苹果MdDRB1基因在番茄中异位表达与功能研究

Ectopic expression and functional research of Apple MdDRB1 gene in tomato

【目的】将苹果Md DRB1基因转入番茄中,探究苹果MdDRB1基因在番茄中的异位表达与功能。【方法】采用农杆菌介导法,将苹果基因Md DRB1转入番茄植物体内,用PCR检测到该基因已整合到番茄总DNA中。对获得的5个转基因番茄株系进行半定量RT-PCR分析,挑选MdDRB1基因表达水平由低到高的3个株系,用实时荧光定量PCR检测与生长素有关的mi RNAs及相应靶基因的相对表达量。并对非转基因和转基因番茄进行3μmol·L-1IAA和5μmol·L-1IAA处理,对植株进行向光性检测。【结果】转基因株系中miR164、miR160、mi R393表达量下调,mi R167表达量上调,同时,它们分别对应的番茄中的靶基因SlNAC1、SlARF10和SlARF16、SlTIR1的相对表达量上调,SlARF8相对表达量下调。IAA处理后,转基因株系幼苗的侧根数目比未转基因植株增多,同时,转基因株系幼苗的向光性也增强。【结论】苹果Md DRB1基因在番茄中的异位表达,通过抑制生长素响应相关的mi R164、miR160、miR393和增加miR167的积累水平,部分解除其对下游靶基因SlNAC1、SlARF10和Sl ARF16、SlTIR1以及增加Sl ARF8的转录后基因沉默作用,进而提高了转基因番茄对生长素响应的敏感性。

【Objective】To explore ectopic expression and function of the Md DRB1 gene in tomato,Apple Md DRB1 was transformed into tomato plants. 【Methods】Apple Md DRB1 gene was genetically trans-formed into tomato with an Agrobacterium-mediated method. PCR analysis verified that Md DRB1 genewas integrated into the genome of tomato. Three among five transgenic lines with different levels of MdDRB1 transcripts were chosen for RT-PCR analysis concerning the expression levels of auxin-related mi-cro RNAs and their target genes. Meanwhile,the seedlings were treated with IAA and detected for the pho-totropism;【Results】The results indicated that the accumulation levels of 4 auxin-related,i.e. mi R164,mi R160,mi R393 reduced and mi R167 increased with the ectopic expression of Md DRB1 in tomato trans-genic seedlings,while the transcripts of their target genes such as Sl NAC1,Sl ARF10,Sl ARF16,Sl TIR1 increased and Sl ARF8 reduced. As a result,transgenic seedlings generated more lateral roots in responseto IAA treatment than the WT control. Meanwhile,the phototropism of the transgenic seedlings was posi-tively correlated with the expression of Md DRB1 gene in transgenic tomatoes;【Conclusion】Our data indi-cated that the ectopic expression of Md DRB1 gene in tomato repressed the accumulation levels of auxin-related mi RNAs including mi R164,mi R160,mi R393 and increased the expression of mi R167,andtherefore partially relieved or enhanced the post-transcriptional gene silencing of their target genes,finally to change the sensitivity to auxin signal.