)-STTM can be used to investigate miRNA function in Nicotiana benthamiana and maize seedlings. The ZMBJ-CMV-2b(N81)-STTM-based downregulation of Nbe-miR165/166 or Nbe-miR159 induced specific and strong miRNA-sequestering phenotypes, and increased the expressions of their predicted target genes. For maize, the ZMBJ-CMV-2b(N81)-STTM based downregulation of zma-miR167 or zma-miR482 caused a decrease of lateral roots growth and a plant stunting phenotypes, respectively. In both cases, the target genes of zma-miR167- or zma-miR482 were increased significantly. Thus, we consider ZMBJ-CMV based VbMS system as a useful tool for high-throughput investigations of miRNA functions in maize.