CN104805097A - Coding sequences and applications of isatis indigotica fortune pinoresinol reductase protein - Google Patents

Abstract

The present invention provides a nucleotide sequence encoding a protein having isatis indigotica fortune lariciresinol reductase activity, wherein the nucleotide sequence is characterized in that the nucleotide sequence is represented by SEQ ID NO.1. The present invention further provides an amino acid sequence of the protein having the isatis indigotica fortune lariciresinol reductase activity, wherein the amino acid sequence is characterized in that the amino acid sequence is represented by SEQ ID NO.2. The present invention further provides applications of the nucleotide sequence encoding the protein having the isatis indigotica fortune lariciresinol reductase activity in enhancement of the isatis indigotica fortune lariciresinol reductase expression of the plant. According to the present invention, the high expression of the isatis indigotica fortune lariciresinol reductase in the plant is easily achieved so as to increase the lariciresinol yield.

Description

Woaded blue rosin spirit coded sequence of reductase enzyme protein and application

Technical field

The present invention relates to a kind of nucleotide sequence and application thereof, the invention still further relates to a kind of sequence of protein, belong to biological technical field.

Background technology

Woaded blue is cress, its root is used as medicine, be conventional Chinese medicine Root of Indigowoad, have clearing heat and detoxicating, the effects such as cool blood relieve sore throat, application is very extensive, and primary formulation has Radix Isatidis granule, Banlangen buccal tablet etc., clinical in influenza, epidemic encephalitis type B, mumps, acute, chronic hepatitis, zoster etc.Chemical composition and pharmacology activity research show that lariciresinol is the important active substances basis that woaded blue plays antiviral efficacy.Therefore, the content improving lariciresinol is the key obtaining the high excellent woaded blue strain of antiviral activity, has important using value.

Plant secondary metabolic engineering utilizes genetically engineered and molecular biology method to illustrate the biosynthetic mechanism of Secondary metabolites, check on the information of pathways metabolism and relevant enzyme, the gene controlling Biosynthetic pathway is regulated and controled, thus reach and transform pathways metabolism on a molecular scale, promote the controlled syntheses of target product, improve the object of specific compound output in plant.Find by literature search, rosin spirit reductase enzyme (PLR) biosynthesizing to lariciresinol being positioned at last step of lariciresinol route of synthesis serves very important regulating and controlling effect, is the important target spot of lignanoid's metabolic engineering.Therefore, be separated from woaded blue and obtain rosin spirit reductase enzyme and utilize it to carry out metabolic regulation, lariciresinol biosynthesizing speed limit bottleneck will be broken, obtain the high-quality woaded blue strain of lariciresinol high yield.Not yet find the relevant report with the woaded blue rosin spirit reductase enzyme protein mentioned by the present invention and application at present.

Summary of the invention

The invention provides the nucleotide sequence that a kind of coding has the albumen of woaded blue rosin spirit reductase activity, it is characterized in that: nucleotide sequence is as shown in SEQ ID NO.1.

In addition, the present invention also provides a kind of protein amino acid sequence with woaded blue rosin spirit reductase activity, it is characterized in that: its sequence is as shown in SEQ ID NO.2.

In addition, the nucleotides sequence that the present invention also provides a kind of coding to have the albumen of woaded blue rosin spirit reductase activity is listed in the application strengthened in expression of plants woaded blue rosin spirit reductase enzyme.

In addition, the present invention also has such feature: adopt Agrobacterium rhizogenes C58C1 as conversion carrier.

In addition, present invention also offers a kind of with the primer of woaded blue total serum IgE needed for template clone nucleotide sequence, it is characterized in that:

With oligonucleotide R1:5'-ATGAGAGAGAATAATAGCGG-3' for forward primer, with oligonucleotide R2:5'-CTAGAGGAATATTTTCAAATA-3' for reverse primer.

In addition, present invention also offers a kind of application improving the content of lariciresinol by turning woaded blue rosin spirit reductase enzyme encoding gene.

Invention effect and effect

The invention provides and a kind ofly there is coding there is the nucleotide sequence of the albumen of woaded blue rosin spirit reductase activity, and corresponding aminoacid sequence, and provide nucleotides sequence and be listed in application in the content improving lariciresinol in woaded blue, there is important practical value.

