CN109337906A - A kind of specifically expressed promoter of wheat endosperm and its application - Google Patents
A kind of specifically expressed promoter of wheat endosperm and its application Download PDFInfo
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Abstract
The invention discloses a kind of specifically expressed promoter of wheat endosperm and its applications.The specifically expressed promoter of wheat endosperm of the invention, the 1107-1724 nucleotide sequences for 3 ' ends at least containing sequence 1 in ordered list, and extend since the 1107th of sequence 1, according to the nucleotide sequence of sequence 1 to 5 ' ends of sequence 1, any one DNA fragmentation that obtained length is 618 to 1724bp.Experiments have shown that, the specifically expressed promoter of wheat endosperm of the invention can specifically drive the expression of target gene in endosperm, can to avoid the target gene in the other tissues of plant continuous expression adverse effect, realize that foreign gene expresses in vegetable nutritorium is precisely controlled.It can be used for carrying out target gene in plant seed the high efficient expression of specificity, this has practice significance to the grain quality improvement of plant and output increased.
Description
Technical field
The present invention relates in field of biotechnology, a kind of specifically expressed promoter of wheat endosperm and its application.
Background technique
Promoter regulates and controls the space-time table of downstream gene by the interaction of itself cis-acting elements and trans-acting factor
It reaches.According to the mode of action and the difference of function, promoter can be divided into composing type, organizing specific type and induction type three classes.Currently,
Common promoter is mostly composition type expression promoter in plant genetic engineering, as used in most of dicot transgenic plants
Cauliflower mosaic virus (CaMV) 35S promoter, unifacial leaf genetically modified plants mainly use the ZmUbi1 from corn to start
Son and OsCc1, OsAct1 promoter from rice.Foreign gene under constitutive promoter driving is in plant different tissues portion
Position and whole stage of development no significant differences, this lasting, efficient expression largely consume the basic substances of plant and cause energy
It measures load and physical waste and even results in death to break metabolic balance, hinder the normal growth of plant and reduce yield.
In order to be precisely controlled foreign gene in the intracorporal expression tissue of crops, it is set to play a positive role to obtain and there is ideal character
Crops, need to go into overdrive development organizations specific expressing promoter.
Gene general under tissue-specific promoter's regulation is transcribed in certain specific organ or tissues.Currently,
Different types of tissue-specific promoter is isolated to from plant, such as arabidopsis root specific expression promoter
Pyk10, lily andro gamete specific expression promoter LGC1, tamato fruit specific expressing promoter 2A11.Specificity promoter
Application tissue specific expression of the foreign gene in plant may be implemented.Wheat endosperm specificity promoter belongs to tissue
Type can drive foreign gene to express in endosperm to improve storage under the premise of not influencing seed development and sprouting by it
The expression quantity for hiding albumen reduces the expression of anaphylactogen, and changes branch/amylose ratio, and final realization is special with small
The target that wheat is cultivated.Therefore, have using endosperm specificity promoter control foreign gene specifically expressing in endosperm important
Practice significance.
Wheat (Triticum aestivium) belongs to cereal crop, is one of Chinese Three major grain crops.However, by
In wheat there are genomes it is huge, life cycle is long and genetic transformation is difficult the problems such as cause it to identify organizing specific starting
Subnumber amount is few, is far from satisfying research and production demand.
Summary of the invention
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) 3 ' 1107-1724 nucleotide sequences of the end at least containing sequence 1 in ordered list, and from the of sequence 1
1107 start, according to sequence 1 nucleotide sequence to sequence 15 ' end extend, obtain length be 618 to 1724bp it is any
One DNA fragmentation；The DNA molecular has promoter function；
B) nucleotide sequence and a) limited has 75% or 75% or more identity, 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 hybridized at 68 DEG C and washes film 2 times in 2 × SSC, the solution of 0.1%SDS.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, such as the side of directed evolution and point mutation
Method is mutated DNA molecular nucleotide sequence of the invention.Those have and separate with the present invention by manually modified
The nucleotide of the DNA molecular nucleotide sequence 75% or higher identity that arrive, as long as maintaining the promoter of expression target gene
Activity is derived from nucleotide sequence of the invention and to be equal to sequence of the invention.
