Application of overexpression of transcription factor AbbHLH29 in improving the biological traits of belladonna

Abstract

This invention discloses the application of overexpression of transcription factor AbbHLH29 in improving the biological traits of belladonna. By overexpressing AbbHLH29 in belladonna, this invention increases the expression level of the H6H gene, iron content, chlorophyll a content, aboveground biomass, or the content of scopolamine and anisodamine. The nucleotide sequence of AbbHLH29 is shown in SEQ ID No. 11. This research is not only significant in elucidating the molecular mechanism by which AbbHLH29 regulates the biosynthesis of belladonna alkaloids and iron homeostasis, but also provides important technical support for future research on belladonna alkaloid metabolic engineering.

Description

Technical Field

[0001] This invention relates to the field of plant transgenic technology, specifically to the application of overexpression of transcription factor AbbHLH29 in improving the biological traits of belladonna. Background Technology

[0002] In plant iron deficiency responses, bHLH-type transcription factors play a crucial role, mediating iron deficiency stress pathways. Researchers have identified three bHLH-type transcription factors—FER-like Deficiency Induced Transcripition Factor (FIT), AtbHLH38, and AtbHLH39—involved in the regulation of plant iron homeostasis (Wu et al., 2012). In Arabidopsis, AtFIT (AtbHLH29) is primarily expressed in roots under iron deficiency induction. FIT mutants exhibit wilting, yellowing, and severely stunted growth under iron-deficient conditions (Iyer et al., 2011). Studies have demonstrated that the iron chelate reductase FRO2 and the iron transporter IRT1 are targets of these three transcription factors, and the transcription of FRO2 and IRT1 is directly regulated by the FIT / AtbHLH38 or FIT / AtbHLH39 complex (Yuan et al., 2008). This suggests that the bHLH family of transcription factors plays an important role in maintaining iron homeostasis, but there are currently no reports on their role in belladonna, especially the regulatory mechanism of AbbHLH29 in the biosynthesis of belladonna alkaloids, which remains unclear. Summary of the Invention

[0003] In view of this, one objective of the present invention is to provide an application of overexpression of transcription factor AbbHLH29 in improving the biological traits of belladonna; another objective of the present invention is to provide a method for improving the biological traits of belladonna by overexpression of transcription factor AbbHLH29.

[0004] To achieve the above objectives, the present invention provides the following technical solution:

[0005] 1. Application of overexpression of transcription factor AbbHLH29 in improving the biological traits of belladonna

[0006] The improved biological traits of belladonna are achieved by increasing the expression level of the H6H gene, increasing iron content, increasing chlorophyll a content, increasing aboveground biomass, or increasing the content of scopolamine and anisodamine.

[0007] As a preferred embodiment of the present invention, the nucleotide sequence of the transcription factor AbbHLH29 is shown in SEQ ID No. 11.

[0008] 2. A method for improving the biological traits of belladonna by overexpressing the transcription factor AbbHLH29 includes the following steps: constructing an AbbHLH29 overexpression vector, transforming belladonna plants under Agrobacterium-mediated transformation, and screening for positive transgenic lines to obtain belladonna with improved biological traits.

[0009] As a preferred embodiment of the present invention, the nucleotide sequence of the transcription factor AbbHLH29 is shown in SEQ ID No. 11.

[0010] As a preferred technical solution of the present invention, the improved biological traits of belladonna include increasing the expression level of the H6H gene, increasing the iron content, increasing the chlorophyll a content, increasing the aboveground biomass, and increasing the content of scopolamine and anisodamine.

[0011] As a preferred technical solution of the present invention, the process of constructing the AbbHLH29 overexpression vector is as follows: the nucleic acid sequence of AbbHLH29 is obtained by PCR amplification using the primers shown in SEQ ID No. 12 and SEQ ID No. 13, and then double digested with XbaI and BamHI, and ligated into the plant expression vector pBI121 to obtain the overexpression vector pBI121-AbbHLH29.

[0012] As a preferred embodiment of the present invention, the Agrobacterium is C58C1 or EHA105.

[0013] The beneficial effects of this invention are as follows: Through tissue expression analysis and spatiotemporal specificity analysis of belladonna, this invention identified an iron-deficiency-induced response bHLH family transcription factor, AbbHLH29, and conducted bioinformatics analysis and iron deficiency induction experiments to determine it as a candidate gene regulating tropane alkaloid synthesis in belladonna for further research. Transgenic belladonna rooted and transgenic lines were obtained using rooting technology and stable plant genetic transformation. Gene level, TAs content, and Fe accumulation levels were analyzed. The results showed that AbbHLH29 promotes the biosynthesis of scopolamine and anisodamine in belladonna by directly regulating the expression of the AbH6H gene; and maintains iron homeostasis in belladonna plants by indirectly regulating the iron chelate reductase FRO2 and the ferrous transporter IRT1. This research is not only significant in revealing the molecular mechanism by which AbbHLH29 regulates belladonna alkaloid biosynthesis and iron homeostasis, but also provides important technical support for future research on belladonna alkaloid metabolic engineering. Attached Figure Description

[0014] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided for illustration:

[0015] Figure 1This is the technical approach of the present invention.

