CN105985936A - Erigeron breviscapus flavanone-3-hydroxylase as well as coding gene and application thereof - Google Patents
Erigeron breviscapus flavanone-3-hydroxylase as well as coding gene and application thereof Download PDFInfo
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- CN105985936A CN105985936A CN201510053624.4A CN201510053624A CN105985936A CN 105985936 A CN105985936 A CN 105985936A CN 201510053624 A CN201510053624 A CN 201510053624A CN 105985936 A CN105985936 A CN 105985936A
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- herba erigerontis
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- scutellarin
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Abstract
The invention belongs to the fields of bio-technology and botanical technology, and in particular relates to a novel scutellarin bio-synthesis pathway metabolic enzyme as well as a coding gene and an application thereof. According to the invention, total RNA of mature erigeron breviscapus is extracted, a gene sequence is obtained by conducting cloning through rapid-amplification of cDNA ends (RACE) and reverse transcription PCR (RT-PCR), and then base composition of the sequence is detected and necessary bioinformatics analysis is completed. According to the invention, the flavanone-3-hydroxylase in the erigeron breviscapus is obtained by cloning for the first time; therefore, the invention lays a foundation for understanding an erigeron breviscapus genetic background and a scutellarin bio-synthesis pathway.
Description
Technical field
The invention belongs to biotechnology and botany technical field, be related specifically to a kind of new scutellarin biosynthesis pathway metabolic enzyme and encoding gene thereof and application.
Background technology
Herba Erigerontis is the dry herb of feverfew Erigeron breviscapus (Vant.) Hand.-Mazz. Erigeron breviscapus (Vant.) Hand-Mazz, has another name called Herba Erigerontis, is distributed mainly on Southwestern China area, especially more with Yunnan.The flavone compound scutellarin (5,6,4-trihydroxyflavone-7-glucuronide) extracted in Erigeron breviscapus (Vant.) Hand.-Mazz. is widely considered to be its main active substances.There is ischemia resisting damage, antiplatelet aggregation, improve many-sided effect such as organ blood flow kinetics and microcirculation, ischemic cardio cerebrovascular diseases is had significant curative effect.
In plant, the biosynthesis pathway of flavonoid generally exists, and produces extremely abundant secondary metabolite, is referred to as flavonoids, and scutellarin (scutellarin) is also a member therein.nullThe most fully the flavone approach of research is by upstream Phenylpropanoid Glycosides approach，Phenylalanine is at PAL (Phenylalanine Ammonia Lyase，PAL)，Cinnamic acid 4-hydroxylase (Cinnamate4-hydroxylase，And 4-coumaric acyl CoA ligase (4-coumarate coenzyme A ligase C4H)，4CL) catalysis phenylalanine generates coumaroyl A，Again by downstream flavone approach，By chalcone synthase (Chalcone Synthase，CHS),Enzyme, namely chalcone isomerase (chalcone isomerase，CHI)，Flavanone 3-hydroxylase (Flavanone 3-hydroxylase，F3H)，Flavone synzyme I (Flavone synthase I，FNS I)，Flavone synthetase II (Flavone synthase II，FNSII) catalytic action generates chromocor compound.And in Herba Erigerontis, be then that flavanone 6-hydroxylase (Flavanone 6-hydroxylase, EbF6H) replaces EbF3H, then generate scutellarin by UDPG aldehyde transferring enzyme (EbF7GAT) catalysis.Therefore EbF3H is the biosynthetic important bypass gene of scutellarin, and further investigation EbF3H is significant to understanding scutellarin biosynthesis, but does not but have the research of EbF3H in Herba Erigerontis at present.The F3H in many plants reported by existing document, and the Flavone metabolism of the Nicotiana tabacum L. such as turning F3H is raised, and improves its saline-alkaline tolerance (Plant Mol Biol, 85 (6): 551-573).Semen Tritici aestivi, Fructus Fragariae Ananssae, the plant such as arabidopsis and Oryza sativa L. also has widely research (Mol Biol, 2013,47 (6): 1028-1030；Plant Physiol Biochem,2014,82:289-298；Plant Physiol Biochem,2008,46(10):833-843；Planta,228(6):1043-1054.)
When Herba Erigerontis is widely used in medical treatment, this wild resource is just plucked in a large number, and considerable economic interests cause Herba Erigerontis resource excessively to be developed, and environment is seriously damaged, and wild resource reduces day by day, gradually cause the present situation of nowadays Herba Erigerontis resource scarcity.But due to problems such as the delayed and genetic background of research of Herba Erigerontis correlated inheritance are narrow; the difficulty of cross-breeding is very big; the most up to the present it is the cultivation of wild strain, offspring's poor stability, manages to Herba Erigerontis large-scale planting and GMP and bring huge challenge.