Term " identity " used herein refers to the sequence similarity with native sequence nucleic acid." identity " includes and this hair
Bright DNA molecular nucleotide sequence is same with 75% or higher or 85% or higher or 90% or higher or 95% or higher
The nucleotide sequence of one property.Identity can with the naked eye or computer software is evaluated.Using computer software, two or more
Identity between a sequence can be indicated with percentage (%), can be used to evaluate the identity between correlated series.
Above-mentioned 75% or 75% or more identity can be 80%, 85%, 90%, 95% or more identity.
Last nucleotide that the DNA molecular 3 ' is held can be the 1724th of sequence 1.
The DNA molecular can for it is following 1) or 2):
1) DNA molecular shown in 1107-1724 of sequence 1 in sequence table；
2) DNA molecular shown in sequence 1 in sequence table.
The present invention also provides the biomaterials for containing the DNA molecular, are following B1) any one of 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 by the DNA molecular, the purpose base of DNA molecular starting expression
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 above-mentioned biomaterial, the carrier can be plasmid, sticking grain, bacteriophage or viral vectors.The plasmid specifically may be used
For carrier pWMB110.
In the recombinant vector, by the expression of DNA molecular starting target gene.In one embodiment of the present of invention
In, the recombinant vector replaces with the DNA especially by by the Ubiquitin promoter in binary expression vector pWMB110
Molecule, and gus reporter gene is inserted into the DNA molecular downstream and is obtained.The target gene is specially gus gene.
In above-mentioned biomaterial, the microorganism can be yeast, bacterium, algae or fungi.Wherein, bacterium can for Agrobacterium or
Escherichia coli.
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 propagation material.
The present invention also provides the DNA moleculars as the application in promoter.
In above-mentioned application, the promoter can be plant tissue specificity promoter
In above-mentioned application, the tissue can be albumen.
The answering in any one of following (a) to (c) the present invention also provides the DNA molecular or the biomaterial
With:
(a) plant variety or strain are cultivated；
(b) expression of target gene is driven in plant；
(c) driving target gene is expressed in albumen.
In above-mentioned application, the plant can be monocotyledon or dicotyledon.The monocotyledon can be grass
Section plant.The gramineae plant can be wheat.
Method specific expressed in albumen that the present invention also provides target gene, which comprises will contain
There is the expression cassette of the DNA molecular and target gene to import in plant, realizes specifically expressing of the target gene in albumen.
In the above method, the plant can be monocotyledon or dicotyledon.The monocotyledon can be grass
Section plant.The gramineae plant can be wheat, barley, rice, corn or jowar.
The primer pair for expanding the DNA molecular overall length or Partial Fragment, also belongs to protection scope of the present invention.
The primer pair can two bars of lists as shown in 10-36 of sequence 2 in sequence table and 10-32 of sequence 3
Chain DNA composition, can also two single stranded DNAs shown in sequence 2 in sequence table and sequence 3 form；It can also be by sequence 5 in sequence table
10-33 and 10-32 of sequence 3 shown in two articles of single stranded DNAs form；It can also be by sequence 5 in sequence table and sequence 3
Shown in two single stranded DNAs composition.
It is demonstrated experimentally that DNA molecular of the invention has promoter function, and target gene can be specifically driven in endosperm
Expression, can to avoid the target gene in the other tissues of plant continuous expression adverse effect, realize external source base
Because that expresses in vegetable nutritorium is precisely controlled；The Reporter gene GUS of DNA molecular starting also may be directly applied to turn base
Because plant is screened, has the advantages that intuitive, high sensitivity.Target gene for being carried out spy by the DNA molecular in plant seed
Anisotropic high efficient expression, this has practice significance to the grain quality improvement of plant and output increased.
