| Hyoscyamine and scopolamine are representative anticholinergic natural drugs of tropane alkaloids(TAs),and widely used in the treatment of motion sickness,Alzheimer’s disease,spasms,and other diseases.The commercial production of medicinal TAs mainly depends on their extraction from the medicinal plants of the Atropa,Datura and Duboisia.Atropa belladonna(A.belladonna)is one of the herbal medicinal plants cultivated on a large area for the production of medicinal TAs.However,the existing A.belladonna varieties have the disadvantages of low TAs contents,large impact by weeds,and higher production costs.Therefore,improving the herbicide resistance of A.belladonna and reducing the production costs of medicinal TAs are major demands of the relevant pharmaceutical industry.Biotechnology is the main method to solve the high production cost of medicinal TAs.Although scientists have successfully used synthetic biotechnology to produce TAs in yeast cells,but their output is extremely low and there is still a great distance from commercial application.Therefore,developing high-yield and herbicide-resistant A.belladonna through genetic engineering technology has important scientific value and good application prospects.This thesis established an A.belladonna genetic transformation system with glyphosate as the screening pressure.And on this basis,the glyphosate-resistant gene(G2-EPSPS),A.belladonna calmodulin gene(AbCaM1),A.belladonna littorine synthase gene(AbLS)and Hyoscyamus niger Hyoscyamine 6β-hydroxylase gene(HnH6H)were used to culture glyphosate-resistant and high TAs yields associated transgenic A.belladonna plants.We obtained hmozygous transgenic A.belladonna lines which overexpressing AbCaM1 and G2-EPSPS,HnH6 H and G2-EPSPS with the genetic breeding method,respectively.Hence,the relative expression levels of related genes,glyphosate resistance and TAs contents were analyzed to evaluate the traits of A.belladonna transgenic lines.Through the above research,this thesis has achieved the following results:1.Establishment the genetic transformation system of A.belladonna with glyphosate as the screening pressureThis thesis first tested the tolerance of A.belladonna cotyledons to different concentrations of glyphosate.Cotyledons were inoculated in MS regeneration medium supplemented with different concentration glyphosate,and observed the survival rates after 14 days.The results showed that:(1)In the MS regeneration medium supplemented with 0.20 m M,0.50 m M,or 1.00 m M glyphosate,the survival rate of cotyledons was 0%,0% and 16%,respectively;when supplemented with 0.05 m M or 0.075 m M glyphosate in the MS medium,the survival rate of cotyledons was100%;when supplemented with 0.10 m M glyphosate,the survival rate was up to 83%.Therefore,it is determined that 0.10 m M glyphosate is the optimal selection pressure for A.belladonna transgenic plants.With the help of restriction enzyme,the Hygr in the pCAMBIA1305.1 was replaced to the G2-EPSPS,and obtained the expression vector pG2.After sequencing verification,introduced into the Agrobacterium-tumefaciens EHA105 to obtain the engineered strain.Agrobacterium-dip in the cotyledons was used for A.belladonna genetic transformation,and the growth of regeneration buds of A.belladonna cotyledons in MS regeneration medium supplemented with 0.10 m M glyphosate was observed after 45 days culture.The results showed that the leaf regeneration rate of A.belladonna was 70.43±2.25%.After the regenerated plants were rooted and refined,the PCR test results for G2-EPSPS showed that the transgene positive rate of the regenerated plants was up to 77.8%.Five independent A.belladonna lines were randomly selected for the testing of glyphosate resistance,and the results showed that they all had good tolerance to 1800 g.a.e/ha glyphosate.After being planted in the field for 120 days,the analysis of agronomic characteristics and TAs contents of the G2-EPSPS-transformed A.belladonna showed that there was no significant difference in agronomic traits between the transgenic wild-type A.belladonna plants,including plant height,crown width,stem thickness,number of branches,leaf length,leaf width and biomass.There is also no significant difference in their TAs contents.The above results indicate that G2-EPSPS can confer A.belladonna glyphosate resistance,but has no effect on the agronomic traits and TAs contents.The establishment of the genetic transformation system of A.belladonna with glyphosate as the screening pressure laid the foundation for the follow-up research in this thesis.2.Development high-yield TAs and glyphosate-resistant homozygous transgenic A.belladonna plants with AbCaM1 and G2-EPSPSAbCaM1 is a calmodulin that is highly expressed in the secondary roots of A.belladonna and regulates the biosynthesis of TAs.It can be used to cultivate A.belladonna with high-yield TAs.Using restriction enzymes,AbCaM1 was inserted into the plant expression vector pG2 to obtain the recombinant vector pGC,which was introduced into Agrobacterium EHA105 to obtain engineering bacteria.Fifteen A.belladonna regenerated plants were obtained after Agrobacterium-mediated transformation.PCR detection results showed that the 35S::AbCaM1 and the35S::G2-EPSPS DNA fragment were specifically