Accompanying drawing explanation

Fig. 1 is the comparison of IiPLR and Arabidopis thaliana PLR albumen;

Fig. 2 is the SDS-PAGE electrophorogram of IiPLR recombinant protein prokaryotic expression in E. coli BL21 (DE3);

Fig. 3 is the Molecular Detection figure of IiPLR transgenosis root of hair;

Fig. 4 is the RT-PCR analysis chart that in IiPLR transgenosis woaded blue root of hair, IiPLR expresses; And

Fig. 5 be in IiPLR transgenosis woaded blue root of hair lariciresinol containing spirogram.

Embodiment

The embodiment of the embodiment of the present invention is described below in conjunction with accompanying drawing

The clone of < embodiment one > woaded blue rosin spirit reductase gene

1. separate tissue

Woaded blue (I.indigotica) leaf harvest, from pharmaceutical college of The 2nd Army Medical College Botanical gardens, after taking material, is placed in liquid nitrogen freezen protective immediately.

The separation of 2.mRNA

Get portion of tissue, grind with mortar, add the 50ml pipe filling and add and fill TRIzol lysate (TRIzol Reagents, Gibco, NY, USA), fully after vibration, then move in glass homogenizer.Move to 50ml after homogenate newly to manage, extracted total RNA.By denaturing formaldehyde gel electrophoresis qualification total serum IgE quality.

3. the full-length clone of gene

According to rosin spirit reductase enzyme conserved sequence in other plant reported, design degenerated primer, utilize homologous genes cloning mechanisms, adopt RACE method (namely being carried out the technology of cDNA end quick clone by PCR), the Gibco BRL test kit of commodity in use.

Carry out cDNA full-length clone, a point three phases carries out:

(1)3'-RACE

PCR (primer is AP+TNX001) obtains sequence 2001TNX3 (514bp), reclaim, be connected on T-Easy carrier, with SP6 or T7 as universal primer, adopt the method stopping thing fluorescent mark (Big-Dye, Perkin-Elmer, USA), ABI377 sequenator (Perkin-Elmer, USA) checks order.Sequencing result GCG software package (Wisconsingroup, USA) the existing database of BLAST and FASTA software search (Genebank+EMBL) in, know that its nucleotide sequence and proteins encoded and known cress homology as the rosin spirit reductase gene of Arabidopis thaliana (Arabidopsis thaliana) is very high, therefore tentatively think that it is a rosin spirit reductase gene.

(2)5'-RACE

First round PCR (AAP+TNX02)

Second takes turns PCR (AUAP+TNXR03) obtains 2001TNX5'(723bp) (process is with (1))

(3) pcr amplification 2001TNX coding region (TNX01+R1) obtains 2001TNX coding region (770bp) (process is with (1)).

The result of BLAST proves that the gene newly obtained from woaded blue is really a rosin spirit reductase gene.By combinationally using above-mentioned three steps, obtain the complete encoding sequence of the woaded blue rosin spirit reductase gene of candidate.Obtain on the basis of total length (at least comprising complete open reading frame) in splicing, further design primer R1:5'-ATGAGAGAGAATAATAGCGG-3' is forward primer, oligonucleotide R2:5'-CTAGAGGAATATTTTCAAATA-3' is reverse primer, with woaded blue total serum IgE for template, carry out RT-PCR amplification, the PCR condition of R1/R2 is 94 DEG C, 5 minutes, thereupon with 94 DEG C, 45 seconds; 58 DEG C, 45 seconds; The reaction process of 72 DEG C, 1 minute carries out 35 circulations, finally 72 DEG C of downward-extensions 5 minutes.Electrophoresis detection pcr amplification product, acquisition expanding fragment length is 1062bp.Then carry out cloning, checking order, the sequence shown in acquisition with pcr amplification product according to a conventional method, see the information of SEQ ID NO.1.

The sequence information of < embodiment two > woaded blue IiPLR gene and homology analysis

The length of woaded blue IiPLR full-length cDNA is 1062bp (Genbank accessionNo.JF264893), comprises the open reading frame (ORF) of 951bp, the terminator codon (tag) of 3bp.This ORF encodes 317 amino acid whose peptide chains.Also have the 5 ' homing sequence of a long 33bp and the 3 ' non-translational region of a long 75bp.Iso-electric point (pI) is 5.64, and molecular size range is about 35.6kDa.

