CN108486112A - A kind of promoter with anther tissue specificity - Google Patents
A kind of promoter with anther tissue specificity Download PDFInfo
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- CN108486112A CN108486112A CN201810329479.1A CN201810329479A CN108486112A CN 108486112 A CN108486112 A CN 108486112A CN 201810329479 A CN201810329479 A CN 201810329479A CN 108486112 A CN108486112 A CN 108486112A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
- C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
- C12N15/823—Reproductive tissue-specific promoters
- C12N15/8231—Male-specific, e.g. anther, tapetum, pollen
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Abstract
The invention discloses a kind of promoters with anther tissue specificity.Promoter provided by the invention be it is following a) or b) or c)：A) DNA fragmentation shown in sequence 1 in sequence table；B) nucleotide sequence and a) limited has 75% or 75% or more homogeneity, and the DNA fragmentation with promoter function；C) under strict conditions with the nucleotide sequence hybridization that a) or b) limits, and the DNA fragmentation with promoter function.It is demonstrated experimentally that the promoter of the present invention has the specifically expressed characteristic in anther tissue, can be used for cultivating genetically modified plants.
Description
Technical field
The present invention relates in biotechnology, a kind of promoter with anther tissue specificity.
Background technology
Exogenous DNA array starts the expression in plant host by being connected to specific promoter, starts subclass
The selection of type determines expression time and the position of gene.Plant gene regulatory is mainly to be carried out on transcriptional level, by a variety of
Cis-acting elements and trans-acting factor it is mutually coordinated.Promoter is important cis-acting elements, is to be located at structure base
The section of DNA sequence for holding the transcription of upstream region controlling gene because of 5 ', can activate RNA polymerase, be allowed to accurately tie with template DNA
It closes, it is ensured that transcription accurately and effectively originates, and plays a crucial role in transcriptional control.Drive the difference of gene expression special according to it
Point, promoter are divided into constitutive promoter and specificity promoter.Constitutive promoter can in all cell or tissues, regardless of
Time and spatially startup transcription.
It is opened at present in widely applied mainly some constitutive promoters of agricultural biological technical field, such as CaMV35S
Mover and corn Ubiquitin-1 promoters, however using these promoters induction target gene conversion Chinese cabbage etc. crops with
When the quality of phase Crop Improvement, often due to the time of destination gene expression (stage of development specificity) or space (organizer
Official's specificity) it cannot control well and lead to improved effect unobvious, or due to these constitutive promoter induced genes
Expression quantity is too high and is impacted to the growth and development of plant, these are all currently with composing type strong promoter binding function base
Because carrying out the obstacle encountered when crop improvement quality.
Specificity promoter can be concentrated in specific condition, position or period and be expressed.Specificity promoter can divide again
For tissue-specific promoter and inducible promoter, wherein inducible promoter does not usually start transcription or transcriptional activity very
It is low, but under the stimulation of certain specific adverse circumstance signals, transcriptional activity can significantly increase.
With transgenic crop being widely applied in the whole world, importance of the promoter in transgenosis obtains widely
Common recognition.Chinese cabbage anther is the organ that Chinese cabbage male gamete pollen generates, with the close phase such as the production of hybrid seeds, breeding and seed production of Chinese cabbage
It closes.Therefore in the research field of Chinese cabbage tissue specific promoter, anther specific promoter is the startup that people pay close attention to the most
One of son.
But the genetic resources for the anther specific promoter that people are had found at present is not very abundant, limits people
Promotion to anther character improvement ability.
Invention content
The object of the present invention is to provide a kind of DNA moleculars with promoter activity.
DNA molecular provided by the invention, for it is following a) or b) or c)：
A) DNA fragmentation shown in sequence 1 in sequence table；
B) nucleotide sequence and a) limited has 75% or 75% or more homogeneity, and the DNA with promoter function
Segment；
C) under strict conditions with the nucleotide sequence hybridization that a) or b) limits, and the DNA fragmentation with promoter function.
