Application and method of overexpressed AbbHLH45 protein in promoting synthesis of tropane alkaloid of belladonna

Abstract

The invention discloses application of overexpressed AbbHLH45 protein in promoting synthesis of tropane alkaloid of belladonna and a method, and belongs to the technical field of plant molecular biology. The amino acid sequence of the AbbHLH45 protein is shown as SEQ ID NO.2. An AbbHLH45 gene overexpression vector is constructed, then leaves of belladonna are transformed under agrobacterium tumefaciens mediation, and the overexpressed AbbHLH45 protein can be used for promoting synthesis of tropane alkaloid of belladonna. A transgenic plant or hairy root with improved tropane alkaloid content is obtained through screening, and compared with a contrast, the content of scopolamine, anisodamine and scopolamine in an over-expression AbbHLH45 transgenic plant or hairy root strain is remarkably improved; the results show that AbMYB93 can promote accumulation of TAs content in atropa belladonna and is a positive regulatory factor for TAs biosynthesis in atropa belladonna.

Description

Technical Field

[0001] The present invention relates to the field of plant molecular biology, and in particular to an application and method of overexpressing AbbHLH45 protein in promoting the synthesis of belladonna tropane alkaloids. Background Art

[0002] Tropane alkaloids (TAs) are a class of alkaloids that contain a basic skeleton of a tropane ring in their structure. Their representative medicinal ingredients include the natural anticholinergic drugs hyoscyamine, anisodamine, and scopolamine that are widely used in clinical practice. At present, the industrial production method of TAs is to extract them from medicinal plants, such as Atropa belladonna, Hyoscyamus niger, and Anisodus acutangulus. The content of TAs in medicinal plants is low and cannot fully meet the market demand. Metabolic engineering is an effective way to increase the content of active ingredients in medicinal materials, and the premise of metabolic engineering is to analyze its biosynthetic pathway or clarify its biosynthetic regulatory mechanism. The biosynthetic pathway of TAs has been completely analyzed, laying the foundation for the study of its transcriptional regulation. However, the study of transcriptional regulation of TAs biosynthesis is still in its infancy. Belladonna is a model species for studying the biosynthesis and metabolic regulation of TAs, and is also the main cultivated medicinal material for producing TAs. The present invention uses Belladonna as a material, and adopts research methods such as bioinformatics, molecular biology, biochemistry and plant biotechnology to study the transcriptional regulation mechanism of TAs biosynthesis. It is found that brassinolide (BR) can increase the content of TAs in Belladonna hairy roots, and the transcription factor AbbHLH4 that responds to BR induction positively regulates TAs biosynthesis, and its regulatory mechanism is studied in depth. Summary of the invention

[0003] In view of this, the object of the present invention is to provide an application of overexpressing AbbHLH45 protein in promoting the synthesis of belladonna tropane alkaloids; the second object of the present invention is to provide an application of biological materials related to the AbbHLH45 protein in promoting the synthesis of belladonna tropane alkaloids; the third object of the present invention is to provide a method for overexpressing the AbbHLH45 gene to promote the synthesis of belladonna tropane alkaloids.

[0004] In order to achieve the above object, the present invention provides the following technical solutions:

[0005] 1. An application of overexpressed AbbHLH45 protein in promoting the synthesis of belladonna tropane alkaloids, wherein the amino acid sequence of the AbbHLH45 protein is shown in SEQ ID NO.2.

[0006] In some embodiments of the present invention, the nucleotide sequence encoding the AbbHLH45 protein is shown as SEQ ID NO.1.

[0007] In some embodiments of the present invention, the tropane alkaloid is hyoscyamine, scopolamine or anisodamine.

[0008] In some embodiments of the present invention, the AbbHLH45 protein belongs to the bHLH transcription factor family, which is induced by BR and can directly bind to the E-box and G-box motifs on the target gene promoter pH6H (-1600 to -1141) sequence to exert its effect. The nucleotide sequence of pH6H (-1600 to -1141) is shown in SEQ ID NO.9.

