CN110029108A - A kind of endogenous full history of life constitutive promoter of seaweed and its application - Google Patents
A kind of endogenous full history of life constitutive promoter of seaweed and its application Download PDFInfo
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- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
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Abstract
The invention belongs to seaweed gene engineering technology field, it is related to a kind of endogenous full history of life constitutive promoter of seaweed and its application.Promoter is nucleotide sequence shown in SEQ ID NO:1；One or more replacing, missing or adding for nucleotide are carried out to nucleotide sequence shown in SEQ ID NO:1 to be obtained；Or, the nucleotide sequence limited with SEQ ID No:1 in sequence table is with 90% or more homology and with the nucleotide sequence of transcription initiation effect.The present invention obtains Enteromorpha actin gene 2 (actin2) promoter using technologies such as chromosome walking, gene cloning, DNA sequencings, has sequence shown in SEQ ID NO:1；2 promoter of the actin gene of clone is inserted into Reporter gene GUS upstream on this basis, building conversion carrier imports Enteromorpha difference Life Stages, detects that gus gene can high efficient expression in vegetative growth phase and reproduction period.Enteromorpha transgene efficiency and biological safety can be improved in the technology, and the breed improvement and gene engineering product exploitation to algae are all of great significance.
Description
Technical field
The invention belongs to seaweed gene engineering technology field, be related to a kind of endogenous full history of life constitutive promoter of seaweed and
It is applied.
Background technique
Tangleweed belongs to rudimentary plant, mainly includes green alga, brown alga and red algae.Many economic species have realized rule
Modelling artificial cultivation, such as Enteromorpha, sea lettuce and the reef film of green algae, kelp, thallus laminariae, sargassum fusifome and the red algae of brown algae
Seaweed, Bangiales, fragrant plant mentioned in ancient texts, agar and the Eucheuma of class.The whole world tangleweed cultivation gross area is more than 3,000,000 mu at present, year
Yield is more than 6,000,000 tons of fresh weights, except part as in addition to nutraceutical, be also used for the important industrial chemicals such as iodine, mannitol, phycocolloid,
And the extraction raw material of marine drug and protein reagent, there is very high economic value.
In recent years, cultivation seaweed is in new energy (such as fermentation producing and ethanol, methane), new material (such as algal polysaccharides weaving fibre
Dimension), the fields such as novel expression system (being prepared by recombinant protein medicaments) also show application potential outstanding.In order to realize seaweed
The needs of slewing breeding, there is an urgent need to develop efficient, safe seaweed technique for gene engineering and a full set of method.Wherein, needle
High efficient expression and directional induction to quiding gene are expressed, and promoter is an important carrier element.
Promoter is a component part of gene, is the DNA sequence dna of RNA polymerase specific recognition and combination, control
The initial time of gene expression (transcription) and the degree of expression.For promoter just as " switch ", itself does not control Gene Activity,
But the transcription of gene is controlled and in conjunction with the protein of referred to as transcription factor.Currently, being with kelp, thallus laminariae, seaweed
The seaweed genetic conversion system of representative is tentatively established, and importing external source functional gene may be implemented to stablize table in seaweed
It reaches.But the promoter element due to using is mostly from terrestrial life, thus there is also many deficiencies.
Firstly, using extensive CaMV35S efficient promoter (coming from cauliflower mosaic virus) in higher plant, in sea
Drive the efficiency of exogenous gene expression generally lower in algae；In addition, most popular SV40 starting subsystem is from spirit in seaweed
Long class simian virus 40, therefore, the use of exogenous promoter bring the inefficient expression of tangleweed transgenosis and presence safety hidden
Suffer from two outstanding problems.Secondly, the seaweed history of life has two stages of vegetative growth phase and reproduction period, the reproduction cell in reproduction period
Often as the acceptor material of genetic transformation.After conversion, if foreign gene is integrated in the single reproduction cell stage, what is grown up to
It is all intracellular all containing the gene imported in new plant；If many cells stage of the foreign gene after reproduction cell sprouting
Integration, then the new plant grown up to is often chimera, i.e., only has in the cell of part in new plant containing the gene imported.Therefore,
If screened in the single reproduction cell stage after conversion, it is possible to prevente effectively from the generation of chimera.But it is used at present
Promoter has only investigated the starting activity in nutrition raw growth period, and does not investigate it in the starting activity in seaweed reproduction period.
Summary of the invention
For the demand of seaweed genetic engineering full history of life constitutive promoter endogenous to algae, the purpose of the present invention is mention
For a kind of endogenous full history of life constitutive promoter of seaweed and its application.
To achieve the above object, the invention adopts a technical scheme as:
A kind of endogenous full history of life constitutive promoter of seaweed, (actin2) promoter of Enteromorpha actin gene 2 are SEQ
Nucleotide sequence shown in ID NO:1；
One or more replacing, missing or adding for nucleotide are carried out to nucleotide sequence shown in SEQ ID NO:1 to be obtained
?；
Or, the nucleotide sequence limited with SEQ ID NO:1 in sequence table is with 90% or more homology and has transcription
The nucleotide sequence of initiation.