BLAST result shows, and IiPLR and equal plant Arabidopsis thaliana PLRs has high similarity as shown in Figure 1, and similarity all reaches more than 80% (see table 1).As can be seen here, there is higher homology in IiPLR gene and model plant Arabidopis thaliana rosin spirit reductase enzyme (PLR) gene, can think that both functionally also have very high similarity.

Fig. 1 is the comparison of IiPLR and Arabidopis thaliana PLR albumen.The consistent sequence black matrix that misuses a word for another to which it bears resemblance represents, the similar sequences of bulk represents with at the bottom of surplus light gray, and dissimilar residue surplus white background represents.Comparison PLRs sequence used is in table 1.

The PLRs sequence used in the analysis of table 1 amino acid alignment

The preparation of < embodiment three >IiPLR albumen

According to the mature protein coding sequence of woaded blue IiPLR, design amplifies the primer of complete coding reading frame, oligonucleotide F:5'-atgagagagaataatagcggcg-3' is forward primer, oligonucleotide R:5'-ctagaggaatattttcaaata-3' is reverse primer, correspond respectively to 20 Nucleotide that encoding sequence 5' and 3' holds, and restriction endonuclease sites is introduced respectively on positive anti-primer, this site is determined, so that construction of expression vector according to the Pet32a carrier selected.Woaded blue IiPLR gene is being ensured to be cloned into Pet32a plasmid expression vector under the prerequisite that reading frame is correct.The plasmid expression vector using the method for PCR to identify utilizes CaCl ₂method proceeds to bacillus coli DH 5 alpha, and Screening and Identification obtains the engineering bacteria DH5 α-pet32a-IiPLR containing pet32a-IiPLR expression vector.

DH5 α-pet32a-IiPLR engineering bacteria jolting overnight incubation in 3ml is containing the LB substratum of 100 μ g/ml penbritins of picking list bacterium colony, draw nutrient solution by the concentration of 1:100 to cultivate about 3 hours in new LB substratum (containing 100 μ g/ml penbritins), after reaching 0.5 to OD600, add IPTG(isopropylthiogalactoside) continue at 37 DEG C to final concentration 1mmol/L and cultivate 2 respectively, 4,6 hours.The 1ml bacterium liquid getting incubation time respectively different is centrifugal.In the bacterial precipitation thing obtained, add lysate, its formula is: 2 × SDS sample-loading buffer 50 μ l, distilled water 45 μ l, 3-mercaptoethanol 5 μ l.Suspendible bacterial precipitation, boils 5 minutes in boiling water bath, centrifugal 1 minute of 10000rpm, gets supernatant and adds in the SDS-PAGE glue of 12% and carry out electrophoresis.Electrophoresis terminates poststaining and observes, and Fig. 2 is that the SDS-PAGE of IiPLR recombinant protein prokaryotic expression in E. coli BL21 (DE3) detects.As shown in Figure 2, M is Protein Marker (Marker), and its molecular weight ranges is 10-170KD.The bacterial strain that the protein content of expection molecular size range increases with IPTG induction time and increases is the engineering bacteria of expressing tag-IiPLR fusion rotein.In Fig. 2, fusion rotein 12 molecular weight is about 55kDa, empty carrier negative control expressing protein 11(TrxTag ^tM, and STag ^tM) molecular weight be about 18kDa, the molecular weight of albumen of IiPLR is 34kDa.

In figure, the 1st swimming lane is the expression strain that band empty carrier is induced without IPTG, 2nd swimming lane is that the expression strain of band empty carrier is induced after 2h through 1mMIPTG, 3rd swimming lane 3 be without induction with destination gene expression bacterial strain, the 4 to 6 swimming lane be with destination gene expression bacterial strain through 1mM IPTG induction 2h, 4h, 6h.Fig. 2 shows that IiPLR gene can successfully utilize the Expression element of carrier to express in pET-32a, and expression amount improves with the prolongation of induction time.