[0016] Figure 2 This experiment investigated the iron deficiency treatment of belladonna. AB: morphology after iron deficiency treatment; CD: chlorophyll a and b content (mg / L) before and after iron deficiency treatment; -FE: iron deficiency treatment; +FE: full culture; scale bar: 2cm, 3cm; error bar: standard error of 3 samples; t-test was used for statistical analysis of significant differences (*P<0.05, **P<0.01).

[0017] Figure 3 The study aimed to determine the expression levels of related genes and metabolites in belladonna after iron deficiency. A: Root and leaf biomass before and after iron deficiency treatment; B: Relative expression levels of FRO2 and IRT1 before and after iron deficiency treatment; C: Alkaloid content before and after iron deficiency treatment; D: AbH6H gene expression level before and after iron deficiency treatment; -FE: Iron deficiency treatment; FE: Whole culture; The error bar represents the standard error of three samples. A t-test was used for statistical analysis of significant differences (*P<0.05, **P<0.01).

[0018] Figure 4 Co-expression analysis of AbbHLH29 in belladonna; co-expression analysis of AbbHLH29 and alkaloid biosynthesis genes in belladonna in primary root, lateral root, mature seed, mature fruit, green fruit, stem, flower, leaf and bud, with colors representing from low relative expression (blue) to high relative expression (red).

[0019] Figure 5 The relative expression level of AbbHLH29 after iron deficiency treatment was shown, with PGK as an internal reference gene.

[0020] Figure 6 The relative expression levels of AbbHLH29 and AbH6H in various tissues of Atropine were represented; PGK was used as an internal reference gene (FB: flower bud; FL: flower; FR: fruit; L: leaf; PR: taproot; S: stem; SR: lateral root); the values ​​are averages, and all results were tested in triplicate.

[0021] Figure 7 To create and detect AbbHLH29-overexpressing belladonna hairy roots by PCR; A: induction of explants; B: single-clone hairy roots; C: expansion culture of hairy roots; DF: detection of 35S::AbbHLH29, NPTII, rolB, and rolC; M: 2000bp marker; P: positive control; N: negative control; 1–5 are individual lines overexpressing hairy roots.

[0022] Figure 8To create and detect interference with AbbHLH29 belladonna hairy roots using PCR; A: induction of explants; B: single-clone hairy roots; C: expansion culture of hairy roots; DF: detection of ibHLH29::OCS, NPTII, rolB, and rolC; M: 2000bp marker; P: positive control; N: negative control; 1–5 are individual lines interfering with hairy roots.

[0023] Figure 9 This study analyzed the gene expression levels of AbbHLH29 and AbH6H in transgenic belladonna hairy roots with overexpression and interference. A–B: Gene expression level of AbbHLH29 in AbbHLH29-overexpressing / interfered AbbHLH29-overexpressing belladonna hairy roots; C–D: Gene expression level of AbbH6H in AbbHLH29-overexpressing / interfered AbbHLH29-overexpressing belladonna hairy roots. The error bar is defined as the standard error of three samples. A t-test was used for statistical analysis of significant differences (*P<0.05, **P<0.01).

[0024] Figure 10 To determine the content of TAs in the hairy roots of AbbHLH29 transgenic belladonna, the following assays were performed: A–C: determination of hyoscyamine (A), scopolamine (B), and hyoscyamine (C) in the hairy roots of AbbHLH29 transgenic and control belladonna; D–F: determination of hyoscyamine (D), scopolamine (E), and hyoscyamine (F) in the hairy roots of AbbHLH29 transgenic and control belladonna. The error bar represents the standard error of three samples. A t-test was used for statistical analysis of significant differences (*P<0.05, **P<0.01).

[0025] Figure 11 To create and detect AbbHLH29-overexpressing belladonna plants using PCR; A: explant induction; B: transgenic belladonna; C: transplantation of transgenic belladonna; DE: detection of 35S::AbbHLH29 and NPTII; M: 2000bp marker; P: positive control; N: negative control; 1–4 are individual lines of transgenic belladonna overexpressing plants.

[0026] Figure 12 To create and detect interference AbbHLH29 belladonna plants by PCR; A: induction of explants; B: transgenic belladonna; C: transplantation of transgenic belladonna; DE: detection of iAbbHLH29::OCS and NPTII; M: 2000bp Marker; P: positive control; N: negative control; 1-4 are individual lines of interference transgenic belladonna plants.

[0027] Figure 13To analyze the gene expression levels of AbbHLH29 and AbH6H in transgenic belladonna plants with overexpression and interference; A–B: gene expression level of AbbHLH29 in transgenic belladonna plants with overexpression / interference with AbbHLH29; C–D: gene expression level of AbbH6H in transgenic belladonna plants with overexpression of AbbHLH29. The error bar is the standard error of three samples, and the t-test was used for statistical analysis of statistical significance differences (*P<0.05, **P<0.01).