amplified in the positive control pGC and 12 regenerated plants.Five lines were randomly selected for further research.The results of qPCR analysis showed that the expression levels of AbCaM1 of the five tested lines were much higher than those in wild-type A.belladonna plants.Southern blot was used to further detect G2-EPSPS in the above five lines.The results showed that they are all independent single copy transgenic A.belladonna lines and can be used as genetic breeding materials to cultivate homozygous transgenic A.belladonna lines.Glyphosate resistance test and TAs contents analysis of T0 transgenic A.belladonna.The transgenic and wild-type A.belladonna were sprayed with glyphosate at a concentration of 1800 g.a.e/ha.After 10 days,all the wild-type A.belladonna died,while all the transgenic A.belladonna plants survived and grew very well.The TAs contents analysis showed that compared with wild-type A.belladonna plants,the contents of hyoscyamine,anisodamine and scopolamine was significantly increased.The above analysis results show that A.belladonna palnts overexpressing AbCaM1 and G2-EPSPS not only has strong glyphosate resistance,but also has enhanced TAs biosynthesis ability.Development of homozygous transgenic A.belladonna lines overexpressing AbCaM1 and G2-EPSPS and analysis their field traits.The T0 transgenic generation T0GC02,T0GC05 and T0GC06 were self-pollinatied to obtain T1 transgenic generation;the seedlings obtained from the germination of T1 generation seeds were further self-pollinatied,and the T1 parent lines were identified three homozygous transgenic lines,which named T1GC02,T1GC05 and T1GC06.Southern blot analysis showed that the homozygous transgenic lines and their corresponding T0 parent have the same DNA hybridization pattern,which can be used to prepare seeds for field trait.Agronomic traits analysis showed that the homozygous transgenic lines had no significant difference with wild-type A.belladonna.Spraying with glyphosate at a concentration of 900 g.a.e/ha,all wild-type A.belladonna were died after 2 weeks,and most of the weeds died,while the transgenic pure line survived well and grew normally.It shows that glyphosate resistance has been stably inherited in the T2 generation transgenic A.belladonna.The TAs contents results showed that the contents of hyoscyamine in all tissues of the homozygous transgenic lines were significantly higher than those in the wild-type A.belladonna plants;the contents of anisodamine and scopolamine in the leaves were significantly higher than those in the control.Taking the main harvested parts primary roots and leaves of A.belladonna plants as an example,the hyoscyamine contents in the homozygous transgenic lines are 2.19～2.58 folds and3.65～4.06 folds of the control,respectively.The contents of anisodamine in the primary roots roots and leaves of the homozygous transgenic lines are are 1.83～2.61 folds and 3.64～4.45 folds of the wild-type.The contents of scopolamine in the leaves of the homozygous transgenic lines are 2.77～5.00 folds of the control;which are not detected in the primary roots of the wild-type control.The results of gene expression analysis showed that the expression level of AbCaM1 in the homozygous transgenic lines was much higher than that in wild-type A.belladonna lines.In order to further explore the regulatory effects of AbCaM1 on TAs biosynthesis,the relative expression levels of all 13 genes involved in TAs biosynthesis were detected,and the expression of ODC,PMT,MPO,PPAR,UGT1,CYP80F1,HDH and H6 H in the homozygous transgenic lines were significantly up-regulated compared with wild-type plants.The above studies show that AbCaM1 selectively up-regulates the expression of TAs biosynthesis genes to promote TAs biosynthesis.3.Study on the function of Littorine synthase in the metabolic engineering of tropane alkaloidsLittorine is an important precursor for the biosynthesis of hyoscyamine and scopolamine.Littorine synthase(LS)catalyzes tropine and phenyllactylglucose to produce littorine by esterification and condensation.However,the contents of littorine in A.belladonna is extremely low,less than one-tenth of hyoscyamine.It is considered to be the rate-limiting enzyme of TAs biosynthesis,and plays an important role in cultivating high-yield A.belladonna with TAs.With the help of two restriction enzymes,AbLS was inserted into the plant expression vector pG2 to obtain the recombinant vector pGL,which was introduced into Agrobacterium EHA105 to obtain engineering bacteria.According to the established genetic transformation system,12 A.belladonna regenerated plants were obtained.PCR detection showed that the 35S::AbLS and the 35S::G2-EPSPS DNA fragment were specifically amplified in the positive control pGC and 10 regenerated plants.qPCR analysis showed that the expression levels of AbLS were much higher than those in wild-type A.belladonna.In the glyphosate resistance experiment,five independent transgenic A.belladonnas lines were randomly selected to spray at a concentration of 1800 g.a.e/ha glyphosate.10 days later,all wild-type A.belladonna lines died,while the transgenic plants grew normally.The measurement of TAs contents showed that the contents of littorine,hyoscyamine,anisodamine and scopolamine