It is preferred that 2 promoter of Enteromorpha actin gene is nucleotide sequence shown in SEQ ID NO:1.
The starting subsystem is obtained from marine green algae.
The starting subsystem is obtained from Ulva (Ulva) plant Enteromorpha (Ulva prolifera).
Further, the chromosome walking of molecular biology (genome walking) technology, gene cloning skill are utilized
Art, DNA sequencing technology are cloned a kind of endogenous full history of life composing type actin gene promotor pUpActin2 of Enteromorpha and are measured
Its sequence；On this basis, the pUpActin2 promoter sequence cloned is inserted respectively into β-glucose using clone technology
The upstream of thuja acid enzyme (GUS) gene and herbicide glufosinate-resistant gene (bar) constructs serial Enteromorpha conversion carrier, warp
Conversion is, it can be achieved that import foreign gene in the high efficient expression of vegetative growth phase and reproduction period.
A kind of seaweed expression system: expression system contains the promoter.
A kind of application of the endogenous full history of life constitutive promoter of seaweed, the promoter drive in seaweed nutritive growth period
Application in exogenous gene high-efficient expressed.
The promoter drives the application in exogenous gene high-efficient expressed in seaweed reproduction period.
Application of the promoter in Enteromorpha genetic breeding.
Application of the promoter in seaweed quality-improving or building seaweed expression system.It is preferred that the Enteromorpha flesh is dynamic
2 promoter of protein gene is nucleotide sequence shown in SEQ ID NO:1.
Actin (Actin) gene of above-mentioned offer is a kind of generally existing constitutive expression gene of living nature, this
The 2 entitled pUpActin2 of (actin2) promoter of Enteromorpha actin gene provided by inventing, from large-scale economic green
Algae --- Enteromorpha (Ulva prolifera).
Compared with the conventional method, the invention has the following beneficial effects:
1. the present invention obtains 2 upstream piece of Enteromorpha actin gene by chromosome walking technology (Genome Walking)
Section further devises specificity amplification primer after activated subfunction verifying, can effectively obtain Enteromorpha actin gene 2 and open
Mover.
2. obtaining the endogenous actin gene 2 of Enteromorpha using the present invention in seaweed transgenic research or application to start
Son, animal virus used at present (such as SV40, CMV) and the viral source (CaMV35S, AMT) of higher plant can be substituted
Promoter avoids the introducing of nucleic acid sequence completely, has higher biological safety, is conducive to transgenosis seaweed in future
Using.
3. actin is the albumen of a high-level constitutive expression, actin gene promotor is an efficient table
The controlling element reached constructs seaweed transgene carrier with 2 promoter of Enteromorpha actin gene of the present invention, drives foreign gene
Expression efficiency can significantly improve.
4. using 2 promoter of Enteromorpha actin gene of the present invention driving selectable marker gene, (such as herbicide glufosinate is anti-
Property Bar gene) expression, by improve resistant gene expression, screening reagent concentration (such as glufosinate) can be increased, thus
The generation of tolerant clon (false positive) is reduced, increases transgenic resistance and clones (true positives) ratio, effectively improve screening efficiency.
5. the seaweed history of life has two stages of vegetative growth phase and reproduction period, the reproduction cell in reproduction period is often as something lost
Pass the acceptor material of conversion.After conversion, if foreign gene is integrated in the single reproduction cell stage, in the new plant grown up to,
It is all intracellular all containing the gene imported；It therefore, can be effective if screened in the single reproduction cell stage after conversion
Avoid the generation of chimera.2 promoter of Enteromorpha actin gene has full Life history, gene can be driven in reproduction period
Expression, driving resistant maker gene are expressed in the reproduction cell stage, for achieving the above object.
Detailed description of the invention
Fig. 1 is 5 ' upstream sequence bioinformatic analysis figures of Enteromorpha actin gene 2 provided in an embodiment of the present invention.
Fig. 2 be Enteromorpha conversion carrier pUPA2-GUS, pUPA2NI-GUS provided in an embodiment of the present invention, pUPA2NH-GUS,
PUPA1-GUS, pBI221 and pSV40-GUS schematic diagram.
Fig. 3 be Enteromorpha provided in an embodiment of the present invention convert pUPA2-GUS, pUPA2NI-GUS, pUPA2NH-GUS,
The quantitative detection result of pUPA1-GUS, pBI221 and pSV40-GUS.
Fig. 4 is Enteromorpha provided in an embodiment of the present invention in vegetative growth phase and reproduction period, actin1 and actin2 gene
RT-PCR testing result.
Fig. 5 is that Enteromorpha conversion carrier pUPA2-bar provided in an embodiment of the present invention constructs schematic diagram.
Fig. 6 is that the PCR for the glufosinate-resistant algae strain that Enteromorpha provided in an embodiment of the present invention converts pUPA2-bar detects knot
Fruit.M:DNA marker Trans2K plus II；1: blank control；2-6: bombardment pUPA2-bar group single plant；7-9: negative right
According to group.
Specific embodiment
Combined with specific embodiments below, element of the invention and element effect achieved are made further specifically
It is bright.