< embodiment four > turns IiPLR gene and improves woaded blue lignanoid content

According to the complete encoding sequence of IiPLR, design amplifies the primer of complete coding reading frame, and restriction endonuclease sites is introduced respectively on positive anti-primer, pcr amplification product is cloned into expression vector pCAMBIA1304, expression vector has been identified under the prerequisite ensureing reading frame, proceeded to again in Agrobacterium rhizogenes C58C1, utilized leaf disk method technical transform woaded blue.

1. select the positive bacterium colony on flat board with sterile toothpick picking YEB, be inoculated in (100mg/LKan(kantlex)+40mg/LRif(Rifampin in 2ml YEB liquid nutrient medium)), at 28 DEG C, 200rpm shaking culture 24-36 hour;

2. 4,000g centrifugal 10min under room temperature;

3. abandon supernatant, thalline 1/2MS liquid nutrient medium suspends, and the 5-20 being diluted to original volume doubly, makes about the OD600=0.5 of bacterium liquid;

4. get the aseptic blade of the growth woaded blue of about two weeks, remove its main lobe arteries and veins, be cut into 1 square centimeter of square vanelets;

5. blade is put into the bacterium liquid prepared, soak 2 to 5min, aseptic filter paper blots bacterium liquid;

6. the blade through infecting is put on MS substratum, 28 DEG C of light culture 48 hours;

7. blade is forwarded to except (1/2B5+Cef500mgL on bacterium culture medium ^-1) in (25 soil 1) DEG C light culture, after about 15d, grow root of hair independent numbering from blade edge of wound, every 7d subculture once;

8. to select after hygromycin selection still well-grown root of hair system, each mono-clonal root of hair carries out independent numbering.Wild-type root of hair number consecutively is CK1, CK2 ..., the root of hair number consecutively turning IiPLR is P1, P2

9. by the root of hair subculture of numbering to 1/2B5+Cef(cephamycin) 250mgL ^-1solid medium on, no longer add screening pressure and be beneficial to hairy root growth, and according to 250mgL ^-1, 150mgL ^-1, 100mgL ^-1, 50mgL ^-1, 0mgL ^-1order constantly reduce Cef concentration, carry out until completely degerming 1/2B5 medium liquid cultivate obtain the genetically modified root of hair system of IiPLR.

The Ri plasmid T-DNA zone of Agrobacterium rhizogenes C58C1 has rolA, rolB, rolC and rolD gene, wherein rolB and rolC gene brings out and maintains root of hair form, by design rolB, rolC gene-specific primer, by PCR method, Molecular Detection is carried out to root of hair, Fig. 3 is the Molecular Detection result of IiPLR transgenosis root of hair, and in figure, M is nucleic acid molecular weight standard DL2000; CK represents wild-type root of hair; P1 ~ P5 represents that different IiPLR transforms root of hair strain.As shown in Figure 3, in all detection samples, all detect rolB and rolC gene, illustrate that root of hair transforms successfully; And use hpt marker gene (hpt gene) to carry out Molecular Detection to IiPLR transgenosis root of hair simultaneously, transform in root of hair strain at the IiPLR of P1 ~ P5 and hpt gene all detected, the IiPLR gene that conversion is described Successful integration in woaded blue genome.

Fig. 4 is that the RT-PCR that in IiPLR transgenosis woaded blue root of hair, IiPLR expresses analyzes.In figure, CK represents wild-type root of hair, and P1 ~ P5 represents that different IiPLR transforms root of hair strain.According to the complete encoding sequence of IiPLR, design Realtime-PCR(real-time quantitative PCR) primer, detect IiPLR expression level in IiPLR transgenosis root of hair, in the transgenic line of P1 ~ P5, P1 strain root of hair gene IiPLR expression amount is the highest, be more than 8 times of wild-type, the expression amount of the IiPLR gene in other strains also slightly raises, but does not have P1 strain obvious.

Fig. 5 is the content of lariciresinol in IiPLR transgenosis woaded blue root of hair, and in figure, CK represents wild-type root of hair, and P1 ~ P5 represents that different IiPLR transforms root of hair strain.

