Primer sequence for amplifying FhSMT1 gene of fritillaria hupehensis and application of primer sequence

Abstract

The invention discloses a primer sequence for amplifying FhSMT1 gene of fritillaria hupehensis and application of the primer sequence, belongs to the technical field of gene engineering, can effectively amplify CDS sequence of FhSMT1 of fritillaria hupehensis, and further discloses a recombinant expression vector primer sequence of FhSMT1, a recombinant expression vector is constructed through the primer, and the FhSMT1 gene of fritillaria hupehensis is obtained. The FhSMT1 can be subjected to subcellular localization after transformation, and the FhSMT1 gene has important significance in amplification of a CDS sequence of the FhSMT1 of the fritillaria hupehensis, analysis of the molecular function of the FhSMT1 and research of the effect of the FhSMT1 in synthesis of isosteroid alkaloids.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and particularly relates to a primer sequence for amplifying the FhSMT1 gene of Fritillaria hupehensis and its application. Background Art

[0002] Steroidal alkaloids are a class of secondary metabolites widely present in medicinal plants, with various pharmacological activities such as blood pressure lowering, cough relieving, asthma relieving, and anti-tumor effects. Their synthesis pathways and metabolic regulation networks have also become one of the research focuses at home and abroad. The chemical structures of the isosteroidal alkaloids of Fritillaria plants are unique, and they have significant activities such as cough relieving, expectorant, anti-inflammatory, and anti-tumor effects, and have become an important resource for new drug development.

[0003] The synthesis of isosteroidal alkaloids is relatively complex, mainly including three parts: the synthesis of the sterol isoprenoid carbon skeleton, the synthesis of cycloartenol, the cyclization product of 2,3-epoxysqualene, and the downstream pathway under the action of structure-modifying enzymes. Cycloartenol is an important branch point of sterol metabolism in higher plants, and is also an important precursor substance and key substrate for the biosynthesis of steroidal compounds. Therefore, the functional analysis of the key enzyme genes involved in the synthesis of cycloartenol has also become a research hotspot in the biosynthesis and metabolism of steroidal alkaloids in Fritillaria plants. Scholars have focused on exploring the effects of key enzymes (such as HMGR, DXR, FPS, CAS, and SQE, etc.) that catalyze the synthesis of cycloartenol on the synthesis of isosteroidal alkaloids in Fritillaria. However, as an important precursor for the biosynthesis of steroidal alkaloids, the structure-modifying enzymes downstream of cycloartenol are more worthy of attention. Only by clarifying the relationship between the function of structure-modifying enzymes and the accumulation of downstream compounds can the downstream synthesis pathway of isosteroidal alkaloids be better elucidated, so as to realize the in vitro synthesis and application of isosteroidal alkaloids.

[0004] Sterol C-24 methyltransferase 1 (SMT1) is a class of methyltransferases that are responsible for transferring one methyl group from S-adenosylmethionine to the C24 position of cycloartenol to form 24-methylenecycloartenol. SMT1 is the first step in catalyzing the synthesis of cycloartenol into steroidal compounds, and is an important rate-limiting enzyme and regulatory target in the synthesis of steroidal compounds, and plays an important role in the synthesis and regulation of plant steroidal compounds. At present, the SMT1 gene has been successfully cloned and functionally studied in plants such as Tripterygium wilfordii and Withania somnifera, but this gene has not been cloned in Fritillaria plants. In addition, the genomes of Fritillaria plants are relatively large (30.15 - 85.38 Gb), and the genome sequencing has not been completed. At the same time, the genomes of its closely related species are quite different, with high polymorphism, resulting in a lack of effective primer sequences for cloning the SMT1 gene in Fritillaria plants, which limits the application of this gene in the synthesis of isosteroidal alkaloids. Summary of the Invention

[0005] In view of this, the present invention discloses a primer sequence for amplifying the FhSMT1 gene of Fritillaria hupehensis and its application.