Endogenous complete 2 promoter sequence of history of life composing type actin gene of Enteromorpha of the present invention and its obtaining transgenosis sea
Application in algae；It can especially realize that foreign gene was expressed and played a role in reproduction period, can effectively improve transgene efficiency, to algae
The breed improvement of kind and gene engineering product exploitation are of great significance.
Embodiment 1: endogenous complete 2 promoter of history of life composing type actin gene of Enteromorpha (U.prolifera)
Clone, sequencing and the bioinformatic analysis of pUpActin2
1. clone and the sequencing of 5 ' upstream sequences of Enteromorpha actin gene 2
Enteromorpha total DNA template is prepared in a conventional manner using the plant genome DNA extracts kit of Tiangeng company, is passed through
0.8% agarose electrophoresis detection, the Enteromorpha genomic DNA band complete display of extraction can satisfy PCR amplification needs.Benefit
With (Genome Walking Kit) chromosome walking kit of TaKaRa company, for the endogenous full history of life composing type of Enteromorpha
5 ' upstream sequences of actin gene 2 design three specific primer (SP1:5 '-CAAGACCGCACCATTGACCTGA-
3'；SP2:5'-GGAGCATCATCGCCAGCAAATC-3'；SP3:5 '-TCAACGAAAGGACGACGAGTGG-3 '), according to examination
Agent box specification carries out chromosome walking, the segment that amplification obtains is carried out Ago-Gel recycling, TA clone connects and carries out
Nucleotide sequencing obtains the total 1739bp of 5 ' upstream sequences of actin gene.
2. the bioinformatic analysis of 5 ' upstream sequences of Enteromorpha actin gene 2
Using plant cis-acting elements on-line analysis tool PLANTCARE (network address http: //
Bioinformatics.psb.ugent.be/webtools/plantcare/html), found through bioinformatic analysis, waterside
There is only some common promoter regulation members such as TATA-Box, CAAT-Box for 5 ' upstream sequences of tongue fur actin gene 2
Part, there is also the particular components (see Fig. 1) such as efficient transcription element (5 ' UTR Py-rich stretch) and 5 ' UTR intrones, can
Tentatively to assert that the sequence has the function of promoter.
Embodiment 2: 2 promoter pUpActin2 of Enteromorpha actin gene drives Reporter gene GUS to grow in seaweed nutritive
Interim efficient constitutive expression
1. the clone of 2 promoter fragment of Enteromorpha actin gene and pUPA2-GUS vector construction
According to 5 ' upstream sequences of the Enteromorpha actin gene 2 that embodiment 1 obtains, for the tool of Bioinformatics Prediction
There is the region of promoter activity, designs 2 promoter pUpActin2 specific primer (actin2- of Enteromorpha actin gene
F14B-HindIII:5’-TGATTACGCCAAGCTTCcacctatcaactatctgcg tctt-3 ', wherein tilted letter represents
The recognition site of restriction enzyme Hind III；actin2-R14B-SmaI:5'-AGGGACTGACCACCCGGGcttgttt
Cagcgcacctcc-3 ', the recognition site capitalization that wherein tilted letter represents restriction endonuclease sma I represent subsequent
It usesHD Cloning Kit is seamlessly connected homologous with carrier end required for Cloning Kit carrier construction
Segment), carry out PCR amplification, amplified fragments are recycled through Ago-Gel, TA clone connects and carries out nucleotide sequencing,
Obtain SEQ ID NO:1 sequence.
With the commercial carrier pBI221 of Clontech company, (CaMV35S promoter drives beta-glucuronidase GUS base
Cause) it is the carrier that sets out, double digestion is carried out to it using restriction enzyme Hind III and Sma I, by gus gene upstream original
CaMV35S promoter is cut away, and through 1% agarose gel electrophoresis, uses OMEGAGel Extraction Kit
Digestion large fragment (carrying gus gene) is recycled.Use TaKaRa High fidelity PCR enzymeMax DNA
III/actin2-R14B-Sma of primer pair actin2-F14B-Hind, I amplified fragments list Polymerase errorless to above-mentioned sequencing
Clone carries out PCR amplification, usesHD Cloning Kit is seamlessly connected Cloning Kit and produces the PCR of acquisition
Hind III and Sma the I double digestion large fragment of object and pBI221 are attached.Then connection product is transformed into Escherichia coli
In Top10 bacterial strain competent cell, positive transformant is screened on the LB plate containing ampicillin.Positive transformant expands
Plasmid is cultivated and extracted, double digestion identification (Hind III and Sma I) is carried out to plasmid, the errorless plasmid of digestion result send sequencing
Company's sequencing.Thus to obtain seaweed conversion carrier pUPA2-GUS (see Fig. 2), β-is driven by 2 promoter of Enteromorpha actin gene
The transcription of glucuronidase gus gene.
