Prunella vulgaris cytochrome oxidase CYP protein and its encoding gene and application

Abstract

The invention discloses a selfheal cytochrome oxidase CYP protein and its encoding gene and application, and belongs to the technical field of plant biology. The selfheal cytochrome oxidase CYP protein disclosed by the invention is a CYP716-4 protein with an amino acid sequence as shown in SEQ ID NO.1, or a CYP716-7 protein with an amino acid sequence as shown in SEQ ID NO.2. The encoding gene of the selfheal cytochrome oxidase CYP protein is a PvμLCYP716-4 gene with a nucleotide sequence as shown in SEQ ID NO.3, or a PvμLCYP716-7 gene with a nucleotide sequence as shown in SEQ ID NO.4. The results of transgenic experiments show that the PvμLCYP716-4 and PvμLCYP716-7 genes oxidize α-amyrin and β-amyrin to generate ursolic acid and oleanolic acid.

Description

Technical Field

[0001] The invention belongs to the technical field of plant biology, and in particular relates to a Prunella vulgaris cytochrome oxidase CYP protein and a coding gene and application thereof. Background Art

[0002] Cytochrome P450 (CYP450) belongs to the superfamily of heme-containing monooxygenases. It is not strictly a cytochrome, but it was named after the carbon monoxide and ferrous iron complex produced when it was first discovered in rat liver microsomes, which has a maximum absorption value at 450nm. The CYP family naming is based on the consistency of the amino acid sequence. If the amino acid sequence similarity exceeds 40%, it belongs to the same family, and if it exceeds 55%, it belongs to the same subfamily. The name is written as the formal abbreviation (CYP) + number (family) + letter (subfamily) + number (isoform), for example, CYP2C5 is the 5 protein in the 2 family C subfamily of CYP.

[0003] The catalytic effect of cytochrome P450 mostly depends on cytochrome P450 reductase (CPR). CPR is composed of three cofactor binding domains: FMN, FAD and NADPH. When NADPH binds to the NADPH binding domain, two electrons are sequentially transferred to FAD and FMN, and finally to the complex bound to the substrate and CYP. The binding site of the substrate and CYP is close to the heme-iron complex. The supplied electrons reduce the trivalent iron in the heme to the ferrous state, and insert the oxygen atom into the substrate. At this point, cytochrome P450 has completed the task of the monooxygenase containing heme.

[0004] Prunella vulgaris L., also known as wheat ear Prunella vulgaris and iron wire Prunella vulgaris, is a perennial herbaceous plant of the genus Prunella in the family Lamiaceae. Prunella vulgaris is rich in triterpenoid compounds, among which ursolic acid and oleanolic acid are the highest content triterpenoid compounds, and ursolic acid is also a marker compound for testing the quality of Prunella vulgaris in the Chinese Pharmacopoeia. Ursolic acid and oleanolic acid are the most common and widely used triterpenoid compounds, so it is very important to study the biosynthesis of ursolic acid and oleanolic acid. However, there is no special study on the biosynthesis of ursolic acid and oleanolic acid in Prunella vulgaris. Therefore, it is necessary to develop and study cytochrome oxidase in Prunella vulgaris, a plant of the family Lamiaceae. Summary of the invention

[0005] In view of the above problems existing in the prior art, the technical problem to be solved by the present invention is to provide a self-squalane cytochrome oxidase CYP protein. Another problem to be solved by the present invention is to provide a gene encoding a self-squalane cytochrome oxidase CYP protein. Another technical problem to be solved by the present invention is to provide an application of a self-squalane cytochrome oxidase CYP protein for producing ursolic acid and oleanolic acid precursor skeletons α-amyrin and β-amyrin. Another technical problem to be solved by the present invention is to provide an application of a gene encoding a self-squalane cytochrome oxidase CYP protein for cyclizing 2,3-oxidosqualene to generate α-amyrin and β-amyrin.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0007] The Prunella vulgaris cytochrome oxidase CYP protein is a CYP716-4 protein with an amino acid sequence as shown in SEQ ID NO.1, or a CYP716-7 protein with an amino acid sequence as shown in SEQ ID NO.2.

