An effective artificial microRNA vector based on Fv-miR166 precursor from strawberry
An effective artificial microRNA vector based on Fv-miR166 precursor from strawberry
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基于草莓 Fv-miR166 前体的有效人工 microRNA 载体
DOI：
10.1016/j.scienta.2019.108643
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发表时间：
2019-10-15
影响因子：
4.3
通讯作者：
Zhang, Zhihong
中科院分区：
文献类型：
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作者：
Li, He;Dong, Xiangxiang;Zhang, Zhihong
人工microRNAs是单链的，大约21nT长，通过取代前miRNAs中双链的成熟miRNA序列而设计。它们通过小RNA生物发生和沉默机制进行处理，并解除对靶基因表达的调控。AmiRNA成为研究基因功能的重要策略，主要集中在敲除植物中的内源mRNAs，少数用于内源miRNA的功能。此外，选择合适的miRNA前体骨架来表达amRNA是至关重要的基础。已发表的论文显示了有效的miRNA载体，大多数是关于拟南芥和水稻的天然miRNA前体。在本研究中，我们首先根据Fragaria Vesca的miR166前体，通过八个人工合成的寡核苷酸的退火法构建了一个简单的amiRNA载体，其中成熟的miR166序列分别被Amir-Gus、Amir-GFP、Amir-NE和amiR390替换。接下来，我们将Amir-GUS、Amir-GFP、Amir-NE和amiR390克隆到含有CaMV 35S启动子的植物表达载体PRI 101-An中，得到了Pamir-GUS、Pamir-GFP、Pamir-NE和PAMIR390载体。草莓果实的瞬时转化表明，在Fv-miR166前体的基础上产生了Amir-GUS和Amir-GFP，导致GUS和GFP的表达水平明显降低。QRT-PCR结果表明，Amir-NE也可以从Fv-miR166主干上加工成成熟的amiRNA。AmiR390能有效靶向内源TAS3基因，促进烟草ta-siRNA3的生物合成。此外，在过量表达amiR390的烟草植株中，发现了明显的表型变化，平均叶长与叶宽的比率变小，幼叶到成虫的相变表型推迟了约10天。结果表明，我们已经开发出一个有效的基于F.Vesca的miRNA前体的amRNA骨架。这是关于草莓amiRNA骨架的第一篇论文。
Artificial microRNAs (amiRNAs) are single-stranded, approximately 21 nt long, and designed by replacing the mature miRNA sequences of duplex within pre-miRNAs. They are processed via small RNA biogenesis and silencing machinery and deregulate target expression. AmiRNA became an important strategy to study gene function, mostly focused on knocking down the endogenous mRNAs in plants, a few used in function of endogenous miRNA. Furthermore, choosing a suitable miRNA precursor backbone used to express amiRNA is vital foundation. The published papers exhibited effective amiRNA vectors most on the natural miRNA precursors of Arabidopsis and rice. In this study, we firstly generated a simple amiRNA vector based on miR166 precursor of Fragaria vesca by annealing of eight synthetic oligonucleotides, in which mature miR166 sequence were replaced by amiR-GUS, amiR-GFP, amiR-NE and amiR390, respectively. Next, we cloned the amiR-GUS, amiR-GFP, amiR-NE and amiR390 into the plant expression vector pRI 101-AN with a CaMV 35S promoter, producing the pAMIR-GUS, pAMIR-GFP, pAMIR-NE and pAMIR390 vectors. The transient transformation of strawberry fruit showed that amiR-GUS and amiR-GFP were produced based on Fv-miR166 precursor, resulting in the obvious low expression levels of GUS and GFP. The qRT-PCR results suggested that amiR-NE could also be processed into mature amiRNA from the Fv-miR166 backbone. amiR390 was effectively targeting the endogenous TAS3 gene for increasing ta-siRNA3 biosynthesis in tobacco. Additionally, in over-expression tobacco plants of amiR390, the obvious phenotype changes were found that the ratio of average leaves length divided by width became smaller and the juvenile-to-adult phase transition phenotypes were delayed about ten days. The results showed that we have developed an effectively amiRNA backbone based on miRNA precursor of F. vesca. This is the first paper about strawberry amiRNA backbone.