Development of enhancer trap lines for functional analysis of the rice genome
Development of enhancer trap lines for functional analysis of the rice genome
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DOI：
10.1046/j.1365-313x.2003.01808.x
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发表时间：
2003-08-01
期刊：
影响因子：
7.2
通讯作者：
Zhang, QF
中科院分区：
文献类型：
--
作者：
Wu, CY;Li, XJ;Zhang, QF
增强子捕获为鉴定新基因和调控元件提供了强有力的策略。在本研究中，我们采用了一个增强子陷阱系统，由GAL 4/VP 16-UAS元件与GUS作为报告基因组成，以产生一个捕获群体的水稻。目前，利用农杆菌介导的T-DNA插入，从两个品种中获得了31443个独立转化子。PCR检测和DNA印迹杂交表明，约94%的转化子含有T-DNA插入片段。转化体平均携带两个拷贝的T-DNA，并且42%的转化体具有单拷贝插入。对约1000株T-0植物的组织化学分析揭示了报告基因表达的各种模式，包括仅在一个组织中表达，以及同时在两个或多个组织中表达。报告基因在T-1家系中的表达模式与T-0植株的表达模式一致，并且在大多数检测的T-1家系中以3：1的孟德尔比率分离。增强子诱捕系中报告基因的表达频率远高于以往报道的基因诱捕系。对近200个转化子的T-DNA插入位点侧翼序列分析表明，几乎所有的T-DNA插入位点侧翼序列与水稻基因组数据库中的序列具有同源性。在田间试验的2679个T-1家系中，约7.5%的家系观察到形态学上明显的突变，超过三分之一的家系分离符合3：1的比例。GAL 4/VP 16-UAS元件为水稻增强子捕获提供了一个有用的系统。
Enhancer trapping has provided a powerful strategy for identifying novel genes and regulatory elements. In this study, we adopted an enhancer trap system, consisting of the GAL4/VP16-UAS elements with GUS as the reporter, to generate a trapping population of rice. Currently, 31 443 independent transformants were obtained from two cultivars using Agrobacterium-mediated T-DNA insertion. PCR tests and DNA blot hybridization showed that about 94% of the transformants contained T-DNA insertions. The transformants carried, on average, two copies of the T-DNA, and 42% of the transformants had single-copy insertions. Histochemical assays of approximately 1000 T-0 plants revealed various patterns of the reporter gene expression, including expression in only one tissue, and simultaneously in two or more tissues. The expression pattern of the reporter gene in T-1 families corresponded well with the T-0 plants and segregated in a 3 : 1 Mendelian ratio in majority of the T-1 families tested. The frequency of reporter gene expression in the enhancer trap lines was much higher than that in gene trap lines reported previously. Analysis of flanking sequences of T-DNA insertion sites from about 200 transformants showed that almost all the sequences had homology with the sequences in the rice genome databases. Morphologically conspicuous mutations were observed in about 7.5% of the 2679 T-1 families that were field-tested, and segregation in more than one-third of the families fit the 3 : 1 ratio. It was concluded that GAL4/VP16-UAS elements provided a useful system for enhancer trap in rice.