CN104805096A - Coding sequences and applications of 4-coumarate coenzyme A ligase protein family of isatis indigotica fortune - Google Patents
Coding sequences and applications of 4-coumarate coenzyme A ligase protein family of isatis indigotica fortune Download PDFInfo
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- CN104805096A CN104805096A CN201410030374.8A CN201410030374A CN104805096A CN 104805096 A CN104805096 A CN 104805096A CN 201410030374 A CN201410030374 A CN 201410030374A CN 104805096 A CN104805096 A CN 104805096A
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- lariciresinol
- woaded blue
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Abstract
The present invention provides three sections of coding sequences of 4-coumarate coenzyme A ligase protein family of isatis indigotica fortune, wherein the coding sequences are respectively represented by SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3. The present invention further provides amino acid sequences of the 4-coumarate coenzyme A ligase protein family of the isatis indigotica fortune, wherein the amino acid sequences are characterized in that the amino acid sequences are represented by SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6. The present invention further provides applications for changing biosynthesis of lariciresinol by regulating the expression of the 4-coumarate coenzyme A family protein of the isatis indigotica fortune. According to the present invention, the important application values are provided in the fields of breeding and biological energy sources.
Description
Technical field
The present invention relates to nucleotide sequence and the application thereof of woaded blue 4-coumaroyl A ligase enzyme protein family, the invention still further relates to the aminoacid sequence of woaded blue 4-coumaroyl A ligase enzyme protein family, 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; Meanwhile, lariciresinol belongs to lignan component, and reduce the content of lariciresinol in woaded blue, reduce lignified degree, effectively can reduce the difficulty using it as feedstock conversion for biofuel, the production for bioenergy is also significant.
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, the controlled syntheses of goal of regulation and control product, go after profits and advoid disadvantages, improve or reduce the object of specific compound output in plant.Find by literature search, 4-coumaroyl A ligase enzyme (4CL) biosynthesizing to lariciresinol 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 4-coumaroyl A ligase enzyme and utilize it to carry out the metabolic regulation of targeted, likely break lariciresinol biosynthesizing speed limit bottleneck on the one hand, obtain the high-quality woaded blue strain of lariciresinol high yield; Likely reduce with lariciresinol the content of the lignanoid being representative on the other hand, reduce the lignified degree of woaded blue, the production for bioenergy provides effective starting material.Not yet find the relevant report with the woaded blue 4-coumaroyl A ligase enzyme albumen mentioned by the present invention and application at present.
Summary of the invention
The invention provides the nucleotide sequence of coding woaded blue 4-coumaroyl A ligase enzyme protein family, it is characterized in that: its nucleotide sequence is respectively as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3.
Present invention also offers the aminoacid sequence of woaded blue 4-coumaroyl A ligase enzyme protein family, it is characterized in that: its sequence is as shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6.
Present invention also offers a kind of expression by regulation and control woaded blue 4-coumaroyl A family protein to change the biosynthetic application of lariciresinol.
Invention effect and effect
The invention provides three sections of encoding sequences of woaded blue 4-coumaroyl A ligase enzyme protein family, respectively as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3.
Present invention also offers the aminoacid sequence of woaded blue 4-coumaroyl A ligase enzyme protein family, it is characterized in that: its sequence is as shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6.
Present invention also offers a kind of expression by regulation and control woaded blue 4-coumaroyl A family protein to change the biosynthetic application of lariciresinol.
Accompanying drawing explanation
Fig. 1 is the comparison of 4CL protein amino acid sequence in the aminoacid sequence of Ii4CL albumen and Arabidopis thaliana;
Fig. 2 is that the SDS-PAGE of Ii4CL1, Ii4CL2, Ii4CL3 fusion rotein prokaryotic expression in E. coli BL21 (DE3) detects;
Fig. 3 is that the SDS-PAGE of the purified fusion rotein of Ii4CL1, Ii4CL2, Ii4CL3 detects;
Fig. 4 is the relative expression quantity suppressing the strain of Ii4CL1 and the Ii4CL1 of WT strain;
Fig. 5 is the content balance suppressing the strain of Ii4CL1 and the lariciresinol of WT strain;
Fig. 6 is the relative expression quantity suppressing the strain of Ii4CL2 and the Ii4CL2 of WT strain;
Fig. 7 is the content balance suppressing the strain of Ii4CL2 and the lariciresinol of WT strain;
Fig. 8 is the relative expression quantity suppressing the strain of Ii4CL3 and the Ii4CL3 of WT strain; And
Fig. 9 is the content balance suppressing the strain of Ii4CL3 and the lariciresinol of WT strain.
