Andrographis transcript factor and its coding gene related to new andrographolide content regulation and application

Abstract

The present application relates to a andrographis transcription factor and its coding gene and application related to new andrographolide content regulation, the andrographis transcription factor has one of the following amino acid residue sequences: a1) the protein consisting of the amino acid sequence shown in sequence 1; a2) the protein derived from a1) by substitution and / or deletion and / or addition of one or several amino acid residues and has new andrographolide content regulation function; a3) the protein consisting of the amino acid sequence shown in sequence 3; a4) the protein derived from a3) by substitution and / or deletion and / or addition of one or several amino acid residues and has new andrographolide content regulation function. Both transcription factors and their coding genes can regulate the new andrographolide content in andrographis, and have important theoretical and practical significance for cultivating high new andrographolide content varieties.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to the application of Andrographis paniculata transcription factors ApMYC2 and ApbZIP46 in regulating the content of neoandrographolide. Background Technology

[0002] Andrographis paniculata ( Andrographis paniculata (Burm. f.) Wall. ex Nees is an important medicinal plant distributed in Southeast Asia and South Asia. It possesses various pharmacological effects and is listed in the Chinese Pharmacopoeia and the United States Pharmacopeia Dietary Supplements. Neoandrographolide is one of the main active diterpenoid compounds in Andrographis paniculata, exhibiting mild cytotoxicity and various pharmacological activities, such as anti-herpes simplex virus, anti-inflammatory, antioxidant, antiparasitic, and hepatoprotective effects. It is also used as a potent chemosensitizer with low cytotoxicity.

[0003] It has been reported that bHLH and bZIPs regulate the complex control of development in various plant types and participate in the abiotic stress regulatory network of terpene biosynthesis. In rice, bHLH026 activates the transcription of downstream genes involved in diterpene antitoxin biosynthesis, thereby affecting the metabolic level of diterpene antitoxins and enhancing disease resistance. ABA and JA-induced AabHLH113 actively regulates artemisinin biosynthesis through interactions with target gene promoters. Furthermore, PgbZIPs activate the biosynthesis of platycodon saponins after MeJA treatment. SmbHLH10 and SmbHLH148 actively regulate the biosynthesis of tanshinone in tanshinone. Summary of the Invention

[0004] The technical problem to be solved by this invention is how to increase the content of neoandrographolide, an active ingredient in Andrographis paniculata.

[0005] This invention provides a transcription factor related to the regulation of andrographolide content, which is a protein having one of the following amino acid residue sequences: a1) A protein consisting of the amino acid sequence shown in Sequence 1; a2) Proteins derived from a1) by substitution and / or deletion and / or addition of one or more amino acid residues and having the function of regulating the content of new andrographolide. a3) A protein composed of the amino acid sequence shown in sequence 3; a4) Proteins derived from a3) by substitution and / or deletion and / or addition of one or more amino acid residues, and which have the function of regulating the content of new andrographolide.

[0006] Furthermore, the transcription factor is derived from Andrographis paniculata.

[0007] The present invention also provides a gene encoding the above-mentioned transcription factor.

[0008] Furthermore, the gene is any of the following DNA molecules: (b1) The nucleotide sequence is the DNA sequence shown in Sequence 2; (b2) A DNA sequence of (b1) with one or more nucleotide substitutions and / or deletions and / or additions; (b3) The nucleotide sequence is the DNA sequence shown in Sequence 4; (b4) A DNA sequence that has undergone substitution and / or deletion and / or addition of one or more nucleotides as described in (b3).

[0009] The present invention also provides recombinant expression vectors, expression cassettes or recombinant bacteria containing the above-mentioned genes.

[0010] The application of the aforementioned transcription factors, genes, recombinant expression vectors, expression cassettes, or recombinant bacteria in regulating the content of neoandrographolide in Andrographis paniculata should also be within the scope of protection of this invention.

[0011] The application of the aforementioned transcription factors, genes, recombinant expression vectors, expression cassettes, or recombinant bacteria in cultivating new Andrographis paniculata with enhanced andrographolide content should also be within the scope of protection of this invention.