[0006] The present invention adopts the following technical solutions:

[0007] A primer sequence for amplifying the FhSMT1 gene of Fritillaria hupehensis, and the primer sequence is shown as follows:

[0008] FhSMT1-F: CTTCCCCACCAAGATCGGTTC,

[0009] FhSMT1-R: GTTTCAACTGTCTGTAGACGATGTCG.

[0010] Further, the CDS sequence of the FhSMT1 gene is as shown in SEQ ID NO.1.

[0011] An application of the above primer sequence in amplifying the CDS sequence of FhSMT1 of Fritillaria hupehensis.

[0012] A recombinant expression vector of the above FhSMT1 gene, and the construction of the recombinant expression vector includes: using pBWA(V)HS-ccdb-GLOSGFP as the vector, designing homologous arm primers, using the recombinant T vector as the template, constructing the recombinant expression vector: pBWA(V)HS-FhSMT1-GLOSGFP, and amplifying the target fragment using a high-fidelity enzyme.

[0013] Further, the homologous arm primers are:

[0014] E5423_0S1(+): AACACGGGGGACTTTGCAACATGTCGAATACCGGGGCTCTGG,

[0015] E5423_0S1(-): CCTGAAGCGGCCGCTGTACACACGTTCCAAGGCTTCACGACATTG.

[0016] Further, the pBWA(V)HS-ccdb-GLOSGFP is double digested with BsaI and Eco31I restriction endonucleases.

[0017] Further, a seamless cloning kit is used to ligate the target fragment and the T vector.

[0018] A protein expression vector of the above-mentioned FhSMT1 gene. The construction of the protein expression vector includes: using PET28a-SUMO-ccdb as the vector, designing primers E5894_0S1 and E5894_1S1, using the recombinant T vector as the template to construct the protein expression vector: PET28a-SUMO-FhSMT1, and using high-fidelity enzyme to amplify the target fragment.

[0019] Further, the primer E5894_0S1 is:

[0020] E5894_0S1(+): GAGAACAGATTGGTGGATCCATGTCGAATACCGGGGCTCTGG,

[0021] E5894_0S1(-): GATGGCTCCAACACGTTCCAAGGCTTCACGACATTG;

[0022] The primer E5894_1S1 is: E5894_1S1(+): TTGGAACGTGTGGAGCCATCCGCAGTTTGAAAAAGG, E5894_1S1(-): TGGTGGTGGTGGTGCTCGAGCTTTTCAAACTGAGGATGGCTCCATGC.

[0023] Further, double digestion of the PET28a-SUMO-ccdb is carried out using BsaI and Eco31I restriction endonucleases, and seamless cloning kit is used to ligate the target fragment and the T vector.

[0024] The beneficial effects of the present invention:

[0025] The present invention provides a primer sequence of FhSMT1 of Fritillaria hupehensis Hsiao et K. C. Hsia, and the CDS sequence of FhSMT1 of Fritillaria hupehensis Hsiao et K. C. Hsia can be effectively amplified by this primer.

[0026] The present invention also provides a primer sequence of a recombinant expression vector of FhSMT1. By using this primer, the recombinant expression vector pBWA(V)HS-FhSMT1-GLOSGFP is constructed, and after being transformed into Agrobacterium, subcellular localization of FhSMT1 can be successfully carried out in tobacco, providing a necessary basis for analyzing the molecular function of FhSMT1.

[0027] The present invention also provides a method for expressing FhSMT1 protein, which can effectively heterologously express FhSMT1 protein of Fritillaria hupehensis Hsiao et K. C. Hsia in Escherichia coli, providing a theoretical basis for studying the role of FhSMT1 in the synthesis of iso-steroidal alkaloids.