[0028] Figure 14 To determine the TAs content in leaves of AbbHLH29 transgenic belladonna plants with overexpression and interference, the following assays were performed: A–C: determination of hyoscyamine (A), anisodamine (B), and scopolamine (C) in leaves of AbbHLH29 transgenic belladonna plants with overexpression; DF: determination of hyoscyamine (D), anisodamine (E), and scopolamine (F) in leaves of AbbHLH29 transgenic belladonna plants with interference. The error bar was defined as the standard error of three samples. A t-test was used for statistical analysis of significant differences (*P<0.05, **P<0.01).

[0029] Figure 15 To determine the TAs content in the roots of AbbHLH29 transgenic belladonna plants with overexpression and interference, the following assays were performed: A–C: determination of hyoscyamine (A), scopolamine (B), and hyoscyamine (C) in the roots of AbbHLH29 transgenic and control belladonna plants; DF: determination of hyoscyamine (D), scopolamine (E), and hyoscyamine (F) in the roots of AbbHLH29 transgenic and control belladonna plants with interference. The error bar represents the standard error of three samples, and a t-test was used for statistical analysis of significant differences (*P<0.05, **P<0.01).

[0030] Figure 16 To determine the iron content of transgenic belladonna plants with overexpression and interference: A: Iron content determination of transgenic belladonna with overexpression of AbbHLH29; B: Iron content determination of transgenic belladonna with interference of AbbHLH29. The error bar is the standard error of 3 samples. A t-test was used for statistical analysis of significant differences (*P<0.05, **P<0.01).

[0031] Figure 17 To determine the chlorophyll content in transgenic belladonna plants with overexpression and interference: A: Comparison of leaves from overexpressed / interference-prone AbbHLH29 plants with wild type; B: Comparison of leaves from overexpressed / interference-prone AbbHLH29 plants with wild type; C and D: Determination of chlorophyll a and b in overexpressed / interference-prone AbbHLH29 plants. The error bar is the standard error of three samples, and the t-test was used for statistical analysis of significant differences (*P<0.05, **P<0.01).

[0032] Figure 18To determine the biomass of transgenic belladonna plants with overexpression and interference: A: Determination of aboveground biomass of AbbHLH29 plants with overexpression / interference; B: Determination of underground biomass of AbbHLH29 plants with overexpression / interference. The error bar is the standard error of three samples, and the t-test was used for statistical analysis of significant differences (*P<0.05, **P<0.01).

[0033] Figure 19 To analyze the gene expression levels of AbFRO2 and AbIRT1 in transgenic belladonna plants with overexpression and interference; A–B: gene expression level of FRO2 in transgenic belladonna plants with overexpression / interference; C–D: gene expression level of IRT1 in transgenic belladonna plants with overexpression / interference. The error bar is the standard error of three samples, and the t-test was used for statistical analysis of statistical significance differences (*P<0.05, **P<0.01). Detailed Implementation

[0034] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0035] The technical route of the present invention is as follows: Figure 1 As shown.

[0036] The plant materials used in this invention are wild-type belladonna and Nicotiana benthamiana, both derived from germplasm resources preserved in our laboratory. The strains are Escherichia coli: DH5α, used for vector construction; and Agrobacterium tumefaciens: EHA105, C58C1, and GV3101, used for belladonna plant genetic transformation experiments, root development genetic transformation experiments, and subcellular localization experiments, respectively.

[0037] Commonly used culture medium preparation

[0038] (1) LB medium: Weigh 10g of sodium chloride, 10g of tryptic peptone and 5g of yeast extract and dissolve them in 1L of ultrapure water. Stir well and autoclave at 121℃ for 20min. Add 15g of agar to prepare solid medium.

[0039] (2) YEP medium: Weigh 8.86g of liquid premixed medium powder and dissolve it in 500mL of ultrapure water. Stir well and autoclave at 121℃ for 20min. To prepare solid medium, add 8.5g of agar.

[0040] (3) MS medium: Weigh 30g of sucrose and 4.43g of MS powder and dissolve them in 1L of ultrapure water. After stirring evenly, adjust the pH to 5.8 with NaOH and autoclave at 121℃ for 20min. To prepare solid medium, add 8g of agar. To prepare gel medium, add 4g of plant gel.

[0041] (4) Belladonna chalazae culture medium: MS agar solid medium + 0.5 mg / L IBA + 200 mg / L Cef;

[0042] (5) Belladonna root tethering medium: MS gel medium + 200 mg / L Cef;

[0043] (6) Belladonna root transformation culture medium: MS liquid medium + 100 mg / L AS;

[0044] (7) Belladonna plant transformation culture medium: MS liquid medium + 3 mg / L 6-BA + 1 mg / L NAA;

[0045] (8) Screening and regeneration medium for belladonna plants: MS agar medium + 3 mg / L 6-BA + 1 mg / L NAA + 200 mg / L L-Cef + 100 mg / L Kana / Hy / Gly

[0046] (9) Belladonna root selection medium: MS gel medium + 200 mg / L Cef + 100 mg / L Kana / Hgy / Gly

[0047] (10) Hogland culture medium: Weigh 1.04g of Hogland dry powder (iron-deficient) mixture, add 2mL of calcium concentrate, stir well and adjust pH to 6.0.