[0009] 2. Use of biological materials related to the AbbHLH45 protein in promoting the synthesis of belladonna tropane alkaloids, wherein the biological materials related to the AbbHLH45 protein include nucleic acid molecules encoding the AbbHLH45 protein, or expression cassettes or recombinant vectors containing nucleic acid molecules encoding the AbbHLH45 protein, or microbial cells containing nucleic acid molecules encoding the AbbHLH45 protein, expression cassettes or recombinant vectors containing nucleic acid molecules encoding the AbbHLH45 protein.

[0010] 3. A method for overexpressing the AbbHLH45 gene to promote the synthesis of belladonna tropane alkaloids, comprising the following steps: constructing an AbbHLH45 gene overexpression vector, then transforming belladonna leaves under the mediation of Agrobacterium, and screening to obtain transgenic hairy roots or plants with increased tropane alkaloid content; the amino acid sequence encoding the AbbHLH45 gene is shown in SEQ ID NO.2.

[0011] In some embodiments of the present invention, the AbbHLH45 gene overexpression vector is obtained by constructing the nucleic acid sequence shown in SEQ ID NO.1 between the Bgl II and BstEII sites of the pG2 plasmid, and the pG2 plasmid is constructed by replacing the hygromycin resistance gene in pCAMBIA1305.1 with the new glyphosate resistance gene G2-EPSPS using Xhol.

[0012] In some embodiments of the present invention, the Agrobacterium is C58C1 strain or EHA105.

[0013] In some embodiments of the invention, the tropane alkaloid is hyoscyamine, scopolamine or anisodamine.

[0014] The beneficial effect of the present invention is that by performing BR treatment on belladonna hairy roots and conducting bioinformatics analysis, the transcription factor AbbHLH45 in belladonna that can be induced by BR and regulate the biosynthesis of TAs in belladonna is identified for the first time, and the specific mechanism of AbbHLH45 regulating the biosynthesis of TAs in belladonna is analyzed by using relevant molecular biology and biochemistry methods, which provides an effective functional gene for the metabolic engineering of tropane alkaloids and provides a new idea for obtaining belladonna germplasm resources that can produce high TAs under BR. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration:

[0016] Figure 1 Expression correlation analysis of BR-induced AbbHLHs and TAs biosynthesis genes in different tissues of Atropaeolum solanum; PR: taproot, SR: fibrous root, L: leaf, S: stem, F: fruit.

[0017] Figure 2 Tissue expression profile analysis of AbbHLHs.

[0018] Figure 3 This is the electrophoresis of the amplified bands of AbbHLH45 and AbbHLH78 coding regions; M: marker 2000bp.

[0019] Figure 4 To create AbbHLH45 transgenic belladonna hairy roots; A: differentiation plate culture, B: induced AbbHLH45 overexpressing hairy roots, C: subculture of AbbHLH45 overexpressing single clone hairy roots, D: AbbHLH45 overexpressing hairy roots cultured in MS liquid medium.

[0020] Figure 5 The expression level of AbbHLH45 gene in AbbHLH45 overexpressing hairy roots.

[0021] Figure 6 The expression levels of TAs biosynthesis genes in AbbHLH45 overexpressing hairy roots.

[0022] Figure 7 The content of TAs in hairy roots overexpressing AbbHLH45; A: hyoscyamine, B: anisodamine, C: scopolamine. Error bars are standard errors (n≥3), and the significant differences were analyzed by t-test (*P<0.05, **P<0.01, ***P<0.001).

[0023] Figure 8 To create transgenic Solanum belladonna plants overexpressing AbbHLH45.

[0024] Fig. 9 is the expression level of AbbHLH45 gene in AbbHLH45 overexpressing belladonna roots.

[0025] Fig.10 The expression levels of TAs biosynthesis genes in AbbHLH45-overexpressing belladonna roots are shown in Table 1. Error bars represent standard errors (n≥3). The significant differences were analyzed using the t-test (*P<0.05, **P<0.01, ***P<0.001).

[0026] Fig.11 The TAs content in leaves and roots of AbbHLH45-overexpressing belladonna plants; error bars are standard errors (n

[0027] ≥3), and the significant differences were analyzed by t test (*P<0.05, **P<0.01, ***P<0.001). DETAILED DESCRIPTION

[0028] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0029] Example 1: In the hairy roots of Atropa belladonna, BR can promote the biosynthesis of TAs.