As shown in Figure 5, HPLC is used to carry out assay discovery to the target compound lariciresinol in root of hair: after IiPLR gene is proceeded to woaded blue root of hair, in P1 strain, the content of lariciresinol is 135.4ug/g, reach 5.7 times of wild-type root of hair, be proportionate with the expression level of IiPLR gene in P1 strain, visible IiPLR transgenosis can significantly improve the content of lariciresinol in woaded blue.For the antiviral effect improving plant, there is important effect.

Sequence involved in the present invention and mark are distinguished as follows:

The information of SEQ ID NO.1

<110> The 2nd Army Medical College

<120> woaded blue rosin spirit coded sequence of reductase enzyme protein and application

<210>1

<211>1062

<212>cDNA

<213> woaded blue (I.indigotica)

<400>1

ggtcctcaaa gaaaacacac atatcttaga gagatgagag agaataatag cggcgagaaa 60

acgcgcgttc tggtggttgg cggtacgggg acgatgggga gaaggatcgt gcgggcgtgt 120

ttggctgagg gacacgagac ttacgttctg cagcagccgg agaccagagt ggacatcgag 180

aaggtgcagc tcctttactc ttacaaaaga ctcggagcac gtcttatcga agcctcattt 240

tccgaccacc agagccttgt ctctgccgtg aagcaagtcg acatagtcgt cgccgccatg 300

tccggtgtcc actttcgcag ccacagcatc cttgttcagc tcaagctcgt tgaagccatc 360

aaagaggccg gtaacatcaa gcgcttttta ccatcagaat ttggaatgga tccatcgcgc 420

atgggacatg cgatgcctcc gggaagagaa acattcgatc aaaaactgga agtacgtaat 480

gcaattgagg ctgccggaat tccccataca tacgtcgtcg gcgcttgctt tgccgcatac 540

ttcgccggaa atttatctca aatgggaact ttgatccctc caaagaaaaa agtcaatatt 600

tatggagacg gaaatgttaa agtggtatat gtagatgaag atgacattgc agaatatacc 660

gcaaagacgc ttgacgatcc tcgcacgatc aataaaacag tgtatgttag acctaccgag 720

aacgttctta cacaaatgga actagttcag atatgggaga aactaacggg aaaagaattg 780

gagaagacca atatttctgc aaatgacttt cttgccgaca ttgaagataa ggagatacca 840

catcaagcgg gattgggaca cttctaccac atcttctacg aaggttgtct cactgatcat 900

gaagtgggag atgacgaaga agcttctaaa ctctaccctg acgtcaagta cactcgcatg 960

gatgaatatt tgaaaatatt cctctagttt ggtctaatcc tgagtacaga catgtaaaag 1020

caaaacacat cgttcccatt aataagtttc ctttcttatt tt 1062

The information of SEQ ID NO.2

<110> The 2nd Army Medical College

<120> woaded blue lariciresinol

<210>3

<211>20

<212>DNA

<213> artificial sequence

<400>1

atgagagaga ataatagcgg 20

The information of SEQ ID NO.4

<110> The 2nd Army Medical College

<120> woaded blue rosin spirit coded sequence of reductase enzyme protein and application <210>4

<211>20

<212>DNA

<213> artificial sequence

<400>1

tagaggaata ttttcaaata 20

Claims (6)

1. coding has a nucleotide sequence for the albumen of woaded blue rosin spirit reductase activity, it is characterized in that:

Described nucleotide sequence is as shown in SEQ ID NO.1.

2. there is a protein amino acid sequence for woaded blue rosin spirit reductase activity, it is characterized in that:

Its sequence is as shown in SEQ ID NO.2.

3. the nucleotides sequence that a coding has the albumen of woaded blue rosin spirit reductase activity is listed in the application strengthened in expression of plants woaded blue rosin spirit reductase enzyme.

4. a conversion carrier for the maturation protein of woaded blue IiPLR, is characterized in that:

Adopt Agrobacterium rhizogenes C58C1 as conversion carrier.

5. with woaded blue total serum IgE for template clones a primer needed for nucleotide sequence as claimed in claim 1, it is characterized in that:

With oligonucleotide R1:5'-ATGAGAGAGAATAATAGCGG-3' for forward primer, with oligonucleotide R2:5'-CTAGAGGAATATTTTCAAATA-3' for reverse primer.

6. one kind is improved the application of the content of lariciresinol by turning woaded blue rosin spirit reductase enzyme encoding gene.