In addition, 5 ' upstream sequences of the Enteromorpha actin gene 2 obtained according to embodiment 1, design special primer
(actin2-F14:5'-CCACCTATCAACTATCTGCGTCTT-3'；actin2-R24:5'-CTTGTTTCAGCGCACCTCC
GGACTAAACTCGGTTGATTCTGTTTCTT CCCTTACACCC-3 ') PCR amplification is carried out, it is dynamic to obtain truncated Enteromorpha flesh
2 upstream sequence of protein gene, 2 upstream initiation codon ATG of sequence deletion Enteromorpha actin gene -251 to -39 is altogether
213 bp (5 ' UTR introne).Further use aforementioned special primer actin2-F14B-HindIII and actin2-R14B-
SmaI carries out PCR amplification to the segment, with pUPA2-GUS with the method building truncated carrier pUPA2NI-GUS of promoter (see figure
2)。
In addition, 5 ' upstream sequences of the Enteromorpha actin gene 2 obtained according to embodiment 1, design special primer
(actin2-F14:5'-CCACCTATCAACTATCTGCGTCTT-3'；actin2-R26:5'-GATCCCGGCAGGGACGTGC
CTGTACACCCAGCTTTCGTGTCG-3'；actin2-F26:5'-CGACACGAAAGCTGGGTGTACAGGCACGTCCCTGC
CGGGATC-3'；Actin2-R14:5 '-CTTGTTTCAGCGCACCTCC-3 ') recombinant PCR amplification is carried out, it obtains truncated
2 upstream sequence of Enteromorpha actin gene, 2 upstream initiation codon ATG -264 of sequence deletion Enteromorpha actin gene to -
255 totally 10 bp (5 ' UTR Py-rich stretch of efficient transcription element).Further use aforementioned special primer
Actin2-F14B-HindIII and actin2-R14B-SmaI carries out PCR amplification to the segment, constructs with pUPA2-GUS with method
The truncated carrier pUPA2NH-GUS of promoter (see Fig. 2).
SEQ ID NO.1pUpActin2
CCACCTATCAACTATCTGCGTCTTCTGAGCAACTTGATAATGATGGAGATGCACCTAGGTTGTCAGAGT
GTTGGACACATGGTACATCACCCATGTTTTGGCACACCAGCTCCTCCTCATACACTTCATCAACAAAACCAAGCAGA
TTTTCAAAGCATCGAGTTTCGACAACAGCTGCATCATTATCGCCTAGGCGATGGAGGTTCATTGTACAGTGTCGTTG
GCAGCGTACTGAATTTCATTGATCTTCTCTTCGCGGTCAAGTTGTCACAGACATTTCACAGCCTCTCCACGACTGTA
GAGGGCTTCGCAGACATAATTTTTATGGCAGCCTCTCTCGGCAAACATTTCAACAACATCACGAGGCGCAACATCAG
TCGCAACTGGCTTTCTGCGCATACTGGAGCCACCAACATCTGCCAAATCAGCCAGATTTGCCTCAACCCAACTTTCA
ATTCGGTCTTGTCTTGTGTCGAGCCTAGAAGTTGCTCACTGCCACATGACAGGTGCACACAGGCTACGGAAACTGAC
TCCCACAGCATGAGCAAGCGACACGGACTTAAGTACTAGGAGCCAGCAACAGGTGTGCTGCGAGTTACATAATGGCC
ACCGTGCTTTGCTAGTGATATTTGTCCTATGAGGTCGGTCAAAGCCATCTGAATACAGCCCTTTTTGGCCTCAACTT
GGATTTTAAAGCACCCTTCACGTTAAAATGCGATAAAGCGAAACGGCCTCTTTAAAGCCTCGAAATCGTTTTAACGT
TAAAACTGTCAAGTAAAATAGCATTTTAAAGTAGGTGAACATGGGCGTAACGTTCAATTTACAGCTCTACTCGCCGT
TTGCTGCGATGGTGCTGAATTCTCTCATATCGACAGCTCGCATGCTGCTTCAAAGCGGATGAAAAGGCGTAGTGACG
TCGGTGTGAAGATTTTCAAAAACATGTCATGTTGAATTTTTCTCGACCCGCACAGCGTGTGTCTGACGAACCACAAA
TACGGAGCAGCCCGGTTATTCCTGCCACGAAGAGGAAACTTTTGCCGGGTCCATTCCCTACGGAGCCCGCAACGACT
CTGTGACGTTCGTACGGTAATGTCCCGCTATTGGTGGCTCAAATCAGTGCCTGTGCGGACGATCAGTGTGCTGCTAA
AACACGACAGAGAAAGCTCGACATTGAAAACGTTACTGCGTCCTGTCTTATCCTGTGGAACCAATAAATAACTCATC
GTTACATTATGCGCACGGTGCGCATCCAGGACCCAGGTGGTGGGGGCCTGGTGGCGACAGGGATTCGTGTCGCGACA
TGTAGCGAATGCAGCTGAATGGTTTTGGCAGGAGGGCAAGGCGAGCAATGCACGAGGCTTAGACCGGTCAACCTTCG
ATTCGAGGTAGCTCCACAAATATGAAGTCGACAGTGCACACGCAAGTTGCATTGGAAGCAGTCGTTTGTGACGTCAA
CGACACGAAAGCTGGGTGTAAGGGAAGAAACAGGCACGTCCCTGCCGGGATCATCCAGAAATGTCACATGATGTCGC
CGCCTTTTGCGGAGTACATGGATTCATGACCGAGCTGCGTATGTGGTTGTGTACTCAAGTGTCGACTTGATGGTGGG
TTGCGCACGTCGAGGCCTTGTTGCAGATGTGCCCGTGACCCGTGAGTGTGCTTTGCAACTTTTTCTGCCATTTGAAT
TGTCTCGAGGTGCAGAATCAACCGAGTTTAGTCCGGAGGTGCGCTGAAACAAG
(a) sequence signature:
* length: 1739 base-pairs
* type: nucleotide
* chain: single-stranded
* topological structure: linear
(b) molecule type: DNA
(c) assume: no
(d) antisense: no
(e) initial source: Enteromorpha (Ulva prolifera)
2. via Particle Bombardment Transformation Enteromorpha
1) preparation of particle gun particulate
It weighs 60mg bronze (the mating consumptive material of Bio-Rad company, U.S. particle gun, 1.0 μm of diameter), the anhydrous second of 1ml is added
Alcohol acutely vibrates 1 minute；10000 revs/min are centrifuged 10 seconds, remove supernatant；1ml sterile water is added to suspend, be centrifugated again
Supernatant is removed, is repeated 3 times altogether, finally bronze is suspended in 1ml sterile water, is dispensed by every part of 50 μ l, 4 DEG C of storages are standby
With.