Detailed description of the invention
Fig. 1 is recombinant bacterium PCR verification result.Wherein, swimming lane M is DNA molecular amount standard, and swimming lane 1,2 and 3 is respectively
The inspection of LBA4404-pWMB-PTaEm2-GUS, LBA4404-pWMB-PTaEm1-GUS and LBA4404-pWMB-PTaEm3-GUS
Survey result.
Fig. 2 is that transgenic plant PCR product detects electrophoresis result.Wherein, swimming lane M is DNA molecular amount standard, swimming lane 1~7
For positive plant；A, b and c be respectively LBA4404-pWMB-PTaEm2-GUS, LBA4404-pWMB-PTaEm1-GUS and
The testing result for the positive transgenic plant that LBA4404-pWMB-PTaEm3-GUS is obtained.
Fig. 3 is the GUS histochemical stain result of transgenic plant different tissues organ.Wherein, a, b and c are respectively
LBA4404-pWMB-PTaEm2-GUS, LBA4404-pWMB-PTaEm1-GUS and LBA4404-pWMB-PTaEm3-GUS are obtained
Positive transgenic plant result；1 is young root, and 2 be young stem, and 3 be lemma, and 4 be glumelle, and 5 be boot leaf, and 6 be little Hua, 7 at
Ripe phase seed longitudal section (white arrow show embryo).
Fig. 4 is the GUS histochemical stain result of transgenic plant endosperm different development stage.Wherein, a is PTaEm2 drive
The positive transgenic plant (i.e. the positive transgenic plant that LBA4404-pWMB-PTaEm2-GUS is obtained) of dynamic GUS, b is
The positive transgenic plant of the GUS of PTaEm1 driving (i.e. plant by the positive transgenic that LBA4404-pWMB-PTaEm1-GUS is obtained
Strain), (i.e. the positive that LBA4404-pWMB-PTaEm3-GUS is obtained turns the positive transgenic plant that c is the GUS of PTaEm3 driving
Gene plant)；It (1) is the sectional view of 8~23 days seeds of Post flowering, (2) are the longitudinal sectional figure of 8~23 days seeds of Post flowering.
Fig. 5 is the GUS histochemical stain result of transgenic plant endosperm specific expression.Wherein: a is PTaEm2 driving
The positive transgenic plant result of GUS；B is the positive transgenic plant result of the GUS of PTaEm1 driving；C is PTaEm3 driving
GUS positive transgenic plant result.
Specific embodiment
The present invention is further described in detail With reference to embodiment, and 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 bit is the 3 ' of corresponding DNA
Terminal nucleotide.
Wheat L03-227 (Zhu JT, Hao PC, Chen GX, Han CX, Li XH, Zeller in following embodiments
FJ,Hsam SLK,Hu YK,Yueming Yan(2014)Molecular cloning,phylogenetic analysis,
and expression profiling of endoplasmic reticulum molecular chaperone BiP
Genes from bread wheat (Triticum aestivum L.) .BMC Plant Biology, 14:260) public can
The biomaterial is obtained from applicant, which only attaches most importance to used in the related experiment of duplicate invention, not can be used as other
Purposes uses.
In following embodiments pWMB110 carrier (Cui XY, Du YT, Fu JD, Yu TF, Wang CT, Chen M,
Chen J,Ma YZ,Xu ZS(2018)Wheat CBL-interacting protein kinase 23 positively
Regulates drought stress and ABA responses.BMC Plant Biology.18:93) public can be from Shen
It asks someone to locate to obtain the biomaterial, which only attaches most importance to used in the related experiment of duplicate invention, not can be used as other purposes
It uses.
Carrier pWMB002 (Zhang Li, Wang Jianfeng, Wang Xiaojie, Kang Zhensheng, Han Dejun (2012) in following embodiments
The building of alfAFP and spCEMA integrative gene expression vector and its to the genetic transformation China Agricultural University journal of wheat, 17
(5): 15-20) public can obtain the biomaterial from applicant, which only attaches most importance to the related experiment institute of duplicate invention
With not can be used as other purposes and use.
