Radix pseudostellariae proline oligopeptidase gene PhPOP1 and application thereof

Abstract

The invention discloses a proline oligopeptidase gene PhPOP1 of radix pseudostellariae and an application of the proline oligopeptidase gene PhPOP1. The nucleotide sequence of the radix pseudostellariae proline oligopeptidase gene PhPOP1 is as shown in SEQ ID NO. 1, and the coded amino acid sequence is as shown in SEQ ID NO. 2. According to the application of the proline oligopeptidase gene PhPOP1 of the radix pseudostellariae in heterologous synthesis of the cyclopeptide HB of the radix pseudostellariae, the proline oligopeptidase gene PhPOP1 of the radix pseudostellariae and a coding gene prePhHB of a linear peptide HB are jointly overexpressed in tobacco, so that heterologous biosynthesis of the cyclopeptide HB of the radix pseudostellariae is realized; according to the application of the proline oligopeptidase gene PhPOP1 in creating the hairy roots of the radix pseudostellariae with high cyclopeptide HB content, the proline oligopeptidase gene PhPOP1 is over-expressed in the hairy roots of the radix pseudostellariae, so that the content of the cyclopeptide HB in the hairy roots of the radix pseudostellariae is increased. The cyclopeptide HB can be heterologously synthesized by applying the enzyme gene and a linear peptide gene prePhHB through a synthetic biological technology, and the heterologously synthesized pseudostellaria heterophylla cyclopeptide HB enzyme has important significance in cultivation of new varieties with high content of pseudostellaria heterophylla cyclopeptide HB and large-scale production of pseudostellaria heterophylla cyclopeptide HB.

Description

Technical Field

[0001] The invention relates to the technical field of plant genetic engineering, and in particular to a Pseudo-P ... Background Art

[0002] Pseudostellaria heterophylla (Mip.) Pax ex Paxet Hoffm. is a dried root of Pseudostellaria heterophylla (Mip.) Pax ex Paxet Hoffm., a plant of the Caryophyllaceae family. It contains chemical components such as cyclic peptides, polysaccharides, amino acids and volatile substances, and has pharmacological effects such as antioxidant, immune promotion and myocardial protection. Cyclic peptide compounds are characteristic chemical components of Pseudostellaria heterophylla and are also an important material basis for the pharmacological activity of Pseudostellaria heterophylla. At present, 19 cyclic peptide components have been isolated and identified from Pseudostellaria heterophylla, including cyclic peptides Heterophyllin AH, Heterophyllin J and Pseudostellarin A-H, Pseudostellarin K, and Pseudostellarin L. Among them, cyclic peptide HB is one of the key evaluation indicators of the quality of Pseudostellaria heterophylla medicinal granules. Modern pharmacological studies have found that Pseudostellaria heterophylla cyclic peptides have anti-inflammatory, anti-tumor, anti-HIV, tyrosinase activity inhibition and angiogenesis promotion effects.

[0003] However, the content of cyclic peptide HB in Radix Pseudostellariae is relatively low, which makes it difficult to meet the market demand for high-quality Radix Pseudostellariae and cyclic peptide HB. The analysis of the biosynthetic pathway of active ingredients is an important prerequisite for improving the quality of medicinal materials, and it is also the basis for the production of medicinal ingredients using metabolic engineering and synthetic biology technologies. Up to now, the biosynthetic pathway of plant cyclic peptides has been preliminarily interpreted. In the early days, it was believed that there were two pathways for the biosynthesis of cyclic peptides. One was that cyclic peptides were completed through non-ribosomal peptide synthesis pathways and were not products encoded by genes; the other was that cyclic peptides were encoded by genes and synthesized into linear peptide precursors by ribosomes, and then cut, cyclized and modified by a series of enzymes to form mature cyclic peptides.

[0004] As the research deepens, more and more experiments show that plant cyclopeptide synthesis is ribosome-dependent. However, in Pseudostellariae Radix, the downstream pathway of cyclopeptide HB biosynthesis has not been resolved, and the enzyme genes involved in the biosynthesis of cyclopeptide HB have not been identified. Therefore, screening the enzyme genes involved in the biosynthesis of Pseudostellariae Radix cyclopeptide HB and identifying their biological functions can not only apply the enzyme gene and the linear peptide gene prePhHB to heterologously and efficiently synthesize cyclopeptide HB through synthetic biology technology, but also has important significance for the cultivation of new varieties with high content of Pseudostellariae Radix cyclopeptide HB and large-scale production. Summary of the invention

[0005] In view of this, one of the objects of the present invention is to provide a Pseudostellaria baicalensis proline oligopeptidase PhPOP1; the second object of the present invention is to provide a Pseudostellaria baicalensis proline oligopeptidase PhPOP1 coding gene; the third object of the present invention is to provide a recombinant expression vector or a recombinant strain containing the Pseudostellaria baicalensis proline oligopeptidase PhPOP1 coding gene; the fourth object of the present invention is to provide the use of the Pseudostellaria baicalensis proline oligopeptidase PhPOP1 coding gene in tobacco to catalyze the Pseudostellaria baicalensis linear peptide HB to produce cyclic peptide HB; the fifth object of the present invention is to provide the use of the Pseudostellaria baicalensis proline oligopeptidase PhPOP1 coding gene in creating Pseudostellaria baicalensis hairy roots with a high cyclic peptide HB content.

[0006] To achieve the above object, the present invention is implemented by the following technical solutions:

[0007] The Pseudo-P ...

[0008] The amino acid sequence of the gene encoding the Pseudoradix Pseudoradix proline oligopeptidase gene PhPOP1 of the present invention is shown as SEQ ID NO.2.

[0009] The Pseudostellariae proline oligopeptidase gene PhPOP1 of the present invention comprises a recombinant expression vector or a recombinant strain.

[0010] The application of the Pseudostellariae proline oligopeptidase gene PhPOP1 in the heterologous synthesis of Pseudostellariae cyclic peptide HB of the present invention is to achieve heterologous biosynthesis of Pseudostellariae cyclic peptide HB by co-overexpressing the Pseudostellariae proline oligopeptidase gene PhPOP1 and the gene prePhHB encoding the linear peptide HB in tobacco. Figure 2 shown.