[0008] The gene encoding the Prunella vulgaris cytochrome oxidase CYP protein is the PvμLCYP716-4 gene with a nucleotide sequence as shown in SEQ ID NO.3, or the PvμLCYP716-7 gene with a nucleotide sequence as shown in SEQ ID NO.4.

[0009] Application of Prunella vulgaris cytochrome oxidase CYP protein CYP716-4 in the preparation of α-amyrin, β-amyrin, ursolic acid and oleanolic acid.

[0010] Application of Prunella vulgaris cytochrome oxidase CYP protein CYP716-7 in the preparation of α-amyrin, β-amyrin, ursolic acid and oleanolic acid.

[0011] Application of the gene PvμLCYP716-4 encoding the Prunella vulgaris cytochrome oxidase CYP protein in the preparation of α-amyrin, β-amyrin, ursolic acid and oleanolic acid.

[0012] The invention discloses an application of the gene PvμLCYP716-7 encoding the Prunella vulgaris cytochrome oxidase CYP protein in the preparation of α-amyrin, β-amyrin, ursolic acid and oleanolic acid.

[0013] The use of the gene encoding PvμLCYP716-4 or PvμLCYP716-7 of Prunella vulgaris cytochrome oxidase CYP protein in the preparation of α-amyrin, β-amyrin, ursolic acid and oleanolic acid is characterized by comprising the following steps:

[0014] 1) constructing an expression vector of Prunella vulgaris cytochrome oxidase CYP gene;

[0015] 2) transforming the constructed expression vector of Prunella vulgaris cytochrome oxidase CYP gene into tobacco;

[0016] 3) Cultivate, screen and obtain transgenic tobacco with increased content of α-amyrin, β-amyrin, ursolic acid and oleanolic acid.

[0017] The expression vector is a plant expression vector.

[0018] The plant expression vector is pCAMBIA1300-PvμLCYP716-4-gfp or pCAMBIA1300-PvμLCYP716-7-gfp.

[0019] The invention discloses an application of pCAMBIA1300-PvμLCYP716-4-gfp or pCAMBIA1300-PvμLCYP716-7-gfp plant expression vector in the preparation of α-amyrin, β-amyrin, ursolic acid and oleanolic acid.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1) The present invention discloses for the first time a cytochrome oxidase from Prunella vulgaris, a plant of the Lamiaceae family, which has good substrate specificity and regional selectivity.

[0022] The Prunella vulgaris cytochrome oxidase CYP protein disclosed in the present invention is a CYP716-4 protein with an amino acid sequence as shown in SEQ ID NO.1, or a CYP716-7 protein with an amino acid sequence as shown in SEQ ID NO.2. The coding gene of the Prunella vulgaris cytochrome oxidase CYP protein is a PvμLCYP716-4 gene with a nucleotide sequence as shown in SEQ ID NO.3, or a PvμLCYP716-7 gene with a nucleotide sequence as shown in SEQ ID NO.4.

[0023] 2) In the experimental groups of the present invention co-injected with PvμLOSC-1 and PvμLCYP716-4 genes and co-injected with PvμLOSC-1 and PvμLCYP716-7 genes, α-amyrin and β-amyrin were not detected at the corresponding positions, but oleanolic acid was detected at 17.901min±0.1min and ursolic acid was detected at 18.351min±0.1min. Compared with the standard samples of α-amyrin, β-amyrin, ursolic acid and oleanolic acid, the peak time and fragment information of the two were consistent.