The stringent condition is to hybridize at 68 DEG C in 2 × SSC, the solution of 0.1%SDS and wash film 2 times.Every time
In the solution of 5min 0.5 × SSC, 0.1%SDS, hybridizes at 68 DEG C and wash film 2 times.Each 15min.
Those of ordinary skill in the art can easily adopt by known method, for example, orthogenesis and point mutation side
Method is mutated the DNA molecular nucleotide sequence of the present invention.Those have and are detached with the present invention by manually modified
The nucleotide of the DNA molecular nucleotide sequence 75% or higher homogeneity that arrive, as long as maintaining the promoter of expression target gene
Activity is the nucleotide sequence derived from the present invention and is equal to the sequence of the present invention.
Term " homogeneity " used herein refers to the sequence similarity with native sequence nucleic acid." homogeneity " includes and this hair
Bright DNA molecular nucleotide sequence have 75% higher or 85% higher or 90% higher or 95% or higher it is same
The nucleotide sequence of one property.Homogeneity can with the naked eye or computer software is evaluated.Using computer software, two or more
Homogeneity between a sequence can use percentage (%) to indicate, can be used for evaluating the homogeneity between correlated series.
Above-mentioned 75% or 75% or more homogeneity can be 80%, 85%, 90%, 95% or more homogeneity.
Any one of it is following B1 the present invention also provides the biomaterial containing the DNA molecular) to B19)：
B1) contain the expression cassette of the DNA molecular；
B2) contain the recombinant vector of the DNA molecular；
B3) contain B1) recombinant vector of the expression cassette；
B4) contain the recombinant microorganism of the DNA molecular；
B5) contain B1) recombinant microorganism of the expression cassette；
B6) contain B2) recombinant microorganism of the recombinant vector；
B7) contain B3) recombinant microorganism of the recombinant vector；
B8) contain the transgenic plant cells system of the DNA molecular；
B9) contain B1) the transgenic plant cells system of the expression cassette；
B10) contain B2) the transgenic plant cells system of the recombinant vector；
B11) contain B3) the transgenic plant cells system of the recombinant vector；
B12) contain the Transgenic plant tissue of the DNA molecular；
B13) contain B1) Transgenic plant tissue of the expression cassette；
B14) contain B2) Transgenic plant tissue of the recombinant vector；
B15) contain B3) Transgenic plant tissue of the recombinant vector；
B16) contain the genetically modified plants organ of the DNA molecular；
B17) contain B1) the genetically modified plants organ of the expression cassette；
B18) contain B2) the genetically modified plants organ of the recombinant vector；
B19) contain B3) the genetically modified plants organ of the recombinant vector.
In above-mentioned biomaterial, the expression cassette can be started the purpose base of expression by the DNA molecular, the DNA molecular
Cause and transcription terminator composition；The DNA molecular is connect with functional way with the target gene, and the purpose
Gene is connect with the transcription terminator.In one embodiment of the invention, the target gene is specially gus gene.
In the recombinant vector, start the expression of target gene by the DNA molecular.In one embodiment of the present of invention
In, the recombinant vector is specially the multiple cloning sites insertion DNA molecular in pC1301 carriers (pCambia1301 carriers)
Obtained recombinant plasmid.The target gene is specially gus gene.
Above-mentioned expression cassette or recombinant vector can pass through pronuclear microinjection method, embryonic stem cell mediated method, reverse transcription disease
Poisonous carrier method, the gene transfer of Sperm-mediated, nuclear transfer transgemic approach, body-cell neucleus transplanting method, mitochondria mediated method etc. are conventional
Biological method transformed animal organ or tissue or cell obtain genetically modified plants object cell or tissue or organ.
In above-mentioned biomaterial, the transgenic plant cells system, the Transgenic plant tissue and the transgenosis are planted
Sundries official does not include propagating materials.
The present invention also provides the DNA moleculars as the application in promoter.