[0030] Exogenous application of BR plays an important role in the accumulation of secondary metabolites. Through preliminary experiments, 0.1 μM BR was used to treat the hairy roots of Atropa belladonna for 7 days, and the solvent 95% ethanol was used as a control to treat the hairy roots of Atropa belladonna. LC-MS detected the content of TAs in its hairy roots. The results showed that after the hairy roots of Atropa belladonna were treated with BR, the contents of hyoscyamine, anisodamine and scopolamine were significantly increased. Among them, the content of hyoscyamine in the hairy roots after BR treatment was 1.66 times that of the control group, the content of anisodamine was 1.80 times that of the control group, and the content of scopolamine was 2.31 times that of the control group. This shows that 0.1 μM BR promotes the biosynthesis of TAs in hairy roots of Atropa belladonna.

[0031] The content of TAs in belladonna hairy roots was significantly increased after treatment with 0.1μM BR. To further verify the results of this study, belladonna hairy roots were treated with 0.1μM BR, 4μM BRZ and solvent 95% ethanol for 4 days, 5 days, 7 days and 10 days. The statistical analysis of TAs content showed that the contents of hyoscyamine, anisodamine and scopolamine in belladonna hairy roots were upregulated after BR treatment. After BRZ treatment, the contents of hyoscyamine, anisodamine and scopolamine in belladonna hairy roots were decreased. These results further indicate that BR can promote the biosynthesis of TAs in belladonna hairy roots.

[0032] Example 2: Effect of BR on the expression level of TAs biosynthesis genes in hairy roots of belladonna

[0033] After treating belladonna hairy roots with 0.1μM BR, it was found that BR could promote the biosynthesis of TAs in belladonna hairy roots. RT-qPCR was used to further analyze the expression levels of TAs biosynthesis genes after BR treatment of belladonna hairy roots. The belladonna hairy roots were taken at 0h, 3h, 6h and 12h after BR treatment, and PGK was used as the internal reference gene, and RT-qPCR was used to analyze the expression levels of TAs biosynthesis genes. The results showed that after BR treatment, the TAs synthesis pathway genes PMT, MPO, PYKS, CYP82M3, TRI, AT4, UGT, LS, CYP80F1, HDH and H6H were significantly upregulated at 3h of BR treatment, and only the expression levels of ODC and PPAR genes did not change significantly; TAs synthesis genes PYKS, UGT and CYP80F1 were still significantly upregulated at 6h of BR treatment. These results indicate that BR can promote the expression of TAs synthesis genes.

[0034] The expression levels of most TAs biosynthesis genes were significantly increased in the hairy roots of belladonna treated with BR for 3h, so the hairy roots of belladonna treated with BR for 3h were selected for transcriptome sequencing. According to the transcriptome data analysis, it was found that TAs biosynthesis genes responded to BR induction. PMT, MPO, PYKS, CYP82M3, TRI, AT4, UGT, LS, CYP80F1, HDH and H6H were all expressed in the hairy root materials treated with BR, and the transcriptome sequencing data were consistent with the RT-qPCR data.

[0035] Example 3: Finding transcription factors that are induced by BR and may regulate TAs biosynthesis

[0036] In order to study the AbbHLH transcription factors involved in TAs biosynthesis in response to BR, the induced expression patterns of these genes in response to BR were analyzed based on the transcriptome data of hairy roots treated with BR for 3 h. Through the induced expression patterns, 17 AbbHLH transcription factors were found to be induced by BR. Given that TAs content was significantly upregulated after BR treatment of hairy roots of A. belladonna, the AbbHLHs transcription factors induced by BR became the focus of this study.

[0037] Since TAs biosynthesis genes are specifically expressed in the fibrous roots of Atropa belladonna, in order to study the transcription factors related to TAs biosynthesis, based on the published transcriptome data of different tissues of Atropa belladonna, we performed tissue expression correlation analysis on 17 AbbHLHs and TAs biosynthesis genes induced by BR and found that 8 AbbHLHs and TAs biosynthesis genes had a high expression correlation ( Figure 1). The relative transcription levels of the eight AbbHLHs in different tissues of A. belladonna were further quantitatively analyzed by RT-qPCR. The results showed that the expression levels of the eight AbbHLHs in fibrous roots were higher than those in other tissues, but AbbHLH1, AbbHLH17, AbbHLH23, AbbHLH80, AbbHLH118 and AbbHLH132 were also highly expressed in other tissues of A. belladonna; only AbbHLH45 and AbbHLH78 were highly expressed in fibrous roots ( Figure 2 ); therefore, we selected AbbHLH45 (SEQ ID NO.1-2) and AbbHLH78 (SEQ ID NO.3-4) as our candidate research genes.

