CN103374582A - Lonicera japonica thunb cinnamate-4-hydroxylase (LJC4H) gene as well as product coded by same and application of gene - Google Patents

Abstract

The invention discloses a honeysuckle cinnamic acid-4-hydroxylase (LJC4H) gene, its coded protease and application. The honeysuckle cinnamic acid-4-hydroxylase (LJC4H) gene provided by the present invention has the nucleotide sequence shown in SEQ ID NO.1 or a homologous sequence with addition, substitution, insertion or deletion of one or more nucleotides Or its alleles and their derived nucleotide sequences. The protein encoded by the gene has the amino acid sequence shown in SEQ ID NO.2 or a homologous sequence with addition, substitution, insertion or deletion of one or more amino acids.

Description

Lonicerae cinnamic acid-4-hydroxylase (LJC4H) gene and its encoded product and application

technical field

The invention belongs to the field of biotechnology, and mainly relates to the use of a full-length cDNA library to clone cinnamic acid-4-hydroxylase gene and its encoded product and application, in particular to biosynthesis of organic acids and flavonoid enzyme genes and their The coding product and application belong to the field of genetic engineering of medicinal plants. the

Background technique

The formation of medicinal plant active ingredients is the product of a unique group of genes in plant secondary metabolic pathways. With the extensive and in-depth study of plant functional genomes, the study of functional genes related to secondary metabolism synthesis of medicinal plants with unique characteristics and broad application prospects has gradually become a research hotspot. It provides a theoretical basis for the biosynthetic pathway and its regulation mechanism and explains the formation of the quality of medicinal materials, and at the same time brings a broad application space for the use of biotechnology to increase the content of target components or directly produce active components or intermediates. the

The chemical composition of medicinal plants is complex and there are many kinds, which mainly contain organic acids, flavonoids, volatile oils, terpenes, diterpenes, trace elements and other components. Lonicera japonica Thunb is a commonly used traditional Chinese medicine, mainly used in various A kind of febrile venereal disease, such as the treatment of symptoms such as body heat, rash, spot, heat toxin sore carbuncle, and sore throat. The main active ingredients of honeysuckle include chlorogenic acid and luteolin, among which chlorogenic acid belongs to organic acids, and luteolin belongs to flavonoids. the

Cinnamate-4-hydroxylase (C4H), also known as trans-cinnamate-4-monooxygenase, is a member of the cytochrome monooxygenase P450 superfamily and belongs to the CYP73 subfamily , can catalyze the hydroxylation of cinnamic acid to produce 4-coumarate, which can be further converted into coumarin, chlorogenic acid, etc., and can also form CoA ester, which can be further converted into flavonoids such as luteolin, which is the phenylpropane pathway Second key enzyme after phenylalanine ammonia lyase. The cloning of honeysuckle cinnamic acid-4-hydroxylase gene provides an important basis for using genetic engineering to increase the content of active ingredients in honeysuckle. Before the publication of the present invention, there has not been any disclosure or report of the cinnamic acid-4-hydroxylase gene and its amino acid sequence in Flos Lonicerae mentioned in this patent application. the

Contents of the invention

The object of the present invention is to provide a honeysuckle cinnamic acid-4-hydroxylase gene (LJC4H). the

The second object of the present invention is to provide the protein encoded by the gene. the

The invention also provides recombinant vector and host cell containing the gene. the

Another object of the present invention is to provide the application of the gene. the

The honeysuckle cinnamic acid-4-hydroxylase (LJC4H) gene provided by the present invention is one of the following nucleotide sequences:

(1) have the nucleotide sequence shown in SEQ ID NO.1;

(2) A homologous sequence of one or more nucleotides added, substituted, inserted or deleted in the nucleotide sequence shown in SEQ ID NO.1 or its allele and its derived nucleotide sequence. the

The protein encoded by this gene is honeysuckle cinnamic acid-4-hydroxylase (LJC4H) gene, which is one of the following amino acid sequences:

(1) have the amino acid sequence shown in SEQ ID NO.2;

(2) A homologous sequence of one or more amino acids added, substituted, inserted or deleted in SEQ ID NO.2. the

The honeysuckle cinnamic acid-4-hydroxylase (LJC4H) gene provided by the present invention is cloned from the honeysuckle flower for the first time, and in vitro experiments show that the LJC4H has the activity of catalyzing the conversion of cinnamic acid. Utilizing the invention, the content of organic acid chlorogenic acid and flavonoid luteolin in plants such as honeysuckle can be increased through genetic engineering technology. the

Description of drawings:

Figure 1: LJC4H functional domain prediction analysis (from NCBI database);

Figure 2: LJC4H phylogenetic tree (neighbor-joining method);

Figure 3: Construction of expression vector pYEUra3-LJC4H;

Figure 4: HPLC detection results of enzymatic reaction products. A, pYEUra3 catalytic product; B, pYEUra3-LJC4H catalytic product (without NADPH); C, pYEUra3-LJC4H catalytic product; D, 1,4-coumaric acid; 2, trans-cinnamic acid standard. 