Embodiment
The clone of < embodiment one > woaded blue 4-coumaroyl A ligase enzyme family 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 lysate, fully after vibration, then move in glass homogenizer.Move to 50ml after homogenate newly to manage, use TRIzol (TRIzol Reagents, Gibco, NY, USA) reagent extracted total RNA.By denaturing formaldehyde gel electrophoresis qualification total serum IgE quality.
3. gene clone (Cloning of the gene)
3.1. the synthesis of the first chain cDNA
3.2. the pcr amplification of woaded blue 4-coumaroyl A ligase enzyme family gene (Ii4CL1, Ii4CL2, Ii4CL3) coding region
According to encoding sequence (the Genbank accessionNo.GU937875 of the 4CL gene family that GenBank logs in, Genbank accession No.KC430622, Genbank accession No.KC430623), the upstream and downstream primer that design amplifies complete encoder block is respectively:
4CL1-F:ATGGAGAAATCCGGCTACGG;(SEQ ID No.7)
4CL1-R:TCACATCTTGGATCTT ACTT;(SEQ ID No.8)
4CL2-F:ATGGTGCTCCAACAACAACA;(SEQ ID No.9)
4CL2-R:CTACTTAGGGTACTCG GTTT;(SEQ ID No.10)
4CL3-F:ATGCAGAAACAGAGCAGCAA;(SEQ ID No.11)
4CL3-R:TTAGTTCACCAACCCA GTTG(SEQ ID No.12)
And on upstream and downstream primer, introduce restriction endonuclease sites (this is determined by the carrier selected) respectively, so that construction of expression vector.With the first chain cDNA for template, check order after pcr amplification.A sequencing adopts 3730 automatic sequencers to complete by rich Asia, Shanghai or Jing Tai biotechnology Services Co., Ltd.Sequencing result shows, the sequence of cloning and the woaded blue 4CL gene coded sequence consistent (table 1 is to 3) that GenBank logs in.
The full-length cDNA of table 1. woaded blue Ii4CL1 gene and aminoacid sequence
The full-length cDNA of table 2. woaded blue Ii4CL2 gene and aminoacid sequence
The full-length cDNA of table 3. woaded blue Ii4CL3 gene and aminoacid sequence
The sequence information of < embodiment two > woaded blue Ii4CL gene family and homology analysis
Ii4CL1 full-length cDNA is 1967bp (GenBank accession number: GU937875
), comprise the open reading frame (ORF) of a 1629bp, 543 amino acid of encoding, iso-electric point (pI) is 8.59, and molecular size range is about 59.5kDa;
Ii4CL2 full-length cDNA is 1975bp(Genbank accession number: KC430622
), comprise the ORF of a 1692bp, 564 amino acid of encoding, iso-electric point (pI) is 5.09, and molecular size range is about 62.2kDa;
The full-length cDNA of Ii4CL3 is 1932bp(Genbank accession number: KC430623), comprise the ORF of a 1629bp, 543 amino acid of encoding, iso-electric point (pI) is 5.52, and molecular size range is about 59.1kDa.
BLAST result shows, and the 4CLs albumen of Ii4CL1, Ii4CL2, Ii4CL3 and other plant has higher similarity.Fig. 1 is the comparison of 4CL protein amino acid sequence in the aminoacid sequence of Ii4CL albumen and Arabidopis thaliana.In figure, the peculiar conserved positions black box of 4CL albumen marks.
By ClustalX software, the result display of full length sequence comparison, Ii4CL1, Ii4CL2 and Ii4CL3 have the distinctive structural domain of 4CL albumen, the Motif1 that 11 amino-acid residues (SSGTTGISKGV) as being positioned at 197-207 position on Ii4CL peptide chain form, this structural domain is the binding site of AMP; 7 amino-acid residues (GEIWVRG) being positioned at 389-395 position on Ii4CL peptide chain form Motif2, and this structural domain is the catalytic site of 4CL albumen.Can infer thus, in Ii4CL1, Ii4CL2 and Ii4CL3 and other plant, 4-coumaroyl A ligase enzyme (4CL) functionally also has very high similarity.
The preparation of < embodiment three > woaded blue Ii4CL family protein and enzyme activity determination
According to the albumen coded sequence of woaded blue Ii4CL1, Ii4CL2 and Ii4CL3, design amplifies the primer (corresponding respectively to more than about 20 Nucleotide that encoding sequence 5' and 3' holds) of complete coding reading frame respectively, and on positive anti-primer, introduce restriction endonuclease sites (this determines according to the Pet32a carrier selected) respectively, so that construction of expression vector.Ii4CL1, Ii4CL2 and Ii4CL3 are being ensured to be cloned into Pet32a carrier under the prerequisite that reading frame is correct.The expression vector identified utilizes CaCl2 method to proceed to bacillus coli DH 5 alpha, and Screening and Identification obtains engineering bacteria DH5 α-pet32a-Ii4CL1, DH5 α-pet32a-Ii4CL2, DH5 α-pet32a-Ii4CL3 respectively containing pet32a-Ii4CL1, pet32a-Ii4CL2, pet32a-Ii4CL3 expression vector.