It takes 1 part of bronze suspension (1 part is 50 μ l) to be transferred to 1.5ml centrifuge tube when conversion to be vibrated, oscillation process
In sequentially add 5 μ l DNA (conversion carrier that i.e. ready-made carrier or above-mentioned steps 1 obtain, including pBI221, pSV40-GUS,
Totally 6 kinds of pUPA1-GUS, pUPA2-GUS, pUPA2NI-GUS or pUPA2NH-GUS, 1 μ g/ μ l), 50 μ l CaCl2(2.5mol/
L), 20 μ l spermidine (0.1mol/L) vibrates 3 minutes；10000 revs/min are centrifuged 10 seconds, abandon supernatant；The rinsing of 250 μ l ethyl alcohol,
Supernatant is removed in centrifugation, is repeated 1 times；Bronze is resuspended in 60 μ l dehydrated alcohols.
2) biolistic bombardment seaweed
Enteromorpha (U.prolifera) thallus for taking culture is cut into the algae section of 2cm long with aseptic operation, and when conversion is used
Round sterile bolting silk (400 mesh, diameter 4cm) is as the supporting body of algae section, and algae section is evenly laid out in center, and formation diameter is about
The circle of 2cm effectively bombardment range, as sample.Each sample bombardment is primary, and bombardment is about with 12 μ l bronze suspension every time.Turn
Changing with high-pressure helium formula particle gun (model: PDS1000/He) is U.S. Bio-Rad Products, bombardment process in desinfection chamber or
It is carried out in super-clean bench；Operating table surface, particle gun surface and inside are sterilized with 70% ethanol, and bombardment consumptive material is that can split film
(Rupture disk), DNA slide glass (Macrocarrier) and blocking net (Stopping screen), it is anhydrous 70% in advance
It impregnates 20 minutes, is dried under ultraviolet lamp stand-by in ethyl alcohol；Transformation Parameters are as follows: recipient cell distance stops net 6.0cm, vacuum degree 28
(inch of mercury).The blank control group that test setting is bombarded with the naked bronze without Plasmid DNA, operating method are identical.
The quantitative detection of 3.GUS gene
After conversion, the seaweed material of conversion group and control group is transferred to VSE culture solution, 15.0 ± 0.5 DEG C of cultivation temperature, light
In dark period 12h/12h, light intensity is about 50mmolm-2·s-1.GUS enzyme can be by the Portugal 4-methyl umbelliferone-β-D- of not fluorescence
Grape glycuronide (4-MUG) catalytic decomposition be the 4-methyl umbelliferone (4-MU) with fluorescence, based on the above principles with standard side
Method can quantitative determine the vigor that GUS enzyme is recombinantly expressed in transgenosis seaweed, to reflect the starting of different promoters
Sub- activity.