[0024] The above results showed that PvμLCYP716-4 and PvμLCYP716-7 genes oxidized α-amyrin and β-amyrin to produce ursolic acid and oleanolic acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the PCR gel image of OSC-1, PvμLCYP716-4 and PvμLCYP716-7 gene cloning;

[0026] Figure 2 Schematic diagram of pCAMBIA1300-GFP vector;

[0027] Figure 3 PCR gel images for gene cloning and vector construction of PvμLCYP716-4 and PvμLCYP716-7;

[0028] Figure 4 This is the PCR gel image of the Agrobacterium culture solution of the target gene;

[0029] Figure 5 GC-MS profiles for identification of injected tobacco products;

[0030] Figure 6 It is a diagram of the molecular structures of α-amyrin, β-amyrin, ursolic acid and oleanolic acid. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. Unless otherwise specified in the following embodiments, the technical means used are conventional means well known to those skilled in the art.

[0032] The plant material Prunella vulgaris selected in the present invention is planted in the Institute of Botany, Chinese Academy of Sciences, Jiangsu Province.

[0033] DNA polymerase was selected from Nanjing Novozyme Biotechnology Co., Ltd. Super-Fidelity DNA Polymerase.

[0034] Example 1

[0035] 1. RNA extraction and cDNA acquisition

[0036] Total RNA was extracted from leaves, stems and roots of Prunella vulgaris using a high-purity total RNA extraction kit (Pudi Biotechnology Product No. AF505B), and reverse transcribed into cDNA using PrimeScript RT reagent Kit with gDNA erase (Takara, Japan).

[0037] 2. Cloning of target gene fragment

[0038] The cDNA obtained by reverse transcription was used as a template and PCR amplified using primers. The primer sequences are as follows:

[0039] OSC-1-F: 5'-ATGTGGAGGCTGAAGGTGGCGGAG-3',

[0040] OSC-1-R: 5'-CAAGCATTGTGATGAAAAGACTCGT-3',

[0041] CYP716-4-F: 5'-ATGGAGATATTCTACGTCTCCCTC-3',

[0042] CYP716-4-R: 5'-AGCTTTGTGAGGGAAAAGTCGAAC-3';

[0043] CYP716-7-F: 5'-ATGGAGATCTTCTACATCTCCCTC-3',

[0044] CYP716-7-R: 5'-ATTAGATTGATTGATTGAGGCTTT-3'.

[0045] The PCR amplification program was as follows: 95°C for 5 min; 94°C for 45 s, 56°C for 1 min, and 72°C for 2 min, for a total of 30 cycles; 72°C for 10 min, and then dropped to 10°C.

[0046] The PCR product was detected by agarose gel electrophoresis, and the DNA was recovered from the agarose gel using the UE DNA gel recovery kit (Suzhou Youyi Landi Biotechnology Co., Ltd.), which was the amplified DNA fragment encoding the gene for the cytochrome oxidase of Prunella vulgaris ( Figure 1 ).

[0047] 3. Target gene fragment connection, transformation and sequencing

[0048] The recovered PCR products were cloned into the pClone007 vector using the pClone007 Blunt Simple Vector Kit (Beijing Qingke Biotechnology Co., Ltd.), and the constructed vectors were named pClone007-PvμLOSC-1, pClone007-PvμLCYP716-4, and pClone007-PvμLCYP716-7. The assembled vectors were transformed into Escherichia coli DH5α cells and cultured overnight in a selective medium (containing 50 μg / mL ampicillin) at 37°C. Colonies were screened by PCR, and the PCR product sequences were determined by Sanger sequencing. The sequencing results showed that the 2,3-oxidosqualene cyclase CYP716-4 of Prunella vulgaris was obtained, and its amino acid sequence was shown in SEQ ID NO.1, and its nucleotide sequence of the encoding gene PvμLCYP716-4 was shown in SEQ ID NO.3; the 2,3-oxidosqualene cyclase CYP716-7 of Prunella vulgaris was obtained, and its amino acid sequence was shown in SEQ ID NO.2, and its nucleotide sequence of the encoding gene PvμLCYP716-7 was shown in SEQ ID NO.4. The 2,3-oxidosqualene cyclase OSC-1 of Prunella vulgaris was obtained, and its amino acid sequence was shown in SEQ ID NO.5, and its nucleotide sequence of the encoding gene PvμLOSC-1 was shown in SEQ ID NO.6;