In above application, the promoter can be tissue-specific promoter.
The tissue can be anther tissue.
The answering in any one of following (a) to (d) the present invention also provides the DNA molecular or the biomaterial
With：
(a) plant variety or strain are cultivated；
(b) Pollination Fertilization ability enhancing plant variety or strain are cultivated；
(c) plant variety or strain that Pollination Fertilization ability slackens are cultivated；
(d) male sterile plants kind or strain are cultivated.
The present invention also provides the DNA moleculars or the biomaterial to start answering in destination gene expression in plant
With.
Method specific expressed in plant anther that the present invention also provides target gene, the method includes：It will contain
There is the expression cassette of the DNA molecular and target gene to import in plant, realizes special table of the target gene in plant anther
It reaches.
The plant can be dicotyledon (such as arabidopsis) or monocotyledon.
The DNA molecular overall length or the primer pair of Partial Fragment are expanded, protection scope of the present invention is also belonged to.
The primer pair can two single stranded DNAs shown in sequence in sequence table 2 and sequence 3 form.
The DNA molecular of the present invention has promoter function, and with the specifically expressed characteristic in anther tissue.It can be with
Improve the growth characteristics of plant anther using genetic modification is carried out to variety of crops by the promoter, such as passes through the promoter
Driving target gene is expressed in plant, for example, to the resistance of environment during anther development can be enhanced, for example resist cold
Property, heat resistance etc., to cultivate ideal genetically modified plants kind；It can also drive more specific expressed in anther
Functional gene or structural gene achieve the purpose that breed improvement, initiative male sterile line.
Description of the drawings
Fig. 1 is GUS coloration results.A is empty vector control plant GUS coloration results, and b is transfer-gen plant GUS dyeing knots
Fruit.
Specific implementation mode
The present invention is further described in detail With reference to embodiment, the embodiment provided is only for explaining
The bright present invention, the range being not intended to be limiting of the invention.Experimental method in following embodiments is unless otherwise specified
Conventional method.Material as used in the following examples, reagent, instrument etc., are commercially available unless otherwise specified.
Quantitative test in following embodiment, is respectively provided with three repeated experiments, and results are averaged.In following embodiments, such as without special
Illustrate, the 1st of each nucleotide sequence is the 5 ' terminal nucleotides of corresponding DNA in sequence table, and last position is the 3 ' of corresponding DNA
Terminal nucleotide.
Chinese cabbage DH system FT (Huang et al., Screening of Chinese cabbage in following embodiments
Mutants produced by 60Co c-ray mutagenesis of isolated microspore cultures,
Plant Breeding,133,480–488(2014)；Huang et al., A new method for generation and
screening of Chinese cabbage mutants using isolated microspore culturing and
EMS mutagenesis, Euphytica (2016) 207:23-33) public can obtain the biomaterial, the life from applicant
Object material is only attached most importance to used in the related experiment of duplicate invention, not can be used as other purposes and is used.
Embodiment 1, ASP1 promoters have anther tissue expression specificity
A DNA fragmentation with promoter function for deriving from Chinese cabbage DH system FT is present embodiments provided, is ordered
Entitled ASP1 promoters, sequence are sequence 1 in sequence table.
One, the structure of recombinant vector and recombinant bacterium
Using Chinese cabbage DH systems FT genomic DNAs template, PCR amplification is carried out using forward primer, reverse primer, is expanded
Increase production object；Utilize restriction endonuclease XbaI (Takara, 1093A) digestion promoter Analysis carrier pC1301 (i.e. pCambia1301, report
Announcement gene is GUS), obtain linearized vector；Ligation amplification product and linearized vector, obtain recombinant vector, to obtained weight
Group carrier is sequenced, and the obtained correct recombinant vector of the sequence containing sequence in ordered list 1 is named as pC1301-
ASP1.The primer sequence is as follows：
Forward primer：5'-CCGGGGATCCTCTAGAATGATTACATGCGTTTGATGATG-3'(sequences 2)；
Reverse primer：5'-ATGAGCTTGCATGCCTTTCTCTTTACAAGTTTATTAGGACTTG-3'(sequences 3).
