Cloning And Functional Characterization Of Hyoscyamine Uptake Permeases Gene AbPUP1

Plant secondary metabolites play an important role in regulating plant responses to biological and abiotic environmental factors,and they have been widely used as a source of natural flavors and medicines.Tropine alkaloids,also known as Anisodamine alkaloids(TAs),are a class of secondary metabolites mainly extracted from Solanaceae plants,including hyoscyamine,anisodamine,and scopolamine.TAs found in Solanaceae plants are known for their anticholinergic and antispasmodic properties that affect the parasympathetic nervous system.In Solanaceae plants,TAs biosynthesis takes place in the roots,and the alkaloids are transported to leaaves for storage.But so far,there have been no reports about TAs transporter and transport forms.Therefore,elucidating the mechanism of TAs transport in plants is of great significance for understanding the formation and accumulation of such compounds in plants.PUP(purine uptake permease)classes as transporters is a small family of transmembrane transporters in nature,and plays an important role in the accumulation and transport of plant secondary metabolites.The PUP transporter gene(NUP1)in tobacco not only specifically transports nicotine,but also regulates the biosynthesis of nicotine,indicating that the PUP family transporter plays an important role in affecting the transport and synthesis of secondary metabolites.In this study,A.belladonna was used as the research material.Based on A.belladonna transcriptome analysis,PUP sequences homologous to tobacco were found from A.belladonna,the gene was determined named AbPUP1.In response to this PUP transport protein,this study used molecular biology,biochemistry and biotechnology to study the biological function of AbPUP1 specific transport of hyoscyamine,and achieved the following results:(1)AbPUP1 transporter specifically transports scopolamineBased on the analysis of the A.belladonna transcriptome data,a sequence annotated as a purine transporter gene was found and named AbPUP1.Digital tissue expression profiles show that the gene is specifically expressed in the roots,with high expression in the lateral roots and a small amount of expression in the primary roots,which is similar to the genes of the synthesis pathway of tropine alkaloids.By constructing the genes of the same family reported in Arabidopsis and tobacco,it was found that AbPUPl and NUP1 belong to homologous genes.The analysis results of membrane protein subcellular localization software TMHMM showed that AbPUPl was localized on the cell membrane.Subcellular localization experiments were carried out in tobacco,and the protoplast was extracted and observed under a laser confocal microscope,which also confirmed that the protein was localized on the cell membrane.The AbPUPl gene was heterologously expressed in a defective yeast system.After culturing in a medium supplemented with scopolamine,HPLC analysis revealed that AbPUP1 had a strong ability to transport hyoscyamine under the cuture conditions with the same concentration of hyoscyamine.Subsequently,AbPUP1 was further expressed in a defective yeast system,cultured in a medium supplemented with 0.5 mM scopolamine,and the content of hyoscymine in yeast at different time from 0 to 12 hours was detected.It was found that as the feeding time increased,The fresh weight of yeast continued to increase,but the content of hyoscymine in the yeast showed a downward trend,reaching the highest absorption peaked at 3h.When the same concentration of hyoscyamine,scopolamine,and anisodamine were added to the medium,It was found that yeast only absorbed hyoscyamine significantly,but not scopolamine and anisodamine;when the medium with a substrate concentration of 0.5 mM scopolamine was added 2.5 times the concentration of scopolamine and anisodamine,respectively After detecting the content of scopolamine in yeast,there was no significant change.This result indicates that AbPUP1 transporter is specific for scopolamine transport.(2)Interfering with the expression of AbPUPl in A.belladonna plants reduced the content of hyoscyamine in the leavesUsing the pBin 19 plasmid as a plant interference expression vector,the constructed recombinant plasmid pBin19-AbPUP1 was transferred into Agrobacterium tumefaciens EHA105 to obtain the engineering strain pBin19-AbPUP1-EYHA105,and then the engineering strain pBin19-A bPUP1-EHA105 was transferred into the leaves of wild-type A.belladonna by the leaf disc method,resistant transgenic plants were obtained by screening with kanamycin.In AbPUP1 transgenic A.belladonna plants,the expression of some genes in the tropane alkaloids synthesis pathway was significantly reduced(p<0.01),and the content of hyoscyamine in the leaves of transgenic plant was also significantly decreased(p<0.01).When interfering with AbPUP1 transgenic plants,the contents of hyoscyamine in the A.belladonna leaves of the Ri-01,Ri-05,Ri-07,and Ri10 strains were 0.16 mg/g DW(Dry Weight),0.42 mg/g DW,0.26 mg/g DW(Dry Weight),0.22mg/g DW,compared with the control group A.belladonna leaf hyoscymine content 1.71mg/gDW decreased by 91%,75%,85%,87%in the leaves of control plants,However,the contents of hyoscyamine in the roots of transgenic plants compared to the control had no significant changes.In summary,this study cloned and studied a gene AbPUP1 that specifically participates in the transport of hyoscyamine in A.belladonna,and conducted an in-depth study on its function,in order to clarify the tropane alkaloids in A.belladonna plants The research of the transport mechanism has laid a foundation,and it also provides a reference for exploring the transport of PUP transport proteins to other plant secondary metabolites.