Bachem C W, van der Hoeven R S, de Bruijn S M, Vreugdenhil D, Zabeau M, Visser R G
Department of Plant Breeding, Graduate School of Experimental Sciences, Wageningen Agricultural University, The Netherlands. Christian.Bachem/users.pv.wau.nl
Plant J. 1996 May;9(5):745-53. doi: 10.1046/j.1365-313x.1996.9050745.x.
Using a highly synchronous in vitro tuberization system, in combination with an amplified restriction fragment polymorphism (AFLP)-derived technique for RNA fingerprinting (cDNA-AFLP), transcriptional changes at and around the time point of potato tuberization have been analyzed. The targeted expression analysis of a specific transcript coding for the major potato storage protein, patatin and a second transcript, coding for ADP-glucose pyrophosphorylase, a key gene in the starch biosynthetic pathway is described. This paper confirms that kinetics of expression revealed by cDNA-AFLP analysis are comparable to those found in Northern analysis. Furthermore, this paper reports the isolation and analysis of two tuber-specific transcript-derived fragments (TDFs) coding for the lipoxygenase enzyme, which are differentially induced around the time point of tuber formation. Analysis of the two lox TDFs demonstrates that it is possible to dissect the expression modalities of individual transcripts, not independently detectable by Northern analysis. Finally, it is shown that using cDNA-AFLP, rapid and simple verification of band identity may be achieved. The results indicate that cDNA-AFLP is a broadly applicable technology for identifying developmentally regulated genes.
利用高度同步的体外块茎形成系统，结合用于RNA指纹分析（cDNA-AFLP）的扩增限制性片段多态性（AFLP）技术，对马铃薯块茎形成时间点及前后的转录变化进行了分析。描述了对编码主要马铃薯贮藏蛋白的特定转录本——马铃薯Patatin蛋白以及编码淀粉生物合成途径关键基因——ADP-葡萄糖焦磷酸化酶的第二个转录本的靶向表达分析。本文证实，cDNA-AFLP分析揭示的表达动力学与Northern分析中发现的动力学相当。此外，本文报道了编码脂氧合酶的两个块茎特异性转录本衍生片段（TDF）的分离和分析，它们在块茎形成时间点前后被差异诱导。对两个脂氧合酶TDF的分析表明，有可能剖析单个转录本的表达模式，而这在Northern分析中是无法独立检测到的。最后，结果表明，使用cDNA-AFLP可以快速、简单地验证条带的同一性。结果表明，cDNA-AFLP是一种广泛应用于鉴定发育调控基因的技术。