Detailed ways

Embodiment 1, the construction of honeysuckle full-length cDNA library

1. Isolation and detection of honeysuckle total RNA

Take 2 g of honeysuckle (Lonicera japonica Thunb) flower buds, quickly grind them into powder with liquid nitrogen in a mortar, and quickly transfer them to 10 mL of extraction buffer (CTAB (W/V) 2%, Tris-HCl (pH 8 .0) 100mmol·L ^-1 , EDTA 25mmol·L ^-1 , NaCl 2.0mol·L ^-1 , PVP40 2%, spermidine 0.5g/L, mercaptoethanol 2%), shake and mix well; Volume chloroform extraction twice, 7500g centrifugation for 15 minutes. Add 1/4 volume of 10M LiCl to the supernatant, mix well and place it at 4°C to precipitate overnight; centrifuge at 7500g for 20 minutes, and use 500 μL SSTE (SDS 0.5%, NaCl 1mol·L ^-1 , Tris-HCl (pH8.0) 10mmol·L ^-1 , EDTA 1mmol·L ^-1 , dissolve at 65°C for 5 minutes. Extract with an equal volume of chloroform, centrifuge at 13,000g for 5 minutes; add 2 times the volume of absolute ethanol to the supernatant, and place at -70°C for 2 hours; 4 Centrifuge at 13000g for 20 minutes, dry the precipitate at room temperature for 10 minutes, and then dissolve it in 100 μL of DEPC-treated water. Use 1.0% agarose electrophoresis to detect the integrity of RNA, and use a GenQuant nucleic acid quantifier to measure the ratio and concentration of A260 and A280. Store at -80°C Refrigerator for spare.

2. Construction of cDNA library

After using the mRNA purification kit (QuichprepTM Micro mRNA PurifiCation Kit, Pharmacia Company) to isolate mRNA, the Creator Smart cDNA Library Construction Kit (Cat. No. 634903) of Clontech Company was used to construct the library. The principle is SMART (switch mechanism at 5′end of mRNA template). the

Embodiment 2: Cloning of honeysuckle-related genes

Randomly pick 5000 single clones for colony PCR identification. Take an appropriate amount of PCR thin-walled tubes, place them on ice, and add 17.3ul of sterilized water to each tube. Use a sterilized 10ul small tip to pick up the monoclonal white spot into sterilized water, shake and mix. Add in order: Taq buffer 2.5 μL, MgCl ₂ (25 mM) 1.8 μL, dNTP (2.5 mM) 1 μL, M13+ primer (10 pmol) 1 μL, M13-primer (10 pmol) 1 μL, Taq enzyme 0.4 μL. After each reagent is added, shake it on the centrifuge to make it sink to the bottom, and place it on the PCR instrument. The PCR reaction conditions were 5 minutes of pre-denaturation at 94°C, 40 seconds at 94°C, 40 seconds at 54°C, 4 minutes at 72°C, 35 cycles of extension at 72°C for 10 minutes, and storage at 4°C. After the PCR reaction enters 4°C, remove the thin-walled PCR tube, take 7ul of the PCR product and add 3ul of Bromine Finland to run electrophoresis, take a picture half an hour later, observe the gel map, and roughly identify the size of the inserted fragment and the small fragment rate according to the gel map. Select the single amplification product of the band and send it to Huada Genomics Company for sequencing to obtain the gene sequence related to honeysuckle.

Example 3, bioinformatics analysis of LJC4H gene

The length of the full-length cDNA of the honeysuckle cinnamic acid-4-hydroxylase (C4H) gene involved in the present invention is 1518 bp, and the detailed sequence is shown in sequence 1 in the sequence list, wherein the open reading frame is located at 1-1518 bp. The full-length cDNA sequence of Lonicerae japonica was searched for nucleotide homology in Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDS translation+PDB+Swissprot+Superdate+PIR databases with BLAST program. The gene has high homology with C4H in other species at the amino acid level, and also has a typical cypX superfamily domain. Figure 1. the