[0012] The application of the aforementioned transcription factors, genes, recombinant expression vectors, expression cassettes, or recombinant bacteria in Andrographis paniculata breeding should also be within the scope of protection of this invention.

[0013] This invention provides a method for cultivating Andrographis paniculata with enhanced andrographolide content, comprising the step of overexpressing the above-mentioned transcription factor in Andrographis paniculata.

[0014] This invention involves transferring the encoding genes of two transcription factors into the leaves of *Andrographis paniculata*, resulting in a significant increase in the content of neoandrographolide compared to leaves transfected with an empty vector. This demonstrates that both transcription factors and their encoding genes can regulate the neoandrographolide content in *Andrographis paniculata*, which has important theoretical and practical significance for cultivating varieties with high neoandrographolide content. Attached Figure Description

[0015] Figure 1 The corresponding expression levels of ApMYC2 in the control (CK) and ApMYC2-overexpressing leaves of Andrographis paniculata; Figure 2 The corresponding expression levels of ApbZIP46 in the control (CK) and ApbZIP46-overexpressing leaves of Andrographis paniculata; Figure 3The transcriptional levels of ApUGT76E1 in leaves of *Andrographis paniculata* overexpressing ApMYC2 were compared with those in the control (CK). Figure 4 The transcriptional levels of ApUGT76E1 in leaves of *Andrographis paniculata* overexpressing ApbZIP46 were compared with those in the control (CK) and ApbZIP46 overexpression. Figure 5 The accumulation of neoandrographolide in leaves of *Andrographis paniculata* (CK and control) and *Andrographis paniculata* overexpressing ApMYC2 was observed. Figure 6 The accumulation of neoandrographolide in leaves of *Andrographis paniculata* (CK and control) and overexpressing ApbZIP46 was observed. Figure 7 These are the results of the interaction verification between ApbZIP46 and ApUGT76E1; Figure 8 This is the result of the interaction verification between ApMYC2 and ApUGT76E1. Detailed Implementation

[0016] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0017] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0018] In the following experiments: PrimeSTAR Max DNA polymerase (Takara Bio Engineering (Dalian) Co., Ltd.) was used to generate first-strand cDNA from total RNA. qRT-PCR analysis was performed on a CFX96 real-time quantitative PCR instrument (Bio-Rad Laboratories, Inc.) using TransScript® Green One-Step-qRT-PCR SuperMix (Beijing TransGen Biotech Co., Ltd.). Primers used for amplifying the selected genes are listed in Table 2. Three biological replicates and three technical replicates were performed with actin as an internal control gene.

[0019] The culture media used in the following examples are: 1) Complete yeast culture medium: YPDA: 1% Yeast extract, 2% Peptone, 2% Glucose, 0.02% Adenine. 2) Yeast defect screening medium: Refer to Clontech's PT3024-1 / Yeast Protocols Handbook. The various culture media are represented by the following symbols: SD-TL: -trp, -leu; SD-TLH: -trp, -leu, -his. 3AT (also written as 3-AT) is a competitive inhibitor of the yeast HIS3 protein. The vector was obtained from Shanghai Zeye Biotechnology Co., Ltd.

[0020] Example 1: Construction of Andrographis paniculata leaf overexpression 1. Obtaining coding genes Using Andrographis paniculata cDNA as a template, the full-length CDS regions of ApMYC2 and ApbZIP46 were amplified using the primers listed in Table 1, yielding the coding gene sequences of ApMYC2 and ApbZIP46. The coding gene sequence of ApMYC2 is shown in Sequence 2, and its encoded protein is shown in Sequence 1; the coding gene sequence of ApbZIP46 is shown in Sequence 4, and its encoded protein is shown in Sequence 3.

[0021] Table 1 Primers required for amplification

[0022] 2. Carrier Construction The sequence between the NcoI and SpeI sites on the pCAMBIA1302 vector was replaced with the encoding gene sequence of ApMYC2 (as shown in Sequence 2), while other sequences remained unchanged, to obtain the recombinant vector pCAMBIA1302-ApMYC2.

