Overexpression Of The AbRac Mutants Promotes The Hyoscyamine Production In Atropa Belladonna

Atropa belladonnais a perennial herb of the Atropa of the family Solanaceae,It is the commerical medicinal plants included by Chinese Pharmacopoeia with hyoscyamine as the main alkaloid.Hyoscymine is specifically synthesized in the root of Atropa belladonna and can be transported to other organs for storage.Hyoscyamine have anticholinergic activity and are used clinically for the treatment of motion sickness,preoperative administration,the treatment of pesticide poisoning and drug addiction.There is a great demand for tropane alkaloids in the pharmaceutical market,Although the hyoscyamine can be synthesized by chemical methods,these methods are too costly to commercialize.Currently,the production of hyoscyamine relies entirely on extraction from natural plants,Plant genetic engineering technology has been proved to be one of the most effective means for cultivating high-value secondary metabolites and high-yield plants.This study is about the small G protein?Rac GTPase,Rac?mutants promotes the content of hyoscyamine in Atropa belladonna.Rac is a type of monomeric protein with GTPase activity,the amino acid sequence of this protein is highly conserved in animals and plants.It not only plays an important role in plant growth and development and other physiological processes,but also regulates the biosynthesis of secondary metabolites such as plant alkaloids and terpenoids.It has been reported that the mutants of G to V or Q to L at the GTPase domain can reduce the activity of GTPase and reduce the hydrolysis of GTP.a gene?AbRac?was cloned from Atropa belladonna and its expression pattern was analyzed.AbRac and its mutants(AbRac^G15V and AbRac^Q64L)were overexpressed in Atropa belladonna and their effects on TAs biosynthesis were also studied.This study obtained the following experimental results:1.Cloning and analysis the sequence of AbRacComparing the well-characterized Scoparia dulcis Rac GTPase in Atropa belladonna transcriptome database to find a small G protein,which is named AbRac.The CDS region was cloned and contained 591 nucleotides,encoding a polypeptide of197 amino acids.AbRac has a very high similarity to the already reported Rac in amino acid sequence.Through bioinformatics analysis,the active site in the active region of GTPase was found and site-directed mutagenesis was performed.The 15th glycine?G?was mutated to valine?V?and the 64th glutamine?Q?was mutated to leucine?L?to yielded mutant genes AbRac^G15V and AbRac^Q64L with lower GTPase activity were obtained.The relative expression levels of AbRac in the main roots,lateral roots,stems,leaves and flowers of Atropa belladonna were detected by real-time fluorescent quantitative PCR.It was found that AbRac expressed the highest amount in the main root,followed by expression in the lateral roots and leaves.Minimum expression in flowers and stems.AbRac has a relative expression in various organizations.2.Breeding and related detection in the transgenic Atropa belladonna of T0generation by overexpressing AbRac mutantThe p2301⁺plasmid was used as a plant expression vector.Recombinant plasmids p2301⁺-AbRac,p2301⁺-AbRac^G15V,and p2301⁺-AbRac^Q64L were constructed by double restriction enzyme digestion.These three recombinant plasmids and empty vector were transferred to Agrobacterium tumefaciens EHA105 by freeze-thawing method,respectively.Bacteria EHA105-p2301⁺-AbRac,EHA105-p2301⁺-AbRac^G15V,EHA105-p2301⁺-AbRac^Q64L,EHA105-p2301⁺.The obtained engineering bacteria were transferred into the wild type Atropa bellamonia plants by the leaf disc method,and plants with kanamycin resistance were obtained.Preliminary overexpression of vector specific primers,resistance genes and Agrobacterium tumefaciens EHA105detection was used to obtain effective transgenic plants.3 transgenic plants by overexpression AbRac,3 transgenic plants by overexpression AbRac^G15V,2 transgenic plants by overexpression AbRac^Q64L.3 p2301⁺independently transformed Atropa belladonna plants were obtained as empty vector control and 11 wild type Atropa belladonna were quickly propagated as non-transgenic controls.PCR results showed that the 795 bp kanamycin resistance gene?NPTII?fragment was detected in the transgenic material,but the fragment was not detected in wild type belladonna;the AbRac and its mutant materials were transfected.The 677 bp fusion fragment of 35S::AbRac^G15V/AbRac^Q64L was detected;and the