Specific steps are as follows: the seaweed thallus of transgenosis is pulverized after liquid nitrogen frozen after conversion 48h
End, be added 1ml protein extract buffer (protein extract buffer be 50mM Na2HPO4(pH 7.0),10mM Na2EDTA
(pH 8.0), 0.1%Triton X-100,10mM beta -mercaptoethanol), after mixing, 4 DEG C of 10000g are centrifuged 15min, supernatant is taken,
Ice bath storage is stand-by.Use the concentration of soluble protein in the Bradford quantification of protein kit detection supernatant of Tiangeng company
Taking 60 μ l supernatants to be added to 37 DEG C of the 540 μ l reaction solutions preheated, (reaction solution is 50mM Na2HPO4(pH 7.0),10mM
Na2EDTA (pH 8.0), 0.1%Triton X-100,10mM beta -mercaptoethanol, 2mM 4-methyl umbelliferone-β-D-Glucose
Aldehydic acid glycosides) in, 100 μ l reaction solutions are taken out in 0,5,15,30,60min respectively to 900 μ l reaction terminating liquid (0.2M Na2CO3)
In, the fluorescent value at 455nm is detected under 365nm exciting light using microplate reader, and calculate GUS enzyme activity, unit is nM MU
min-1(mg protein)-1。
Testing result shows 2 promoter pUpActin2 of Enteromorpha actin gene and Enteromorpha pUpActin1 promoter, height
Efficient promoter CaMV35S, the efficient promoter SV40 in mammal in equal plants can drive gus gene to seek in Enteromorpha
The long-term efficient constitutive expression of health, wherein pUpActin2 is suitable with SV40 promoter activity, about CaMV35S promoter
1.5 times, equal slightly below Enteromorpha pUPA1 promoters (see Fig. 3).In addition, converting the carrier after promoter truncates into Enteromorpha
PUPA2NI-GUS or pUPA2NH-GUS almost drops the results show that the starting vigor of the two is significantly reduced compared with overall length promoter
Down to background level, it is seen that 5 ' UTR intrones and efficient transcription member in 2 promoter pUpActin2 of Enteromorpha actin gene
5 ' UTR Py-rich stretch of part is essential required element for maintaining the high vigor of pUpActin2 promoter
(see Fig. 3).
The above results prove, 5 ' upstreams of obtained Enteromorpha endogenous constitutive actin gene 2 are expanded by embodiment 1
Sequence has promoter function, and 5 ' UTR intrones and 5 ' UTR Py-rich stretch of efficient transcription element are required elements,
The promoter can high efficiency drive foreign gene in Enteromorpha vegetative growth phase realize efficient constitutive expression.
Embodiment 3: Enteromorpha actin gene actin1 and actin2 is in seaweed history of life different phase transcriptional level
Variance analysis
1. the RT-PCR primer of Enteromorpha actin gene actin1 and actin2 design
It is designed according to the cDNA sequence of Enteromorpha actin gene actin1 and actin2 gene and carries out transcriptional level difference
The RT-PCR specific amplification primer of analysis, respectively (ACT1RT-F:5 '-CCGATGGGCAAGTAATCAC-3 '；ACT1RT-R:
5'-TGAAGGTTGTATCATGAACTCC-3'；ACT2RT-F:5'-AATCCCGCACTGTTAGGC-3'；ACT2RT-R:5'-
CAAACATGGTTGTCCCGC-3’)
2. the preparation of vegetative growth phase and reproductive phase material in the Enteromorpha full history of life
Seaweed material is placed in VSE culture solution to cultivate, cultivation temperature is 15.0 ± 0.5 DEG C, light dark period 12h/
12h, light intensity are about 50mmolm-2·s-1.Then, it takes out part seaweed and is cut into 1-5mm2Left and right size induces reproduction.About 60-
After 72h, using micro- sem observation dissection frond, choose the algae section that vegetative cell is all divided into reproduction cell.Finally, will be in
Vegetative growth phase and the seaweed material of reproductive phase are placed on -80 DEG C of refrigerator freezings and save.
3. the RT- of vegetative growth phase and reproductive phase actin gene actin1 and actin2 in the Enteromorpha full history of life
PCR analysis
Described to specifications using the plant RNA extraction kit of Quan Shi King Company, extraction prepares Enteromorpha total serum IgE mould
Plate, through denaturing agarose electrophoresis detection, the Enteromorpha RNA band complete display of extraction can satisfy reverse transcription PCR amplification needs.
Use PrimeScriptTMII 1st Strand cDNA Synthesis Kit prepares Enteromorpha cDNA template.It uses
PrimeScriptTMRT reagent Kit with gDNA Eraser kit carries out fluorescence real-time quantitative PCR reaction.It is interior
Ginseng gene is Enteromorpha 18S gene.The analysis of relative quantification data, which uses, compares threshold method (2-△△CtMethod), every group of processing is arranged 3 and puts down
Row.
RT-PCR analysis the result shows that, Enteromorpha actin gene actin1 transcriptional level in nutrient growth cell is significant
Higher than reproduction cell, and Enteromorpha actin gene actin2 transcriptional level is higher in nutrition and reproductive phase, and without obvious
Difference (see Fig. 4).According to 2 functional verification experimental result of embodiment of this case it is found that the two is efficient in vegetative growth phase
Constitutive promoter；But when Enteromorpha cell is in the reproduction cell stage, pUpActin1 promoter activity is almost reduced to 0,
And pUpActin2 promoter still has high transcriptional activity, is a kind of endogenous full history of life constitutive promoter.
Embodiment 4: 2 promoter pUpActin2 of Enteromorpha actin gene drives selectable marker gene bar in Enteromorpha reproduction
The screening of transformant is realized in expression in cell
1. the clone of Enteromorpha actin gene promotor segment and pUPA2-bar vector construction
According to 5 ' upstream sequences of the Enteromorpha actin gene 2 that embodiment 1 obtains, for the tool of Bioinformatics Prediction
There is the region of promoter activity, designs 2 promoter pUpActin2 specific primer (actin2FB- of Enteromorpha actin gene
PstI:5 '-TGATTACGCCCTGCAGccacctatcaactatctgcg-3 ', wherein tilted letter represents restriction enzyme
The recognition site of Pst I；Actin2RB-SmaI:5 '-TTCTGGGCTCATCCCGGGcttgtttcagcgcacctccgg-3 ',
Wherein tilted letter represents the recognition site of restriction endonuclease sma I；Capitalization represents subsequent useHD
Cloning Kit is seamlessly connected required for Cloning Kit carrier construction and the homologous segment of carrier end), carry out PCR expansion
Increase, amplified fragments are recycled through Ago-Gel, TA clone connects and carry out nucleotide sequencing, are obtained and SEQ ID NO:1
The identical segment of sequence.
