Huperzia serrata HsCAO1b gene and application

Abstract

The invention discloses a huperzia serrata HsCAO1b gene and application thereof, and belongs to the technical field of molecular biology and genetic engineering. Aiming at the problems that huperzia serrata wild resources are scarce, and CAO family genes of the huperzia serrata have unknown functions and are difficult to clone, the invention provides a complete coding sequence SEQ ID NO: 1 of the gene, constructs an expression vector containing the gene, and introduces the expression vector into host cells and plants. The gene can be used for regulating the metabolism of amine substances in plants, reducing the content of 2-phenylethylamine, piperidine or putrescine and increasing the content of spermidine or spermine; alkaloid synthesis is regulated and controlled, and total alkaloid is reduced or nicotine content is increased; the quantity of rosette leaves in the bolting period is increased to regulate and control plant growth and development, and gene resources are provided for production of active ingredients such as huperzine A and the like.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of molecular biology and genetic engineering, and particularly relates to a kind of Huperzia serrata HsCAO1b Gene and application. BACKGROUND

[0002] Copper amine oxidase (CAO) is a class of quinoproteins widely distributed in bacteria, yeast, fungi, plants and animals. The enzyme consists of a homodimer of 70-90 kDa subunits, each subunit contains an active site of copper ion and three conserved histidine sites that bind to copper ions, as well as a covalently bound 2,4,5-trihydroxyphenylalanine quinone (TPQ) cofactor. TPQ is formed by post-translational modification of the conserved tyrosine (Tyr) side chain of CAO, which is located in the consensus sequence Asn-Tyr-Asp / Glu at the active site. Its function is to catalyze the oxidative deamination of primary amines together with the copper ion it carries.

[0003] Studies have shown that CAO plays a key role in the development and metabolic regulation of various tissues and organs in plants, and its functions are widely involved in vascular tissue formation, lateral and adventitious root development, stomatal movement regulation, flowering time regulation, fruit development, and response to adversity. CAO, by specifically catalyzing the oxidative deamination of diamine compounds such as putrescine and cadaverine, is at the key intersection of diamine and polyamine metabolic networks, and plays a core regulatory role. Cadaverine is an important diamine metabolite, and putrescine is a key precursor of polyamine biosynthesis, being a direct substrate for the synthesis of spermidine and spermine. The catalysis of CAO on these two substrates not only regulates the level of diamines in plant cells, but also significantly affects the steady-state balance of downstream polyamines, affecting plant growth and development processes and adversity adaptation mechanisms. The reaction catalyzed by CAO on cadaverine and putrescine generates ammonia, hydrogen peroxide, and the corresponding aldehyde compounds, which play important roles in plant metabolism and signal transduction. Among them, hydrogen peroxide, as an important reactive oxygen species, is involved in the processes of ABA-induced stomatal closure, inhibition of hypocotyl growth under salt stress, jasmonic acid signaling pathway-mediated root lignification, and programmed cell death. 4-aminobutyraldehyde can be further converted into gamma-aminobutyric acid (GABA), which accumulates significantly under abiotic stress, significantly enhancing the environmental adaptability of plants. 5-aminovaleraldehyde, as a precursor of piperidine alkaloids, can form the basic skeleton of the piperidine ring through spontaneous cyclization, and then participate in the biosynthesis of various plant secondary metabolites.

[0004] Huperzia serrata is a plant with important medicinal value, and is one of the main medicinal plants of huperzine A. However, due to its slow growth and difficult propagation in the wild, and over-exploitation, the wild population of H. serrata is threatened, which makes it difficult to continuously obtain related genes and metabolites from natural resources. At present, the key genes involved in the regulation of secondary metabolism in H. serrata are not well known, especially the function of CAO family genes has not been clearly defined, which limits the research and utilization of the genetic resources of the species. Due to the lack of known gene sequence information, it is difficult to clone the gene and obtain its complete coding region, making it difficult to carry out further functional research and application exploration. SUMMARY

[0005] An object of the present application is to solve at least the above problems and provide at least the advantages to be described later.