PC1301-ASP1 and pC1301 are directed respectively into Agrobacterium tumefaciems GV3101, recombinant bacterium is obtained, the weight that will be obtained
Group bacterium is respectively designated as GV3101-pC1301-ASP1 and GV3101-pC1301.
Two, it is expressed in arabidopsis using promoter ASP1 driving Gus reporter genes
Step 1：Arabidopsis is planted
Colombia's wildtype Arabidopsis thaliana that this experiment uses takes 100 seeds to be put into 4 DEG C of refrigerator low-temperature treatments 3-5 days
Point is sowed in 10cm × 10cm nutritive cubes afterwards, and turf is housed in nutritive cube:Vermiculite:Perlite=7:4:2 matrix, and irrigated
Tap water.After the completion, the plastic film for applying layer of transparent is concentrated on alms bowl, culturing room is placed in and cultivates 3-4 days, and seed is fitted after sprouting
When lifting film, leak informaton.Seedling is slightly healthy and strong, removes film, sprinkles profoundly water.Lift pour week about after film it is primary permeable.Wait for that seedling grows 6-8 pieces
Start to pour 1/2MS nutrient solutions when true leaf, pour 10mL every time, nutrient solution and water separately pour, until bolting is bloomed.
Step 2:Agriculture bacillus mediated transgenic experiments
GV3101-pC1301-ASP1 is inoculated in 10mL liquid LB (containing kanamycins), 28 DEG C, 200rpm shakings
48h takes 5 μ L detections after bacterium solution muddiness, takes 1mL to expand culture 48h in 100mL liquid LB (containing kanamycins), cultivated
Product.It waits for that arabidopsis grows to Sheng flower bud phase, carries out infecting experiment.It is sprinkled profoundly water to Arabidopsis plant within first day, the same day will with small scissors
The little Hua opened on Arabidopsis plant and the silique tied all are cut, and the big bud that will be opened is retained.It simultaneously will culture
Product is assigned to 12,000rpm, 10min in 50mL centrifuge tubes and is centrifuged；Abandon supernatant, with 50mL 1/2MS culture mediums (sterilize in advance,
Contain surfactant) thalline is resuspended, then centrifuge and receive bacterium, obtained thalline (is sterilized, contained with 50mL 1/2MS culture mediums in advance
Have surfactant) it is resuspended, obtain thalline suspension (OD600 0.8-1.2)；The inflorescence for the arabidopsis trimmed is dipped in completely
In thalline suspension, culture dish is uniformly rotated back and forth, is taken out after 50s, plant is lain against under dark surrounds, infects 30 successively
Strain.The Arabidopsis plant infected is taken out afterwards for 24 hours, it is vertical to place, it cultivates in suitable environment, to later stage single plant sowing, obtains
To T1 for transgenic seed.
According to the method described above, GV3101-pC1301-ASP1 is replaced with into GV3101-pC1301, prepares empty vector control plant
Strain.
Step 3:Transfer-gen plant screens and GUS dyeing
T1 is screened for transgenic seed using hygromycin, positive transgenic plant is obtained；Wait for that positive transgenic is planted
After strain grows 10-15 piece true leaves, each single plant takes 2-3 piece tender leafs respectively, extracts genomic DNA, with the forward primer of step 1 and
Reverse primer carries out PCR detections, the results show that containing purpose band in positive transgenic plant.
After positive transgenic plant bolting, root, stem, leaf, the inflorescence of choosing same positive single plant carry out GUS dyeing.And
As a contrast using empty vector control plant.
The results are shown in Figure 1, the expression without gus gene in empty vector control plant；And the gus gene in transfer-gen plant
Only there is strongly expressed in anther, the very strong blue that naked eyes can be observed obviously is shown, in other each organ (root, stem and leaves
Piece) in, the not expression of gus gene substantially.This shows that ASP1 promoters of the invention can be in the anther of transfer-gen plant
Start the expression of gus gene downstream, and this expression has anther-specific.