[0048] DNA was extracted from positive transgenic plants using the TPS method, and promoter amplification template DNA was extracted using the CTBA method. Total RNA was extracted from plant materials using the RNAprep Pure Plant Total RNA Extraction Kit (DP432) from Beijing Tiangen Biotech Co., Ltd., following the instructions provided in the kit. Belladonna cDNA was prepared using the EvoM-MLV reverse transcription kit from Aikerui Biotechnology Co., Ltd., and quantitative PCR experiments were performed using the dye method (SYBR Green I) using the 2xSP qPCR Mix kit from Chongqing Baoguang Biotechnology Co., Ltd.

[0049] Example 1. Effects of iron deficiency treatment on growth, development, and alkaloid content of belladonna.

[0050] 1. Phenotypic observation of belladonna after iron deficiency treatment

[0051] Several belladonna seedlings were germinated. After germination, seedlings with uniform growth were transplanted into soil. When the seedlings reached about 20cm in height, uniformly growing wild belladonna seedlings were selected and treated with iron deficiency. The germinated, sterile wild-type belladonna seedlings were then cultured in soil for 14 days. After the soil was thoroughly cleaned, they were placed in different types of Hogrange solutions for cultivation. Physiological indicators of the plants were observed. The plants were divided into a control group and an experimental group. The control group consisted of belladonna seedlings cultured in full-body Hogrange solution, while the experimental group consisted of belladonna seedlings treated with iron-deficient Hogrange solution. Phenotypic changes began to appear after approximately 21 days of continuous cultivation.

[0052] The results are as follows Figure 2 As shown in Figure A, belladonna plants cultured under iron-deficient conditions exhibited inhibited growth, with leaves turning yellow and white, poorly developed and shorter roots with fewer root formations, resulting in stunted overall plant growth. Iron is crucial for plants, as chlorophyll synthesis is inseparable from iron, and the experimental results are consistent with those of iron-deficient plants. As shown in Figure B, under iron-deficient conditions, both chlorophyll a and chlorophyll b in the plant leaves were significantly lower than in the control group, indicating a substantial inhibition of chlorophyll production. This demonstrates that iron deficiency significantly inhibits the growth and development of belladonna plants and chlorophyll synthesis.

[0053] 2. Determination of gene content, biomass, and alkaloids in belladonna after iron deficiency treatment.

[0054] Fresh belladonna material from the above-described grouped treatments was placed in 1.5 mL centrifuge tubes and flash-frozen in liquid nitrogen. RNA was extracted and reverse-engineered into cDNA for qPCR experiments of relevant genes (primers are shown in Table 1). The plant height and root length of the remaining materials in the two experimental groups were measured using calipers. The materials were washed and dried to facilitate biomass statistics and metabolite determination.

[0055] Table 1 Primers for Quantitative Real-Time PCR

[0056]

[0057] Quantitative PCR using the SYBR Green I dye method was performed using the 2xSP qPCR Mix kit from Chongqing Baoguang Biotechnology Co., Ltd. The reaction steps are as follows:

[0058] (1) Use enzyme-free water to dilute 20 times the reverse transcribed cDNA as the amplification template for real-time quantitative PCR;

[0059] (2) Use 96-well plates to conduct experiments. Prepare the following reaction systems. Set up 3 biotechnological replicates for each sample. The entire experiment was conducted on ice.

[0060] Table 2. Quantitative PCR reaction system

[0061]

[0062]

[0063] (3) Real-time quantitative PCR reaction program: 95℃, 3 min; 95℃, 10 s; 60℃, 30 s; Steps 2 and 3 were set to 40 cycles; then the melting curve was plotted, with the program set as follows: starting temperature 65℃, ending temperature 90℃. Each cycle was increased by 0.5℃ and the reaction time was set to 10 s. The obtained data results were used as internal references for AbPGK gene, and the relative expression level of the gene was calculated according to the 2-ΔΔCt statistical analysis method.

[0064] The results showed that the metabolism and physiological state of belladonna plants were altered when exposed to iron deficiency. Biomass analysis of the iron-deficient plants revealed that iron deficiency led to poor plant growth; the leaves and roots of plants grown under iron-deficient conditions were approximately twice the weight of those grown under full-culture conditions. Figure 3 (A). Furthermore, a literature review revealed that iron chelate reductase (FRO2) and ferrous transporter (IRT1) play crucial roles in iron transport and absorption in Arabidopsis thaliana. Therefore, qPCR was used to determine the gene expression levels of FRO2 and IRT1, and it was found that under iron deficiency conditions, the gene levels of FRO2 and IRT1 were induced. Figure 3 (B) This experimental result is consistent with the literature reports.