[0006] Another object of the present application is to provide a H. serrata HsCAO1b The CDS of the gene is 2094 bp in length, encoding a protein composed of 697 amino acid residues, with a predicted molecular weight of about 176.11 kDa and a theoretical isoelectric point (pI) of 4.95.

[0007] To achieve these objects and other advantages of the present application, a H. serrata HsCAO1b The sequence of the gene is shown as SEQ ID NO. 1.

[0008] An expression vector comprising the H. serrata HsCAO1b gene.

[0009] A preparation method of the expression vector, wherein the expression vector takes the expression vector pCAMBIA1301 as a skeleton, and the H. HsCAO1b gene is inserted downstream of the promoter of pCAMBIA1301, and the insertion site is between the SacI and XbaI restriction endonuclease enzyme cleavage sites.

[0010] A host cell comprising the expression vector, wherein the host cell includes prokaryotic cells and eukaryotic cells.

[0011] A transgenic plant, wherein the genome of the plant integrates the H. HsCAO1b serrata gene.

[0012] The H. HsCAO1b serrata gene is used for regulating the metabolism of plant amines.

[0013] Preferably, the H. HsCAO1bGenes for reducing the content of 2-phenylethylamine, piperidine or putrescine in plants, or increasing the content of spermidine or spermine in plants The Huperzia serrata HsCAO1b Application of the genes in regulating the synthesis of alkaloids in plants.

[0014] Preferably, the Huperzia serrata HsCAO1b Genes for reducing the content of total alkaloids in plants, or increasing the content of nicotine in plants, or regulating the synthesis of huperzine A in Huperzia serrata. The Huperzia serrata HsCAO1b Application of the genes in regulating the growth and development of plants, specifically increasing the number of rosette leaves during the bolting stage.

[0015] The present application at least includes the following advantages: First, the present application successfully cloned the full-length coding sequence (SEQ ID NO: 1) of the Huperzia serrata HsCAO1b gene for the first time, providing key molecular materials for the functional research and application of the gene, and filling the gap in the research of CAO family genes in this species.

[0016] Second, the present application constructs HsCAO1b plant overexpression vectors of the gene and transforms model plants, confirming that the expression of the gene can significantly regulate the metabolic balance of amines (such as reducing the content of 2-phenylethylamine, piperidine, putrescine, and increasing the content of spermidine, spermine) and alkaloids (such as reducing the content of total alkaloids, and increasing the content of nicotine) in plants, providing a new way for directional regulation of plant secondary metabolism using genetic engineering methods.

[0017] Third, the expression of the gene of the present application can promote the increase of the number of rosette leaves in plants, indicating that it has potential application value in regulating the growth and development of plants, and laying a foundation for genetic improvement of Huperziaceae plants and biosynthesis of active alkaloids such as huperzine A.

[0018] Other advantages, objects and features of the present application will be embodied partly through the following description, and partly will be understood by those skilled in the art through research and practice of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 Huperzia serrata HsCAO1b Phylogenetic tree analysis of CAO family proteins of other species; Figure 2 Huperzia serrata HsCAO1b Arabidopsis thaliana, Huperzia serrata, Lens culinaris, Glycine max, P. tetrastichus Homology alignment of 9 CAO proteins; Figure 3 pCAMBIA1301 vector plasmid map; Figure 4 (A) and (B) are HsCAO1b Electrophoretic identification of DNA and cDNA of the gene in T3 generation transgenic *Nicotiana benthamiana*; (C), (D), and (E) are respectively Col-0 wild-type *Arabidopsis thaliana*, 35S:: HsCAO1b Phenotypic diagrams of transgenic Arabidopsis strains 10 and 18; (F) Statistical results of the number of rosette leaves at bolting; (G) 2-phenylethylamine content; (H) Piperidine content.