<110>Agricultural University Of Shenyang
<120>A kind of promoter with anther tissue specificity
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 2147
<212> DNA
<213>Chinese cabbage
<400> 1
atgattacat gcgtttgatg atgtttgtag actgggattg aacccaagtt tgtcttgttc 60
tttgattgtc ctgaggaaga gatggagagg cgcctcttgg gaagaaacca ggttagtgtg 120
atatgaaaag ctctgtttct tttactctgt gtagcctctt gtttcatgta tgttgattgt 180
tgcatccgtt tctggtgttg aaggggagag aggatgacaa tatagagact ataaggaagc 240
gtttcaaggt gtttcttgaa tctagcttgc cagtgattca gtactacgaa gctaagggca 300
aagttcgcaa ggtgaaaatc tttttttctt ctagttttta ctgtattacg gtccagcaat 360
tgtaagttgt gtaacaaagt tttgttgttt ctaatacttg gctttagatt catgccgcaa 420
agcctattga agatgtgttt caggaggtga aggcagtctt ttctcctgaa gctgagaagg 480
taaaaagaaa acaatagttg tgccattctt taatatctaa tagtcagtag aaaattaaag 540
actccaatga actacctgta atgtagaaac ttgttactaa tggttactga tttgaatcat 600
gaatacatga ttgttctgta aagagaaaga atcatgcagc tatgatggca ttattttatc 660
acttaagtta ttgctaagag ctgaaaaaaa aaaaaagaaa agttattgct aagattctca 720
agttaacgat tctgtgaacg ttttatttag gttgaagcct aggcagcagc atcgtaaaga 780
gatacctgat acattggtca ggtatgtcta gcagccaatg atggttgaac gtttgtttat 840
gtgtgttcaa acacaactca gcttttgatc tgtaaaagta aaaaaaaaaa gctacttgaa 900
tctgaaataa aaggctgtgt ggaagcatgt tctgatgtat ctagttctgt tatagaatct 960
tgaggactaa atgagtatgt gatggtgatg attaatgtct gtttcctttg aaactttgta 1020
taggtaagct taggggttgc atctttacaa ggaagtctct gcttggttgt attttttctt 1080
cttctatata acccatatga ttactgttat tattattata cacctcttta cctattatat 1140
atttttgttt ttttttgtat ttgatgaaat tttgtgattt gtactttctg taatttcatt 1200
tgaactaatg agattttata atccttttat aatataccta gattaatgaa atccgtgtgt 1260
tgtgtcttgt gtggtctttc ttgttgttct tgttcccatt gtttatttct gctgtaactt 1320
ttgtttttca ccaaatccat attattgagc taagtatcgg aaagagttac atagtgtaaa 1380
taaatacatc acatgtctta ttcagtaaaa taaatcaata ctagtaagct actctagttt 1440
gaaattttta atgagaccta ggatgtgact tcattttcgt tttgccagac aacaacagta 1500
acttaatatc tgacatgatt caaattcaga gtatgtttcg tctcaaatct ttcttttaac 1560
tatcactact tgttacattt taaatatatt tttgacaaca aattttttac taaagaatta 1620
ttaatggttc tgcatctcaa gagatcttag tttcaaattt cagaaaaaaa cgttgttaat 1680
ggagacaata tttactttac tggttaagga ttatgcagtc atatgtgcat ttttgtaaga 1740
caagtgtatt gactatcggc cacggaatcg tcaatataat acttttttaa tagtttataa 1800
tatcaaagtt aattaggaga atacaaatcc tacatgatct gatcatttcg atcatgtact 1860
tgctatatat tttcatagtt ttaataactc ttttcacaat atgaaaatcc aacaatagtt 1920
agccaatcct atttatgtca aactacacag aaatcacaaa tctctattca aagtcaaacc 1980
aaacaaaaat agcaaacaac aaaaacacta cacacacaaa tatactaaat atagaggcaa 2040
agatttagta tataaacgcc ttcttctcca tccacctatt tgcacatctc atatctatca 2100
ataataatta aataaaacac aagtcctaat aaacttgtaa agagaaa 2147
Claims (10)
1.DNA molecules, for it is following a) or b) or c)：
A) DNA fragmentation shown in sequence 1 in sequence table；
B) nucleotide sequence and a) limited has 75% or 75% or more homogeneity, and the DNA fragmentation with promoter function；
C) under strict conditions with the nucleotide sequence hybridization that a) or b) limits, and the DNA fragmentation with promoter function.