[0065] The extraction of alkaloids in this invention follows the method described by Bedewitz et al. (2014). Dried belladonna material is ground into a fine powder using a mortar and pestle. 25 mg of the powder is weighed into each 10 mL centrifuge tube, and 1 mL of 20% HPLC-grade methanol (containing 0.1% formic acid) is added. The mixture is then incubated at 37°C with shaking for 3 hours. The material is then centrifuged at room temperature for 15 minutes (12000 rpm). The supernatant is collected, filtered, diluted 20 times, and placed into a sample vial.

[0066] Alkaloids were detected using a Thermo Fisher Scientific liquid chromatography-mass spectrometry (UPLC-MS) system to determine the contents of hyoscyamine, anisodamine, and scopolamine in hairy root materials, transgenic plants, and iron-deficient belladonna. The chromatographic column was a Hypersil Gold C18 column (2.1 × 100 mm, 1.9 μm); the column temperature was set at 35 °C; the injection volume was 4 μL; the mobile phase consisted of acetonitrile (phase A) and 0.1% formic acid (phase B); the flow rate was 0.4 mL / min; and a binary gradient elution was used for the sample.

[0067] Studies have shown that ferrous ions are essential coenzyme factors in the biosynthetic pathway of secondary metabolites, thus it is speculated that iron deficiency may be involved in alkaloid synthesis. This invention measured the alkaloid content of iron-deficient belladonna plants and found that the contents of hyoscyamine, scopolamine, and anisodamine all decreased under iron-deficient conditions, with the decrease in hyoscyamine content being the most significant. Figure 3 (C). Our research group has previously conducted a comprehensive analysis of the synthetic pathways of TAs, among which AbH6H belongs to the α-ketoglutarate / ferric-dependent dioxygenase family. Gene expression level analysis revealed the following results: Figure 3 As shown in Figure D, the expression level of the gene AbH6H in the alkaloid synthesis pathway of belladonna was significantly upregulated under iron deficiency.

[0068] Example 2. Screening and Cloning of Candidate Genes

[0069] 1. Transcriptome analysis of belladonna

[0070] Based on bioinformatics analysis and relevant domestic and international literature reports on iron deficiency response transcription factors, the candidate gene AbbHLH29 was selected for this study. Specific primers targeting the CDS region of the AbbHLH29 electronic sequence were designed using Snapgene 1.1.3 software. Using iron-deficient belladonna cDNA as a template, amplification was performed using Primer Star Max DNA Polymerase. PCR amplification results were detected by agarose gel electrophoresis. The purified product was ligated into the pJET1.2 / blunt vector, transformed into DH5α culture, and after two days of culture, positive colonies were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing to obtain the physical sequence of AbbHLH29 (SEQ ID No. 11). Electronic sequences of the Arabidopsis bHLH family were downloaded from NCBI and aligned using MEGA multiple sequence alignment. DNAMAN was used for phylogenetic tree construction.

[0071] Co-expression analysis of the belladonna bHLH gene family and alkaloid synthesis pathway genes in different tissues showed that aba_locus_128992 and the belladonna alkaloid synthesis pathway gene AbH6H had similar expression patterns. Figure 4 Phylogenetic analysis was performed on the protein sequence of aba_locus_128992 and members of the bHLH protein family in the model plant Arabidopsis thaliana. The results showed that aba_locus_128992 is closely related to AtbHLH29 in Arabidopsis, therefore it was named AbbHLH29. AtbHLH29 in Arabidopsis is an iron-deficiency-inducible transcription factor. Based on previous iron-deficiency induction experiments and the fact that AbH6H requires iron ions as a coenzyme factor for catalysis, AbbHLH29 was identified as a candidate gene for this study, and further investigation will be conducted to determine whether it participates in the biosynthesis of belladonna alkaloids.

[0072] 2. AbbHLH29 is induced by iron deficiency

[0073] By subjecting plants to iron deficiency treatment, changes in the iron deficiency response transcription factor AbbHLH29 were measured. Figure 5 As shown, the expression level of AbbHLH29 in belladonna was significantly upregulated in response to iron deficiency. These results indicate that AbbHLH29, like AbH6H, is induced by iron deficiency, suggesting that AbbHLH29 may be involved in the synthesis of alkaloids in belladonna.

[0074] 3. Expression pattern analysis of AbbHLH29 and AbH6H in belladonna tissue

[0075] qPCR-specific primers for AbbHLH29 and AbH6H were designed, and quantitative real-time PCR was used to measure their tissue expression patterns in seven tissues: flower, bud, leaf, stem, taproot, lateral root, and flower bud. Simultaneously, the induced expression of the AbbHLH29 gene in two belladonna experimental groups was measured and analyzed. Three biological replicates and three technical replicates were set up for each group.

[0076] The relative expression levels of AbbHLH29 and AbH6H genes in different tissues of belladonna plants were analyzed by qPCR. Figure 6 As shown, AbbHLH29 and AbH6H have extremely high tissue expression similarity. The expression level of AbbHLH29 in the lateral root, the site of alkaloid synthesis, is relatively higher than that in other tissues. Therefore, it is preliminarily speculated that AbbHLH29 may affect the alkaloid content in belladonna by regulating the expression level of the AbH6H gene.