With the carrier p35S-bar of laboratory oneself building, (CaMV35S promoter drives herbicide glufosinate-resistant gene
Bar it is) carrier that sets out, double digestion is carried out to it using restriction enzyme Pst I and Sma I, by CaMV35S before bar gene
Promoter is cut away, and after 1% agarose gel electrophoresis, uses OMEGAGel Extraction Kit is to digestion
Large fragment is recycled.Use TaKaRa High fidelity PCR enzymeMax DNA Polymerase is to above-mentioned sequencing
Errorless actin2FB-PstI and actin2RB-SmaI segment monoclonal carries out PCR amplification, usesHD
Cloning Kit is seamlessly connected Cloning Kit, and Pst I and Sma the I double digestion of the PCR product of acquisition and p35S-bar is big
Segment is attached.Then connection product is transformed into Escherichia coli Top10 bacterial strain competent cell, is containing ammonia benzyl mould
Positive transformant is screened on the LB plate of element.Positive transformant, which expands, cultivates and extracts plasmid, carries out double digestion identification to plasmid
(Pst I and Sma I), the errorless plasmid of digestion result send sequencing company to be sequenced.Thus to obtain seaweed conversion carrier pUPA2-bar
(see Fig. 5) drives bar genetic transcription by 2 promoter of Enteromorpha actin gene.
2. via Particle Bombardment Transformation Enteromorpha reproduction cell
1) preparation of particle gun particulate
Referring to the step 1 in embodiment 2, preparation carries the gold of two kinds of plasmids of pUPA1-bar and pUPA2-bar respectively altogether
Powder.
2) biolistic bombardment seaweed reproduction cell
Reproduction induction is carried out to seaweed first referring to 3 step 2 of embodiment, via Particle Bombardment Transformation is carried out in sporangial stage, turns
Change process is referring to 2 step 2 of embodiment.
The detection of 3.bar gene
After conversion, frond restores 8h in the dark, and herbicide glufosinate is then added into culture solution, makes its effective component
The final concentration of 40 μ g/ml of PPT, screening time are one week, and the reproduction cell during which diffused out is transferred to VSE culture solution after diluting
It expands culture.The result shows that bombardment pUPA1-bar group, does not obtain resistant plant, and pUPA2-bar group is bombarded, obtained
Resistant plant.When with enough biomass, its genome DNA template is prepared, and specifically draw according to the design of bar gene region
Object (bar1-F:5 '-TCTGCACCATCGTCAACCACTACA-3 '；Bar1-R:5 '-TCAAATCTCGGTGACGGGCAGGAC-
3 ') PCR detection is carried out, wild type algae strain PCR result is feminine gender, and positive (see Fig. 6) is presented in resistance algae strain.
The above results prove, in the case where the high dose of herbicide glufosinate selects pressure, 2 promoter of Enteromorpha actin gene
Can be successfully driven reproduction cell stage of the bar gene in the Enteromorpha history of life is transcribed and is expressed, so that positive transformants algae
Strain successfully obtains the resistance to glufosinate, can be used for stablizing the acquisition of building and the homozygote algae strain of expression system.