For scutellarin biosynthesis pathway, only upstream gene is cloned (PLoS One, 2014,9 (6): e100357), downstream gene and bypass gene are almost without any progress, and this is to regulate and control, from molecular level, the purpose reaching to improve scutellarin content further to cause puzzlement greatly.Therefore it is highly important for cloning, analyze and regulate and control the unknown gene in metabolic pathway.
There is no the cloning process of EbF3H, sequence analysis and related application in document report Herba Erigerontis at present.
Summary of the invention
It is an object of the invention to provide important metabolic enzyme and encoding gene, the flavanone-3-hydroxylase (EbF3H) that this metabolic enzyme specifically extracts from Herba Erigerontis of a kind of new scutellarin biosynthesis pathway bypass.Another object of the present invention is to provide this Herba Erigerontis flavanone-3-hydroxylase and application of encoding gene thereof.
The main technical schemes of the present invention is: by planting seed in soil, take growth 90 days Herba Erigerontis plant as process object, extract total serum IgE reverse transcription cDNA, by RACE and RT-PCR clone obtain EbF3H, order-checking obtains nucleotide sequence, and carries out bioinformatic analysis.
Specifically comprise the following steps that
A. the sprouting of seed: by Herba Erigerontis planting seed in soil, cover transparent plastic film and keep moisture, in illumination 12 hour/day, intensity of illumination 2500-3500Lux, cultivating under the conditions of preferably 3000Lux, temperature is 25 DEG C, removes plastic sheeting after germinateing, color is obtained glossy in emerald green, eugonic seedling after 30 days；
B. seedling is transplanted: every strain seedling replanting is to independent soil environment, (illumination 12 hour/day under similarity condition, intensity of illumination 3000Lux, temperature 25 DEG C) cultivate 60 days (without plastic sheeting), choose the Herba Erigerontis plant grown fine as object；
C. taking a strain and grow 90 days ripe Herba Erigerontis plant, extract total serum IgE (TotalRNA), reverse transcription obtains cDNA, be stored in-20 DEG C standby；
D. design primer, obtained the nucleotide sequence of EbF3H by the method for RACE and RT-PCR；
F. necessary comprehensively bioinformatic analysis is carried out.
Wherein in step C and D, the experiment reaction related to all is carried out according to the step of offer in industrialization test kit.
Wherein in step F, the software related to and website using method are all according to explanation operation.
Wherein needing to carry out the process of seed before sowing, process step, particularly as follows: dry oil lamp flower seed 30mg is soaked in 30min in pure water, removes the impurity of floating surface.
Term is explained, " Lux " refers to intensity of illumination herein.
" independent soil environment " refers to plant shoots separately plantation herein, but other conditions include soil, illumination, and temperature all keeps constant.
A first aspect of the present invention, there is provided a kind of new scutellarin biosynthesis pathway metabolic enzyme and encoding gene thereof.
The invention provides a kind of Herba Erigerontis flavanone-3-hydroxylase EbF3H, the aminoacid sequence of described EbF3H is as shown in SEQ ID NO:1 (362aa).
The invention provides the encoding gene of a kind of Herba Erigerontis flavanone-3-hydroxylase EbF3H, the nucleotide sequence of the encoding gene of described EbF3H is (1327bp, wherein ORF1086bp is SEQ ID NO:11) as shown in SEQ ID NO:2.
A second aspect of the present invention, there is provided above-mentioned Herba Erigerontis flavanone-3-hydroxylase and the application of encoding gene thereof.
Described application, refers to utilize this Herba Erigerontis flavanone-3-hydroxylase and encoding gene biosynthesis scutellarin thereof.
Described application, refers to the quality utilizing this Herba Erigerontis flavanone-3-hydroxylase and encoding gene thereof to improve Herba Erigerontis further.
The described quality improving Herba Erigerontis, specifically refers to improve the content of scutellarin in Herba Erigerontis.
Beneficial effects of the present invention is as follows:
The present invention completes clone and the analysis of biological information of Herba Erigerontis EbF3H, the perfect biosynthetic understanding of scutellarin, attempts metabolic engineering for carrying out the regulation and control of molecular level further, improves scutellarin content and lay a good foundation.
Present invention clone obtains a new functional gene participating in scutellarin synthesis, and the genetic background understanding of Herba Erigerontis is had actively impact.
Accompanying drawing explanation
The bioinformatic analysis of Fig. 1: EbF3H, A) the domain analysis of EbF3H；B) secondary structure analysis of EbF3H albumen；C) the tertiary structure analysis of EbF3H albumen；D) the also cell positioning analysis of EbF3H.