[0023] The sequence between the NcoI and SpeI sites on the pCAMBIA1302 vector was replaced with the gene sequence encoding ApbZIP46 (as shown in Sequence 4), while other sequences remained unchanged, to obtain the recombinant vector pCAMBIA1302-ApbZIP46.

[0024] 3. Plant transfection The pCAMBIA1302 vector (as a blank control), the recombinant vectors pCAMBIA1302-ApMYC2 and pCAMBIA1302-ApbZIP46 were transformed into EHA105 competent cells (Shanghai Weidi Biotechnology Co., Ltd.) for injection into Andrographis paniculata leaves. Three injection sites were performed for each leaf, with six biological replicates. The injected plants were cultured for 5 days to obtain Andrographis paniculata leaves overexpressing ApMYC2, leaves overexpressing ApbZIP46, and the blank control (CK). Leaves were collected for subsequent analysis.

[0025] Example 2 1. Overexpression levels of ApMYC2 and ApbZIP46 in Andrographis paniculata leaves The expression levels of transcription factors ApbZIP46 and ApMYC2 in Andrographis paniculata leaves overexpressing ApMYC2, Andrographis paniculata leaves overexpressing ApbZIP46, and the blank control plant (CK) were measured respectively. The results are as follows: Figure 1 and Figure 2 As shown, CK is the blank control. This invention constructs overexpression vectors for ApMYC2 and ApbZIP46 to transiently overexpress ApMYC2 and ApbZIP46 in plants. Compared with the control plant CK, the expression level of ApMYC2 in the leaves of *Andrographis paniculata* overexpressing ApMYC2 increased by 4.19 times; the expression level of ApbZIP46 in the leaves of *Andrographis paniculata* overexpressing ApbZIP46 increased by 5.74 times.

[0026] 2. Expression level of ApUGT76E1 overexpression in Andrographis paniculata leaves The expression levels of the gene ApUGT76E1 were measured in the leaves of *Andrographis paniculata* overexpressing ApMYC2, leaves overexpressing ApbZIP46, and the blank control plant CK, respectively. CK served as the blank control. The gene encoding ApUGT76E1 is shown in sequence 6, and the amino acid sequence of the corresponding protein is shown in sequence 5. The results are as follows: Figure 3 and Figure 4 As shown, compared with the control plant CK, the expression level of glycosyltransferase ApUGT76E1 in leaves of Andrographis paniculata overexpressing ApMYC2 increased by 2.37 times; and the expression level of glycosyltransferase ApUGT76E1 in leaves of Andrographis paniculata overexpressing ApbZIP46 increased by 1.33 times.

[0027] The above expression levels were detected using qRT-PCR analysis, and the required primers are shown in Table 2.

[0028] Table 2 Primers required for qRT-PCR analysis

[0029] 3. Determination of Andrographolide Content The content of neoandrographolide in leaves of *Andrographis paniculata* overexpressing the drug was determined by liquid chromatography-mass spectrometry (LC-MS). The LC-MS was performed using a Waters Acquity I-Class UPLC (Waters Corporation, USA) with a Waters Acquity UPLC BEH C18 column (2.1 × 50 mm, 1.7 μm) at 40℃. The injection volume was 1 μL. The mobile phase flow rate was 0.4 mL / min. The mobile phase consisted of solutions A and B. Solution A was a 0.1% (v / v) formic acid aqueous solution. Solution B was acetonitrile. Gradient elution was performed: from 0 to 15 min, the volume fraction of solution B in the mobile phase decreased linearly from 90% to 40%.

[0030] The results are as follows Figure 5-6 As shown, compared with the control, the content of neoandrographolide in Andrographis paniculata leaves overexpressing ApMYC2 and Andrographis paniculata leaves overexpressing ApbZIP46 increased significantly by 3.05 times and 3.88 times, respectively.

[0031] The above results indicate that ApMYC2 and ApbZIP46 are positive regulators of the biosynthesis of andrographolide diterpenes (especially neoandrographolide).

[0032] Example 3 Further transient overexpression experiments and Y1H assays confirmed that ApMYC2 and ApbZIP46 act as positive regulators of diterpenoid accumulation by binding to ApUGT76E1, a key diterpenoid biosynthesis-related gene.