DH5 α-pet32a-Ii4CL1, the DH5 α-pet32a-Ii4CL2 of difference picking list bacterium colony and DH5 α-pet32a-Ii4CL3 engineering bacteria jolting overnight incubation in 3ml is containing the LB substratum of 100 μ g/ml penbritins, 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 to final concentration 1mmol/L continue at 37 DEG C respectively cultivate 4 hours.Get 1ml bacterium liquid centrifugal, lysate (2 × SDS sample-loading buffer 50 μ l is added in bacterial precipitation thing, distilled water 45 μ l, 3-mercaptoethanol 5 μ l), suspendible bacterial precipitation, boil 5 minutes in boiling water bath, centrifugal 1 minute of 10000rpm, precipitation and supernatant add electrophoresis in 12%SDS-PAGE glue respectively.Fig. 2 is that the SDS-PAGE of Ii4CL1, Ii4CL2, Ii4CL3 fusion rotein prokaryotic expression in E. coli BL21 (DE3) detects.SDS-PAGE detects as shown in Figure 2, find with the positive colony bacterial strain of goal gene under the condition having IPTG to induce, all there is the high expression level of special target protein, Ii4CL1 molecular weight is about 78kDa, Ii4CL2 molecular weight is about 80kDa, Ii4CL3 molecular weight is about 77kDa, because empty carrier negative control expresses label protein (TrxTag
tM, His
and STag
tM) molecular weight is about 18kDa, expressing protein removes the molecular weight of label protein and the Ii4CL1 molecular weight of prediction is about 60kDa, Ii4CL2 molecular weight is about 62kDa, Ii4CL3 molecular weight is about 59kDa and conforms to, illustrate that Ii4CL1, Ii4CL2, Ii4CL3 gene can successfully utilize the Expression element of carrier to express in pET-32a, and all have the expression of fusion rotein in precipitation and supernatant.
Great expression woaded blue Ii4CL1, Ii4CL2, Ii4CL3 albumen as stated above, collects the NTA-agarose (Qiagen, Germany) that supernatant adds certain volume, and 4 DEG C adsorb 1 hour.Then with containing the His buffer solution twice of 100nM imidazoles, with containing 20mM N, N'-bis-piperazine, the buffer solution elution of 500mM NaCl, 300mM imidazoles is to obtain the albumen of purifying.Fig. 3 is that SDS-PAGE detects the purified fusion rotein of Ii4CL1, Ii4CL2, Ii4CL3, as shown in Figure 3, the fusion rotein after purified and desalination detects through SDS-PAGE, presents clear obvious single band, and molecular size range is correct, proves to obtain the higher fusion rotein of purity.The purity of woaded blue Ii4CL1, Ii4CL2, Ii4CL3 albumen of Bradford method Detection and Extraction.
Enzyme is lived the mensuration of correlation parameter: choose styracin, 4-coumaric acid, coffic acid, forulic acid, sinapinic acid be substrate, in 500ul reaction system: 2.5mMATP(Triphosaden), 2.5mM magnesium chloride, 0.2mM coenzyme A, 100mM pH=7.5Bis – Tris – propane damping fluid, 0.2-0.4mM substrate, reacts in appropriate purifying protein one by one, condition is pH=7.5,30 DEG C.Wherein, the reaction times of styracin, 4-coumaric acid, forulic acid is 5 minutes, and the reaction times of coffic acid, sinapinic acid is 2 minutes, with 10umol Glacial acetic acid termination reaction.After question response terminates, the generation of corresponding coenzyme A ester is judged by the increase of absorbancy under UV spectrophotometer measuring respective wavelength (styracin-311nm, 4-coumaric acid-333nm, coffic acid-346nm, forulic acid-345nm, sinapinic acid-352nm), and optical extinction coefficient is followed successively by 22000,21000,18000,19000 and 20000mL mmol
-1cm
-1, can be used to the concentration calculating the ester formed.According to Lineweaver – Burk graphing method, try to achieve K
m, k
cat, k
enz.Result is as shown in table 4: in woaded blue, each 4CL has different enzymic activitys, and has substrate selective.Ii4CL2 can with kind of the substrate catalyzed combination of four except styracin, and catalytic capability is all stronger; Ii4CL3 and coffic acid, forulic acid have stronger binding ability, and catalysis styracin ability is more weak, can not catalysis 4-coumaric acid and sinapinic acid; Ii4CL1 and 5 kind of substrate all can not catalyzed combination.