[0011] The application of the Pseudostellariae proline oligopeptidase gene PhPOP1 of the present invention in creating Pseudostellariae hairy roots with high cyclic peptide HB content, by overexpressing the proline oligopeptidase gene PhPOP1 in the Pseudostellariae hairy roots, thereby promoting the content of cyclic peptide HB in the Pseudostellariae hairy roots, as described in Figure 3 shown.

[0012] The method of the present invention for heterologously synthesizing Pseudostellariae cyclopeptide HB by co-expressing the Pseudostellariae proline oligopeptidase gene PhPOP1 and the coding gene prePhHB of the linear peptide HB in tobacco comprises the following steps:

[0013] S1, inserting the coding sequence of Pseudoradix heterophylla proline oligopeptidase gene PhPOP1 into the plant expression vector pEAQ-HT through AgeI and XhoI restriction endonuclease sites to construct the plant overexpression vector pEAQ-PhPOP1-HT;

[0014] S2, inserting the coding sequence of the gene prePhHB encoding the linear peptide HB into the plant expression vector pCAMBIA3301 through the BamHI and XindIII restriction endonuclease sites to construct the plant overexpression vector pCAMBIA3301-prePhHB;

[0015] S3, the plant overexpression vectors pEAQ-PhPOP1-TA and pCAMB IA3301-prePhHB described in steps S1 and S2 are respectively transferred into the host strain Agrobacterium GV3101 to obtain an engineered strain containing the plant overexpression vector, the bacterial solution is evenly mixed and then injected into tobacco to obtain transiently transformed tobacco with co-expression of PhPOP1 and prePhHB genes, and the content of cyclopeptide HB in tobacco leaves is detected.

[0016] The method for transferring Agrobacterium into the host strain in step S3 of the present invention is a freeze-thaw method.

[0017] The method for constructing a transgenic Pseudostellariae hairy root overexpressing the Pseudostellariae proline oligopeptidase gene PhPOP1 of the present invention comprises the following steps:

[0018] 1) The coding sequence of the Pseudo-Phenothiazine proline oligopeptidase gene PhPOP1 was inserted into the plant expression vector pBI121 through the XbaI and SacI restriction endonuclease sites to construct a plant overexpression vector pBI121-PhPOP 1;

[0019] 2) The plant overexpression vector pBI121-PhPOP1 described in 1) is transferred into the host strain Agrobacterium rhizogenes K599 to obtain an engineered strain containing the plant overexpression vector, and transgenic Pseudostellaria heterophylla hairy roots are obtained after Agrobacterium-mediated transformation;

[0020] 3) Screening the transgenic Pseudostellariae hairy roots described in step 2) to obtain overexpressed transgenic hairy roots with significantly increased cyclic peptide HB content.

[0021] The method of transferring the host strain Agrobacterium in step 2) of the present invention is freeze-thaw method.

[0022] In step 3) of the present invention, the screening is performed by using antibiotic screening, and the antibiotic is kanamycin.

[0023] In step 3) of the present invention, the screening further comprises PCR detection, and the primers for PCR detection are the forward detection primer shown in SEQ ID NO.11 and the reverse detection primer shown in SEQ ID NO.12.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1. The present invention cloned the sequence of the proline oligopeptidase gene PhPOP1, and co-expressed it with the gene encoding the linear peptide HB, prePhHB, in tobacco to produce the cyclic peptide HB, thus realizing the heterologous biosynthesis of the Pseudostellaria heterophylla cyclic peptide HB;

[0026] 2. The transgenic P. heterophylla hairy roots overexpressing the proline oligopeptidase gene PhPOP1 of the present invention can achieve the regulation of the increase of the cyclic peptide HB content, which is of great significance for the large-scale production of cyclic peptide HB using metabolic engineering technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 :1% agarose gel electrophoresis of Pseudostigma heterophylla proline oligopeptidase PhPOP1 gene.

[0028] Figure 2 : Analysis of the content of P. heterophylla cyclic peptide HB in tobacco leaves transiently transformed with PhPOP1 and prePhHB, wherein (A): chromatogram of cyclic peptide HB in transiently transformed tobacco; (B): the content of cyclic peptide HB in transiently transformed tobacco; error bars are standard errors (n=3), and t-test statistical analysis was performed (** indicates: P<0.01).

[0029] Figure 3 : Obtaining hairy roots of Pseudostellaria baicalensis overexpressing PhPOP1 and analyzing the gene expression level and cyclic peptide HB content in the hairy roots, including (A): inducing rooting of Pseudostellaria baicalensis cut-root seedlings; (B): sterilization of transgenic Pseudostellaria baicalensis hairy roots; (C): expansion culture of transgenic Pseudostellaria baicalensis hairy roots; (DE): PCR detection of 35S::PhPOP1 and Kana specific fragments; (F): chromatogram of cyclic peptide HB in hairy roots; (G): expression level of PhPOP1 in hairy roots; (H): content of cyclic peptide HB in hairy roots; error bars are standard errors (n=3), t-test statistical analysis (* indicates: P<0.05, ** indicates: P<0.01). DETAILED DESCRIPTION

[0030] 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.

[0031] Example 1 Cloning of the proline oligopeptidase gene PhPOP1 in Radix Pseudostellariae

[0032] Extract RNA from Pseudostellaria heterophylla, convert it into cDNA, and design PCR amplification primers based on the transcriptome of Pseudostellaria heterophylla:

[0033] The upstream primer is: 5′-ATGACTTCCTCCACCTTCTC-3′ (SEQ ID NO. 3);

[0034] The downstream primer is: 5′-TTAATCTGTCCAAGAGGCGT-3′ (SEQ ID NO. 4);

[0035] The PCR amplification conditions were: 98°C pre-denaturation for 1 minute; 98°C denaturation for 15 seconds, 56°C annealing for 15 seconds, 72°C extension for 60 seconds, for a total of 32 cycles; and finally 72°C extension for 2 minutes. After the PCR amplification was completed, the PCR product was detected by electrophoresis in a 1% agarose gel. The results were as follows: Figure 1 As shown; the target fragment was gel-recovered to obtain the full-length CDS sequence of the proline oligopeptidase gene PhPOP1. The coding region sequence of the gene was 2202bp in length, encoding 733 amino acids. Its nucleotide sequence was shown in SEQ ID NO.1, and its encoded amino acids were shown in SEQ ID NO.2.

