关键词: 核桃, 报告基因, 红色荧光蛋白, DsRED, 体细胞胚, 再生植株

Abstract:

Objective: Reporter gene is a gene encoding protein or enzyme which can be detected easily. DsRED, a fluorescent reporter gene, has been widely expressed in eukaryotic cells including animals, plants and yeast. At present, DsRED gene has been transformed into walnut(Juglans regia) somatic embryos as a reporter gene successfully. The growth and proliferation of transformed somatic embryos, tissue-cultured plantlets and 3-year-old green-house regenerated plants are growing well. The research detected the expression of DsRED gene in somatic embryos and regenerated plants of walnut to explore whether the expression of DsRED as a reporter gene was stable in the regenerated walnut plants and whether it had influence on the subsequent growth and development of regenerated plants, and to provide evidence for further broadening the application scope of DsRED as a reporter gene. Method: Detection of fluorescence expression in somatic embryos, tissue-cultured plantlets and 3-year-old plants of walnut by fluorescence microscopy. The expression of DsRED gene and protein in walnut somatic embryos and tissue-cultured plantlets were detected by PCR, qRT-PCR and Western Blot. Moreover, to confirm the reliability and stability of DsRED expression, the morphological indices of tissue-cultured plantlets and 3-year-old plants were measured as well. Result: The appearance of DsRED somatic embryos were pink-to-red under white light while they showed bright red under fluorescent light. Compared to the control, transformed DsRED somatic embryos exhibited globular, heart-shaped, torpedo and cotyledonary embryo development stages with normal morphology. The appearance of DsRED tissue-cultured plantlets and 3-year-old plants showed bright red and control showed totally dark under fluorescent field while they are similar under the bright field. The difference of growth parameters of stems and leaves between DsRED and control were not significant. The microscopy detection showed that DsRED gene was expressed highly in the vascular bundles of vegetative organs in DsRED tissue-cultured plantlets and 3-year-old plants. DsRED gene expression of somatic embryos and tissue-cultured plantlets were identified by PCR, qRT-PCR and Western Blot. The results showed that the expected band size (681 bp) was obtained from DsRED somatic embryos and tissue-cultured plantlets. There was no significant difference in the expression of DsRED mRNA between DsRED somatic embryos and tissue-cultured plantlets, while the DsRED mRNA expression level in control was zero. Western Blot analysis showed that there were strong positive bands at 26 kDa in DsRED somatic embryos and tissue-cultured plantlets, while no specific bands in the control. The analysis on the gray scale of the Western Blot bands indicated that the relative expressions had no significant differences between the DsRED somatic embryos and tissue-cultured plantlets, while the expression of the control was zero. Conclusion: DsRED gene can be expressed and translated stably in walnut somatic embryos, tissue-cultured plantlets and 3-year-old plants. The DsRED is an ideal genetic transformation report gene of walnut. This study provides a basis for the application of DsRED as a reporter gene in fruit crops.

Key words: Juglans regia, reporter gene, red fluorescent protein, DsRED, somatic embryo, regenerated plant