Any one of 2. it is following B1 the biomaterial containing DNA molecular described in claim 1) to B19)：
B1 the expression cassette) containing DNA molecular described in claim 1；
B2 the recombinant vector) containing DNA molecular described in claim 1；
B3) contain B1) recombinant vector of the expression cassette；
B4 the recombinant microorganism) containing DNA molecular described in claim 1；
B5) contain B1) recombinant microorganism of the expression cassette；
B6) contain B2) recombinant microorganism of the recombinant vector；
B7) contain B3) recombinant microorganism of the recombinant vector；
B8) the transgenic plant cells system containing DNA molecular described in claim 1；
B9) contain B1) the transgenic plant cells system of the expression cassette；
B10) contain B2) the transgenic plant cells system of the recombinant vector；
B11) contain B3) the transgenic plant cells system of the recombinant vector；
B12 the Transgenic plant tissue) containing DNA molecular described in claim 1；
B13) contain B1) Transgenic plant tissue of the expression cassette；
B14) contain B2) Transgenic plant tissue of the recombinant vector；
B15) contain B3) Transgenic plant tissue of the recombinant vector；
B16) the genetically modified plants organ containing DNA molecular described in claim 1；
B17) contain B1) the genetically modified plants organ of the expression cassette；
B18) contain B2) the genetically modified plants organ of the recombinant vector；
B19) contain B3) the genetically modified plants organ of the recombinant vector.
3. DNA molecular described in claim 1 is as the application in promoter.
4. application according to claim 3, it is characterised in that：The promoter is tissue-specific promoter.
5. application according to claim 4, it is characterised in that：It is described to be organized as anther tissue.
6. biomaterial answering in any one of following (a) to (d) described in DNA molecular or claim 2 described in claim 1
With：
(a) plant variety or strain are cultivated；
(b) Pollination Fertilization ability enhancing plant variety or strain are cultivated；
(c) plant variety or strain that Pollination Fertilization ability slackens are cultivated；
(d) male sterile plants kind or strain are cultivated.
7. biomaterial described in DNA molecular or claim 2 described in claim 1 starts in plant in destination gene expression
Using.
8. target gene method specific expressed in plant anther, including：DNA molecular described in claim 1 will be contained
It is imported in plant with the expression cassette of target gene, realizes specifically expressing of the target gene in plant anther.
9. expanding DNA molecular overall length or the primer pair of Partial Fragment described in claim 1.
10. primer pair according to claim 9, it is characterised in that：The primer pair is by sequence in sequence table 2 and sequence 3
Shown in two single stranded DNAs composition.
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| CN110643608A (en) * | 2019-11-14 | 2020-01-03 | 潍坊兴旺生物种业有限公司 | Promoter with anther tissue specificity |
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| CN110283821B (en) * | 2019-06-21 | 2022-07-12 | 山东玄康种业科技有限公司 | Promoter with anther tissue specificity |
| CN110643608A (en) * | 2019-11-14 | 2020-01-03 | 潍坊兴旺生物种业有限公司 | Promoter with anther tissue specificity |
| CN110643608B (en) * | 2019-11-14 | 2022-12-20 | 山东玄康种业科技有限公司 | Promoter with anther tissue specificity |
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