[0028] The present invention is of great significance for amplifying the CDS sequence of FhSMT1 from Fritillaria hupehensis, analyzing the molecular function of FhSMT1, and studying the role of FhSMT1 in the synthesis of iso-steroidal alkaloids. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0030] Figure 1 : Schematic diagram of the electrophoresis results of PCR identification (A) of the FhSMT1 gene and PCR (B) of positive colonies of the present invention;

[0031] Figure 2 : Schematic diagram of the prediction of protein domains of the FhSMT1 amino acid sequence by InterProscan of the present invention;

[0032] Figure 3 : Schematic diagram of the prediction analysis of the transmembrane structure of FhSMT1 by TMHMM of the present invention;

[0033] Figure 4 : Schematic diagram of the prediction of the secondary domain of the FhSMT1 sequence by SOMPA of the present invention;

[0034] Figure 5 : Schematic diagram of the prediction of the tertiary domain of the FhSMT1 sequence by Swiss-model of the present invention;

[0035] Figure 6 : Schematic diagram of the phylogenetic tree constructed by the Neighbor-joining method for FhSMT1 and SMT1 and SMT2 proteins of other species of the present invention;

[0036] Figure 7 : Schematic diagram of the pBWA(V)HS-FhSMT1-GLOSGFP expression vector constructed by the present invention;

[0037] Figure 8 : Schematic diagram of the verification of the EcoRV endonuclease digestion of the recombinant pBWA(V)HS-FhSMT1-GLOSGFP expression vector of the present invention;

[0038] Figure 9 : Schematic diagram of the subcellular localization of FhSMT1 of the present invention;

[0039] Figure 10: Schematic diagram of the PET28a-SUMO-FhSMT1 expression vector constructed in the present invention;

[0040] Figure 11 : Schematic diagram of the digestion verification of the recombinant PET28a-SUMO-FhSMT1 expression vector of the present invention by Esp3I endonuclease;

[0041] Figure 12 : Schematic diagram of the SDS-PAGE detection of the small-scale expression of His-FhSMT1-sumo protein of the present invention;

[0042] Figure 13 : Schematic diagram of the SDS-PAGE detection of the purified His-FhSMT1 protein of the present invention. Detailed implementation manners

[0043] For a better understanding of the technical solution of the present invention, the embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0044] It should be clear that the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the scope of protection of the present invention.

[0045] Example 1

[0046] A primer sequence for amplifying the FhSMT1 gene of Fritillaria hupehensis, and the primer sequence is shown as follows: FhSMT1-F: CTTCCCCACCAAGATCGGTTC,

[0047] FhSMT1-R: GTTTCAACTGTCTGTAGACGATGTCG.

[0048] Furthermore, the CDS sequence of the FhSMT1 gene is as shown in SEQ ID NO.1.

[0049] The specific SEQ ID NO.1 is:

[0050] ATGTCGAATACCGGGGCTCTGGATCTTGCTTTAAGCGTTGGTGGCAAGATCGATGGGAAGGAAGTGCAATCGGCCGTCAAGCAGGACGAGGAGTCGAGAAAGGTCAACTACAATGATATGGTGAACAAGTACTATGATCTTGCCACCAGCTTCTACGAGTTTGGGTGGGGAGAGTCTTTTCACTTTGCTCCTAGGTTTAAAGATAAGACATTACGCGAAAGCATCAAGCGTCATGAGCAGTTCATTGCCTTGCAGCTAGGCTTGAAAAGAGGAATGAAGGTATTGGATGTGGGATGCGGTATTGGTGGACCACTAAGAGAAATTTCTAGATTCAGCTTCACATCCATTACTGGATTGACCAACAATGATTATCATATATTTAGGGGCACGGAACTAAACCGCTTAGCAGGACTGAGCGAGTCTTGCAACTTTGTTAAGGTATACAGTCCGCTGGGCAATGTCGTGAAGCCTTGGAACGTGTAG。

[0051] Use of the above primer sequence in amplifying the CDS sequence of FhSMT1 from Fritillaria hupehensis.

[0052] A recombinant expression vector of the above FhSMT1 gene, the construction of the recombinant expression vector includes: using pBWA(V)HS-ccdb-GLOSGFP as the vector, designing homologous arm primers, using the recombinant T vector as the template, constructing the recombinant expression vector: pBWA(V)HS-FhSMT1-GLOSGFP, and using a high-fidelity enzyme to amplify the target fragment.

[0053] Furthermore, the homologous arm primers are:

[0054] E5423_0S1(+): AACACGGGGGACTTTGCAACATGTCGAATACCGGGGCTCTGG,

[0055] E5423_0S1(-): CCTGAAGCGGCCGCTGTACACACGTTCCAAGGCTTCACGACATTG.

