An experimental design for transient gene expression in lotus petals
[Objective]Transient gene expression is a rapid and effective cellular and genetic technique.It is particularly valuable for swiftly assessing gene function,with wide-ranging applications in plant research.To fully understand the application of genetic engineering in the field of plant secondary metabolite research and achieve research feedback teaching,a comprehensive experiment concerning transient gene expression and its impacts on the accumulation of secondary metabolites in plants was designed.[Methods]Compared with the model plants used in transient expression assay traditionally,Nelumbo nucifera(lotus),a non-model-horticultural plant,was selected as the research object.Lotus is a perennial aquatic plant that is edible,has extremely important medicinal value and contains a large amount of secondary metabolites such as alkaloids and flavonoids,making it a very suitable material for studying the biosynthesis of secondary metabolites and their regulationin plants.In this study,transcription factors NnWRKY70 and NnMYB5 involved in lotus alkaloid biosynthesis and anthocyan in accumulation were cloned,and the full-length coding sequences of NnWRKY70 and NnMYB5 were inserted into the binary vector pMDC83 to generate pMDC83-Nn WRKY70 and pMDC83-NnMYB5 constructs.Transient overexpressions of NnWRKY70 and NnMYB5 were conducted in the lotus petals of the'Qiuxing'and'Zhigaowushang'varieties 3 days before blooming.Agrobacterium GV3101 strain carrying the pMDC83-NnWRKY70 and pMDC83-NnMYB5 vectors were infiltrated on the lower side of petals using a needleless syringe.The lotus petals were sampled 2 days after infiltration for subsequent gene expression analysis using real-time quantitative polymerase chain reaction,and the alkaloid content in the petals was quantitatively measured by high-performance liquid chromatography(HPLC).Also,the accumulation of anthocyanins in the petals was qualitatively observed.[Results]The results showed that the expression level of NnWRKY70 in the'Qiuxing'petals after transient transformation increased significantly,2.75 times higher than that of the control,indicating the successful transient expression of NnWRKY70 in the petals.The HPLC detection indicated that the overexpression of NnWRKY70 promoted a significant increase in total alkaloids in petals at 2.25 times higher than the control,suggesting that the NnWRKY70 transcription factor might play a positive role in the regulation of alkaloid biosynthesis in lotus.Similarly,the transient expression of NnMYB5 in the'Zhigaowushang'petals led to the upregulation of this gene and the anthocyanin accumulation in the white lotus petals,demonstrating the role of NnMYB5 transcription factor in the control of petal coloration.[Conclusions]This experiment achieved a breakthrough in transient gene expression on lotus petals and preliminarily verified the function of the transcription factors NnWRKY70 and NnMYB5 in regulating the biosynthesis of secondary metabolites in aquatic plants.The introduction of this experiment to the experimental teaching system for plant biology and physiology courses was successful,enabling students to better understand and master the principle of instantaneous gene expression and its application in plant research and fully stimulating students'enthusiasm for scientific research and higher-order thinking.Moreover,this experimental design is an expansion and improvement of previous experiments in plant physiology,thus effectively improving the quality and effectiveness of experimental teaching and laying the foundation for cultivating comprehensive and high-quality professionals in biology.
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