[0077] Example 3. Determination of gene expression levels and alkaloid content in the roots of AbbHLH29 transgenic belladonna.

[0078] 1. Construction of AbbHLH29 overexpression and interference vector

[0079] Based on experimental requirements, the plant expression vector pBI121 was selected as the expression vector for overexpressing plant materials. Specific upstream and downstream primers were designed based on the electronic sequence obtained from sequencing (SEQ ID No. 11) (Table 3). After amplifying the target fragment, double digestion with XbaI and BamHI was performed, followed by recovery and overnight T4 ligation to transform E. coli competent cells to obtain positive colonies. After successful sequencing, plasmids were extracted for subsequent experiments.

[0080] Table 3. Primers for PCR construction of overexpression vectors

[0081]

[0082] Based on experimental requirements, the plant expression vectors pHANNIBAL and pBin19 were selected as expression vectors for interfering with plant materials. Specific upstream and downstream primers were designed based on the electronic sequence obtained from sequencing (SEQ ID No. 11) (Table 4). First, two interfering fragments, one forward and one reverse, were amplified and ligated to the intermediate vector pHANNIBAL. After amplifying the target fragment, double digestion with enzymes was performed, and the overnight T4 solution was recovered and ligated to transform competent E. coli cells to obtain positive colonies. After successful sequencing, the plasmid was extracted. The successfully sequenced intermediate vector was double digested with Sac I and Spe I (isosaccharides of Xba I), and the final vector pBin19 was double digested with Sac I and Xba I, respectively. After recovery, the overnight T4 solution was recovered and ligated to transform competent E. coli cells to obtain positive colonies. After successful sequencing, the plasmid was extracted for subsequent experiments.

[0083] Table 4. PCR primers for constructing the interference vector

[0084]

[0085] The successfully sequenced overexpression vector and interference vector plasmids were transformed into C58C1 and EHA105, respectively, to obtain transgenic overexpression roots and plants.

[0086] 2. Obtaining AbbHLH29 transgenic hairy roots

[0087] To investigate the regulatory role of transcription factor AbbHLH29 in the alkaloid synthesis pathway in belladonna, this study overexpressed and interfered with AbbHLH29, successfully obtaining transgenic hairy roots. The vectors pBI121-AbbHLH29 and pBin19-AbbHLH29 constructed in this example were transformed into *Agrobacterium rhizogenes* C58C1 to obtain engineered strains. These engineered strains were genetically transformed using leaves from sterile belladonna seedlings as explants. Roots were induced from the leaves on MS gel medium containing 400 mg / L cephalosporin and 50 mg / L kanamycin. Figure 7 A; Figure 8 A) Single clones were inoculated into gel medium containing 200 mg / L cephalosporin and cultured under suitable conditions until they were ready for subsequent experiments. Figure 7 B; Figure 8 B). Positive lines were obtained by extracting genomic DNA from the hairy roots of belladonna and performing positive detection. Figure 7 D; Figure 8 (D). Cut 3-5 hairy roots, approximately 3 cm long (with root tips), from the positive hairy roots and inoculate them into MS liquid medium. After 28 days of culture on a shaker in a dark incubator, harvest the hairy roots for subsequent testing. Figure 7 C; Figure 8(C). Three biological replicates were set up for each line. The blank control consisted of hairy roots obtained by transforming C58C1 with pBI121 and pBin19 blank vectors.

[0088] 3. Detection of gene expression levels in alkaloid synthesis pathways in AbbHLH29 transgenic hairy roots

[0089] In this experiment, RNA was extracted from monoclonal positive hairy roots and reverse-engineered into cDNA. Using cDNA as a template and the belladonna gene AbPGK as an internal control, real-time quantitative PCR was used to detect the expression levels of AbbHLH29 and genes involved in the alkaloid synthesis pathway in belladonna hairy roots. Analysis of the results showed that the expression level of AbbHLH29 in belladonna hairy roots overexpressing AbbHLH29 was significantly higher than that in the control. Figure 9 The expression level of the synthetic pathway gene AbH6H was increased by 5.22–7.01 times compared to the control. Figure 9 The expression level of AbbHLH29 increased by 3.86–6.03 times in the hairy roots of belladonna with AbbHLH29 interference compared to the control, with a decrease of 63%–77%. Figure 9 The expression levels of AbH6H were also significantly reduced, decreasing by 60%–76%. Figure 9 (D). The above experimental results indicate that transcription factor AbbHLH29 is a positively regulating transcription factor in the synthesis of belladonna alkaloids.

[0090] 4. Extraction and content determination of root alkaloids from transgenic belladonna.