[0038] Example 4: Gene cloning

[0039] In this experiment, the root of Solanum belladonna was used as the experimental material, and the Tiangen total RNA extraction kit was used to obtain cDNA. According to the electronic full-length sequences of AbbHLH45 and AbbHLH78, specific primers (AbbHLH45-F / R, AbbHLH78-F / R) were designed. Using cDNA as a template, the HyperFusion DNA polymerase produced by APExBIO was used, and the amplification conditions were 98℃ pre-denaturation for 2 minutes; 98℃ denaturation for 15 seconds, 56℃ annealing for 15 seconds, 72℃ extension for 30 seconds, a total of 32 cycles; finally 72℃ extension for 5 minutes, 4℃ storage; amplify the respective physical full-length sequences ( Figure 3 ), the primers used are shown in Table 1 below.

[0040] Table 1. Primers for gene amplification

[0041]

[0042]

[0043] Example 5: Cloning and analysis of TAs biosynthetic gene promoters

[0044] In order to study whether the candidate genes are directly involved in the transcriptional regulation of TAs biosynthesis, yeast one-hybrid system (Y1H) was used to study the candidate genes. Previous studies have shown that bHLH family transcription factors bind to E-box (5'-CANNTG-3') and G-box (5'-CACGTG-3') motifs, thereby regulating the expression of downstream genes. For example, in Artemisia annua, AabHLH1 can bind to the E-box of the promoters of amorpha-4,11-diene synthase (ADS) and P450 monooxygenase (CYP71AV1), thereby increasing the content of artemisinin. Therefore, based on the genome data of A. belladonna, we analyzed the promoters of TAs biosynthesis genes and obtained the promoter sequences of TAs biosynthesis genes. These promoter sequences were predicted by the online website New PLACE, and it was found that there were a large number of E-boxes and G-boxes in the promoters of TAs biosynthesis genes. These results further suggest that transcription factors of the AbbHLH family may be directly involved in the transcriptional regulation of TAs biosynthesis. There are a large number of E-boxes and G-boxes in the promoters of TAs biosynthesis genes. These results further suggest that transcription factors of the AbbHLH family may be directly involved in the transcriptional regulation of TAs biosynthesis. Y1H was used to verify whether there was a binding effect between AbbHLH45 and AbbHLH78 and the promoters of TAs biosynthesis genes. First, the truncated fragments of the TAs biosynthesis gene promoter mentioned in Example 2 were constructed in the pLacZ vector respectively; AbbHLH45 and AbbHLH78 were constructed in the vector pB42AD respectively to form AbbHLH45-pB42AD and AbbHLH78-pB42AD. AbbHLH78-pB42AD and the promoter truncation vector were co-transformed into the yeast strain EGY48. The yeast colonies co-transformed with AbbHLH78-pB42AD and the truncated fragment of the promoter of the TAs biosynthesis gene did not turn blue on the yeast single hybrid color plate, indicating that there was no binding interaction between AbbHLH78 and the promoters of these enzyme genes. AbbHLH45-pB42AD and the promoter truncation vector were co-transformed into the yeast strain EGY48. The yeast colonies co-transformed with AbbHLH45 and pH6H (-1600 to -1141) (SEQ ID NO.9) turned blue on the yeast single hybrid color plate, indicating that AbbHLH45 had a binding interaction with pH6H.In addition, when AbbHLH45 was co-transformed with the promoter regions of other enzymes in the TA pathway (such as pODC, pPMT, pMPO, pPYKS, pTRI, pAT4, pPPAR, pCYP82M3, pUGT, pLS, pCYP80F1, and pHDH) into yeast colonies, the color development plate did not turn blue, indicating that there was no binding interaction between AbbHLH45 and the promoters of these enzyme genes.