[0056] Furthermore, the pBWA(V)HS-ccdb-GLOSGFP is double digested with BsaI and Eco31I restriction endonucleases.

[0057] Further, a seamless cloning kit was used to ligate the target fragment and the T vector.

[0058] A protein expression vector of the above-mentioned FhSMT1 gene. The construction of the protein expression vector includes: using PET28a-SUMO-ccdb as the vector, designing primers E5894_0S1 and E5894_1S1, using the recombinant T vector as the template, constructing the protein expression vector: PET28a-SUMO-FhSMT1, and using a high-fidelity enzyme to amplify the target fragment.

[0059] Further, the primer E5894_0S1 is:

[0060] E5894_0S1(+): GAGAACAGATTGGTGGATCCATGTCGAATACCGGGGCTCTGG,

[0061] E5894_0S1(-): GATGGCTCCAACACGTTCCAAGGCTTCACGACATTG;

[0062] The primer E5894_1S1 is:

[0063] E5894_1S1(+): TTGGAACGTGTGGAGCCATCCGCAGTTTGAAAAAGG,

[0064] E5894_1S1(-): TGGTGGTGGTGGTGCTCGAGCTTTTCAAAC TGAGGATGGCTCCATGC.

[0065] Further, PET28a-SUMO-ccdb was double digested with restriction enzymes BsaI and Eco31I, and a seamless cloning kit was used to ligate the target fragment and the T vector.

[0066] Example 2

[0067] 1. Cloning of the FhSMT1 gene.

[0068] Extract the RNA from the bulbs of Fritillaria hupehensis, reverse transcribe to obtain the cDNA of the FhSMT1 gene, and use it as a cloning template. According to the CDS sequence of FhSMT1 obtained by transcriptome sequencing, design specific primers with Primer Premier 5.0 software: FhSMT1-F: CTTCCCCACCAAGATCGGTTC, FhSMT1-R: GTTTCAACTGTCTGTAGACGATGTCG. Use high-fidelity enzyme to amplify the target fragment, detect the target band by 0.8% agarose gel electrophoresis (electrophoresis conditions: 1×TBE electrophoresis buffer, 120V, 35 min), and use a DNA gel recovery kit to cut and recover the target band to obtain the purified target gene. The recovered product was ligated to the pClone007 Versatile SimpleVector vector according to the TA cloning ligation kit, transformed into DH5α competent cells, and then the transformed bacterial solution was spread on an LB plate containing AMP resistance and cultured overnight at 37°C. Pick monoclonal colonies for bacterial solution, and perform PCR test with M13F / R as primers. The electrophoresis pattern is as shown in Figure 1 . Sequence the positive strains. Finally, an FhSMT1 gene sequence with a length of 750 bp and a CDS length of 483 bp was obtained, named FhSMT1.

[0069] The CDS sequence of the FhSMT1 gene is SEQ ID NO.1:

[0070] ATGTCGAATACCGGGGCTCTGGATCTTGCTTTAAGCGTTGGTGGCAAGATCGATGGGAAGGAAGTGCAATCGGCCGTCAAGCAGGACGAGGAGTCGAGAAAGGTCAACTACAATGATATGGTGAACAAGTACTATGATCTTGCCACCAGCTTCTACGAGTTTGGGTGGGGAGAGTCTTTTCACTTTGCTCCTAGGTTTAAAGATAAGACATTACGCGAAAGCATCAAGCGTCATGAGCAGT TCATTGCCTTGCAGCTAGGCTTGAAAAGAGGAATGAAGGTATTGGATGTGGGATGCGGTATTGGTGGACCACTAAGAGAAATTTCTAGATTCAGCTTCACATCCATTACTGGATTGACCAACAATGATTATCATATATTTAGGGGCACGGAACTAAACCGCTTAGCAGGACTGAGCGAGTCTTGCAACTTTGTTAAGGTATACAGTCCGCTGGGCAATGTCGTGAAGCCTTGGAACGTGTAG

[0071] The encoded amino acid sequence is SEQ ID NO.2:

[0072] MSNTGALDLALSVGGKIDGKEVQSAVKQDEESRKVNYNDMVNKYYDLATSFYEFGWGESFHFAPRFKDKTLRESIKRHEQFIALQLGLKRGMKVLDVGCGIGGPLREISRFSFTSITGLTNNDYHIFRGTELNRLAGLSESCNFVKVYSPLGNVVKPWNV*

[0073] 2. Protein structure prediction.

