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 lonicera japonica thunb cinnamate-4-hydroxylase (LJC4H) gene as well as a protease coded by the same and application of the gene. The LJC4H gene has nucleotide sequences shown in SEQ ID NO.1 or homologous sequences obtained through addition, substitution, insertion or deletion of one or a plurality of nucleotides or nucleotide sequences derived from alleles of the gene and the gene. A protein coded by the gene has amino acid sequences shown in SEQ ID NO.2 or homologous sequences obtained through addition, substitution, insertion or deletion of one or a plurality of amino acids.

Description

Japanese Honeysuckle styracin-4-hydroxylase (LJC4H) gene and coded product and application

Technical field

The invention belongs to biological technical field, relate generally to and utilize cDNA library clone's styracin-4-hydroxylation enzyme gene and coded product and application, relating in particular to biosynthesizing has the machine acids of pharmacological component, flavones fermentoid gene and coded product and application, belongs to the Gene Engineering of Medicinal Plants field.

Background technology

The formation of medicinal plant activeconstituents is the product of peculiar gene group in the Secondary Metabolism of Plant approach.Along with plant functional genomics research extensively with deeply, show unique characteristics and have the research of the synthetic correlation function gene of medicinal plant secondary metabolism of broad prospect of application to become gradually the focus of research, these gene clonings will be provided fundamental basis for the formation that biosynthetic pathway and the regulatory mechanism thereof of parsing medicinal plant activeconstituents are conciliate release material quality, bring wide application space for utilizing biotechnology to improve target component content or direct production effective constituent or intermediate simultaneously.

The complex chemical composition of medicinal plant, of a great variety, wherein mainly contain the Multiple components such as organic acid, flavonoid, volatile oil, terpene, diterpenes, trace element, Japanese Honeysuckle Lonicera japonica Thunb is conventional Chinese medicine simply, be mainly used in various febrile diseases, such as the treatment of the symptoms such as body heat, dermexanthesis, a spot, hot carbuncle carbuncle, swelling and pain in the throat.The main active ingredient of Japanese Honeysuckle comprises chlorogenic acid and wooden slippers grass glycosides etc., and its Content of Chlorogenic Acid belongs to organic acid, and galuteolin belongs to flavonoid compound.

Styracin-4-hydroxylase (cinnamate-4-hydroxylase, C4H), claim again trans-cinnamic acid-4-mono-oxygenase, be one of member of cytopigment monooxygenase P450 superfamily, belong to the CYP73 subfamily, can produce 4-tonka-bean hydrochlorate by catalysis styracin hydroxylation, can be further converted to tonka bean camphor, chlorogenic acid etc., also can form the CoA ester, be further converted to the flavonoids such as wooden slippers grass glycosides again, be second key enzyme behind the phenylpropyl alcohol alkane approach relaying phenylalanine ammonia lyase.The clone of Japanese Honeysuckle styracin-4-hydroxylation enzyme gene provides important foundation for utilizing genetically engineered to improve the honey suckle active component content.Before the present invention comes forth, any styracin in the Japanese Honeysuckle mentioned in the present patent application-4-hydroxylation enzyme gene and aminoacid sequence thereof that disclose or reported arranged not yet.

Summary of the invention

The object of the present invention is to provide a kind of Japanese Honeysuckle styracin-4-hydroxylation enzyme gene (LJC4H).

Second purpose of the present invention provides the protein of this genes encoding.

The present invention also provides recombinant vectors and the host cell that contains this gene.

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

Japanese Honeysuckle styracin provided by the present invention-4-hydroxylase (LJC4H) gene is one of following nucleotide sequence:

(1) has the nucleotide sequence shown in the 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.

The protein of this coded by said gene is Japanese Honeysuckle styracin-4-hydroxylase (LJC4H) gene, 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.

Japanese Honeysuckle styracin provided by the present invention-4-hydroxylase (LJC4H) gene is cloned from Japanese Honeysuckle first, and experiment in vitro shows that LJC4H has the activity that the catalysis styracin transforms.Utilize the present invention can improve by genetic engineering technique the content of organic acid substance chlorogenic acid, Flavonoid substances galuteolin in the plants such as Japanese Honeysuckle.

