A novel expression cassette for the efficient visual selection of transformed tissues in florist's chrysanthemum ( Chrysanthemum morifolium Ramat.)
A novel expression cassette for the efficient visual selection of transformed tissues in florist's chrysanthemum ( Chrysanthemum morifolium Ramat.)
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DOI：
10.4314/ajb.v10i51
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发表时间：
2011-09
影响因子：
--
通讯作者：
J. Mao;G. Stoopen;M. Jongsma;Caiyun Wang
中科院分区：
文献类型：
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作者：
J. Mao;G. Stoopen;M. Jongsma;Caiyun Wang
携带视觉报告基因的构建体与高效启动子结合，可以促进抗性作物中稳定转化体的鉴定和选择。本文报道了一种利用核酮糖-1,5-二磷酸羧化酶（rcs）启动子与绿色荧光蛋白（GFP）报告基因结合的新结构，在花菊（chrysanthemum morifolium Ramat.）中启动了绿色荧光蛋白的高表达。以该表达盒为基础，建立了以叶片为外植体的农杆菌转化菊花1581基因型的再生方案，获得了7%的转化效率。比较了两种不同的GFP结构在细胞质或质体上的表达。两种GFP构建体的表达水平都很高，在叶组织中绿色荧光占主导地位，即使没有GFP过滤器，也可以很容易地观察和显微解剖转化组织。在正常光照下，GFP靶向质体的植株由于GFP的高表达而呈现出浅绿色表型。定量逆转录PCR分析表明，内含子的质体靶向构建体的GFP mRNA稳态转录水平显著提高。这种新的表达盒可能允许在广泛的植物物种中独立于抗生素选择的转化组织的直接视觉选择。
Constructs carrying visual reporter genes coupled with efficient promoters could facilitate the process of identification and selection of stable transformants in recalcitrant crops. Here, a novel construct utilizing a ribulose-1,5-bisphosphate carboxylase (RbcS) promoter combined with the green fluorescent protein (GFP) reporter gene to initiate very high expression of GFP in florist's chrysanthemum (Chrysanthemum morifolium Ramat.) was described. Based on this expression cassette, a new regeneration protocol using leaf discs as explants was developed for the Agrobacterium-mediated transformation of Chrysanthemum genotype ‘1581’, and a transformation efficiency of 7% was obtained. The expression of two different GFP constructs targeted to either cytosol or plastids was compared in transgenic lines. Both GFP constructs were expressed at such a high level that the green fluorescence dominated red fluorescence in the leaf tissues, allowing easy observation and microdissection of transformed tissues even without a GFP filter. Under normal light, plants with GFP targeted to plastids had a light green phenotype deriving from the high GFP expression. Quantitative reverse transcriptional PCR analysis showed that the plastid targeted construct with intron had significantly higher steady state transcript levels of GFP mRNA. This novel expression cassette may allow direct visual selection of transformed tissues independent of antibiotic selection in a wide range of plant species.