Effect Of Rol Gene Expression In Atropa Belladonna Root On Tropine Alkaloid Synthesis

Tropine alkaloids(Tropane alkaloids,TAs)are a class of anticholinergic drugs commonly used in clinical practice,and have a good effect in the treatment of analgesia,anesthesia,anti-motion sickness,detoxification,Parkinson's disease,pesticide poisoning and other treatments.At present,tropine alkaloids are mainly extracted from plants such as the natural plants Atropa belladonna,Datura stramonium,Anisodus tanguticus,et al.The market demand is huge,and the cost of industrial synthesis is high.Atropa belladonna is included in the Chinese Pharmacopoeia and mainly provides tropine alkaloids as a commercial source plant.Natural belladonna has very low levels of tropane alkaloids.It is of great research significance to use gene metabolic engineering to increase the content of tropane alkaloids in Atropa belladonna.In this study,the key enzyme gene promoter H6H-p of the medicinal plant Belladonna downstream was obtained by cloning the belladonna genomic DNA;the rolC gene was cloned from the C58C1 Agrobacterium rhizogenes pRiA4 plasmid.The rolC gene was cloned and constructed into the GUS coding region of pBI121 to obtain a CaMV35S-driven expression vector;similarly,pBI121 was used as the expression vector,H6H-p was the tissue-localized promoter and the rolC gene was constructed to obtain the H6H-p-driven rolC gene Location expression vector;EHA105-pBI121-CaMV35S-rolC and EHA105-pBI121-H6H-p-rolC engineered bacteria are obtained by transformation,and EHA105-pCaMV35S-rolC and EHA105-pH6H-p-rolC transgenic belladonna are obtained through genetic transformation..The phenotype analysis of 4 transgenic seedlings of EHA105-pCaMV35S-rolC and EHA105-pH6H-p-rolC respectively showed that compared with the control,the roots of the rolC gene transgenic seedlings driven by CaMV35 S grew densely,and the stem segments above ground The time is shortened,the leaves increase,and the tissue culture period blooms earlier;after two months of outdoor growth,there will be two different phenotypes from the control group,one is the leaf shrinkage,and the other is the slender leaf.At the same time,under outdoor growing conditions,the flowers still bloom in advance,the internodes are shortened,the apex dominance decreases,and the leaf shape changes.There was no obvious phenotypic difference and variation between the rolC transgenic vaccine driven by H6H-p and the control.This shows that CaMV35 S driving the rolC gene expression will cause the phenotype of the above-ground part of belladonna to change,while H6H-p drives the rolC gene expression and will not cause the above-ground part of the mutation.Further testing of the alkaloids and gene expression levels of the transgenic seedlings showed that the contents of hyoscyamine and anisodamine in the 4 transgenic seedlings of EHA105-pCaMV35S-rolC increased,and the content of anisodamine detected in the C-7 transgenic plants The highest(69.831 ?g/g),which is 4.553 times of the wild-type belladonna control(15.336 ?g/g),and 3.484 times of the empty transgenic belladonna control(20.045 ?g/g);C-1 transgenic plants have the highest hyoscyamine content(149.787 ?g/g),which is 3.784 times higher than that of the wild-type belladonna(39.588 ?g/g)control,and 4.014 times higher than that of the unloaded transgenic belladonna(37.32 ?g/g)control;for rolC gene expression and synthesis pathway genes The expression analysis showed that the rolC gene was highly expressed in the four transgenic roots and leaves,and the higher the expression,the more obvious the phenotype difference of the gene,while the pathway genes PMT,TRI,H6 H were only detected in the roots.Among them,the expression levels of H6 H and TRI genes related to the synthesis of hyoscyamine and anisodamine are higher than the control,indicating that the expression of rolC gene will increase the expression of belladonna TAs synthesis pathway genes,thereby promoting the metabolic flow to hyoscyamine and anisodamine Synthesis direction shift.Similarly,the detection of alkaloids of 4 transgenic EHA105-pH6H-p-rolC strains showed that the content of hyoscyamine and anisodamine was also increased,and the content of anisodamine(60.379 ?g/g)in the H-C-2 transgenic plants increased the most.Significantly,7.072 times higher than that of wild-type belladonna(8.538 ?g/g),and 3.167 times higher than that of the unloaded transgenic belladonna(19.066 ?g/g)control.Fluorescence quantitative PCR detection of expression levels showed that,compared with the control,the 4 transgenes driven by H6H-p detected rolC gene expression in roots,but no expression in leaves,indicating that H6H-p has achieved rolC gene localization the expression of H6 H in the 4 EHA105-pH6H-p-rolC transgenic seedlings was higher than that of the control.The test of alkaloids showed that the content of anisodamine in the 4 transgenic seedlings increased,which is the same as the key enzyme that catalyzes its synthesis.The expression level of gene H6 H is the same;while only two lines of PMT and TRI have higher gene expression levels than the control,correspondingly,in the 4 seedlings elevated by hyoscyamine,the expression of PMT and TRI genes of H-C-1 and H-C-2 lines The amount is higher than the control expression.It shows that the expression of rolC gene driven by H6H-p can also increase the expression of TAs synthesis pathway genes,thereby increasing the content of hyoscyamine and anisodamine,but the efficiency is weaker than the expression of rolC gene driven by CaMV35 S,but it will not make plants Variation occurs in the above-ground part,which affects plant growth.The research results of this subject concluded that the expression of rolC gene in belladonna can effectively increase the expression of TAs alkaloid synthesis pathway genes,and increase the content of hyoscyamine and anisodamine in belladonna,but it will have a variability in plant phenotype;And the rolC gene expressed through H6H-p tissue positioning,while increasing alkaloids,does not affect belladonna plant phenotypic growth.The realization of the selective control of the rolC gene provides a new method for further cultivating high-yielding alkaloid belladonna strains in the medicinal field of belladonna,and further developing the TAs alkaloid genetic engineering metabolism research of belladonna.