Description of drawings:

Fig. 1: LJC4H functional domain forecast analysis (deriving from ncbi database);

Fig. 2: LJC4H systematic evolution tree (adjacent method);

Fig. 3: expression vector pYEUra3-LJC4H makes up;

Fig. 4: enzymatic reaction product HPLC detected result.A, the pYEUra3 catalysate; B, pYEUra3-LJC4H catalysate (without NADPH); C, the pYEUra3-LJC4H catalysate; D, Isosorbide-5-Nitrae-coumaric acid; 2, the trans-cinnamic acid standard substance.

Embodiment

The structure of embodiment 1, Japanese Honeysuckle cDNA library

1, the separation and detection of the total RNA of Japanese Honeysuckle

Extracting honeysuckle (Lonicera japonica Thunb) bud 2g, in mortar with the quick grind into powder of liquid nitrogen, (CTAB (W/V) 2%, Tris-HCl (pH8.0) 100mmolL in the 10mL Extraction buffer of fast transfer to 65 ℃ preheating ^-1, EDTA 25m molL ^-1, NaCl 2.0molL ^-1, PVP40 2%, spermidine 0.5g/L, mercaptoethanol 2%), mixing fully vibrates; With equal-volume chloroform extracting twice, centrifugal 15 minutes of 7500g.Supernatant liquor adds the 10M LiCl of 1/4 volume, places 4 ℃ of precipitations behind the mixing and spends the night; Centrifugal 20 minutes of 7500g, (SDS 0.5%, NaCl 1molL with 500 μ L SSTE for precipitation ^-1, Tris-HCl (pH8.0) 10mmolL ^-1, EDTA 1mmolL ^-1, 65 ℃ of dissolvings 5 minutes.With the extracting of equal-volume chloroform, centrifugal 5 minutes of 13000g; Supernatant liquor adds 2 times of volume dehydrated alcohols, places 2h for-70 ℃; Centrifugal 20 minutes of 4 ℃ of 13000g, the precipitation drying at room temperature is dissolved in after 10 minutes in the water that 100 μ L DEPC process, and detects the integrity of RNA with 1.0% agarose electrophoresis, with GenQuant nucleic acid quantification instrument mensuration A260, A280 ratio and concentration.Place-80 ℃ of refrigerators for subsequent use.

2, the structure of cDNA library

Adopt mRNA purification kit (QuichprepTM Micro mRNA PurifiCation Kit, Pharmacia company) behind the separating mRNA, adopt the Creator Smart cDNA Library Construction Kit (Cat.No.634903) of Clontech company to build the storehouse, principle is SMART (switch mechanism at 5 ' end of mRNA template).

Embodiment 2: the clone of Japanese Honeysuckle genes involved

5000 mono-clonals of picking carry out bacterium colony PCR evaluation at random.Get an amount of PCR thin-walled tube, place on ice, every pipe adds first the aqua sterilisa of 17.3ul.10ul lancet choicest with the bacterium of going out is got the mono-clonal hickie to aqua sterilisa, the vibration mixing.Add successively: Taq buffer 2.5 μ L, MgCl ₂(25mM) 1.8 μ L, dNTP (2.5mM) 1 μ L, M13+ primer (10pmol) 1 μ L, M13-primer (10pmol) 1 μ L, Taq enzyme 0.4 μ L.Each reagent gets rid of on the whizzer after all adding well, makes it to sink to the bottom, and places on the PCR instrument.The PCR reaction conditions is 94 ℃ of denaturations after 5 minutes, 94 ℃ 40 seconds, 54 ℃ 40 seconds, 72 ℃ 4 minutes, 4 ℃ of preservations were extended in rear 72 ℃ of 35 circulations 10 minutes.After the PCR reaction enters 4 ℃, take off the PCR thin-walled tube, get the 7ulPCR product and add the leakage of electricity swimming of 3ul bromine Finland, take a picture after half an hour, observe glue figure, identify roughly size and the small segment rate of Insert Fragment according to glue figure.Select the single amplified production of band to deliver to the large genome company of China and check order, obtain Japanese Honeysuckle genes involved sequence.