[0036] SEQ ID NO.1 Gene sequence of Pseudo-P ...

[0037] >PhPOP1

[0038]

[0039] SEQ ID NO.2 Amino acid sequence of Pseudostigma heterophylla proline oligopeptidase PhPOP1

[0040] >PhPOP1

[0041] MTSSTFSKPLHYPPVRRDDSVVDDYFGVKIPDPYRWLEDPDSEETKEFVQNQIKLADSVLEECDSRDKIKKKITDFINFPRCGVPFKRGDKCFHFYNSGLQAQNVLHIQDDVEAKPEVLLDPNLIVNGKAGLHVHSEDAKYIAYGLPLGLTEWVTIKVMRIEDREILPDTLSWVKFSGCYW THDCKGFFYCPYPPRNEAQEEDSETKTSTFDTSSSLNQTVSYHFLGTDQSEDILCWRDLENPLQHFKIDVTADGKYLLIYIHVSSGVMNKVYYVDLTTLPNGLEGYRGREDLLPFVKFIDDYDATYTAVANDDSVFIFLTNKDAPNNKLVRVDLNNPDIWTDVIPHSEKEVLESANAVNGNQL LVRYLSDVKHVLEVRDLESGSLLHRIPSGIGSVGGVNARREDSVVFFKFTSFLTPGIIYQCDLKDAVPQLKIFQESVVPEFDRSEFEVNQVFFPSKDGTKIPMFIVARKGISLDGSHPCELHGYGGFNINLMPNFSASRIVWLKHLGGVFCLPNIRGGGEYGDEWHKAGMLDKKQNVFDDFIS AAEFLISNGYTAPTKLGIEGGSNGGLLVATCINQRPDLFACAMANCGVMDMLRFHKFTMGYLWTADYGCSEKEEDFNWLIKYSPLHNVRRPWEESENKQLQYPATMILAADHDDRVVPLHSFKLLATMQYLLCTTLEDSPQKNPLIARIECKASHFGRATMLQIEEVTDRYAFLAKAVNASWTD

[0042] Example 2 Construction of plant expression vector containing PhPOP1 and prePhHB genes

[0043] 1. Construction of plant heterologous expression vectors pEAQ-PhPOP1-HT and pCAMBIA3301-prePhHB

[0044] (1) Based on the obtained PhPOP1 gene sequence, the restriction sites in its coding region and the pEAQ-HT vector were analyzed, and AgeI and XhoI were selected as restriction endonuclease sites for the construction of the pEAQ-HT vector. An AgeI restriction site was introduced upstream of the full-length PhPOP1, and an XhoI restriction site was introduced downstream of the full-length PhPOP1. The primer sequences are as follows:

[0045] PhPOP1-F-AgeI:5'-GC ACCGGT ATGACTTCCTCCACCTTCTC-3' (SEQ I

[0046] D NO.5)

[0047] PhPOP1-R-XhoI:5'-GC CTCGAG TTAATCTGTCCAAGAGGGCT-3'(SEQ ID NO.6)

[0048] (2) According to the gene sequence of prePhHB (GeneBank: MH699110) downloaded from NCBI, the restriction sites in its coding region and pCAMBIA3301 vector were analyzed, and BamHI and XindIII were selected as restriction endonuclease sites for the construction of pCAMBIA3301 vector. The BamHI restriction site was introduced upstream of the full-length prePhHB, and the XindIII restriction site was introduced downstream of the full-length prePhHB. The primer sequences are as follows:

[0049] prePhHB-F-BamHI:5'-GC GGATCC ATGTCTACTATTTCAGCCATCCAC-3'(SEQ ID NO.7)

[0050] prePhHB-R-XindIII:5'-GC AAGCTT TTACACCATGAGGGAAATATCATCT C-3'(SEQ IDNO.8)

[0051] 2. Construction of plant overexpression vector pBI121-PhPOP1

[0052] According to the obtained PhPOP1 gene sequence, the restriction sites in its coding region and pBI121 vector were analyzed, and XbaI and SacI were selected as restriction endonuclease sites for vector construction. XbaI restriction site was introduced upstream of the full-length PhPOP1, and SacI restriction site was introduced downstream of the full-length PhPOP1. The primer sequences are as follows:

[0053] PhPOP1-F-XbaI:5'-GC TCTAGA ATGACTTCCTCCACCTTCTC-3'(SEQ ID NO.9)

[0054] PhPOP1-R-SacI:5'-GC GAGCTC TTAATCTGTCCAAGAGGGCT-3'(SEQ ID NO.10)

[0055] Example 3 Application of Pseudostellariae Proline Oligopeptidase Gene PhPOP1 in Heterologous Synthesis of Pseudostellariae Cyclic Peptide HB

[0056] 1. Obtaining the engineered strain GV3101 Agrobacterium

[0057] The empty vectors pEAQ-HT and pCAMBIA3301 and the plant heterologous expression vectors pEAQ-PhPOP1-HT and pCAMBIA3301-prePhHB were respectively transferred into the engineering strain Agrobacterium GV3101 by freeze-thaw method to obtain the engineering strains GV3101-pEAQ-HT, GV3101-pCAMBIA3301, GV3101-pEAQ-PhPOP1-HT, and GV3101-pCAMBIA3301-prePhHB.

[0058] 2. Instantaneous transformation of tobacco

[0059] The positive strains of the above four engineering strains were inoculated into 5 ml YEP liquid medium containing 5 mg / L rifampicin (Rif) + 20 mg / L gentamicin (Gen) + 50 mg / L kanamycin (Kan), and cultured overnight at 28°C, 220 rpm / min; the cells were collected by centrifugation at 4000 rpm / min for 10 min; the cells were resuspended in MS liquid medium and the OD of the bacterial solution was adjusted. 600 The value was 0.6, 200 mmol / L acetosyringone (AS) and 10 mmol / L MES (PH = 5.8) were added, and the mixed Agrobacterium culture was placed at 28°C for 3 hours.

