人工microRNAs对拟南芥At1g13770和At2g23470基因的特异沉默

Specific gene silencing of At1g13770 and At2g23470 by artificial microRNAs in Arabidopsis

DUF647(Domain of unknown function 647)蛋白家族是在真核生物中广泛存在的、高度保守的蛋白家族。拟南芥中该基因家族共有6个成员,迄今为止拟南芥DUF647家族中4个成员的功能尚不清楚。文章以拟南芥内源MIR319a前体为骨架,构建了敲减DUF647家族中2个基因At1g13770和At2g23470表达的人工microRNAs(Artifical microRNAs,amiRNAs)。利用WMD(Web microRNA designer)平台设计分别靶向At1g13770和At2g23470基因的amiRNAs序列,通过重叠PCR置换拟南芥MIR319a前体序列。构建融合amiRNAs前体的植物表达载体pCHF3-amiRNAs,在农杆菌介导下转化拟南芥。RT-PCR分析表明,amiRNAs能够显著抑制At1g13770和At2g23470基因的表达,获得了抑制效果明显的转基因株系。At2g23470-amiRNA转基因植株At2g23470转录水平的下调导致育性严重下降。文章为进一步研究这两个基因的功能奠定了良好的基础。

DUF647（domain of unknown function 647） protein family is found in diverse eukaryotic organisms and highly conserved in eukaryotes.It has 6 members in Arabidopsis genome.So far,the function of 4 members of Arabidopsis DUF647 family is unknown.In this report,using an endogenous Arabidopsis MIR319a precursor as the backbone,we constructed two artificial microRNAs（amiRNAs） to knock down the expression of two DUF647 family genes At1g13770 and At2g23470.Using the WMD（Web microRNA Designer） platform,we designed two amiRNAs targeting At1g13770 and At2g23470 genes,respectively.Both amiRNAs sequences were engineered into the MIR319a precursor using overlapping PCR and the amiRNAs backbones were transferred into the binary vector pCHF3.The resulting plasmids that harbor amiRNAs stem loop fragments were transformed into Arabidopsis by Agrobacterium-mediated floral diping.Upon constitutive expression of these two amiRNAs,the target genes were efficiently down-regulated in transgenic line.The decreased level of At2g23470 transcript in At2g23470-amiRNA transgenic plants resulted in severe sterility.This work will facilitate the functional analysis of At1g13770 and At2g23470 genes in Arabidopsis growth and development.