Agrobacterium strains LBA4404 and auxiliary bacteria strain HB101 (Escherichia coli) in following embodiments are documented in document
(the structure of Bai Xuemei, Tao Baiqiu, Zhang Yan (2013) alfalfa drought resisting correlation mitogen-activated protein kinase plant expression vector
Build the Inner Mongol petrochemical industry, 1:30-32) in, the public can obtain these biomaterials from applicant, these biomaterials are only
Used in the related experiment of duplicate of attaching most importance to invention, it not can be used as other purposes and use.
Embodiment 1, PTaEm1 promoter have endosperm expression specificity
The DNA fragmentation with promoter function for deriving from wheat L03-227 is present embodiments provided, is named
For PTaEm1 promoter, sequence is 1107-1724 of sequence 1 in sequence table.
One, the building of recombinant vector and recombinant bacterium
1, the building of pWMB-PTaEm1
Using wheat L03-227 genomic DNA as template, PCR amplification is carried out using the primer pair that primer 1 and primer 2 form,
Obtain amplified production；Primer sequence is as follows:
Primer 1 (upstream primer): 5 '-AAAAAGCTTCGCGTCATAGCATAGATAGATGTTGTG-3 ' (sequence 2, lower stroke
Line is the identification sequence of Hind III)；
Primer 2 (downstream primer): 5 '-AAAGGATCC(sequence 3, underscore are CTCGGTGGACTGTCGGTGAATTG-3 '
The identification sequence of BamH I).
By amplified production using Hind III and BamH I digestion, digestion products are obtained；Digestion products and pWMB110 are carried
Body is connected through Hind III with the skeleton carrier that BamH I digestion obtains, and obtains recombinant vector, obtained sequence is correctly weighed
Group carrier is named as pWMB-PTaEm1, shown in 1107-1724 containing sequence 1 in ordered list of pWMB-PTaEm1
PTaEm1 promoter.
PTaEm1 promoter is shortened, it is obtained and truncates DNA fragmentation, which is denoted as PTaEm2, the sequence of PTaEm2
It is classified as 1428-1724 of sequence 1 in sequence table；The 5 ' of PTaEm1 promoter are held to its upstream and is extended, DNA piece is obtained
The DNA fragmentation is denoted as PTaEm3 by section, and the sequence of PTaEm3 is sequence 1 in sequence table.Building respectively containing PTaEm2 and
The recombinant vector of PTaEm3, verifies whether the two DNA fragmentations have promoter function.The building of recombinant vector is as follows:
Using wheat L03-227 genomic DNA as template, PCR amplification is carried out using the primer pair that primer 3 and primer 2 form,
Obtain amplified production；Primer 3 (upstream primer): 5 '-AAAAAGCTTgaactaggattaagccgattacgtg-3 ' (sequence 4,
Underscore is the identification sequence of Hind III).
By amplified production using Hind III and BamH I digestion, digestion products are obtained；Digestion products and pWMB110 are carried
Body is connected through Hind III with the skeleton carrier that BamH I digestion obtains, and obtains recombinant vector, obtained sequence is correctly weighed
Group carrier is named as pWMB-PTaEm2, shown in 1428-1724 containing sequence 1 in ordered list of pWMB-PTaEm2
PTaEm2。
Using wheat L03-227 genomic DNA as template, PCR amplification is carried out using the primer pair that primer 4 and primer 2 form,
Obtain amplified production；Primer 4 (upstream primer): 5 '-AAAAAGCTTACCCCAACAACACCAACAAAGCAG-3 ' (sequence 5, under
It is marked as the identification sequence of Hind III).
By amplified production using Hind III and BamH I digestion, digestion products are obtained；Digestion products and pWMB110 are carried
Body is connected through Hind III with the skeleton carrier that BamH I digestion obtains, and obtains recombinant vector, obtained sequence is correctly weighed
Group carrier is named as pWMB-PTaEm3, and pWMB-PTaEm3 is containing PTaEm3 shown in sequence 1 in ordered list.