[0060] The engineered strain GV3101-pEAQ-PhPOP1-HT was mixed with GV3101-pCAMBIA3301-pre PhHB and GV3101-pCAMBIA3301, and GV3101-pEAQ-HT was mixed with GV3101-pCAMBI A3301-prePhHB and GV3101-pCAMBIA3301 in a 1:1 ratio, and then injected into tobacco leaves grown for 35 days by injection infection. The tobacco leaves were cultured in the dark for 4 days. The tobacco leaves were taken and dried, and the content of Pseudostellaria heterophylla cyclic peptide HB in the leaves was detected.

[0061] The results are as follows Figure 2 As shown in the results, when the prePhHB gene was expressed in tobacco leaves, the production of cyclic peptide HB was detected in the transgenic materials. When PhPOP1 and prePhHB were co-expressed in tobacco leaves, the production of cyclic peptide HB was also detected in the transgenic materials, and the content of cyclic peptide HB was about 6.5 times higher than that in tobacco leaves expressing the prePhHB gene alone. In tobacco leaves expressing PhPOP1 alone, the production of cyclic peptide HB was not detected. The above results show that PhPOP1 can produce cyclic peptide HB in tobacco using the Pseudostellariae linear peptide prePhHB as a substrate.

[0062] Example 4 Application of Pseudostellariae Proline Oligopeptidase Gene PhPOP1 in Creating Pseudostellariae Hairy Roots with High Cyclopeptide HB Content

[0063] 1. Obtaining the engineered strain K599 Agrobacterium rhizogenes

[0064] The empty vector pBI121 and the plant expression vector pBI121-PhPOP1 were respectively transferred into the engineering strain Agrobacterium rhizogenes K599 by freeze-thaw method to obtain the engineering strains K599-pBI121 and K599-pBI121-PhPOP1.

[0065] 2. Germination of sterile seedlings of Pseudostellaria heterophylla

[0066] The seeds of Radix Pseudostellariae were soaked in clean water for 12-20 hours, and the seeds were sterilized with 75% ethanol for 1 minute, washed with sterile water for 2-3 times, and then disinfected with an appropriate amount of 1:1 sterilized water for 6 minutes. After washing with water for 4-5 times, the morphologically complete embryos of Radix Pseudostellariae were obtained by dissection, inoculated on a culture dish of MS culture medium, and cultured in a sterile culture room with 16 hours of light, 8 hours of darkness, 25°C, and 75% humidity for 14 days. The peeled seedlings grown in the culture medium were cut and rooted for transformation.

[0067] 3. Induction of Hairy Roots of Pseudostellaria heterophylla

[0068] The obtained engineered strains K599-pBI121 and K599-pBI121-PhPOP1 were respectively applied to LB solid culture dishes (50mg / L Kan+100mg / L Stre) containing corresponding resistance, placed in a 28°C incubator for 48h, and then used to infect Pseudostellaria heterophylla root-cut seedlings. The roots of the sterile Pseudostellaria heterophylla seedlings cultured for about 14d were cut, and appropriate amounts of cultured K599-pBI121 and K599-pBI121-PhPO P1 engineered bacterial colonies were scraped from the cut wounds, respectively, and placed in MS liquid culture medium tissue culture bottles with filter paper, 23°C, 16h light, 8h dark culture until new Pseudostellaria heterophylla roots grew from the wounds coated with Agrobacterium, and the screening culture medium was replaced every 10 days. After one month, the Pseudostellaria heterophylla roots were transferred to the liquid culture medium containing MS liquid for expansion culture.

[0069] 4. PCR detection of transgenic Pseudostellaria heterophylla hairy roots

[0070] According to the gene sequence of PhPOP1 and the plant pBI121 vector sequence, the forward detection primer and the reverse detection primer were designed respectively. The primer sequences are as follows:

[0071] 35S:5'-GACGCACAATCCCACTATCC-3'(SEQ ID NO.11)

[0072] PhPOP1-R:5'-TTAATCTGTCCAAGAGGCT-3'(SEQ ID NO.12)

[0073] According to the gene sequence of PhPOP1, fluorescent quantitative PCR detection primers were designed to detect the expression of PhPOP1 gene in the hairy roots of transgenic P. heterophylla. The primer sequences are as follows:

[0074] PhPOP1-qF:5'-CGTGCATTCTGTAAGCGAGGACGC-3'(SEQ ID NO.13)

[0075] PhPOP1-qR:5'-CAGTCATGGGTCCAATAACAACCAC-3'(SEQ ID NO.14)

[0076] 5. Extraction of cyclic peptide HB from transgenic Pseudostellaria heterophylla hairy roots

[0077] Take out the well-grown positive hairy roots and put them in a 40℃ oven to dry overnight to constant weight. Grind the dried hairy roots of Pseudostellaria heterophylla into uniform powder, weigh about 0.100g and put it into a 10mL ep tube, add 5mL methanol to the ep tube, and put it into an ultrasonic instrument for 45min (temperature 30℃, power 50%). After the ultrasonication, centrifuge the sample at 8000rpm for 10min, take the supernatant into a new ep tube, put it into a nitrogen blower at 60℃ to dry, add 1mL methanol to the ep tube and ultrasonicate it in an ultrasonic instrument for 15min to re-dissolve. Absorb the solution and filter it with a 0.45μm filter head and transfer it to the injection bottle.

[0078] 6. Determination of the content of cyclic peptide HB

[0079] The extracted samples were tested using the Waters high performance liquid chromatography detection system, and the chromatographic column was Sy Column (4.6mm×250mm, 5μm); mobile phase is acetonitrile and 0.1% phosphoric acid water, volume ratio is 60%:40%, flow rate is 1.0mL / min, column temperature is 30℃, detection wavelength is 202nm. 20μL of standard sample is injected, 20μL of each experimental sample is injected, and the injection is repeated 3 times. The content of cyclopeptide HB in the sample is calculated according to the concentration and peak area of ​​the standard, and then divided by the dry weight of the hairy root powder of Pseudostellaria heterophylla to calculate the content of cyclopeptide HB in the dry weight of the sample.