[0074] ProtParam was used to predict the protein. The FhSMT1 protein encodes 160 amino acids with a molecular formula of C 805 H 1253 N 221 O 237 S5, relative molecular mass is 17979.44, theoretical isoelectric point is 8.85, positively charged amino acid residues (Arg+Lys) are 21, negatively charged amino acid residues (Asp+Glu) are 18. The protein stability coefficient is 42, and it is presumed to be an unstable protein.

[0075] The protein domain prediction of the FhSMT1 amino acid sequence was carried out using InterProscan. This protein contains an SMT domain and belongs to the SMT family. The predicted protein domains are as Figure 2 shown.

[0076] The transmembrane structure of FhSMT1 was analyzed using TMHMM. This protein has no transmembrane region. The predicted transmembrane domains are as Figure 3 shown.

[0077] The secondary domain prediction of the FhSMT1 sequence was carried out using SOMPA. The results showed that the secondary structure of FhSMT1 consists of 56.25% α-helix, 10.00% extended strand, and 33.75% random coil. The predicted secondary structure of the FhSMT1 protein is as Figure 4 shown.

[0078] The tertiary domain prediction of the FhSMT1 sequence was carried out using Swiss-model. The results showed that its spatial structure is formed by a large number of α-helices. The predicted tertiary structure of the FhSMT1 protein is as Figure 5 shown.

[0079] The phylogenetic tree of FhSMT1 and SMT1 and SMT2 proteins of other species was constructed using the Neighbor-joining method, as Figure 6 shown. The results showed that FhSMT1 of Fritillaria hupehensis has the closest genetic relationship with sterol C-24 methyltransferase 1 of Dendrobium catenatum.

[0080] 3. Subcellular localization of FhSMT1 protein

[0081] (1) Construction of the pBWA(V)HS-FhSMT1-GLOSGFP expression vector.

[0082] Using pBWA(V)HS-ccdb-GLOSGFP as the expression vector, homologous arm primers E5423_0S1(+): AACACGGGGGACTTTGCAACATGTCGAATACCGGGGCTCTGG’ and E5423_0S1(-): CCTGAAGCGGCCGCTGTACACACGTTCCAAGGCTTCACGACATTG were designed. Using the recombinant T vector as the template, the target fragment was amplified using high-fidelity enzyme. The pBWA(V)HS-ccdb-GLOSGFP vector was double digested with BsaI and Eco31I restriction endonucleases, and the target fragment and the linearized vector were ligated using a seamless cloning kit to construct the pBWA(V)HS-FhSMT1-GLOSGFP expression vector, asFigure 7 As shown. Subsequently, the recombinant pBWA(V)HS-FhSMT1-GLOSGFP expression vector was transformed into DH5α competent cells. Then, the transformed bacterial solution was spread on an LB plate containing Kan resistance and cultured overnight at 37°C. Single colonies were picked and sequenced using the pBWA(V)HS-ccdb-GLosgfp identification primers (HS)35seq: TTCATTTGGAGAGAACACGGGGGAC and GFP-40R: TCGCCGTCGAGCTCCACGAGG. At the same time, the recombinant expression vector was digested with EcoRV endonuclease, and the results showed that the actual fragment size of the recombinant expression vector was consistent with the theoretical value, as Figure 8 shown.

[0083] (2) Subcellular localization.