Detailed description of the invention
In conjunction with embodiment and accompanying drawing, the present invention is described in detail, but the enforcement of the present invention is not limited only to this.
The experimental technique of unreceipted actual conditions in the following example, generally according to normal condition, or according to the condition proposed by manufacturer.
Embodiment 1:
One, the collection of sample
1. the process of seed: dry oil lamp flower seed 30mg is soaked in 30min in pure water, removes the impurity of floating surface, is uniformly laid in the soil of pre-wetting by the seed after screening, covers one layer of soil then at surface.
2. germinate: on soil, cover the plastic sheeting of layer of transparent, to preserve moisture.Being placed in intensity of illumination is cultivation under the conditions of 3000Lux, illumination 12 hour/day, and temperature is 25 DEG C, removes plastic sheeting, obtain color glossy in emerald green, eugonic seedling after 30 days after germinateing.
3. cultivate: by every strain seedling replanting to independent soil environment, continue similarity condition and cultivate 60 days.
4. sampling: herb samples, aluminium-foil paper is packed, and is stored in-80 DEG C and saves backup after liquid nitrogen flash freezer.
Two, the clone of EbF3H
1. take the Herba Erigerontis sample met in above-mentioned Part I.
The extraction of 2.RNA: extract test kit according to RNAThe illustration method of Reagent, Tian Gen company (TianGen) extracts the TotalRNA (total serum IgE) of Herba Erigerontis.
The structure in 3.RACE library: build 3 ' and 5 ' RACE libraries according to the illustration method of RACE library construction Kit (SMARTTM RACE cDNA Amplification Kit, precious biological engineering, Japan, TAKARA, JAPAN).
4.RACE: by 5 ' and 3 ' ends of 5 ' and 3 ' RACE clone genes, using Phusion to surpass fidelity dna polymerase and carry out reacting (NEW ENGLAND BIOLABS, AMERICA), the primer of use is shown in Table 1.
The primer that table 1RACE uses
5.RT-PCR: design primer according to RACE result, uses Phusion to surpass fidelity dna polymerase and carries out reacting (NEW ENGLAND BIOLABS, AMERICA) and carry out RT-PCR and obtain EbF3H total length, and condition is: 98 DEG C, 30s, 1 circulation；98 DEG C, 10s, 57 DEG C, 10s, 72 DEG C, 60s, 40 circulations；72 DEG C, 5min, 1 circulation.The primer used is shown in Table 2, and the nucleotide sequence obtained, as shown in SEQ ID NO:2, entrusts genewiz biotech firm (Suzhou) to check order successfully.
The primer that table 2qRT-PCR uses
Embodiment 2: express the vector construction of EbF3H-GFP fusion protein
Experimental technique: expand EbF3H by the primer (sequence is as follows) with restriction enzyme site SalI and SpeI, use Phusion to surpass fidelity dna polymerase to carry out reacting (NEW ENGLAND BIOLABS, AMERICA) carry out RT-PCR and obtain the sEbF3H total length with restriction enzyme site, condition is: 98 DEG C, 30s, 1 circulation；98 DEG C, 10s, 55 DEG C, 10s, 72 DEG C, 60s, 40 circulations；72 DEG C, 5min, 1 circulation.Again by restricted enzyme SalI and SpeI (Takara, Japan) enzyme action sEbF3H and pA7-GFP carrier.Being attached in 16 DEG C by digestion products finally by T4 ligase (Takara, Japan), condition is according to explanation operation.The EbF3H-Pa7-GFP carrier obtained is that the experiment of follow-up Subcellular Localization is standby.
The primer that table 3EbF3H-Pa7-GFP vector construction uses
Embodiment 3: the fusion protein that embodiment 2 obtains carries out bioinformatic analysis
nullThe domain analysis of 1.EbF3H: use MAGA 5.05 software (http://www.megasoftware.net/) that with F3H in other plant, EbF3H is carried out homogeneous assays，Obtain 20G-FeII-Oxy,Tri-domains of PLNO03176 and PLNO02515，The F3H sequence information used is all from NCBI website，Sequence information is respectively: Gynura bicolor (GbF3H,BAJ17667.1),Dahlia pinnata(DaF3H,BAJ21534.1),Chrysanthemum x morifolium(CmF3H,AAB97310.1),Pilosella officinarum(PoF3H,ACB56921.1)and Carthamus tinctorius(CtF3H,AEG64806.1)，As shown in Figure 1A.
The secondary structure analysis of 2.EbF3H: use SOPMA software (https: //npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl？Page=/NPSA/npsa_sopma.html) carrying out the secondary structure analysis of EbF3H albumen, result shows that albumen is irregularly crimped by 35.36% alpha-helix, 21.82% extended configuration, 9.67% β-corner and 33.15%, and result is as shown in Figure 1B.