[0080] The results are as follows Figure 3 As shown, the expression level of PhPOP1 gene in the transgenic hairy roots of P. heterophylla overexpressing PhPOP1 gene was significantly increased, and the content of cyclic peptide HB was also significantly increased, and the differences were significant.

[0081] In order to further verify the feasibility and effectiveness of the embodiments of the present invention, the inventors conducted a series of experiments, as follows:

[0082] 1. Cloning of the proline oligopeptidase gene PhPOP1 from Pseudostellaria heterophylla

[0083] 1.1 Test materials

[0084] The seedlings of Pseudostellariae used in the experiment were planted in a culture pot filled with organic nutrient soil and placed in an artificial climate chamber for growth, with a light / dark period of 16h / 8h and a temperature of 23±2°C. Tobacco seeds were sown directly in the nutrient soil, and the growth conditions were consistent with those of Pseudostellariae plants. In this study, 30-day-old tobacco seedlings were used for the heterologous synthesis of Pseudostellariae cyclic peptide HB experiments.

[0085] 1.2 Test methods

[0086] 1.2.1 Total RNA from Pseudostellaria heterophylla

[0087] Total RNA was extracted from Pseudostellaria heterophylla roots using Promega Super Total RNA Extraction Kit ( Super Total RNA Extraction Kit, LS1040) was used to extract total RNA from Pseudostellaria heterophylla tuber roots according to the kit instructions. The steps are as follows:

[0088] (1) Place the root tuber of Pseudostellaria heterophylla in a frozen grinder and grind it with liquid nitrogen until it is homogenized. Take about 100 mg of the sample and put it into a sterile 1.5 mL eppendorf tube;

[0089] (2) Add 500 μL of lysis buffer and diluent, mix well with a pipette, and leave at room temperature for 5 min;

[0090] (3) Centrifuge at 12,000 rpm for 5 min and transfer the supernatant to a new sterile EP tube;

[0091] (4) Add 0.5 times the volume of anhydrous ethanol and pipette rapidly 20 times until the liquid becomes light blue and turbid with white foam;

[0092] (5) Transfer the mixture to a centrifuge column, centrifuge at 12,000 rpm for 1 min, and discard the filtrate;

[0093] (6) Add 600 μL RNA washing solution, centrifuge at 12,000 rpm for 45 s, and discard the filtrate;

[0094] (7) Add 50 μL of DNase I incubation solution to the center of the adsorption membrane and leave at room temperature for 15 min;

[0095] (8) Add 600 μL RNA washing solution, centrifuge at 12,000 rpm for 45 s, and discard the filtrate;

[0096] (9) Add 600 μL RNA washing solution, centrifuge at 12,000 rpm for 45 seconds, and discard the filtrate. Replace the centrifuge column in the collection tube and centrifuge at 12,000 rpm for 2 minutes;

[0097] (10) Repeat step 8;

[0098] (11) Transfer the spin column to the elution tube, add 70 μL of nuclease-free water to the center of the spin column membrane, let stand at room temperature for 2 min, and centrifuge at 12,000 rpm for 1 min;

[0099] (12) After centrifugation, place the sample on ice immediately for RNA quality assessment and concentration determination, or store at -80°C for later use.

[0100] 1.2.2 Obtaining cDNA

[0101] Takara's PrimeScript RT reagent kit with gDNA Eraser kit was used for RNA reverse transcription. The steps are as follows:

[0102] (1) Add the following reagents to a 200 μL ep (ep1) tube:

[0103]

[0104] (2) Gently mix the mixture and centrifuge it;

[0105] (3) Incubate at 42°C for 2 min. Place the reaction mixture on ice immediately after the incubation.

[0106] (4) Prepare the following reaction solution in a new RNAsae ep (ep2) tube:

[0107]

[0108] (5) Gently pipette and mix the mixture, and centrifuge it;

[0109] (6) Pipette 10 μL of the above mixture into ep1 and mix thoroughly by pipetting;

[0110] (7) Then put the reaction mixture into the PCR instrument, and the reaction program is: 37℃, 15min; 85℃, 5s. After the reaction is completed, the sample is immediately placed on ice for a few minutes and then stored in a -20℃ refrigerator for later use.

[0111] 1.2.3 Design PCR amplification primers based on Pseudostellaria heterophylla transcriptome:

[0112] The upstream primer is: 5′-ATGACTTCCTCCACCTTCTC-3′ (SEQ ID NO. 3);

[0113] The downstream primer is: 5′-TTAATCTGTCCAAGAGGCGT-3′ (SEQ ID NO. 4);

[0114] The PCR amplification conditions were: 98°C, 1 min; 98°C, 15 s; 56°C, 15 s; 72°C, 60 s, for a total of 32 cycles; and finally 72°C, 2 min. The gene sequence encoding the proline oligopeptidase PhPOP1 was obtained. The coding region sequence of the gene was 2,202 bp in length, encoding 733 amino acids. Its nucleotide sequence is shown in SEQ ID NO.1, and its encoded amino acids are shown in SEQ ID NO.2.

[0115] SEQ ID NO.1 Gene sequence of Pseudo-P ...

[0116] >PhPOP1

[0117]

[0118] SEQ ID NO.2 Amino acid sequence of Pseudostigma heterophylla proline oligopeptidase PhPOP1

[0119] >PhPOP1

[0120] MTSSTFSKPLHYPPVRRDDSVVDDYFGVKIPDPYRWLEDPDSEETKEFVQNQIKLADSVLEECDSRDKIKKKITDFINFPRCGVPFKRGDKCFHFYNSGLQAQNVLHIQDDVEAKPEVLLDPNLIVNGKAGLHVHSEDAKYIAYGLPLGLTEWVTIKVMRIEDREILPDTLSWVKFSGCYW THDCKGFFYCPYPPRNEAQEEDSETKTSTFDTSSSLNQTVSYHFLGTDQSEDILCWRDLENPLQHFKIDVTADGKYLLIYIHVSSGVMNKVYYVDLTTLPNGLEGYRGREDLLPFVKFIDDYDATYTAVANDDSVFIFLTNKDAPNNKLVRVDLNNPDIWTDVIPHSEKEVLESANAVNGNQL LVRYLSDVKHVLEVRDLESGSLLHRIPSGIGSVGGVNARREDSVVFFKFTSFLTPGIIYQCDLKDAVPQLKIFQESVVPEFDRSEFEVNQVFFPSKDGTKIPMFIVARKGISLDGSHPCELHGYGGFNINLMPNFSASRIVWLKHLGGVFCLPNIRGGGEYGDEWHKAGMLDKKQNVFDDFIS AAEFLISNGYTAPTKLGIEGGSNGGLLVATCINQRPDLFACAMANCGVMDMLRFHKFTMGYLWTADYGCSEKEEDFNWLIKYSPLHNVRRPWEESENKQLQYPATMILAADHDDRVVPLHSFKLLATMQYLLCTTLEDSPQKNPLIARIECKASHFGRATMLQIEEVTDRYAFLAKAVNASWTD