[0049] Example 2

[0050] 1. Construction of recombinant expression vector

[0051] The primer sequences are as follows:

[0052] OSC-1-1300-GFP-F:

[0053] 5'-CTCGATACACCAAATCGACTCTAGAATGTGGAGGCTGAAG-3',

[0054] OSC-1-1300-GFP-R:

[0055] 5'-GCCCTTGCTCACCATGGTACCCAAGCATTGTGATGAAAA-3';

[0056] CYP716-4-1300-gfp-F:

[0057] 5'-CTCGATACACCAAATCGACTCTAGAATGGAGATATTCTACGTC-3',

[0058] CYP716-4-1300-gfp-R:

[0059] 5'-GCCCTTGCTCACCATGGTACCAGCTTTGTGAGGGAAAAG-3';

[0060] CYP716-7-1300-gfp-F:

[0061] 5'-CTCGATACACCAAATCGACTCTAGAATGGAGATCTTCTACATC-3',

[0062] CYP716-7-1300-gfp-R:

[0063] 5'-GCCCTTGCTCACCATGGTACCATTAGATTGATTGATTGA-3';

[0064] 1300-F:

[0065] 5'-CTGAATTTCATAACCAATTCGATACA-3',

[0066] pEGFP-N3:

[0067] 5'-GTCGCCGTCCAGCTCGACCAG-3'.

[0068] The pCAMBIA1300-GFP vector ( Figure 2 ) for enzyme digestion, and the digestion products were gel purified.

[0069] The target gene fragment was cloned from pClone007-PvμLOSC-1, pClone007-PvμLCYP716-4 and pClone007-PvμLCYP716-7 vectors using primers OSC-1-1300-gfp-F / R, CYP716-4-1300-gfp-F / R, and inserted into the (XbaI / KpnI) pCAMBIA1300-GFP transformation plasmid using isothermal DNA assembly technology. The assembled plasmid reaction was transformed into Escherichia coli DH5α competent cells (Sangon Biotech) and cultured overnight at 37°C on selective LB agar plates (containing 50μg / mL kanamycin). Using primers 1300-F and pEGFP-N3, PCR was used to screen colonies, and the PCR product sequences were determined by Sanger sequencing to obtain positive transformants containing expression vectors pCAMBIA1300-PvμLOSC-1-gfp, pCAMBIA1300-CYP716-4-gfp, and pCAMBIA1300-CYP716-7-gfp ( Figure 3 ).

[0070] 2 mL of liquid LB culture was inoculated with positive transformants and then shaken overnight at 37 °C. The plasmid of the positive transformant was extracted using the UE plasmid miniprep kit (Suzhou Youyi Landi Biotechnology Co., Ltd.), and the plasmid containing the target gene was transformed into Agrobacterium GV3101 competent cells (Sangon Biotechnology) using the heat shock method and cultured at 30 °C for 2 days on selective LB agar plates (kanamycin 50 μg / mL and rifampicin 50 μg / mL). Positive transformants were verified by colony PCR ( Figure 4 ), and then the positive transformants were inoculated into 2 mL of liquid LB culture, cultured at 30°C for 1 day, and then stored in 25% glycerol at -80°C.

[0071] 2. Tobacco transient transfection and product analysis

[0072] The Agrobacterium strain containing the target plasmid construct was cultured in LB medium (kanamycin 50 μg / mL + rifampicin 50 μg / mL) at 30°C with shaking at 200 rpm for 16 h; the cells were then centrifuged at 8000 g for 5 min, and then resuspended in an equal volume of Agrobacterium induction buffer (pH 5.6) containing 10 mM MES (2-(N-morpholino)ethanesulfonic acid monohydrate), 10 mM MgCl2 and 150 μM acetosyringone, and incubated at room temperature for 3 hours. The optical density OD 600 Measure the concentration of the cell resuspension and divide the strain combinations into the OD values ​​of each strain. 600The above strain mixture was infiltrated into the back of 4-5 week old Nicotiana benthamiana leaves using a needleless syringe, cultured in dark for 1 day, and then cultured in light.

