Xue Zhong-Tian, Holefors Anna, Welander Margareta
Department of Crop Science, The Swedish University of Agricultural Sciences, PO Box 55, S-230 53 Alnarp, Sweden.
J Plant Physiol. 2008;165(5):544-52. doi: 10.1016/j.jplph.2006.11.010. Epub 2007 May 8.
The rolA gene encoded on the Ri plasmid of Agrobacterium rhizogenes causes developmental alterations, including dwarfing characteristics in the transgenic plants. In an attempt to introduce dwarfing characteristics into apple rootstocks for breeding purposes, the rolA gene was incorporated into the apple rootstock M26 and obtained four transgenic clones. All the clones exhibited reduced growth compared to untransformed control plants but different degree of dwarfing and wrinkled leaves. In the present study, expression of the rolA gene was further investigated by analysing the structure of the rolA transcript and the levels of the rolA mRNAs from these clones. The nucleotide (nt) sequence of the rolA transcript showed two forms of the transcript: one, the unspliced form, was co-linear with the rolA sequence in the genomic DNA; the other was spliced mRNA in which an 85-base pair (bp) intron sequence in the 5' untranslated region (5'UTR) was spliced out. The position of splicing is different from that in Arabidopsis thaliana but similar to the splicing site found in tobacco. The transcription start region of the rolA gene in apple was 206bp upstream of that in Arabidopsis and 277bp upstream to Nicotiana tabacum transcription start. A hairpin-like secondary structure and an upstream open reading frame (uORF) were revealed in the rolA 5'UTR. The levels of the rolA mRNA in the apple transgenic clones were analysed by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). The results showed slight variation in the shoot tissues of the transgenic clones.
发根农杆菌Ri质粒上编码的rolA基因会引起发育改变，包括转基因植物中的矮化特征。为了将矮化特征引入苹果砧木用于育种目的，将rolA基因导入苹果砧木M26中，并获得了4个转基因克隆。与未转化的对照植株相比，所有克隆的生长均受到抑制，但表现出不同程度的矮化和叶片皱缩。在本研究中，通过分析rolA转录本的结构以及这些克隆中rolA mRNA的水平，进一步研究了rolA基因的表达。rolA转录本的核苷酸（nt）序列显示出两种转录本形式：一种是未剪接形式，与基因组DNA中的rolA序列共线性；另一种是剪接后的mRNA，其中5'非翻译区（5'UTR）中的一个85碱基对（bp）内含子序列被剪接掉。剪接位置与拟南芥中的不同，但与烟草中发现的剪接位点相似。苹果中rolA基因的转录起始区域比拟南芥中的转录起始区域上游多206bp，比烟草转录起始区域上游多277bp。在rolA 5'UTR中发现了一个发夹样二级结构和一个上游开放阅读框（uORF）。通过半定量逆转录-聚合酶链反应（RT-PCR）分析了苹果转基因克隆中rolA mRNA的水平。结果表明，转基因克隆的茎组织中存在轻微差异。