[0121] 2. Construction of plant expression vector containing PhPOP1 and prePhHB genes

[0122] 2.1 Construction of plant heterologous expression vectors pEAQ-PhPOP1-HT and pCAMBIA3301-prePhHB

[0123] (1) Based on the obtained PhPOP1 gene sequence, the restriction sites in its coding region and the pEAQ-HT vector were analyzed, and AgeI and XhoI were selected as restriction endonuclease sites for the construction of the pEAQ-HT vector. An AgeI restriction site was introduced upstream of the full-length PhPOP1, and an XhoI restriction site was introduced downstream of the full-length PhPOP1. The primer sequences are as follows:

[0124] PhPOP1-F-AgeI:5'-GC ACCGGT ATGACTTCCTCCACCTTCTC-3' (SEQ I

[0125] D NO.5)

[0126] PhPOP1-R-XhoI:5'-GC CTCGAG TTAATCTGTCCAAGAGGGCT-3'(SEQ ID NO.6)

[0127] (2) According to the gene sequence of prePhHB (GeneBank: MH699110) downloaded from NCBI, the restriction sites in its coding region and pCAMBIA3301 vector were analyzed, and BamHI and XindIII were selected as restriction endonuclease sites for the construction of pCAMBIA3301 vector. The BamHI restriction site was introduced upstream of the full-length prePhHB, and the XindIII restriction site was introduced downstream of the full-length prePhHB. The primer sequences are as follows:

[0128] prePhHB-F-BamHI:5'-GC GGATCC ATGTCTACTATTTCAGCCATCCAC-3'(SEQ ID NO.7)

[0129] prePhHB-R-XindIII:5'-GC AAGCTT TTACACCATGAGGGAAATATCATCT C-3'(SEQ IDNO.8)

[0130] 2.2 Construction of plant overexpression vector pBI121-PhPOP1

[0131] According to the obtained PhPOP1 gene sequence, the restriction sites in its coding region and pBI121 vector were analyzed, and XbaI and SacI were selected as restriction endonuclease sites for vector construction. XbaI restriction site was introduced upstream of the full-length PhPOP1, and SacI restriction site was introduced downstream of the full-length PhPOP1. The primer sequences are as follows:

[0132] PhPOP1-F-XbaI:5'-GC TCTAGA ATGACTTCCTCCACCTTCTC-3'(SEQ ID NO.9)

[0133] PhPOP1-R-SacI:5'-GC GAGCTC TTAATCTGTCCAAGAGGGCT-3'(SEQ ID NO.10)

[0134] 3. Tobacco transient transformation and identification of PhPOP1 function using synthetic biology

[0135] 3.1 Obtaining the engineered strain GV3101 Agrobacterium

[0136] The empty vectors pEAQ-HT and pCAMBIA3301 and the plant heterologous expression vectors pEAQ-PhPOP1-HT and pCAMBIA3301-prePhHB were respectively transferred into the engineering strain Agrobacterium GV3101 by freeze-thaw method to obtain the engineering strains GV3101-pEAQ-HT, GV3101-pCAMBIA3301, GV3101-pEAQ-PhPOP1-HT, and GV3101-pCAMBIA3301-prePhHB.

[0137] 3.2 Transient transformation of tobacco

[0138] The positive strains of the above four engineering strains were inoculated into 5 ml YEP liquid medium containing 5 mg / L rifampicin (Rif) + 20 mg / L gentamicin (Gen) + 50 mg / L kanamycin (Kan), and cultured overnight at 28°C, 220 rpm / min; the cells were collected by centrifugation at 4000 rpm / min for 10 min; the cells were resuspended in MS liquid medium and the OD of the bacterial solution was adjusted. 600 The value was 0.6, 200 mmol / L acetosyringone (AS) and 10 mmol / L MES (PH = 5.8) were added, and the mixed Agrobacterium culture was placed at 28°C for 3 hours.

[0139] The engineered strain GV3101-pEAQ-PhPOP1-HT was mixed with GV3101-pCAMBIA3301-pre PhHB and GV3101-pCAMBIA3301, and GV3101-pEAQ-HT was mixed with GV3101-pCAMBI A3301-prePhHB and GV3101-pCAMBIA3301 in a 1:1 ratio, and then injected into tobacco leaves grown for 35 days by injection infection. The tobacco leaves were cultured in the dark for 4 days. The tobacco leaves were taken and dried, and the content of Pseudostellaria cyclotide HB in the leaves was detected. The results are shown in Tables 1 and Figure 2 .

[0140] Table 1 Comparison of Pseudostellariae Cyclotide HB Content in Tobacco Leaves Co-expressing PhPOP1 and prePhHB

[0141]

[0142] Note: Compared with pEAQ-HT+prePhHB, **P<0.01.

[0143] From Table 1 and Figure 2 It can be seen that when the prePhHB gene is expressed in tobacco leaves, the generation of cyclic peptide HB can be detected in the transgenic material. When PhPOP1 and prePhHB are co-expressed in tobacco leaves at the same time, the generation of cyclic peptide HB can also be detected in the transgenic material, and the content of the generated cyclic peptide HB is about 6.5 times higher than that in the material with a single preP hHB gene. In tobacco leaves expressing PhPOP1 alone, the generation of cyclic peptide HB could not be detected. The above results show that PhPOP1 can produce cyclic peptide HB in tobacco using the Pseudostellariae linear peptide prePhHB as a substrate. It shows that the simultaneous co-expression of PhPOP1 and prePh HB genes in tobacco can generate Pseudostellariae cyclic peptide HB, and the content is significantly higher than the content of cyclic peptide HB in tobacco overexpressing prePhHB alone. This shows that the Pseudostellariae proline oligopeptidase gene PhPOP1 obtained by the present invention can be used for heterologous synthesis of Pseudostellariae cyclic peptide HB in tobacco.