results of qPCR showed that the expression of AbRac/AbRac^G15V/AbRac^Q64L was greatly increased in the AbRac and its mutant materials.The material of the transgenic AbRac/AbRac^G15V/AbRac^Q64L was subjected to rapid propagation,and each of the independently transformed plants had not less than3 rapid propagation materials.After the transplanted seedlings were transplanted,the hyoscyamine in the roots and leaves was separately extracted and detected by HPLC.The results showed that the content of hyoscyamine in the roots of wild-type,empty plasmid control and AbRac?three different types of control,respectively?was at the same level,and there was no significant difference;The content of hyoscyamine in the roots of transgenic plants by overexpressing AbRac^G15V/AbRac^Q64L64L was significantly higher than that of the roots of the three control plants.The content of hyoscyamine in the leaves of the three control plants was also at the same level,the content of scopolamine in the leaves of transgenic plants by overexpressing AbRac^G15V/AbRac^Q64L was significantly higher than that of the control.The expression of PMT and TRI in these materials involved in hyoscyamine biosynthesis was analyzed by qPCR.The results showed that the expression level of TRI gene was greatly increased in the materials by overexpressing AbRac^G15V/AbRac^Q64L,but the expression level of PMT,CYP80F1 gene was not increased.The above results indicated that there was no significant change in the content of hyoscyamine in the empty plasmid control and overexpressing AbRac compared with the wild type plants;and the overexpression of AbRac^G15V or AbRac^Q64L64L could increase the content of hyoscyamine in the Atropa belladonna.3.Breeding and related detection in the transgenic Atropa belladonna of T1generation by overexpressing AbRac mutantIn order to further study whether the increase of hyoscyamine content in transgenic materials has similar performance in sexual reproductive progeny,three overexpressing AbRac^G15V strains and two overexpressing AbRac^Q64L strains were bagged and selfed,and T1 generation was obtained.seed.After germination and seeding,the gene expression and the content of hyoscyamine were detected.Since the T0 generation results showed no significant difference in the content of hyoscyamine in the three controls,in the T1 generation test,the wild type belladonna seedlings were used as control.Molecular detection results showed that 795 bp NPTII fragment and 677 bp 35S::AbRac^G15V/AbRac^Q64L fragment were detected in overexpressing AbRac^G15V/AbRac^Q64L64L Atropa belladonna of T1 generation seedlings,but not in wild type plants.These gene fragments were detected;qPCR results showed that the expression level of AbRac^G15V/AbRac^Q64L Atropa belladonna of T1 generation seedling roots was much higher than that of wild type;meanwhile,the expression level of TRI in transgenic belladonna roots was also significantly improved.The content of hyoscyamine in Atropa belladonna roots and leaves was also analyzed by HPLC.The results showed that the content of hyoscyamine in roots and leaves of AbRac^G15V/AbRac^Q64L Atropa belladonna of T1 was significantly higher than that of wild type belladonna.The results of T1 generation transgenic belladonna materials showed that the gene expression and purine content of the transgenic AbRac mutant material were consistent with the results of the T0 generation,indicating that the phenotype brought by the transgene was genetically stable.In summary,This study cloned a small GTPase gene in Atropa belladonna and made two site directed mutations successfully,The gene was expressed in various organs of Atropa belladonna,It was found that overexpression of its mutants AbRac^G15V/AbRac^Q64L could increase the expression level of TRI gene in Atropa belladonna,and eventually lead to an increase in the content of hyoscyamine in the transgenic Atropa belladonna.In summary,This study cloned a small GTPase gene in Atropa belladonna and made two site directed mutations successfully,The gene was expressed in various organs of Atropa belladonna,and its expression level in the root was higher than that in the aerial part.The overexpression of AbRac in Atropa belladonna had no effect on the TAs content,while overexpression its mutants AbRac^G15V/AbRac^Q64L could increase the content of hyoscyamie,which laid the foundation for further development of new varieties of Atropa belladonna with high TAs yield based on these transgenic materials.