Phosphomannose isomerase, pmi, as a selectable marker gene for durum wheat transformation
Phosphomannose isomerase, pmi, as a selectable marker gene for durum wheat transformation
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DOI：
10.1016/j.jcs.2005.06.004
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发表时间：
2006-01-01
影响因子：
3.8
通讯作者：
Blanco, A
中科院分区：
文献类型：
--
作者：
Gadaleta, A;Giancaspro, A;Blanco, A
转基因技术提供了一个强大的工具，开发性状，否则难以实现通过传统方法。转基因植物的开发需要使用选择标记基因，因为植物转化的效率对于许多重要物种来说不是最佳的，特别是对于单子叶植物如硬粒小麦（Tritictan turgidum var. Durum）。许多人对目前使用的标记基因在用于田间栽培的植物中的持久性表示关注。为了维持这一领域的进一步进展，需要替代的有效选择方法。最近的发展是使用选择性基因，与未转化的细胞相比，这些基因使转化的细胞具有代谢优势（阳性选择），未转化的细胞缓慢饥饿，伴随着生长和活力的降低。这种选择策略与传统的阴性选择相反，在传统的阴性选择期间，转基因细胞能够在选择性培养基上存活，而非转基因细胞被选择剂主动杀死。我们比较了“选择效率”的一个常用的负选择方法，采用吸水链霉菌酒吧基因赋予抗除草剂双丙氨膦，积极选择采用大肠杆菌磷酸甘露糖异构酶（pmi）基因作为可选择的基因和甘露糖作为选择剂。利用基因枪技术，分别用bar和pmi基因轰击硬粒小麦品种Svevo的幼胚愈伤组织。通过PCR分析和氯酚红试验分别证实了两个基因在To代中的整合和表达。选择效率，计算为表达植物的数目除以总再生体的数目，当pini用作选择标记基因时（90.1%）比当bar用作选择标记基因时（26.4%）高。因此，建立了一个有效的选择方法，硬粒小麦转化，避免了除草剂抗性基因的使用。(c)2005爱思唯尔有限公司保留所有权利。
Transgene technology provides a powerful tool for developing traits that are otherwise difficult to achieve through conventional methods. The development of transgenic plants requires the use of selectable marker genes, as the efficiency of plant transformation is less than optimal for many important species, especially for monocots such as durum wheat (Tritictan turgidum var. durum). Many concerns have been expressed about the persistence of currently used marker genes in plants used for field cultivation. To sustain further progress in this area, alternative efficient selection methods are desirable. A recent development is the use of selective genes that give transformed cells a metabolic advantage (positive selection) compared to untransformed cells, which are slowly starved with a concomitant reduction in growth and viability. This selection strategy is in contrast to traditional negative selections during which the transgenic cells are able to survive on a selective medium whereas the non-transgenic cells are actively killed by the selective agent. We compared the 'selection efficiency' of a commonly used negative selection method that employs the Streptornyces hygroscopicus bar gene to confer resistance to the herbicide bialaphos, to a positive selection employing the Escherichia coli phosphomannose isomerase (pmi) gene as the selectable gene and mannose as the selective agent. Calli derived from immature embryos of the durum wheat cultivar Svevo were bombarded separately with bar and pmi genes using a biolistic system. The integration and expression of the two genes in the To generation were confirmed by PCR analysis with specific primers for each gene and the chlorophenol red assay, respectively. The selection efficiency, calculated as the number of expressing plants divided by the number of total regenerants, was higher when pini was used as the selectable marker gene (90.1%) than when bar was used (26.4%). Thus, an efficient selection method for durum wheat transformation was established that obviates the use of herbicide resistance genes. (c) 2005 Elsevier Ltd. All rights reserved.