[0144] 4. Obtaining transgenic hairy roots of P. heterophylla overexpressing the PhPOP1 gene and determining the cyclic peptide HB content

[0145] 4.1 Obtaining the engineered strain K599 Agrobacterium rhizogenes

[0146] The empty vector pBI121 and the plant expression vector pBI121-PhPOP1 were respectively transferred into the engineering strain Agrobacterium rhizogenes K599 by freeze-thaw method to obtain the engineering strains K599-pBI121 and K599-pBI121-PhPOP1.

[0147] 4.2 Germination of sterile seedlings of Pseudostellaria heterophylla

[0148] The seeds of Radix Pseudostellariae were soaked in clean water for 12-20 hours, and the seeds were disinfected with 75% ethanol for 1 minute, washed with water 2-3 times, and then disinfected with an appropriate amount of 1:1 disinfectant for 6 minutes. After washing with water 4-5 times, the morphologically complete embryos of Radix Pseudostellariae were dissected and inoculated on a culture dish of MS culture medium, and cultured in a sterile culture room with 16 hours of light, 8 hours of darkness, 25°C, and 75% humidity for 14 days. The peeled seedlings grown in the culture medium were cut and rooted for transformation.

[0149] 4.3 Induction of Pseudostellaria heterophylla hairy roots

[0150] The engineered bacterial solution containing the pBI121-PhPOP1 plant expression vector in Implementation 1 was applied to an LB solid culture dish (50mg / L Kan+100mg / L Stre) containing the corresponding resistance, placed in a 28°C incubator for 48 hours, and then used to infect Pseudostellaria heterophylla root-cut seedlings. The sterile Pseudostellaria heterophylla seedlings that had been cultured for about 14 days were root-cut, and an appropriate amount of cultured pBI121-PhPOP1-K599 Agrobacterium colonies were scraped from the cut wounds and placed in a MS liquid culture medium tissue culture bottle with filter paper. The culture was carried out at 23°C, 16 hours of light, and 8 hours of darkness until new Pseudostellaria heterophylla roots grew out of the wounds coated with Agrobacterium. The screening culture medium was replaced every 10 days. After one month, the Pseudostellaria heterophylla roots were transferred to a liquid culture medium containing MS liquid for expansion culture. The results are as follows: Figure 3 As shown in AC.

[0151] 4.4 PCR detection of transgenic Pseudostellaria heterophylla hairy roots

[0152] According to the gene sequence of PhPOP1 and the plant pBI121 vector sequence, the forward detection primer and the reverse detection primer were designed respectively. The primer sequences are as follows:

[0153] 35S:5'-GACGCACAATCCCACTATCC-3'(SEQ ID NO.11)

[0154] PhPOP1-R:5'-TTAATCTGTCCAAGAGGCT-3'(SEQ ID NO.12)

[0155] In order to identify the positive clones of transgenic hairy roots of Pseudospermum heterophyllum, genomic DNA extracted by TPS simple method was used as template and 35S:5' and PhPOP1-R were used as upstream and downstream primers.

[0156] Identification of PCR positive clones. The 35S:PhPOP1 fusion fragment and the screening marker gene Kana were detected in the hairy roots of P. heterophylla overexpressing PhPOP1. The results of agarose gel electrophoresis showed that the PCR product of Kana in the positive control had a specific band at around 560 bp, which was consistent with the reference sequence size ( Figure 3 D), indicating that the PCR amplification results are valid. The PCR detection results for the marker gene Kana in the transgenic hairy roots of P. heterophylla showed that a specific band of about 2202 bp could be detected in the hairy root strains overexpressing PhPOP1 ( Figure 3 -E). These results indicate that the T-DNA region carrying the PhPOP1 gene has been successfully integrated into the genome of P. heterophylla, and the hairy roots obtained in this experiment are PhPOP1-overexpressing transgenic P. heterophylla hairy roots.

[0157] According to the gene sequence of PhPOP1, fluorescent quantitative PCR detection primers were designed to detect the expression of PhPOP1 gene in the hairy roots of transgenic P. heterophylla. The primer sequences are as follows:

[0158] PhPOP1-qF:5'-CGTGCATTCTGTAAGCGAGGACGC-3'(SEQ ID NO.13)

[0159] PhPOP1-qR:5'-CAGTCATGGGTCCAATAACAACCAC-3'(SEQ ID NO.14)

[0160] 4.5 Extraction of cyclic peptide HB from transgenic Pseudostellaria heterophylla hairy roots

[0161] Take out the well-grown positive hairy roots and put them in a 40℃ oven to dry overnight to constant weight. Then grind the dried hairy roots of Pseudostellaria heterophylla into uniform powder, weigh about 0.100g and put it into a 10Ml ep tube, add 5mL methanol to the ep tube, and put it into an ultrasonic instrument for 45min (temperature 30℃, power 50%). After the ultrasonication, centrifuge the sample at 8000rpm for 10min, take the supernatant into a new ep tube, put it into a nitrogen blower at 60℃ to dry, add 1mL methanol to the ep tube and ultrasonicate it in the ultrasonic instrument for 15min to re-dissolve. Absorb the solution and filter it with a 0.45um filter head and transfer it to the injection bottle.

[0162] 4.6 Determination of cyclic peptide HB content

[0163] The extracted samples were tested using the Waters high performance liquid chromatography detection system, and the chromatographic column was Sy Column (4.6mm×250mm, 5μm); mobile phase is acetonitrile and 0.1% phosphoric acid water, volume ratio is 60%:40%, flow rate is 1.0mL / min, column temperature is 30℃, detection wavelength is 202nm. 20μL of standard sample is injected, 20μL of each experimental sample is injected, and the injection is repeated 3 times. The peak time of the cyclopeptide HB standard is 21.021min, and the retention time of the sample is 21.122min. The content of cyclopeptide HB in the sample is calculated according to the concentration and peak area of ​​the standard, and then divided by the dry weight of the hairy root powder of Pseudostellaria heterophylla to calculate the content of cyclopeptide HB in the dry weight of the sample.