[0033] 1. Carrier Construction The coding sequence of ApUGT76 was replaced with the sequence between EcoRI and SacI on the pHIS2 vector to obtain the vector pHIS2-ApUGT76E1pro.

[0034] The coding sequences of ApMYC2 and AbZIP46 were replaced with the sequences between EcoRI and SacI on the pGADT7 vector to obtain the vectors pGADT7-ApMYC2 and pGADT7-AbZIP46, respectively.

[0035] pHIS2-ApUGT76E1pro was co-expressed with pGADT7-ApMYC2, pGADT7-AbZIP46, and pGADT7 (blank) in yeast, respectively, to obtain pHIS2-QD+pGADT7-AppMYC2, pHIS2-QD+pGADT7-AbZIP46, and pHIS2-QD+pGADT7 strains.

[0036] 2. Construction and culture of recombinant yeast The pHIS2-QD+pGADT7-AppMYC2 and pHIS2-QD+pGADT7-AbZIP46 strains were inoculated onto different yeast selection media (SD-TL, SD-TLH, SD-TLH+10 mM 3AT, and SD-TLH+20 mM 3AT), respectively, with strains carrying the blank vector as positive controls. Their growth was observed, and the results are as follows: Figure 7 and Figure 8 As shown.

[0037] As can be seen from the images, pHIS2-QD+pGADT7-AppMYC2 and pHIS2-QD+pGADT7-AbZIP46 can grow normally on SD-TL, SD-TLH, SD-TLH+10 mM 3AT, and SD-TLH+20 mM 3AT plates.

[0038] The control group pHIS2-ApUGT76E1+pGADT7 grew normally on SD-TL and SD-TLH plates, grew weakly on SD-TLH+10 mM 3AT plates, and did not grow on SD-TLH+20 mM 3AT plates.

[0039] The above experiments show that pHIS2-ApUGT76E1 interacts with pGADT7-APBZIP46 and pGADT7-APMYC2.

[0040] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. Transcription factors associated with the regulation of andrographolide content are proteins having one of the following amino acid residue sequences: a1) A protein consisting of the amino acid sequence shown in Sequence 1; a2) Proteins derived from a1) by substitution and / or deletion and / or addition of one or more amino acid residues and having the function of regulating the content of new andrographolide. a3) A protein composed of the amino acid sequence shown in sequence 3; a4) Proteins derived from a3) by substitution and / or deletion and / or addition of one or more amino acid residues, and which have the function of regulating the content of new andrographolide.

2. The transcription factor according to claim 1, characterized in that, The transcription factor was derived from Andrographis paniculata.

3. A gene encoding the transcription factor of claim 1.

4. The gene according to claim 3, characterized in that, The gene is any of the following DNA molecules: (b1) The nucleotide sequence is the DNA sequence shown in Sequence 2; (b2) A DNA sequence of (b1) with one or more nucleotide substitutions and / or deletions and / or additions; (b3) The nucleotide sequence is the DNA sequence shown in Sequence 4; (b4) A DNA sequence of (b3) with one or more nucleotide substitutions and / or deletions and / or additions.

5. A recombinant expression vector, expression cassette, or recombinant bacteria containing the gene described in claim 3 or 4.

6. The use of the transcription factor of claim 1 or 2, the gene of claim 3 or 4, the recombinant expression vector, expression cassette, or recombinant bacteria of claim 5 in regulating the content of neoandrographolide in Andrographis paniculata.

7. The use of the transcription factor of claim 1 or 2, the gene of claim 3 or 4, the recombinant expression vector, expression cassette, or recombinant bacteria of claim 5 in the cultivation of new Andrographis paniculata with enhanced Andrographolide content.

8. The application of the transcription factor of claim 1 or 2, the gene of claim 3 or 4, the recombinant expression vector, expression cassette or recombinant bacteria of claim 5 in the breeding of Andrographis paniculata.

9. A method for cultivating new Andrographis paniculata with enhanced andrographolide content, characterized in that, The step includes overexpressing the transcription factor of claim 1 or 2 in Andrographis paniculata.

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