Key Laboratory of Horticultural Plant Biology (Ministry of Education), National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan, China.
Protoplasma. 2011 Jul;248(3):531-40. doi: 10.1007/s00709-010-0200-2. Epub 2010 Aug 24.
Polyploidization is an important speciation mechanism for all eukaryotes, and it has profound impacts on biodiversity dynamics and ecosystem functioning. Green fluorescent protein (GFP) has been used as an effective marker to visually screen somatic hybrids at an early stage in protoplast fusion. We have previously reported that the intensity of GFP fluorescence of regenerated embryoids was also an early indicator of ploidy level. However, little is known concerning the effects of ploidy increase on the GFP expression in citrus somatic hybrids at the plant level. Herein, allotetraploid and diploid cybrid plants with enhanced GFP (EGFP) expression were regenerated from the fusion of embryogenic callus protoplasts from 'Murcott' tangor (Citrus reticulata Blanco × Citrus sinensis (L.) Osbeck) and mesophyll protoplasts from transgenic 'Valencia' orange (C. sinensis (L.) Osbeck) expressing the EGFP gene, via electrofusion. Subsequent simple sequence repeat (SSR), chloroplast simple sequence repeat and cleaved amplified polymorphic sequence analysis revealed that the two regenerated tetraploid plants were true allotetraploid somatic hybrids possessing nuclear genomic DNA of both parents and cytoplasmic DNA from the callus parent, while the five regenerated diploid plants were cybrids containing nuclear DNA of the leaf parent and with complex segregation of cytoplasmic DNA. Furthermore, EGFP expression was compared in cells and protoplasts from mature leaves of these diploid cybrids and allotetraploid somatic hybrids. Results showed that the intensity of GFP fluorescence per cell or protoplast in diploid was generally brighter than in allotetraploid. Moreover, same hybridization signal was detected on allotetraploid and diploid plants by Southern blot analysis. By real-time RT-PCR and Western blot analysis, GFP expression level of the diploid cybrid was revealed significantly higher than that of the allotetraploid somatic hybrid. These results suggest that ploidy level conversion can affect transgene expression and citrus diploid cybrid and allotetraploid somatic hybrid represents another example of gene regulation coupled to ploidy.
多倍化是所有真核生物的一个重要物种形成机制，它对生物多样性动态和生态系统功能有深远的影响。绿色荧光蛋白（GFP）已被用作原生质体融合早期体细胞杂种的有效标记物，进行肉眼筛选。我们之前曾报道过，再生胚状体的 GFP 荧光强度也是倍性水平的早期指标。然而，对于柑橘体细胞杂种中倍性增加对 GFP 表达的影响，人们知之甚少。在此，通过电融合，从‘Murcott’橘柚（Citrus reticulata Blanco ×Citrus sinensis (L.) Osbeck）胚性愈伤组织原生质体和转 EGFP 基因‘ Valencia’甜橙（C. sinensis (L.) Osbeck）叶肉原生质体融合产生的杂种中，再生了增强 GFP（EGFP）表达的同源四倍体和二倍体杂种植株。随后的简单重复序列（SSR）、叶绿体简单重复序列和切割扩增多态性序列分析表明，再生的两个四倍体植株是真正的异源四倍体体细胞杂种，具有双亲的核基因组 DNA 和愈伤组织亲本的细胞质 DNA，而再生的五个二倍体杂种是含有叶亲本核 DNA 并具有细胞质 DNA 复杂分离的杂种。此外，比较了这些二倍体杂种和异源四倍体体细胞杂种成熟叶片细胞和原生质体中的 EGFP 表达。结果表明，二倍体中每个细胞或原生质体的 GFP 荧光强度普遍比四倍体强。此外，通过 Southern blot 分析，在异源四倍体和二倍体植株上均检测到相同的杂交信号。通过实时 RT-PCR 和 Western blot 分析，揭示了二倍体杂种的 GFP 表达水平明显高于异源四倍体体细胞杂种。这些结果表明，倍性水平的转换会影响转基因的表达，并且柑橘二倍体杂种和异源四倍体体细胞杂种是基因调控与倍性相关的另一个例子。