[0164] The results are shown in Tables 2, 3 and Figure 3 .

[0165] Table 2 Comparison of relative expression levels of PhPOP1 gene in transgenic Pseudostellaria heterophylla hairy roots

[0166]

[0167] Note: Compared with WT, *P<0.05, **P<0.01.

[0168] As shown in Table 2, in the four Pseudostellariae rooting lines OE-PhPOP1-2, OE-PhPOP1-3, OE-PhPOP1-4 and OE-PhPO P1-15, the expression level of PhPOP1 was significantly higher than that of the control group (CK); in the Pseudostellariae rooting line OE-PhPOP1-7, the expression level of PhPOP1 was significantly higher than that of the CK group. This shows that the method of the present invention successfully created transgenic Pseudostellariae hairy roots with overexpression of PhPOP1.

[0169] Table 3 Comparison of cyclic peptide HB content in hairy roots of Pseudostellaria heterophylla overexpressing PhPOP1

[0170]

[0171] Note: Compared with WT, *P<0.05, **P<0.01.

[0172] As can be seen from Table 3, in the three Pseudostellariae rooting strains OE-PhPOP1-3, OE-PhPOP1-4 and OE-PhPOP1-7, the content of cyclic peptide HB was significantly higher than that of the control group (CK); while in the two Pseudostellariae rooting strains OE-PhPOP1-2 and OE-PhPOP1-15, the content of cyclic peptide HB was significantly higher than that of the CK group. This shows that this method of overexpressing PhPOP1 in the roots of Pseudostellariae can greatly promote the synthesis and accumulation of cyclic peptide HB in the roots of Pseudostellariae. Figure 3 As shown, the gene expression level of PhPOP1 in the hairy roots of transgenic P. heterophylla overexpressing the PhPOP1 gene was significantly increased, and the content of cyclic peptide HB was also significantly increased, and the differences were significant.

[0173] Although the present invention has been described in detail above by means of general description, specific implementation methods and experiments, it is obvious to those skilled in the art that some modifications or improvements can be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A Pseudo-P ... The nucleotide sequence of the Pseudoradix heterophylla proline oligopeptidase gene PhPOP1 is shown in SEQ ID NO.

1.

2. The coding gene of the Pseudostellariae proline oligopeptidase gene PhPOP1 according to claim 1, characterized in that: The amino acid sequence of the coding gene is shown in SEQ ID NO.

2.

3. The Pseudo-P ... Contains a recombinant expression vector or a recombinant strain.

4. The use of the Pseudostellariae proline oligopeptidase gene PhPOP1 as claimed in claim 1 in the heterologous synthesis of Pseudostellariae cyclic peptide HB, by co-overexpressing the Pseudostellariae proline oligopeptidase gene PhPOP1 and the gene prePhHB encoding the linear peptide HB in tobacco, thereby achieving the heterologous biosynthesis of Pseudostellariae cyclic peptide HB.

5. Use of the Pseudostellariae proline oligopeptidase gene PhPOP1 as claimed in claim 1 in creating Pseudostellariae hairy roots with high cyclic peptide HB content, by overexpressing the proline oligopeptidase gene PhPOP1 in the Pseudostellariae hairy roots, thereby promoting the content of cyclic peptide HB in the Pseudostellariae hairy roots.

6. A method for heterologously synthesizing Pseudostellariae cyclic peptide HB by co-expressing the Pseudostellariae proline oligopeptidase gene PhPOP1 and the gene prePhHB encoding the linear peptide HB in tobacco, characterized in that: The steps include: S1, inserting the coding sequence of Pseudoradix heterophylla proline oligopeptidase gene PhPOP1 into the plant expression vector pEAQ-HT through AgeI and XhoI restriction endonuclease sites to construct the plant overexpression vector pEAQ-PhPOP1-HT; S2, inserting the coding sequence of the gene prePhHB encoding the linear peptide HB into the plant expression vector pCAMBIA3301 through the BamHI and XindIII restriction endonuclease sites to construct the plant overexpression vector pCAMBIA3301-prePhHB; S3, the plant overexpression vectors pEAQ-PhPOP1-TA and pCAMBIA3301-prePhHB described in steps S1 and S2 are respectively transferred into the host strain Agrobacterium GV3101 to obtain an engineered strain containing the plant overexpression vector, the bacterial solution is evenly mixed and then injected into tobacco to obtain transiently transformed tobacco with co-expression of PhPOP1 and prePhHB genes, and the content of cyclopeptide HB in tobacco leaves is detected.

7. The method according to claim 6, characterized in that The method for transferring into host strain Agrobacterium is freeze-thaw method.

8. A method for constructing transgenic Pseudostellariae hairy roots overexpressing the Pseudostellariae proline oligopeptidase gene PhPOP1, characterized in that: The steps include: 1) The coding sequence of the Pseudo-Phenothiazine proline oligopeptidase gene PhPOP1 was inserted into the plant expression vector pBI121 through the XbaI and SacI restriction endonuclease sites to construct a plant overexpression vector pBI121-PhPOP 1; 2) The plant overexpression vector pBI121-PhPOP1 described in 1) is transferred into the host strain Agrobacterium rhizogenes K599 to obtain an engineered strain containing the plant overexpression vector, and transgenic Pseudostellaria heterophylla hairy roots are obtained after Agrobacterium-mediated transformation; 3) Screening the transgenic Pseudostellariae hairy roots described in step 2) to obtain overexpressed transgenic hairy roots with significantly increased cyclic peptide HB content.

9. The construction method according to claim 8, characterized in that: In step 2), the method of transferring into the host strain Agrobacterium is freeze-thaw method.

10. The construction method according to claim 8, characterized in that: In step 3), the screening further includes PCR detection, and the primers for PCR detection are the forward detection primer shown in SEQ ID NO.11 and the reverse detection primer shown in SEQ ID NO.12.

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