[0020] Figure 5 (A) and (B) are HsCAO1b Electrophoretic identification of DNA and cDNA of the gene in transgenic tobacco Benzoylus; (C) Nicotine content; (D) Total alkaloid content; (E) Putrescine content; (F) Spermine content; (G) Spermine content. Detailed Implementation

[0021] The present invention will now be described in further detail with reference to the accompanying drawings, so that those skilled in the art can implement it based on the description.

[0022] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0023] Example 1 Huperzia serrata HsCAO1b Cloning of genes 1. Total RNA extraction and reverse transcription Total RNA was extracted using the Fastpure Universal Plant TotalRNA Isolation Kit provided by Novizan Biosciences Co., Ltd., and reverse transcription was performed using the TransScript One-Step gDNA Removal and cDNASynthesisSuperMix Kit, following the instructions provided.

[0024] 2. Extraction of total DNA Total DNA was extracted using the FastPure Plant DNA Isolation MiniKit-BOX2 kit provided by Novizan Biosciences Co., Ltd., following the instructions.

[0025] 3. HsCAO1b Cloning of gene sequences Predictions were made using NCBI's ORFfinder online software (http: / / www.ncbi.nlm.nih.gov / orffinder / ). HsCAO1bThe CDS sequence was obtained. Primers were designed based on the CDS sequence, and the full-length gene was amplified by PCR using the extracted DNA as a template. The primer sequences are as follows: 5' primer (SEQ ID NO.2): 5'-atgatccccacctttccctggaac-3' 3' primer (SEQ ID NO.3): 5'-ttatctctcatcaacgtgagaac-3' After optimizing the PCR conditions based on the primer Tm values, amplification was performed according to the following procedure: The PCR system is as follows: The PCR reaction program was as follows: pre-denaturation at 95°C for 5 minutes, followed by 32 cycles (denaturation at 95°C for 30 seconds, annealing at 60°C for 15 seconds, extension at 72°C for 1.2 minutes), and final extension at 72°C for 5 minutes, and finally storage at 4°C.

[0026] The obtained PCR product was purified using the Fastpure Gel DNA Extraction Mini Kit agarose gel extraction kit, following the manufacturer's instructions. The purified product was ligated into the Blunt-Zero vector and transformed into *E. coli*. HsCAO1b The CDS amplification primers were used to perform PCR detection on single colonies. Clones with the target band were sent for sequencing, and the gene sequence is shown in SEQ ID NO.1. The final result was... HsCAO1b The full-length CDS of the gene is 2094 bp, encoding a protein composed of 697 amino acid residues. The predicted molecular weight of this protein is approximately 176.11 kDa, and the theoretical isoelectric point (pI) is 4.95.

[0027]

[0028] Will HsCAO1b Protein sequence with a variety of species CAO members of phylogenetic analysis, found that it is closely related to the HsCAO1 Protein homologous relationship in the closest, suggesting that they may have similar functions ( Figure 1 ).

[0029] To HsCAO1b Protein sequence and homologous from Arabidopsis, snake foot Huperzia, bean ( Lens culinaris ), home mountain bean ( Lathyrus sativus ) and four column Huperzia ( Phlegmariurus tetrastichus ) and other nine CAO family members, the results show that HsCAO1b Typical CAO conservation characteristics, including a conserved consensus sequence (Asn-Tyr-Asp / Glu), three copper ligand sites, and four amino acid residues Tyr, and lysine (lysine, Lys), Asp and Asn (Asn-Tyr-Asp / Glu) play a key role in the formation of the catalytic active center TPQ ( Figure 2 ) Example 2 Huperzia serrata HsCAO1b Construction of plant expression vector 1, according to the obtained CDS sequence of HsCAO1b , design in 5' and 3' end respectively add SacI and XbaI restriction sites of primers, construct overexpression vector. Expression vector for pCAMBIA1301, map is Figure 3 . The primer sequences are as follows: Overexpression primer F (SEQ ID NO. 4): 5'-catgagctcatgatccccacctttccctg-3' Overexpression primer R (SEQ ID NO. 5): 5'-gctctagattatctctcatcaacgtgag-3' cDNA as a template, PCR amplification. PCR reaction system is the same as the system of amplifying CDS full-length gene, other reaction conditions are the same.