[0045] Example 6. Construction of AbbHLH45 overexpression vector

[0046] pG2 (pG2 plasmid is a plant binary expression vector modified by Zhang et al. that has a novel glyphosate resistance gene G2-EPSPS in eukaryotes. It was constructed by replacing the hygromycin resistance gene in pCAMBIA1305.1 with G2-EPSPS using Xhol, see the literature Engineering tropane alkaloid production and glyphosate resistance by overexpressing AbCaM1 and G2-EPSPS in Atropabelladonna) was double-digested with BglII and BstEII, respectively, and the pG2 digested fragment was recovered. Using the primers in Table 2 and the AbbHLH45 gene as a template, Takara's PrimeSTAR Max DNA Polymerase kit was used for amplification. The amplification conditions were denaturation at 98°C for 10 seconds, annealing at 55°C for 5 seconds, and extension for 5 seconds to obtain the AbbHLH45 gene fragment with homology arms. The gene fragment was recovered, and the AbbHLH45 gene fragment and the vector fragment were homologously recombined using a recombinase to construct the AbbHLH45 overexpression vector AbbHLH45-pG2.

[0047] Table 2. Primers for constructing AbbHLH45 overexpression vector

[0048]

[0049] Example 7: Creation of transgenic belladonna hairy roots of AbbHLH45-OE

[0050] The results of the previous experiments showed that AbbHLH45 binds to the promoter of the TAs biosynthesis gene H6H. To further verify the function of AbbHLH45 in plants, the AbbHLH45-pG2 plant expression vector was constructed and transformed into Agrobacterium strain C58C1. The pG2 empty vector was also transformed as a control group. The positive strains were selected to infect the leaves of Azalea belladonna and transferred to the screening culture plate after 2 days of co-culture. Figure 4, A); replace the new screening culture plate every 2 weeks, and continue to culture for one month before hairy roots grow from the leaf cuts ( Figure 4 , B); monoclonal hairy roots with good growth and normal morphology were selected and subcultured to MS solid culture plates for three weeks ( Figure 4 , C); After obtaining hairy roots with consistent growth, they were inoculated into MS liquid culture and cultured at 24°C and 110 rpm in a shaking incubator for one month ( Figure 4 , D), the material is used for the detection of gene expression and TAs content.

[0051] In order to identify positive single clones of transgenic hairy roots, this study used DNA of AbbHLH45-pG2 transgenic hairy roots as template, wild-type belladonna hairy roots as negative control, water as blank control, and AbbHLH45-pG2 recombinant plasmid diluted 50 times as positive control. Molecular detection was performed by PCR, and electrophoresis results showed that a 35S::AbbHLH45 gene fragment of about 837 bp was detected in AbbHLH45-pG2 transgenic hairy roots, which was consistent with the predicted band size, and was not detected in the negative control and blank control; G2-EPSPS::G2-EPSPS of about 1500 bp was detected in some transgenic hairy roots of AbbHLH45-pG2 and pG2, which was consistent with the target band size, and was not detected in the negative control and blank control. RT-qPCR was used to further verify the changes in the expression level of the target gene AbbHLH45 in transgenic hairy roots. The cDNA of AbbHLH45-pG2 positive transgenic hairy roots and pG2 positive transgenic hairy roots were obtained. RT-qPCR specific binding primers were designed. The results showed that the expression of AbbHLH45 gene in the positive transgenic hairy roots overexpressing AbbHLH45 was significantly increased, and the increase of AbbHLH45 in L-17 was the largest, reaching 114 times ( Figure 5 ).

[0052] Example 8: Detection of gene expression levels of TAs biosynthesis pathway in transgenic belladonna hairy roots

[0053] RT-qPCR was used to further verify the changes in the expression levels of TAs biosynthesis genes in transgenic hairy roots. The AbbHLH45-pG2 positive transgenic hairy root cDNA was used as a template, and the pG2 positive transgenic hairy root cDNA was used as a control. RT-qPCR specific binding primers were designed (Table 3-Table 4). The results showed that the TAs biosynthesis genes CYP82M3, TRI, UGT and H6H were significantly upregulated in the positive transgenic hairy roots overexpressing AbbHLH45, and there was no significant change in the expression of ODC, PMT, MPO, PYKS, AT4, PPAR, LS, CYP80F1 and HDH genes ( Figure 6).