The bioinformatic analysis of embodiment 3, LJC4H gene

The length of the Japanese Honeysuckle styracin that the present invention relates to-4-hydroxylase (C4H) full length gene cDNA is 1518bp, and detailed sequence is seen the sequence 1 in the sequence table, and wherein opening code-reading frame is positioned at 1-1518bp.The Japanese Honeysuckle full length cDNA sequence is carried out the nucleotide homology retrieval with blast program in Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDS translation+PDB+Swissprot+Superdate+PIR database.This gene on amino acid levels with other species in C4H higher homology is arranged, have simultaneously typical cypX superfamily structural domain.Such as Fig. 1.

The research of embodiment 4, LJC4H gene function

1. the structure of expression vector

Take LJC4H cDNA as template, utilize primer P1:5 '-GTCGACATGGATCTTCTCCTCTTAGA-3 ', P2:5 '-GTCGACTCAAAAAGATCTCTT-3 ' carries out the PCR reaction, get the 5ul amplified production and add the leakage of electricity swimming of 3ul bromine Finland, take a picture after half an hour, observe glue figure, amplified fragments is 1500bp.Cut amplified production 2 hours with Sal I enzyme, utilize recovery test kit (Takara company, China) purifying enzyme to cut product.Utilize simultaneously Sal I to cut the pYEUra3 carrier 2 hours at 37 ℃ of lower enzymes, add 5ul bromine Finland and carry out agarose gel electrophoresis, observe glue figure, and utilize and reclaim test kit recovery 6100bp fragment.

The two linked enzyme spends the night 16 ℃ of connections.Connect product and cut 2h with Xho I enzyme, reclaim about 7600bp fragment to reclaim test kit, 16 ℃ of connections are spent the night.Electric shock transforms the bacillus coli DH 5 alpha competent cell, screens recon at the 2% glucose USM flat board that contains penbritin.The pYEUra3 plasmid that contains the C4H clone is identified and dna sequence analysis through PCR and digestion with restriction enzyme electrophoresis, preserves the recombinant plasmid pET-LJC4H with correct target sequence and transforms for expressing.This plant expression vector called after pYEUra3-LJC4H (Fig. 3).

2. abduction delivering and enzyme biopsy are surveyed

With pYEUra3-LJC4H plasmid electric shock transform portion enzymolysis yeast host bacterium AB1380, cultivate and screened afterwards positive yeast in 5 days and contain on the USM substratum of 2% glucose in 2ml, choose in mono-clonal and the 2mL USM liquid nutrient medium 30 ℃ of concuss overnight incubation after 4-5 days.The centrifugal 5min of 5000rpm abandons supernatant, dilutes 20 times with the USM liquid nutrient medium that contains 2% glucose, and 30 ℃ of concuss are cultivated 24h.Extract the culturing cell total protein, detect the heme peroxidase activity.After choosing 30 ℃ of concuss of USM liquid nutrient medium that the high expression level transformant contains 2% glucose with 50mL and cultivating 20h, be inoculated into 30 ℃ of concuss of USM liquid nutrient medium that 1L do not contain glucose and cultivate 40h, reclaim thalline, the separating particles body.

Get 30-50mg microsomal protein 30 ℃ of incubation 10min in 600mL C4H enzymatic reaction system (the NADPH final concentration is 0.5mM, Na3PO3 concentration 100mM, pH 7.4, styracin concentration is 0.1mm).With 40mL 6M HCl termination reaction, with twice of 600 μ L ethyl acetate extraction, with make organic phase volatilization in the vacuum freezing drying oven, and be dissolved in the 800 μ L acetonitriles, use high-efficient liquid phase chromatogram technique analysis, condition is 3.9 * 300mm (10 μ m) μ Bondapak 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%, 30 ℃ of column temperatures the results are shown in Figure 4.

The research of embodiment 5, sudden change LJC4H gene function

Take LJC4H cDNA as template, utilize primer P3:5 '-GTCGACATGGATCTTCTCCTCTTCGA-3 ', P4:5 '-GTCGACTCAAAAAGATCTCTT-3 ' carries out the PCR reaction, get and take a picture after the 5ul amplified production adds 3ul bromine Finland leakage of electricity half an hour, observe glue figure, obtain nonsynonymous mutation LJC4H.Expression vector establishment, abduction delivering and enzyme biopsy survey are identical with embodiment 4, and the result shows that sudden change LJC4H gene function and LJC4H do not have significant difference.

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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