[0030] 2, the obtained PCR product purification, purification using Fastpure Gel DNA Extraction Mini Kit agarose gel recovery kit, according to the instructions. Purified product and Blunt-Zero vector ligation, transformation of E. coli. Transformation method according to the Blunt-Zero vector instructions. Use HsCAO1b Expression vector amplification primer PCR detection of single colonies, have the purpose of band cloning sequencing.

[0031] 3. The plasmid of the correct monoclonal was extracted. The gene was cut from the Blunt-Zero vector by two restriction enzymes Sac I and Xba I, and was connected with pCAMBIA1301 digested by the same enzymes. The ligation product was transformed into DH5a cells, and then was cultured on LB solid plate containing 100 mg / L kanamycin. The colonies were identified by PCR and restriction enzyme analysis of plasmid DNA. The constructed recombinant was named as pCAMBIA1301-PhyA-PhyB. 35S:: HsCAO1b .

[0032] Example 3 Functional identification of Phyllosobryum HsCAO1b 1. Transgenic HsCAO1b Arabidopsis thaliana lines were identified for their functions: 1) Col-0 wild-type Arabidopsis thaliana plants were planted in a culture room (22°C, 16 h light / 8 h dark, light intensity 120 μmol m -2 s -1 ) and were allowed to bloom.

[0033] 2) The expression vector 35S:HsCAO1b was transformed into Agrobacterium GV3101, and the identified Agrobacterium monoclonal was picked and cultured in LB liquid medium containing 50 mg / L kanamycin and 20 mg / L rifampicin at 28°C.

[0034] 3) The Agrobacterium was collected by centrifugation at 5000 rpm for 5 min at room temperature, and the precipitate was suspended in transformation liquid (5% sucrose, 0.02% Silwet) to an OD 600 of 0.8-1.0.

[0035] 4) The inflorescences of the plants watered the previous day were soaked in the transformation liquid with the bacterial suspension for 1 min, and were cultured in the dark for 24 h, and then were moved to grow under weak light.

[0036] 5) The mature seeds of the plants were collected after 3-5 times of transformation once a week.

[0037] 6) The collected seeds were sterilized with 0.1% mercuric chloride for 10 min, and then were sowed on MS solid plate medium containing 30 mg / L hygromycin for resistance screening, to obtain T1 generation positive transgenic plants.

[0038] 7) The T1 generation seeds were collected, and were again subjected to resistance screening on MS solid plate medium containing 30 mg / L hygromycin, to obtain T2 generation positive transgenic plants with trait segregation.

[0039] ​8) Continue sowing the T2 generation and observe whether the T3 phenotype segregates and identify homozygous plants at the molecular level. Metabolite content determination and phenotypic observation revealed a significant decrease in the content of amines 2-phenylethylamine and piperidine in the T3 generation transgenic positive lines, and an increase in the number of rosette leaves at bolting. For example... Figure 4 As shown.

[0040] 2. Instantaneous expression HsCAO1b Functional identification of the Benedict's tobacco gene: 1) In the culture room (23℃, 16 h light / 8 h dark, light intensity 120 μmol m²), -2 s -1 Planting wild-type Tobacco Benedict plants takes about one and a half months.

[0041] 2) Expressive vector 35S:: HsCAO1b Agrobacterium GV3101 was transformed, and identified Agrobacterium colonies were picked and cultured in LB broth containing 50 mg / L kanamycin and 20 mg / L rifampin at 28°C with shaking for approximately 16 h. The bacterial culture was then transferred to fresh LB broth containing 50 mg / L kanamycin and 20 mg / L rifampin and cultured until OD500 reached. 600 Approximately 0.6~0.8.

