摘要
【目的】报告基因是编码易被检测的蛋白质或酶的基因。红色荧光蛋白基因DsRED因其优异的适用特性已作为报告基因广泛应用于动植物以及酵母等真核细胞内基因表达的研究。课题组前期已将DsRED成功转入核桃体细胞胚,阳性体胚生长及增殖正常、阳性再生植株长势良好。本研究通过对核桃体细胞胚和再生植株中DsRED基因的跟踪研究,探讨DsRED作为报告基因在核桃再生植株中的表达是否稳定以及对再生植株的后续生长和发育是否具有影响,同时为进一步拓宽DsRED作为报告基因的应用范围提供参考。【方法】通过荧光显微镜检测核桃体细胞胚、继代培养3年的组培苗以及3年生温室苗的DsRED荧光表达的稳定性,并通过PCR、qRT-PCR以及Western Blot技术检测DsRED基因及其编码蛋白在核桃体细胞胚和组培苗中表达的稳定性。同时,通过对DsRED组培苗及3年生苗的形态指标进行测定,从而明确DsRED作为外源报告基因的可靠性。【结果】核桃DsRED体细胞胚在白光下呈粉红至红色,荧光下呈明亮的红色;与对照相似,DsRED体细胞胚发育也经历了球形胚、心形胚、鱼雷胚和子叶胚阶段,且形态正常。DsRED组培苗和3年生苗在荧光下植株表面呈现明亮的红色,而对照无激发,视野中呈现黑色,且白光下二者表型一致。DsRED组培苗和3年生苗的茎、叶生长参数与对照相比无显著差异。显微结构荧光检测发现,DsRED在组培苗和3年生苗营养器官的维管束部位表达量较高。对核桃DsRED体胚和组培苗进行PCR检测发现DsRED体胚和组培苗均可扩增获得目的条带,与预期条带大小一致(681 bp);qRT-PCR检测结果显示,DsRED组培苗各组织及DsRED体胚的DsRED mRNA表达量无显著性差异,而对照体胚和组培苗中表达量为0。Western Blot检测结果表明,DsRED组培苗和体胚有26 kDa强阳性条带,对照植株中无特异性条带,进一步分析条带灰度,结果表明DsRED组培苗各组织间及DsRED体胚的DsRED表达量无显著性差异,而对照相应结构中表达量均为0。【结论】DsRED基因转入核桃体胚后,可在体胚和继代培养3年的组培苗中正常翻译和稳定表达,3年生温室苗DsRED表达稳定且植株表型正常,表明DsRED是核桃理想的遗传转化报告基因。研究结果可为DsRED作为报告基因在果树中的应用提供参考。
【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.
作者
任飞
张佳琦
胡恒康
梁璧
黄有军
娄和强
张启香
Ren Fei;Zhang Jiaqi;Hu Hengkang;Liang Bi;Huang Youjun;Lou Heqiang;Zhang Qixiang(State Key Laboratory of Subtropical Silviculture School of Forestry&Biotechnology,Zhejiang A&F University,Hangzhou 311300)
出处
《林业科学》
EI
CAS
CSCD
北大核心
2020年第12期166-176,共11页
Scientia Silvae Sinicae
基金
国家自然科学基金项目(31670682,31800563,31600547,31971672)
浙江省自然科学基金项目(LY18C150002)
浙江省农业(果品)新品种选育重大科技专项(2016C02052-13)。
关键词
核桃
报告基因
红色荧光蛋白
DSRED
体细胞胚
再生植株
Juglans regia
reporter gene
red fluorescent protein
DsRED
somatic embryo
regenerated plant