2, the building of pWMB-PTaEm1-GUS
Using carrier pWMB002 as template, PCR amplification is carried out using the primer pair that primer 5 and primer 6 form, is expanded
Product；Primer sequence is as follows:
Primer 5 (upstream primer): 5 '-AAAGGATCCATGTTACGTCCTGTAGAAACCCCAAC-3 ' (sequence 6, lower stroke
Line is the identification sequence of BamH I)；
Primer 6 (downstream primer): 5 '-AAAGAGCTC(sequence 7, underscore are TCATTGTTTGCCTCCCTGCTG-3 '
The identification sequence of Sac I).
Amplified production is utilized into BamH I and Sac I digestion, obtains gus gene segment；The pWMB-that step 1 is obtained
PTaEm1 utilizes BamH I and Sac I digestion, recycles skeleton carrier；Gus gene segment and skeleton carrier are connected, recombination is obtained and carries
The correct recombinant vector of sequence is named as pWMB-PTaEm1-GUS by body.PWMB-PTaEm1-GUS is containing sequence 1 in ordered list
1107-1724 shown in gus gene shown in PTaEm1 promoter and sequence 8, PTaEm1 promoter is located at gus gene
Upstream.
Amplified production is utilized into BamH I and Sac I digestion, obtains gus gene segment；The pWMB-that step 1 is obtained
PTaEm2 utilizes BamH I and Sac I digestion, recycles skeleton carrier；Gus gene segment and skeleton carrier are connected, recombination is obtained and carries
The correct recombinant vector of sequence is named as pWMB-PTaEm2-GUS by body.PWMB-PTaEm2-GUS is containing sequence 1 in ordered list
1428-1724 shown in gus gene shown in PTaEm2 and sequence 8, PTaEm2 is located at gus gene upstream.
Amplified production is utilized into BamH I and Sac I digestion, obtains gus gene segment；The pWMB-that step 1 is obtained
PTaEm3 utilizes BamH I and Sac I digestion, recycles skeleton carrier；Gus gene segment and skeleton carrier are connected, recombination is obtained and carries
The correct recombinant vector of sequence is named as pWMB-PTaEm3-GUS by body.PWMB-PTaEm3-GUS is containing sequence 1 in ordered list
Shown in gus gene shown in PTaEm3 and sequence 8, PTaEm3 is located at gus gene upstream.
3, the building of recombinant bacterium
PWMB-PTaEm1-GUS is imported in Escherichia coli Trans5 α, recombinant bacterium Trans5 α-pWMB-PTaEm1- is obtained
GUS。
By agrobacterium strains LBA4404 in 28 DEG C of 36~40h of culture, auxiliary bacteria strain HB101 cultivates in 37 DEG C to 12~
16h, by Trans5 α-pWMB-PTaEm1-GUS in 37 DEG C of 12~16h of culture；The bacterium that three kinds of cultures terminate respectively is taken into 100 μ L bacterium
Liquid is added in the centrifuge tube of 1.5mL, mixes, 4000rpm, and centrifugation 3min collects thallus, while there was only agrobacterium strains
The mixed bacteria liquid of LBA4404 and auxiliary bacteria strain HB101 are control；The not antibiotic LB Liquid Culture of 50 μ L is added into thallus
Base is resuspended thallus, obtains thallus suspension；Thallus suspension is dripped on not antibiotic LB solid medium, keeps thallus agglomerating,
After plate is dry, 28 DEG C of inversion cultures 16~for 24 hours；The a small amount of thallus of picking, in the LB containing rifampin, gentamicin and kanamycins
It crosses on solid medium, 28 DEG C of 36~40h of culture identify positive colony using the primer pair that primer 7 and primer 8 form.Draw
Object sequence is as follows:
Primer 7 (upstream primer): 5 '-AGCCGATTACGTGGCTTTAGCAG-3 ' (sequence 9),
Primer 8 (downstream primer): 5 '-GCATCGGCGAACTGATCGTTAAAAC-3 ' (sequence 10).