[0042] 3) Collect the Agrobacterium precipitate by centrifugation at 4000 rpm for 10 min at room temperature. Resuspend the bacterial cells in injection buffer (50 mmol / L 2-(N-morpholino)ethanesulfonic acid, 10 mmol / L MgCl2, 0.2 mmol / L acetylsuccine), and centrifuge again at 4000 rpm for 10 min. Discard the supernatant and repeat this washing step twice. Finally, resuspend the bacterial cells in injection buffer and adjust the bacterial concentration to OD. 600 It is 0.1~0.2.

[0043] 4) Select robust, dark green, and thick tobacco plants that have not yet flowered, and water them thoroughly the day before injection. Use a disposable syringe to inject the resuspended bacterial solution into the tobacco leaf mesophyll tissue. After injection, use absorbent paper to remove excess bacterial solution from the leaf surface, mark the injection site, and incubate the plants in the dark for 48 hours.

[0044] 5) Tobacco leaves were collected 48 h after injection. A portion was used to detect the expression level of the target gene or protein, while the other portion was dried at 60℃ for the determination of amine and alkaloid content. Metabolite analysis results showed that transient expression... HsCAO1b The contents of putrescine and total alkaloids in tobacco leaves decreased significantly, but the contents of spermidine, spermine, and nicotine increased significantly. Figure 5 As shown.

[0045] The above results fully demonstrate that HsCAO1bIt is likely to be a key gene involved in plant development, amine and alkaloid synthesis. This study provides an important theoretical basis for artificially fine regulation of the growth and development of Huperziaceae plants and amine metabolism, as well as the biosynthesis of active alkaloids such as HupA by genetic engineering, and has important practical significance.

[0046] Although embodiments of the present application have been disclosed as above, it is not limited only to the use listed in the specification and embodiments, and can be fully applied to various fields suitable for the present application, and additional modifications can be easily made by those skilled in the art, and thus the present application is not limited to specific details and the figures shown and described herein, without departing from the general concept defined by the claims and the equivalent scope.

Claims

1. A gene of Huperzia serrata HsCAO1b characterized in that, The sequence of the gene is shown as SEQ ID NO.

1.

2. An expression vector, characterized in that, A gene comprising the sequence of claim 1 HsCAO1b gene.

3. The method for preparing the expression vector as described in claim 2, characterized in that, The expression vector takes the expression vector pCAMBIA1301 as a skeleton, and inserts the HsCAO1b The gene is inserted downstream of the pCAMBIA1301 promoter, and the insertion site is between the Sac I and Xba I restriction endonuclease enzyme cleavage sites.

4. A host cell, characterized in that, The host cell includes prokaryotic cells and eukaryotic cells.

5. A transgenic plant, characterized in that, The plant genome has integrated therein the gene of claim 1 HsCAO1b Gene.

6. The Huperzia serrata of claim 1 HsCAO1b Use of the gene in regulating plant amine metabolism.

7. Use according to claim 6, characterized in that, said huperzia serrata HsCAO1b genes for reducing the content of 2-phenylethylamine, piperidine or putrescine in a plant, or for increasing the content of spermidine or spermine in a plant.

8. The Huperzia serrata of claim 1 HsCAO1b Use of the gene in regulating plant alkaloid synthesis.

9. Use according to claim 8, characterized in that, huperzia serrata HsCAO1b genes for reducing the total alkaloid content in plants, or for increasing the nicotine content in plants, or for modulating the synthesis of huperine A in h. serrata.

10. The Huperzia serrata of claim 1 HsCAO1b The application discloses a gene and application thereof in regulating plant growth and development, and belongs to the field of plant genetics and biotechnology. The regulation of the growth and development of the plant specifically increases the number of rosette leaves in the bolting stage of the plant.

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