[0084] The constructed recombinant expression vector was transferred into Agrobacterium tumefaciens GV3101 by electroporation and cultured at 30°C for 2 days. The GV3101 Agrobacterium was scraped off from the solid culture dish with an inoculation loop and inoculated into 10 mL of YEB liquid medium with hygromycin resistance and cultured at 170 rpm / min for 1 h. Centrifuged at 4000 rpm / min for 4 min, the supernatant was removed, and the cells were resuspended with a suspension of 10 mM MgCl2 (containing 120 μM AS), and the OD600 was adjusted to about 0.6. The tobacco leaves were injected and after two days of dark culture, observed using a laser confocal microscope. The results showed that FhSMT1 was localized in the nucleus and cytoplasm, as Figure 9 shown.

[0085] 4. Protein expression of FhSMT1.

[0086] (1) Construction of the PET28a-SUMO-FhSMT1 expression vector.

[0087] Using PET28a-SUMO-ccdb as the expression vector, primers E5894_0S1 and E5894_1S1 were designed. Primer E5894_0S1:

[0088] E5894_0S1(+): GAGAACAGATTGGTGGATCCATGTCGAATACCGGGGCTCTGG, E5894_0S1(-): GATGGCTCCAACACGTTCCAAGGCTTCACGACATTG;

[0089] Primer E5894_1S1:

[0090] E5894_1S1(+): TTGGAACGTGTGGAGCCATCCGCAGTTTGAAAAAGG;

[0091] E5894_1S1(-): TGGTGGTGGTGGTGCTCGAGCTTTTCAAAC TGAGGATGGCTCCATGC;

[0092] Using the recombinant T-vector as a template, the target fragment was amplified using a high-fidelity enzyme. The PET28a-SUMO-ccdb vector was double-digested with BsaI and Eco31I restriction endonucleases, and the target fragment and the linearized vector were ligated using a seamless cloning kit to construct the PET28a-SUMO-FhSMT1 expression vector, as Figure 10 shown. Subsequently, the recombinant PET28a-SUMO-FhSMT1 expression vector was transformed into DH5α competent cells. Then, the transformed bacterial solution was spread on an LB plate containing Kan resistance and cultured overnight at 37°C. Single colonies were picked and sequenced using the PET28a-SUMO-ccdb identification primers PET28a-Sumo-J292F: GTCGGACTCAGAAGTCAATCAAG and PET28a-sumo-J155R: CCGGATATAGTTCCCTCTTTCAG. At the same time, the recombinant expression vector was digested with EcoRV endonuclease. The results showed that the actual fragment size of the recombinant expression vector was consistent with the theoretical value. The schematic diagram of the Esp3I endonuclease digestion verification of the recombinant PET28a-SUMO-FhSMT1 expression vector is as Figure 11 shown.

[0093] (2) Small-scale protein expression test

[0094] The recombinant PET28a-SUMO-FhSMT1 expression vector was transferred into the expression strain BL21, and colony PCR was performed for detection. The colonies with correct band sizes were cultured on a large scale. A part of the culture was used to preserve the bacterial solution with 20% glycerol at a final concentration, and the remaining was inoculated into 8 mL of LB medium for culture. When the OD600 reached about 0.6, 1 mL of the bacterial solution was collected for standby, and IPTG (0.2 mM) was added to the remaining bacterial solution; induction expression was carried out at 16°C / 37°C for 16 h, the bacterial cells were collected by centrifugation, and an appropriate volume of Lysis buffer was added to resuspend the bacterial cells (5 mL of Lysis buffer was added to 1 g of bacterial cells). The supernatant and precipitate were collected by ultrasonic disruption, and the precipitate was resuspended with the same volume of Lysis buffer as the supernatant. 30 μL of the sample was taken, 10 μL of SDS Sample Buffer (4x) was added and mixed well, boiled at 100°C for 10 min, loaded onto the gel, and SDS-PAGE was performed for detection. The results showed that the target protein His-FhSMT1-sumo was highly expressed in the supernatant at 16°C, as Figure 12 shown.