Sequence table
<110>Institute of Oceanology of the Chinese Academy of Sciences
<120>a kind of endogenous full history of life constitutive promoter of seaweed and its application
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1739
<212> DNA
<213>Enteromorpha (Ulva prolifera)
<400> 1
ccacctatca actatctgcg tcttctgagc aacttgataa tgatggagat gcacctaggt 60
tgtcagagtg ttggacacat ggtacatcac ccatgttttg gcacaccagc tcctcctcat 120
acacttcatc aacaaaacca agcagatttt caaagcatcg agtttcgaca acagctgcat 180
cattatcgcc taggcgatgg aggttcattg tacagtgtcg ttggcagcgt actgaatttc 240
attgatcttc tcttcgcggt caagttgtca cagacatttc acagcctctc cacgactgta 300
gagggcttcg cagacataat ttttatggca gcctctctcg gcaaacattt caacaacatc 360
acgaggcgca acatcagtcg caactggctt tctgcgcata ctggagccac caacatctgc 420
caaatcagcc agatttgcct caacccaact ttcaattcgg tcttgtcttg tgtcgagcct 480
agaagttgct cactgccaca tgacaggtgc acacaggcta cggaaactga ctcccacagc 540
atgagcaagc gacacggact taagtactag gagccagcaa caggtgtgct gcgagttaca 600
taatggccac cgtgctttgc tagtgatatt tgtcctatga ggtcggtcaa agccatctga 660
atacagccct ttttggcctc aacttggatt ttaaagcacc cttcacgtta aaatgcgata 720
aagcgaaacg gcctctttaa agcctcgaaa tcgttttaac gttaaaactg tcaagtaaaa 780
tagcatttta aagtaggtga acatgggcgt aacgttcaat ttacagctct actcgccgtt 840
tgctgcgatg gtgctgaatt ctctcatatc gacagctcgc atgctgcttc aaagcggatg 900
aaaaggcgta gtgacgtcgg tgtgaagatt ttcaaaaaca tgtcatgttg aatttttctc 960
gacccgcaca gcgtgtgtct gacgaaccac aaatacggag cagcccggtt attcctgcca 1020
cgaagaggaa acttttgccg ggtccattcc ctacggagcc cgcaacgact ctgtgacgtt 1080
cgtacggtaa tgtcccgcta ttggtggctc aaatcagtgc ctgtgcggac gatcagtgtg 1140
ctgctaaaac acgacagaga aagctcgaca ttgaaaacgt tactgcgtcc tgtcttatcc 1200
tgtggaacca ataaataact catcgttaca ttatgcgcac ggtgcgcatc caggacccag 1260
gtggtggggg cctggtggcg acagggattc gtgtcgcgac atgtagcgaa tgcagctgaa 1320
tggttttggc aggagggcaa ggcgagcaat gcacgaggct tagaccggtc aaccttcgat 1380
tcgaggtagc tccacaaata tgaagtcgac agtgcacacg caagttgcat tggaagcagt 1440
cgtttgtgac gtcaacgaca cgaaagctgg gtgtaaggga agaaacaggc acgtccctgc 1500
cgggatcatc cagaaatgtc acatgatgtc gccgcctttt gcggagtaca tggattcatg 1560
accgagctgc gtatgtggtt gtgtactcaa gtgtcgactt gatggtgggt tgcgcacgtc 1620
gaggccttgt tgcagatgtg cccgtgaccc gtgagtgtgc tttgcaactt tttctgccat 1680
ttgaattgtc tcgaggtgca gaatcaaccg agtttagtcc ggaggtgcgc tgaaacaag 1739
Claims (7)
1. a kind of endogenous full history of life constitutive promoter of seaweed, it is characterised in that: Enteromorpha actin gene 2 (actin2) opens
Mover is nucleotide sequence shown in SEQ ID NO:1；
One or more replacing, missing or adding for nucleotide are carried out to nucleotide sequence shown in SEQ ID NO:1 to be obtained；
Or, the nucleotide sequence limited with SEQ ID NO:1 in sequence table is with 90% or more homology and has transcription initiation
The nucleotide sequence of effect.
2. the endogenous full history of life constitutive promoter of seaweed according to claim 1, it is characterised in that: the starting subsystem sea
It is obtained in foreign green alga.
3. a kind of seaweed expression system, it is characterised in that: expression system is containing promoter described in claim 1.
4. a kind of application of the endogenous full history of life constitutive promoter of seaweed described in claim 1, it is characterised in that: described to open
Mover drives the application in exogenous gene high-efficient expressed in seaweed nutritive growth period.
5. the application of the endogenous full history of life constitutive promoter of seaweed according to claim 4, it is characterised in that: the starting
Son drives the application in exogenous gene high-efficient expressed in seaweed reproduction period.
6. pressing the application of the endogenous full history of life constitutive promoter of seaweed described in claim 4 or 5, it is characterised in that: described
Application of the promoter in Enteromorpha genetic breeding.
7. pressing the application of the endogenous full history of life constitutive promoter of seaweed described in claim 4 or 5, it is characterised in that: described
Application of the promoter in seaweed quality-improving or building seaweed expression system.
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| CN201910325865.8A CN110029108B (en) | 2019-04-23 | 2019-04-23 | Endogenous whole-life-history constitutive promoter of seaweed and application thereof |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103497951A (en) * | 2013-09-05 | 2014-01-08 | 上海海洋大学 | Kelp gametophyte lhcf 6 gene promoter and application thereof |
| CN105907761A (en) * | 2016-01-04 | 2016-08-31 | 云南农业大学 | Paddy metallothionein gene OsMT2bL promoter and application thereof |
| CN106520766A (en) * | 2016-05-31 | 2017-03-22 | 中国科学院海洋研究所 | Seaweed endogenesis constructive promoter and application thereof |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103497951A (en) * | 2013-09-05 | 2014-01-08 | 上海海洋大学 | Kelp gametophyte lhcf 6 gene promoter and application thereof |
| CN105907761A (en) * | 2016-01-04 | 2016-08-31 | 云南农业大学 | Paddy metallothionein gene OsMT2bL promoter and application thereof |
| CN106520766A (en) * | 2016-05-31 | 2017-03-22 | 中国科学院海洋研究所 | Seaweed endogenesis constructive promoter and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| WANDONG FU等: "Molecular Cloning and Analysis of a Cytosolic Hsp70 Gene from Enteromorpha prolifera (Ulvophyceae, Chlorophyta)", 《PLANT MOL BIOL REP》 * |
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