were significantly increased in the transgenic A.belladonna plants.Taking the A.belladonna leaves as an example,the contents of hyoscyamine in the transgenic A.belladonna plants are 1.32～2.10 folds of the control;the contents of anisodamine are 2.20～3.35 folds of the control;The contents of scopolamine are 2.53～4.23 folds than that in the control.No littorine is detected in the transgenic A.belladonna leaves,but the contents of littorine in the roots of transgenic A.belladonnais are 2.37～4.37 folds of the control.The above results indicate that the transgenic A.belladonna plants overexpressing AbLS and G2-EPSPS not only have strong glyphosate resistance,but also have higher TAs yield.AbLS can be used as a potential target for genetic engineering of TAs biosynthesis.4.Development high-yield scopolamine and glyphosate-resistant homozygous transgenic A.belladonna plants with HnH6 H and G2-EPSPSH6H catalyzes the conversion of hyoscyamine to scopolamine,which is the key rate-limiting enzyme of TAs biosynthesis,and widely used in the genetic engineering technology to cultivate high-yield transgenic materials of scopolamine.With the help of two restriction enzymes HnH6 H was inserted into the plant expression vector pG2 to obtain the recombinant vector pGH.Thirty A.belladonna regenerated plants were obtained according to the genetic transformation.Nine of G2-EPSPS and HnH6 H genes were successfully integrated into the genome of transgenic plants and had high expression.Five independent lines were randomly selected for southern blot assay to make molecular detection of G2-EPSPS.The results showed that T0GH03 and T0GH06 are independent single copy transgenic A.belladonna lines and can be used as genetic breeding materials to cultivate homozygous transgenic A.belladonna lines.Glyphosate resistance test and TAs contents analysis of T0 transgenic A.belladonna plants,two weeks after spraying with glyphosate at a concentration of 1800 g.a.e/ha,all wild-type A.belladonna plants died,while transgenic A.belladonna plants survived and grew normally.The results of TAs contents analysis showed that compared with wild-type A.belladonna plants,the contents of scopolamine are significantly increased.The above results show that the transgenic A.belladonna plants with high yield of scopolamine and glyphosate resistance was successfully obtained.The homozygous lines of transgenic A.belladonna overexpressing HnH6 H and G2-EPSPS were developed and its field traits were analyzed.The T0 transgenic generation T0GH03 and T0GH06 were respectively self-pollinatied to obtain T1 transgenic generation;the seedlings obtained from the germination of T1 generation seeds were further self-pollinatied,and the T1 parent lines were identified two homozygous transgenic lines,which named T1GH03 and T1GH06.And these two homozygous transgenic lines were used to prepare seeds and seedlings to carry out field traits analysis.Agronomic traits analysis showed that the homozygous transgenic lines had no significant difference with wild-type A.belladonna plants.Spraying with glyphosate at a concentration of 900 g.a.e/ha,all wild-type A.belladonna were died after 2 weeks,and most of the weeds died,while the transgenic pure line survived well and grew normally.It shows that glyphosate resistance has been stably inherited in the T2 generation transgenic A.belladonna.The TAs contents in the roots and leaves of A.belladonna plants were analyzed.The results showed that the contents of scopolamine were significantly increased.In roots,the scopolamine of homozygous transgenic lines were 5.89 and 5.94 folds of the wild-type lines;in leaves,the scopolamine of the homozygous transgenic lines was 11.85 and 15.30-fold of the wild-type.In summary,this thesis has successfully established an A.belladonna genetic transformation system using glyphosate as the selection pressure and glyphosate resistance gene G2-EPSPS as the selection marker gene.On this basis,AbCaM1 and G2-EPSPS were used to develop homozygous transgenic A.belladonna lines with high-yield TAs and strong glyphosate resistance;overexpression of AbCaM1 caused the up-regulation of seven key enzyme genes in the TAs biosynthetic pathway,strengthening the transgenic A.belladonna TAs biosynthesis capacity,thus increasing the contents of TAs.The application of LS and G2-EPSPS to cultivate TAs high-yield and glyphosate-resistant transgenic A.belladonna,proving that the littorine biosynthesis catalyzed by LS is the rate-limiting step in the TAs biosynthetic pathway,and LS can be used as a candidate gene for metabolic engineering to enhance the biosynthesis of TAs.The genes of HnH6 H and G2-EPSPS were used to develop homozygous transgenic A.belladonna plants with high scopolamine production and resistance to glyphosate.The increase in the scopolamine contents of transgenic A.belladonna plants was due to the overexpression of HnH6 H which caused more hyoscyamine to be converted into scopolamine.The genetically modified A.belladonna cultivated in this thesis not only has higher TAs contents,but also has the ability to glyphosate resistence,which can effectively reduce the production cost of TAs,and has good application and development value. |
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