The kinetic parameter of table 4Ii4CL albumen
< embodiment four > changes the biosynthesizing of lariciresinol by the expression of regulation and control woaded blue Ii4CL family protein
According to the complete encoding sequence of Ii4CL1, Ii4CL2 and Ii4CL3, design amplifies the primer of complete coding reading frame, and restriction endonuclease sites is introduced respectively on positive anti-primer, pcr amplification product is oppositely inserted into expression vector pCAMBIA1304, build gene inhibition expression vector, proceeded to respectively in Agrobacterium rhizogenes C58C1 again, 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 2ml YEB liquid (100mg/L Kan+40mg/L Rif), 28 degree, 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 about 1 square centimeter of square vanelets;
5. blade is put into the bacterium liquid prepared, soak 2-5min, 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 WT1, WT2 The root of hair number consecutively turning Ii4CL1 is D1, D2 The root of hair number consecutively turning Ii4CL2 is E1, E2 The root of hair number consecutively turning Ii4CL3 is F1, F2
9. by the root of hair subculture of numbering to 1/2B5+Cef250mgL
-1solid medium on, no longer add screening pressure and be beneficial to hairy root growth, and constantly reduce Cef concentration (250mgL
-1→ 150mgL
-1→ 100mgL
-1→ 50mgL
-1→ 0mgL
-1), carry out 1/2B5 medium liquid until completely degerming and cultivate the acquisition genetically modified root of hair system of Ii4CL.
Respectively according to the complete encoding sequence of Ii4CL1, Ii4CL2 and Ii4CL3, the primer of design real-time quantitative Realtime-PCR, detects the expression level of each Ii4CL in transgenosis root of hair; And with HPLC, assay is carried out to target compound lariciresinol in root of hair.Result is as shown in Fig. 4 to Fig. 9, and in figure, WT represents wild-type root of hair; D, E, F strain represents the transgenosis root of hair suppressing to express Ii4CL1, Ii4CL2 and Ii4CL3 respectively.Fig. 4 and Fig. 5 shows the content of suppression expression 4CL1 on lariciresinol almost not to be affected; Fig. 6 and Fig. 7 shows and suppresses to express the content that 4CL2 significantly improves lariciresinol in transgenosis root of hair, and improving maximum strain content and reach 92.2 μ g/g, is about 2.5 times of WT strain; Fig. 8 and Fig. 9 shows and suppresses to express 4CL3 and significantly can reduce the content of lariciresinol, and the minimum strain of content only has 13.8 μ g/g, is about 0.4 times of WT strain.Therefore the lariciresinol of different accumulating level can be obtained in application by the expression level regulating and controlling different 4CL genes.
Regulating the meaning of lariciresinol to be by aforesaid method: on the one hand, lariciresinol is important antiviral activity composition, by suppressing to express the content that 4CL2 improves lariciresinol, obtaining the elite plant strain that antiviral effective constituent is high.
On the other hand, lariciresinol belongs to lignan component, suppresses to express the content that 4CL3 significantly can reduce lariciresinol, reduces lignified degree, thus effectively to reduce it as feedstock conversion be the difficulty of biofuel, the production for bioenergy is significant.
Claims (3)
1. the nucleotide sequence of coding woaded blue 4-coumaroyl A ligase enzyme protein family, is characterized in that:
Wherein, described nucleotide sequence is respectively as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3.
2. the aminoacid sequence of woaded blue 4-coumaroyl A ligase enzyme protein family, is characterized in that:
Its sequence is as shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ IDNO.6.
3. the expression passing through regulation and control woaded blue 4-coumaroyl A family protein is to change the biosynthetic application of lariciresinol.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106148355A (en) * | 2016-06-30 | 2016-11-23 | 中国人民解放军第二军医大学 | The application in regulation and control Lignanoids compounds synthesis of the Isatis indigotica Fort. IiAP2/ERF049 gene |
| CN111647571A (en) * | 2020-05-26 | 2020-09-11 | 上海中医药大学 | IiPLR1 amino acid site-directed mutant protein and coding gene and application thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106148355A (en) * | 2016-06-30 | 2016-11-23 | 中国人民解放军第二军医大学 | The application in regulation and control Lignanoids compounds synthesis of the Isatis indigotica Fort. IiAP2/ERF049 gene |
| CN106148355B (en) * | 2016-06-30 | 2019-08-06 | 中国人民解放军第二军医大学 | Application of the woaded blue IiAP2/ERF049 gene in regulation Lignanoids compounds synthesis |
| CN111647571A (en) * | 2020-05-26 | 2020-09-11 | 上海中医药大学 | IiPLR1 amino acid site-directed mutant protein and coding gene and application thereof |
| CN111647571B (en) * | 2020-05-26 | 2023-02-28 | 上海中医药大学 | IiPLR1 amino acid site-directed mutant protein and coding gene and application thereof |
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Application publication date: 20150729 |