Embodiment 4, the research of LJC4H gene function

1. Construction of expression vector

Using LJC4H cDNA as a template, use primers P1: 5′-GTCGACATGGATCTTCTCCTCTTAGA-3′, P2: 5′-GTCGACTCAAAAAGATCTCTT-3′ to carry out PCR reaction, take 5ul of the amplified product and add 3ul of bromo-Finland running electrophoresis, take pictures half an hour later, and observe the gel Figure, the amplified fragment is 1500bp. The amplified product was digested with Sal I for 2 hours, and the digested product was purified using a recovery kit (Takara, China). At the same time, the pYEUra3 vector was digested with Sal I at 37°C for 2 hours, and 5ul of brominated Finnish was added for agarose gel electrophoresis, the gel pattern was observed, and the 6100bp fragment was recovered with a recovery kit. the

The two were ligated overnight at 16°C by ligase. The ligation product was digested with Xho I for 2 hours, a fragment of about 7600 bp was recovered with a recovery kit, and ligated overnight at 16°C. Escherichia coli DH5α competent cells were transformed by electric shock, and recombinants were screened on 2% glucose USM plates containing ampicillin. The pYEUra3 plasmid containing the C4H clone was identified by PCR and restriction endonuclease electrophoresis and DNA sequence analysis, and the recombinant plasmid pET-LJC4H with the correct target sequence was saved for expression transformation. The plant expression vector was named pYEUra3-LJC4H (Fig. 3). the

2. Induced expression and enzyme activity detection

Use the pYEUra3-LJC4H plasmid to transform the partially enzymatic yeast host strain AB1380 by electric shock. After culturing for 5 days, screen the positive yeast on 2ml USM medium containing 2% glucose. After 4-5 days, pick a single clone and put it in 2mL USM liquid medium, at 30°C Incubate overnight with vigorous shaking. Centrifuge at 5000rpm for 5min, discard the supernatant, dilute 20 times with USM liquid medium containing 2% glucose, and incubate vigorously at 30°C for 24h. The total protein of cultured cells was extracted, and the activity of heme peroxidase was detected. Select high-expressing transformants and culture them with 50 mL of USM liquid medium containing 2% glucose at 30°C with vigorous shaking for 20 hours, then inoculate them into 1L of USM liquid medium without glucose at 30°C for 40 hours with vigorous shaking, recover bacteria, and isolate microsomes. the

Take 30-50 mg microsomal protein and incubate in 600 mL C4H enzymatic reaction system (NADPH final concentration 0.5 mM, Na3PO3 concentration 100 mM, pH 7.4, cinnamic acid concentration 0.1 mm) at 30 °C for 10 min. Terminate the reaction with 40mL 6M HCl, extract twice with 600μL ethyl acetate, evaporate the organic phase in a vacuum freeze-drying oven, dissolve it in 800μL acetonitrile, and analyze it by high performance liquid chromatography, the condition is 3.9×300mm (10μm) μBondapak C18 column (Waters), 996photodiode array detector (Waters, Milford, MA)), mobile phase A: 0.85% phosphoric acid [w/v]; mobile phase B: acetonitrile. Gradient elution 0-30min A 80%-48% , B 20%-52%, column temperature 30°C, the results are shown in Figure 4. the

Embodiment 5, research on the function of the mutant LJC4H gene

Using LJC4H cDNA as a template, use primers P3: 5′-GTCGACATGGATCTTCTCCTCTTCGA-3′, P4: 5′-GTCGACTCAAAAAGATCTCTT-3′ for PCR reaction, take 5ul of the amplified product and add 3ul of bromo-Finland to run for half an hour, then take pictures and observe the gel map , to obtain the non-synonymous mutation LJC4H. The expression vector construction, induced expression and enzyme activity detection were the same as in Example 4, and the results showed that the function of the mutant LJC4H gene was not significantly different from that of LJC4H. the

Claims (3)

1. Japanese Honeysuckle styracin-4-hydroxylase (LJC4H) gene is characterized in that it is one of following nucleotide sequences:

(1) dna sequence dna of SEQ ID No.1;

(2) nucleotide sequence that add, replace, insert or delete the homologous sequence of one or more Nucleotide or its allelotrope and derive of the nucleotide sequence shown in the SEQ ID No.1.

2. Japanese Honeysuckle styracin-4-hydroxylase (LJC4H) is characterized in that, is one of following aminoacid sequence:

(1) has the aminoacid sequence shown in the SEQ ID No.2;

(2) SEQ ID No.2 add, replace, insert or delete one or more amino acid whose homologous sequences.

3. recombinant vectors is characterized in that: contain Japanese Honeysuckle styracin claimed in claim 1-4-hydroxylase (LJC4H) gene complete sequence or partial sequence.

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