Generation and characterization of a tomato DCL3-silencing mutant
Generation and characterization of a tomato DCL3-silencing mutant
复制标题
DOI：
10.1016/j.plantsci.2014.02.007
复制
发表时间：
2014-05-01
期刊：
影响因子：
5.2
通讯作者：
Arazi, Tzahi
中科院分区：
文献类型：
--
作者：
Kravchik, Michael;Damodharan, Subha;Arazi, Tzahi
DICER-like 3 (DCL3) 是异染色质 24 核苷酸 (nt) 小 RNA (sRNA) 和长 microRNA (ImiRNA) 生物发生的主要参与者，高等植物 DCL3 突变体已从拟南芥和水稻中鉴定出来。在这里，通过使用反式激活的人工 miRNA 生成了番茄 DCL3 (SIDCL3) 突变体并进行了表征。组成型反式激活使 SIDCL3 水平降低 64%，导致 24-nt sRNA 水平显着降低，21-和 22-nt sRNA 显着增加。后者与 S1DCL4 和 S1DCL2b 的特异性上调相关，S1DCL4 和 S1DCL2b 分别在 21-nt 和 22-nt sRNA 的生物发生中发挥作用。此外，在大多数产生sRNA的基因组位点上，观察到对照幼苗和沉默幼苗的小RNA特征之间几乎完全重叠，这表明这些位点上24-nt sRNA的减少被来自相同双链RNA底物的21和22-nt sRNA的生物发生所补偿。此外，生物信息学分析和 S1DCL3 沉默幼苗中表达的减少鉴定了四种新的番茄 lmiRNA，其中两种被发现受到发育调控。综上所述，这些结果确定了 SIDCL3 对番茄中 24-nt sRNA 和 lmiRNA 生物发生的要求，并表明 SIDCL4 和 S1DCL2b 作为 S1DCL3 的替代品。 (C) 2014 Elsevier Ireland Ltd. 保留所有权利。
DICER-like 3 (DCL3) is a major player in heterochromatic 24-nucleotide (nt) small RNA (sRNA) and long microRNA (ImiRNA) biogenesis, and higher plant DCL3 mutants have been characterized from Arabidopsis thaliana and rice. Here, a tomato DCL3 (SIDCL3) mutant was generated through the use of trans-activated artificial miRNA and characterized. Constitutive trans-activation knocked down SIDCL3 levels by 64%, resulting in dramatically decreased 24-nt sRNA levels and a significant increase in 21- and 22-nt sRNAs. The latter was correlated with specific upregulation of S1DCL4 and S1DCL2b, which function in the biogenesis of 21- and 22-nt sRNAs, respectively. Moreover, at the majority of sRNA-generating genomic loci, an almost complete overlap between small RNA signatures of control and silenced seedlings was observed, suggesting that the reductions in 24-nt sRNAs at these loci were compensated for by biogenesis of 21and 22-nt sRNAs from the same double-stranded RNA substrates. In addition, bioinformatic analysis and reduced expression in S1DCL3-silenced seedlings identified four novel tomato lmiRNAs, two of which were found to be developmentally regulated. Taken together, these results establish the requirement of SIDCL3 for the biogenesis of 24-nt sRNAs and lmiRNAs in tomato and suggest SIDCL4 and S1DCL2b as surrogates for S1DCL3. (C) 2014 Elsevier Ireland Ltd. All rights reserved.