[0054] Table 3. qRT-PCR reaction system

[0055]

[0056] Table 4. qRT-PCR primers

[0057]

[0058]

[0059] Example 9: Detection of TAs content in transgenic belladonna hairy roots

[0060] In order to study the changes in TAs content in hairy roots after AbbHLH45 overexpression, TAs were extracted from hairy roots of AbbHLH45 overexpression and control group and detected by LC-MS. The results showed that the contents of hyoscyamine, anisodamine and scopolamine were significantly upregulated in transgenic hairy roots overexpressing AbbHLH45. In L1, L17, L32, L34 and L45 where AbbHLH45 was overexpressed, the contents of hyoscyamine were 2.19, 1.80, 1.63, 1.62 and 1.97 times of those in the control group, respectively; the contents of anisodamine were 2.78, 2.87, 2.76, 2.09 and 2.87 times of those in the control group, respectively; the contents of scopolamine were increased by 2.12, 2.26, 2.44, 3.14 and 2.12 times, respectively ( Figure 7 ).

[0061] Example 10: Creation of transgenic AbbHLH45-OE belladonna plants

[0062] To further study the effect of AbbHLH45 overexpression on TAs in transgenic belladonna plants. The constructed AbbHLH45-pG2 vector was transformed into Agrobacterium strain EHA105, and the pG2 empty vector was transformed as a control group; sterile belladonna seedlings grown for two weeks after belladonna seeds germinated were used to create transgenic plants. After the leaves and hypocotyls of belladonna seedlings were infected with the bacterial liquid, they were placed on co-cultivation plates in a dark room (24°C), transferred to screening plates for culture after 2 days, and replaced with new screening plates after 1 week; the screening plates were replaced every 2 weeks thereafter; after one month of culture on the screening plates, the belladonna explants were incised to differentiate into regenerated buds; the differentiated regenerated buds were cut with scissors and transferred to rooting medium for culture, and a large number of roots were differentiated after 4 weeks ( Figure 8). The root-differentiated belladonna seedlings were transferred to the artificial climate chamber (24°C, 16 / 8 light / dark) in the soil culture area for seedling hardening. In order to identify the positive strains of transgenic belladonna, this study used the DNA of transgenic belladonna AbbHLH45-pG2 as a template, the DNA of wild-type belladonna as a negative control, water as a blank control, and the recombinant plasmid AbbHLH45-pG2 diluted 50 times as a positive control. Molecular detection was performed by PCR. The electrophoresis results showed that a 35S::AbbHLH45 gene fragment of about 837 bp was detected in AbbHLH45-pG2 transgenic belladonna, which was consistent with the predicted band size. It was not detected in the negative control and blank control. G2-EPSPS::G2-EPSPS of about 1500 bp was detected in some transgenic belladonna of AbbHLH45-pG2 and pG2, which was consistent with the predicted band size. The band of G2-EPSPS::G2-EPSPS was not detected in the negative control and blank control. RT-qPCR was used to further verify the changes in the expression level of the target gene AbbHLH45 in transgenic belladonna. The cDNA of AbbHLH45-pG2 positive transgenic belladonna and pG2 positive transgenic belladonna was obtained. RT-qPCR specific binding primers were designed. The results showed that the expression of AbbHLH45 gene in the positive transgenic belladonna with overexpression of AbbHLH45 was significantly increased, and the increase of AbbHLH45 in L-13 was the largest, which was 8.8 times that of the control group ( Fig. 9 )

[0063] Example 11: Increased expression of TAs biosynthesis genes in AbbHLH45-OE belladonna plants

[0064] RT-qPCR was used to further verify the changes in the expression levels of TAs biosynthesis genes in transgenic belladonna. The AbbHLH45-pG2 positive transgenic belladonna cDNA was used as a template, and the pG2 positive transgenic hairy root cDNA was used as a control group. RT-qPCR specific binding primers were used. The results showed that the TAs biosynthesis pathway enzyme genes CYP82M3, TRI, UGT and H6H were significantly upregulated in the AbbHLH45 overexpressed positive transgenic belladonna, and the expression levels of ODC, PMT, MPO, PYKS, AT4, PPAR, LS, CYP80F1 and HDH genes did not change significantly, which was consistent with the expression trend in the AbbHLH45 overexpressed transgenic hairy roots ( Fig.10 ).