The tertiary structure analysis of 3.EbF3H: using Phyre2 software (http://www.sbg.bio.ic.ac.uk/phyre2/html) to carry out EbF3H albumen and carry out tertiary structure analysis, result is as shown in Figure 1 C.
The Subcellular Localization of 4.EbF3H: with rice protoplast as medium, expresses EbF3H-GFP fusion protein in carrier pA-7, observes the GFP fluorescin expressive site at cell, finds that EbF3H-GFP fusion protein is mainly expressed in Cytoplasm, result such as Fig. 1 D.
Above domain, with tertiary structure analysis result, two grades show that it has the feature of F3H, in addition Subcellular Localization description of test its mainly express in Cell sap, this mainly matches with Flavone metabolism in Cell sap, and therefore clone the EbF3H that obtains is the flavanone 3-hydroxylase in Herba Erigerontis.
The ultimate principle of the present invention, principal character and advantages of the present invention have more than been shown and described.Skilled person will appreciate that of the industry; the present invention is not restricted to the described embodiments; the principle that the present invention is simply described described in above-described embodiment and description; the present invention also has various changes and modifications without departing from the spirit and scope of the present invention, and these changes and improvements both fall within scope of the claimed invention.Claimed scope is defined by appending claims and equivalent thereof.
Claims (6)
1. a Herba Erigerontis flavanone-3-hydroxylase, its aminoacid sequence is as shown in SEQ ID NO:1.
2. an encoding gene for Herba Erigerontis flavanone-3-hydroxylase as claimed in claim 1, its nucleotides sequence
Row are as shown in SEQ ID NO:2.
3. a Herba Erigerontis flavanone-3-hydroxylase as claimed in claim 1 is in biosynthesis scutellarin
Application.
4. a Herba Erigerontis flavanone-3-hydroxylase as claimed in claim 1 is in the quality improving Herba Erigerontis
Application, the described quality improving Herba Erigerontis refers to improve the content of scutellarin in Herba Erigerontis.
5. the encoding gene of a Herba Erigerontis flavanone-3-hydroxylase as claimed in claim 2 is at biosynthesis lamp
Application in small cup B prime.
6. the encoding gene of a Herba Erigerontis flavanone-3-hydroxylase as claimed in claim 2 is improving Herba Erigerontis
Quality in application, the described quality improving Herba Erigerontis refers to improve containing of scutellarin in Herba Erigerontis
Amount.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112458065A (en) * | 2020-11-04 | 2021-03-09 | 江南大学 | Silybum marianum-derived flavone 3-hydroxylase and application thereof |
Citations (4)
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|---|---|---|---|---|
| WO1999043825A1 (en) * | 1998-02-25 | 1999-09-02 | E.I. Du Pont De Nemours And Company | Plant flavanone-3-hydroxylase |
| CN1314943A (en) * | 1999-06-17 | 2001-09-26 | Basf公司 | Method for producing plants with increased flavonoid and phenolic compound content |
| WO2003106633A2 (en) * | 2002-06-13 | 2003-12-24 | E.I. Du Pont De Nemours And Company | Method to increase isoflavonoid levels in plants and plants producing increased levels of isoflavonoids |
| CN103614348A (en) * | 2012-10-31 | 2014-03-05 | 上海交通大学 | Tulip flavanone-3-hydroxylase TfF3H protein and encoding gene thereof |
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2015
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999043825A1 (en) * | 1998-02-25 | 1999-09-02 | E.I. Du Pont De Nemours And Company | Plant flavanone-3-hydroxylase |
| CN1314943A (en) * | 1999-06-17 | 2001-09-26 | Basf公司 | Method for producing plants with increased flavonoid and phenolic compound content |
| WO2003106633A2 (en) * | 2002-06-13 | 2003-12-24 | E.I. Du Pont De Nemours And Company | Method to increase isoflavonoid levels in plants and plants producing increased levels of isoflavonoids |
| CN103614348A (en) * | 2012-10-31 | 2014-03-05 | 上海交通大学 | Tulip flavanone-3-hydroxylase TfF3H protein and encoding gene thereof |
Non-Patent Citations (2)
| Title |
|---|
| NI-HAO JIANG,ET AL: "Analysis of the Transcriptome of Erigeron breviscapus Uncovers Putative Scutellarin and Chlorogenic Acids Biosynthetic Genes and Genetic Markers", 《PLOS ONE》 * |
| 张广辉等: "灯盏花黄酮合成酶II 基因克隆及其生物信息学分析", 《中国中药杂志》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112458065A (en) * | 2020-11-04 | 2021-03-09 | 江南大学 | Silybum marianum-derived flavone 3-hydroxylase and application thereof |
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