[0091] To further investigate the effect of AbbHLH29 on the synthesis of tropane alkaloids, this study analyzed the contents of anisodamine and scopolamine in the roots of the blank control and transgenic belladonna using liquid chromatography-mass spectrometry (LC-MS). The extraction and determination of alkaloids followed the method of Bedewitz (Bedewitz et al., 2014). Compared with the control, the anisodamine content in the hairy roots increased by 1.84–2.28 times after overexpression, and the scopolamine content increased by 1.90–2.05 times. Figure 10 (AC); the content of scopolamine in hairy roots decreased by 39%–60% and the content of hyoscyamine decreased by 22%–57% after interference. Figure 10 ,DF). The content detection of alkaloids in transgenic hairy roots showed that AbbHLH29 can positively regulate the increase of scopolamine and anisodamine content in belladonna hairy roots.

[0092] Example 4. Determination of gene expression levels and alkaloid content in AbbHLH29 transgenic belladonna plants

[0093] 1. Obtaining AbbHLH29 transgenic plants

[0094] The vectors pBI121-AbbHLH29 and pBin19-AbbHLH29 constructed in Example 5 were transformed into Agrobacterium EHA105 to obtain engineered strains. The engineered strains were then used as explants from sterile belladonna seedlings for stable genetic transformation. The leaves were induced to sprout on MS medium containing 50 mg / L kanamycin and 200 mg / L cephalosporin, plus 3 mg / L 6-BA and 1 mg / L NAA. Figure 11 A; Figure 12 A) After sprouting, belladonna was inoculated into a container of MS agar medium containing 0.5 mg / L IBA and cultured until it was ready for subsequent experiments. Figure 11 ,BC; Figure 12 , BC). Genomic DNA was extracted from belladonna plants and positive lines were obtained through positive detection. Figure 11 DE; Figure 12 DE).

[0095] 2. Detection of gene expression levels in alkaloid synthesis pathways in AbbHLH29 transgenic belladonna plants

[0096] In this experiment, RNA was extracted from transgenic positive plants and reverse-engineered into cDNA. Real-time quantitative PCR was used to detect the expression levels of genes involved in the alkaloid synthesis pathway in AbbHLH29 and belladonna plants. Analysis of the results showed that the expression level of AbbHLH29 in belladonna plants overexpressing AbbHLH29 was significantly higher than that in the control group. Figure 13 The expression level of the synthetic pathway gene AbH6H was increased by 5.22–7.01 times compared to the control. Figure 13 The expression level increased to 3.86–6.03 times. Furthermore, in belladonna plants with AbbHLH29 interference, the expression level of AbbHLH29 was significantly reduced compared to the control, decreasing by 74%–89% (13, B), and the expression level of AbH6H was also significantly reduced, decreasing by 56%–85% ( ). Figure 13 (D). The above experimental results indicate that transcription factor AbbHLH29 is a positively regulating transcription factor in the synthesis of belladonna alkaloids.

[0097] 3. Detection of alkaloids in AbbHLH29 transgenic belladonna plants

[0098] This study used liquid chromatography-mass spectrometry (LC-MS) to analyze the contents of anisodamine and scopolamine in wild-type and transgenic belladonna plants. Compared with the wild type, the contents of anisodamine and scopolamine in the aerial parts of transgenic belladonna plants overexpressing AbbHLH29 were significantly increased, with anisodamine content increasing by 2.15–2.87 times. The content of scopolamine in the leaves showed a larger increase, averaging 2.92–3.81 times. Figure 14 (AC); The aerial parts of transgenic belladonna interfering with AbbHLH29 showed a significant decrease in the content of both anisodamine and scopolamine, with anisodamine content decreasing by 41%–67% and scopolamine content decreasing by 46%–55%. Figure 14 ,DF).

[0099] Overexpression increased the content of scopolamine in the underground parts of the plant by 1.44–1.77 times and the content of anisodamine by 1.69–2.64 times. Figure 15 The content of scopolamine in the underground parts of AbbHLH29 plants decreased by 51%–66%, and the content of hyoscyamine decreased by 43%–60%. Figure 15 ,DF).

[0100] In summary, the expression of anisodamine and scopolamine in transgenic plants showed significant increases in both leaves and roots, with scopolamine showing a higher increase in leaves; the expression of anisodamine and scopolamine in transgenic plants showed significant inhibition in both leaves and roots.

[0101] 4. Determination of iron content in AbbHLH29 transgenic plants

[0102] AbbHLH29 is a closely related gene of Arabidopsis thaliana, one of the important transcription factors involved in the regulation of iron homeostasis. Iron deficiency induction experiments have confirmed that AbbHLH29 is indeed induced by iron deficiency. To investigate whether the overexpression or deletion of AbbHLH29 affects the iron content of plants, the iron content of young shoots from transgenic plants was measured. The specific procedures for iron content measurement are as follows:

[0103] (1) Select healthy transgenic positive plants and wild-type plants, and place them in aluminum foil at -80℃ for rapid freezing;

[0104] (2) After freeze-drying the material for 2 days, grind it with a mortar and pestle.