[0095] (3) Protein purification

[0096] The above-preserved bacterial solution was activated and then inoculated into 400 mL of LB medium for cultivation until the OD600 reached about 0.6. 0.2 mM IPTG was added, and induction expression was carried out at 16 °C for 16 h. The bacterial cells were collected by centrifugation. The bacterial cells were resuspended with Lysis buffer (5 mL of Lysis buffer was added to 1 g of bacterial cells), and then ultrasonicated. The cell lysate was collected and filtered. The filtered cell lysate was incubated with the corresponding beads at 4 °C for 3 h. The beads were collected (the supernatant could be saved first at this time). Wash buffer was added to the beads for impurity washing, and gradient elution was carried out using Elution buffer (imidazole). The eluate was collected. 30 μL was taken and added to 10 μL of SDS Sample Buffer (4x), boiled for 10 min, loaded after centrifugation, and detected by SDS-PAGE. The results showed that the His-FhSMT1 protein concentration eluted by the 100 mM imidazole eluate was the highest, which was 1.3 μg / μL, and the protein purity was about 98.3%, as Figure 13 shown.

[0097] The above has introduced the embodiments of the present invention in detail. Specific examples are used in this article to elaborate on the principle and implementation manner of the present invention. The description of the above embodiments is only used to help understand the method and its core idea of the present invention; at the same time, for those of ordinary skill in the art, according to the idea of the present invention, there will be changes in the specific implementation manner and application scope. In summary, the content of this specification should not be construed as a limitation to the present invention.

Claims

1. A primer sequence for amplifying the FhSMT1 gene of Fritillaria hupehensis, characterized in that, The primer sequences are as follows: FhSMT1-F: CTTCCCCACCAAGATCGGTTC, FhSMT1-R: GTTTCAACTGTCTGTAGACGATGTCG.

2. The primer sequence according to claim 1, wherein The CDS sequence of the FhSMT1 gene is shown as SEQ ID NO.

1.

3. The application of the primer sequence according to any one of claims 1 and 2 in amplifying the CDS sequence of FhSMT1 of Fritillaria hupehensis Hsiao et K. C. Hsia.

4. A recombinant expression vector of the FhSMT1 gene as described in claim 2, characterized in that, The construction of the recombinant expression vector includes: using pBWA(V)HS-ccdb-GLOSGFP as the vector, designing homologous arm primers, using the recombinant T vector as the template, constructing the recombinant expression vector: pBWA(V)HS-FhSMT1-GLOSGFP, and amplifying the target fragment using high-fidelity enzyme.

5. The recombinant expression vector according to claim 4, wherein The homologous arm primers are: E5423_0S1(+): AACACGGGGGACTTTGCAACATGTCGAATACCGGGGCTCTGG, E5423_0S1(-): CCTGAAGCGGCCGCTGTACACACGTTCCAAGGCTTCACGACATTG.

6. The recombinant expression vector according to claim 5, wherein, Use BsaI and Eco31I restriction endonucleases to perform double digestion on the pBWA(V)HS-ccdb-GLOSGFP.

7. The recombinant expression vector according to claim 6, wherein, Use a seamless cloning kit to ligate the target fragment and the T vector.

8. A protein expression vector of the FhSMT1 gene as described in claim 2, characterized in that, The construction of the protein expression vector includes: using PET28a-SUMO-ccdb as the vector, designing E5894_0S1 primer and E5894_1S1 primer, using the recombinant T vector as the template, constructing the protein expression vector: PET28a-SUMO-FhSMT1, and amplifying the target fragment using high-fidelity enzyme.

9. The protein expression vector according to claim 8, wherein The E5894_0S1 primer is: E5894_0S1(+): GAGAACAGATTGGTGGATCCATGTCGAATACCGGGGCTCTGG, E5894_0S1(-): GATGGCTCCAACACGTTCCAAGGCTTCACGACATTG; The E5894_1S1 primer is: E5894_1S1(+): TTGGAACGTGTGGAGCCATCCGCAGTTTGAAAAAGG, E5894_1S1(-): TGGTGGTGGTGGTGCTCGAGCTTTTCAAAC TGAGGATGGCTCCATGC.

10. The protein expression vector according to claim 9, wherein, Use BsaI and Eco31I restriction endonucleases to perform double digestion on the PET28a-SUMO-ccdb, and use a seamless cloning kit to ligate the target fragment and the T vector.

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