[0065] Example 12: Increased TAs content in AbbHLH45-OE belladonna plants

[0066] In order to study the changes in TAs content in AbbHLH45 overexpressed belladonna, TAs were extracted from AbbHLH45 overexpressed and control belladonna and detected by LC-MS. The results showed that the contents of hyoscyamine, anisodamine and scopolamine were significantly upregulated in transgenic belladonna overexpressed with AbbHLH45. The content of TAs in the leaves of transgenic belladonna was analyzed. In the L-2, L-9, L-13 and L-15 lines overexpressing AbbHLH45, the content of hyoscyamine was 1.41 times, 1.97 times, 1.79 times and 1.87 times that of the control group, respectively; the content of anisodamine was 1.45 times, 1.63 times, 1.36 times and 1.63 times that of the control group, respectively; the content of scopolamine was 3.12 times, 3.73 times, 3.76 times and 3.50 times that of the control group, respectively. The contents of TAs in the roots of transgenic AbbHLH45-overexpressing L-2, L-9, L-13, and L-15 plants were analyzed. The contents of hyoscyamine were 1.45, 1.68, 1.61, and 1.77 times, respectively, of that in the control group; the contents of anisodamine were 1.75, 1.86, 1.46, and 1.68 times, respectively, of that in the control group; and the contents of scopolamine were 4.03, 3.44, 3.54, and 2.22 times, respectively, of that in the control group. Fig.11 ).

[0067] The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. An application of overexpressing AbbHLH45 protein in promoting the synthesis of belladonna tropane alkaloids, characterized in that: The amino acid sequence of the AbbHLH45 protein is shown in SEQ ID NO.

2.

2. The use according to claim 1, characterized in that: The nucleotide sequence encoding the AbbHLH45 protein is shown in SEQ ID NO.

1.

3. The use according to claim 1, characterized in that: The tropane alkaloid is hyoscyamine, scopolamine or anisodamine.

4. The use according to claim 1, characterized in that: The AbbHLH45 protein belongs to the bHLH transcription factor family, is induced by BR, and can directly bind to the E-box and G-box motifs on the target gene promoter pH6H (-1600 to -1141) sequence to exert its effect. The nucleotide sequence of pH6H (-1600 to -1141) is shown in SEQ ID NO.

9.

5. Use of a biomaterial related to the AbbHLH45 protein of claim 1 in promoting the synthesis of belladonna tropane alkaloids, characterized in that: The biological material includes a nucleic acid molecule encoding AbbHLH45 protein, or an expression cassette or recombinant vector containing a nucleic acid molecule encoding AbbHLH45 protein, or a microbial cell containing a nucleic acid molecule encoding AbbHLH45 protein, an expression cassette or recombinant vector containing a nucleic acid molecule encoding AbbHLH45 protein.

6. A method for overexpressing the AbbHLH45 gene to promote the synthesis of belladonna tropane alkaloids, characterized in that: The method comprises the following steps: constructing an AbbHLH45 gene overexpression vector, then transforming belladonna leaves under the mediation of Agrobacterium, and screening to obtain transgenic hairy roots or plants with increased tropane alkaloid content; the amino acid sequence encoding the AbbHLH45 gene is shown in SEQ ID NO.

2.

7. The method according to claim 6, characterized in that The AbbHLH45 gene overexpression vector is obtained by constructing the nucleic acid sequence shown in SEQ ID NO.1 between the Bgl II and BstEII sites of the pG2 plasmid. The pG2 plasmid is constructed by replacing the hygromycin resistance gene in pCAMBIA1305.1 with a new glyphosate resistance gene G2-EPSPS using Xhol.

8. The method according to claim 6, characterized in that The Agrobacterium is C58C1 strain or EHA105 strain.

9. The method according to claim 6, characterized in that The tropane alkaloids are hyoscyamine, scopolamine or anisodamine.

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