[0105] (3) Weigh 10 mg of each sample into a 2 mL centrifuge tube, add 60 μL of concentrated nitric acid to each sample, nitrate at 70 °C for 12 h, add 140 μL of ultrapure water after nitrification, centrifuge for 10 min (2000 r / min), pass through a water film, centrifuge again under the same conditions, and then take the supernatant and put it into a sample bottle.

[0106] (4) The metal concentration was measured using an inductively coupled plasma mass spectrometer (ICP-MS) (wavelength: 238.204 nm).

[0107] The results are as follows Figure 16 As shown, the iron content of overexpression lines was increased to some extent, with the OE7 line showing a two-fold increase; the iron content of belladonna plants decreased significantly after interference. These results demonstrate that AbbHLH29 is involved in regulating iron absorption by belladonna plants.

[0108] 5. Determination of leaf phenotype and chlorophyll content of AbbHLH29 transgenic belladonna overexpression / interference

[0109] Through the creation of transgenic plants, this study cultured transgenic belladonna that overexpressed and interfered with the expression of [the plant species]. Figure 17 It can be clearly observed that there are certain differences in the color of their leaves. Figure 17 A) The leaves of the interfered type were yellower than those of the wild type, while the leaves of the overexpressed type were greener than those of the wild type. Figure 17 (B) It is speculated that the expression of AbbHLH29 affects the chlorophyll content of belladonna plants. The chlorophyll content was determined according to the method of Ran Hairong (Ran Hairong, 2022).

[0110] Analysis of the results revealed that the chlorophyll content in belladonna plants overexpressing AbbHLH29 was increased, with chlorophyll a content increasing by 1.34–1.76 times, while the content of chlorophyll b showed no significant difference. The total chlorophyll content increased by 1.24–1.66 times. Figure 17 (C); The chlorophyll content in belladonna plants affected by AbbHLH29 was reduced, with chlorophyll a content decreasing by 50%–75%, chlorophyll b content decreasing by 49%–68%, and total chlorophyll content decreasing by 50%–72%. Figure 17 The above experimental results indicate that the transcription factor AbbHLH29 affects the synthesis of chlorophyll in the leaves of belladonna plants.

[0111] 6. Statistical analysis of biomass from AbbHLH29 transgenic belladonna overexpression / interference

[0112] pass Figure 17 In B, a clear difference in plant biomass was observed. The biomass of the transgenic belladonna after freeze-drying was statistically analyzed, and the results are as follows: Figure 18As shown, the overall dry weight of the aboveground parts of the plants after interference was 60% lower than that of the wild type, and the aboveground part weight of the overexpressing plants was approximately 1.28 times that of the wild type. The overall dry weight of the underground parts of the plants after interference was 40% lower than that of the wild type, while there was no significant difference in the overexpressing plants. These results indicate that interference with AbbHLH29 significantly reduced the biomass of both the aboveground and underground parts of the plants, while overexpression only affected the aboveground biomass.

[0113] 7. Detection of downstream target genes of AbbHLH29

[0114] Previous studies have demonstrated that AbFRO2 and AbIRT1 are significantly induced by iron deficiency. To investigate whether AbbHLH29 affects the gene expression of AbFRO2 and AbIRT1, qPCR detection was performed on existing transgenic plants, and the results were as follows: Figure 19 As shown, the expression levels of AbFRO2 and AbIRT1 did not change significantly in overexpression plants, but in interference plants, the expression levels of both AbFRO2 and AbIRT1 decreased significantly. These results indicate that interfering with AbbHLH29 expression directly affects the expression of AbFRO2 and AbIRT1, but overexpression has no effect, suggesting that other genes may be involved in the process.

[0115] The above-described embodiments are merely preferred embodiments provided to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

Claims

1. The application of overexpression of transcription factor AbbHLH29 in improving the biological traits of belladonna, characterized in that, The improved belladonna biological traits are to increase the iron content, chlorophyll a content, aboveground biomass, and the content of scopolamine and anisodamine in belladonna. The nucleotide sequence of the transcription factor AbbHLH29 is shown in SEQ ID No.

11.

2. A method for improving the biological traits of belladonna by overexpressing transcription factor AbbHLH29, characterized in that, The procedure includes the following steps: constructing an AbbHLH29 overexpression vector, transforming belladonna plants under Agrobacterium-mediated transformation, and screening for positive transgenic lines to obtain belladonna with improved biological traits. The nucleotide sequence of the transcription factor AbbHLH29 is shown in SEQ ID No.

11. The improved biological traits are: increasing iron content, increasing chlorophyll a content, increasing aboveground biomass, and increasing the content of scopolamine and anisodamine.

3. The method according to claim 2, characterized in that, The process of constructing the AbbHLH29 overexpression vector is as follows: the nucleic acid sequence of AbbHLH29 is obtained by PCR amplification using the primers shown in SEQ ID No. 12 and SEQ ID No. 13, and then double digested with XbaI and BamHI, and ligated into the plant expression vector pBI121 to obtain the overexpression vector pBI121-AbbHLH29.

4. The method according to claim 2, characterized in that, The Agrobacterium is C58C1 or EHA105.

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