[0073] Four days after tobacco injection, tobacco leaves were taken and frozen with liquid nitrogen, freeze-dried, soaked in 5 times dry weight of methanol for 24 hours, extracted by low temperature ultrasonic for 30 minutes every 8 hours, then oscillated and mixed, centrifuged at 12000xg for 10 minutes, and the supernatant was taken for subsequent analysis. 50μL of supernatant was concentrated and dried at 50℃, then 250μL of N-methyl-N-(trimethylsilyl) trifluoroacetamide was added and mixed, and derivatized in a water bath at 80℃ for 40 minutes (10.1002 / 1873-3468.12074), and the obtained samples were subsequently subjected to gas chromatography-mass spectrometry analysis. A gas chromatograph (Agilent 8860) was connected to an inert MSD system (Agilent 5977B), using an HP-5MS capillary column (30m×0.25mm×0.25um, Aglient), helium as the carrier gas, a flow rate of 1mL / min, an injection temperature of 250℃, an interface temperature of 320℃, a gas chromatography-mass spectrometry program of 80℃ for 1 minute, rising to 320℃ at a rate of 20℃ / min, and maintaining for 17 minutes, an injection split ratio of 1:5, and a scanning range of 20-750. The contents of α-amyrin, β-amyrin, oleanolic acid, and ursolic acid in the samples were verified by comparing the retention time and fragmentation molecular weight of the standards.

[0074] The results are as follows Figure 5 As shown, in the experimental groups co-injected with PvμLOSC-1 and PvμLCYP716-4 genes and co-injected with PvμLOSC-1 and PvμLCYP716-7 genes, α-amyrin and β-amyrin were not detected at the corresponding positions, but oleanolic acid was detected at 17.901min±0.1min and ursolic acid was detected at 18.351min±0.1min. Compared with the standard samples of α-amyrin, β-amyrin, ursolic acid and oleanolic acid, the peak time and fragment information of the two were consistent.

[0075] The above results show that PvμLCYP716-4 and PvμLCYP716-7 genes oxidize α-amyrin and β-amyrin to produce ursolic acid and oleanolic acid. The molecular structures of α-amyrin, β-amyrin, ursolic acid and oleanolic acid are shown in Figure 2. Figure 6 shown.

[0076] In addition, it should be understood that after reading the above content of the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. Gene encoding Prunella vulgaris cytochrome oxidase CYP protein PvμLCYP716-4 The application in the preparation of ursolic acid and oleanolic acid is characterized in that: Construction of Prunella vulgaris cytochrome oxidase PvμLCYP716-4 Prunella vulgaris cytochrome oxidase PvμLCYP716-4 Gene expression vectors and PvμLOSC-1 The gene expression vectors are simultaneously co-injected and transformed into tobacco; PvμLCYP716-4 The gene nucleotide sequence is shown in SEQ ID NO. 3; Pvμ LOSC-1 The nucleotide sequence of the gene is shown in SEQ ID NO.

6.

2. Gene encoding Prunella vulgaris cytochrome oxidase CYP protein PvμLCYP716-7 The application in the preparation of ursolic acid and oleanolic acid is characterized in that: Construction of Prunella vulgaris cytochrome oxidase PvμLCYP716-7 Prunella vulgaris cytochrome oxidase PvμLCYP716-7 Gene expression vectors and PvμLOSC-1 The gene expression vectors are simultaneously co-injected and transformed into tobacco; PvμLCYP716-7 The gene nucleotide sequence is shown in SEQ ID NO. 4; Pvμ LOSC-1 The nucleotide sequence of the gene is shown in SEQ ID NO. 6.

Images