Young J C, Krysan P J, Sussman M R
Biology Department, Western Washington University, Bellingham, Washington 98225, USA.
Plant Physiol. 2001 Feb;125(2):513-8. doi: 10.1104/pp.125.2.513.
The sequencing of the Arabidopsis plant genome is providing a fuller understanding of the number and types of plant genes. However, in most cases we do not know which genes are responsible for specific metabolic and signal transduction pathways. Analysis of gene function is also often confounded by the presence of multiple isoforms of the gene of interest. Recent advances in PCR-based reverse genetic techniques have allowed the search for plants carrying T-DNA insertions in any gene of interest. Here we report preliminary screening results from an ordered population of nearly 60,470 independently derived T-DNA lines. Degenerate PCR primers were used on large DNA pools (n = 2,025 T-DNA lines) to screen for more than one gene family member at a time. Methods are presented that facilitated the identification and isolation of isoform-specific mutants in almost all members of the Arabidopsis H(+)-proton ATPase gene family. Multiple mutant alleles were found for several isoforms.
拟南芥植物基因组测序使人们对植物基因的数量和类型有了更全面的了解。然而，在大多数情况下，我们并不清楚哪些基因负责特定的代谢和信号转导途径。对基因功能的分析也常常因感兴趣基因存在多种同工型而变得复杂。基于PCR的反向遗传学技术的最新进展使得能够在任何感兴趣的基因中寻找携带T-DNA插入的植物。在此，我们报告了来自近60470个独立衍生的T-DNA系的有序群体的初步筛选结果。使用简并PCR引物对大型DNA池（n = 2025个T-DNA系）进行筛选，以便一次筛选一个以上的基因家族成员。本文介绍了一些方法，这些方法有助于在拟南芥H(+)-质子ATP酶基因家族的几乎所有成员中鉴定和分离同工型特异性突变体。在几种同工型中发现了多个突变等位基因。