Can expand and obtain the clone of purpose band is positive colony (swimming lane 2, Fig. 1), and control, will be containing just without purpose band
The recombinant bacterium of true purpose band (sequence of sequencing display purpose band is sequence 11) is named as LBA4404-pWMB-PTaEm1-
GUS。
According to the method described above, pWMB-PTaEm1-GUS is replaced with into pWMB-PTaEm2-GUS and pWMB-PTaEm3- respectively
Obtained positive colony is denoted as LBA4404-pWMB-PTaEm2-GUS (swimming lane 1, Fig. 1) and LBA4404-pWMB-by GUS respectively
PTaEm3-GUS (swimming lane 3, Fig. 1).
Two, the building and identification of transgenic wheat
Utilize the LBA4404-pWMB-PTaEm1-GUS of step 1 by pWMB-PTaEm1-GUS by agrobacterium-mediated transformation
Transformed wheat Fielder (Ishida Y, Tsunashima M, Hiei Y, Komari T. (2015) Wheat (Triticum
aestivum L.)Transformation Using Immature Embryos.In:Wang K(eds)Agrobacterium
Protocols.Methods in Molecular Biology, vol 1223.Springer, New York, NY) in, it obtains
Transgenic wheat.Wheat Transformation realizes (Ishida Y, Tsunashima M, Hiei using PureWheat Wheat Transformation method
Y,Komari T.(2015)Wheat(Triticum aestivum L.)Transformation Using Immature
Embryos.In:Wang K(eds)Agrobacterium Protocols.Methods in Molecular Biology,
vol 1223.Springer,New York,NY)。
According to the method described above, LBA4404-pWMB-PTaEm1-GUS is replaced with into LBA4404-pWMB-PTaEm2- respectively
GUS and LBA4404-pWMB-PTaEm3-GUS carries out transgenic experiments, obtains two kinds of transgenic wheats.
The identification of transgenic wheat:
Using the transgenic wheat leaf DNA of extraction as template, PCR amplification is carried out with primer 7 and primer 8, can expand and arrive mesh
The plant of segment be positive transgenic plant.Fig. 2 is that the PCR of each 7 positive transgenic plant of 3 kinds of transgenic wheats is detected
As a result.
Three, gus gene detection of expression
The different times and different tissues for the positive transgenic plant for taking step 2 to detect carry out GUS histochemical stain.
Specific steps are as follows:
Step 1: organization material being put into test tube, suitable GUS fixer is added, mildly shakes 30min at room temperature, is used
The sodium phosphate buffer (pH7.0) of 50nmol/L washes 10~15min, is repeated 3 times, and removes remaining fixer in tissue；
Step 2: 1min vacuumize process being carried out to dyeing liquor, each tissue is placed in 6h in GUS dyeing liquor；Group will be dyed again
It knits and is first placed in the rinsing decoloration of 75% ethyl alcohol, then with 50% and 20% ethyl alcohol successively soaking 20min；It takes pictures under anatomical lens, tissue
It is displayed in blue as GUS expressive site.
The histochemical stain result of transgenic plant different tissues gus gene is as shown in Figure 3, in which: 1 is young root, and 2 are
Young stem, 3 be lemma, and 4 be glumelle, and 5 be boot leaf, and 6 be little Hua, and 7 be maturity period seed longitudal section (white arrow show embryo).As a result
Display: PTaEm1 promoter can drive gus gene to express in endosperm with PTaEm3, in other tissues (young root, children stem,
Lemma, glumelle, little Hua, boot leaf, embryo) in cannot drive the expression of gus gene, and PTaEm2 cannot drive in any tissue
The expression of gus gene.
The GUS histochemical stain of transgenic plant endosperm different development stage is as shown in Figure 4, in which: (1) is Post flowering
The sectional view of 8~23 days seeds, (2) are the longitudinal sectional figure of 8~23 days seeds of Post flowering.As the result is shown: with PTaEm3 in Post flowering 8
The expression of gus gene can be driven within~23 days always, and PTaEm1 promoter can drive gus gene table in 14 days endosperm of Post flowering
It reaches, until seed maturation；And PTaEm2 is unable to always gus gene expression.
The GUS histochemical stain of transgenic plant endosperm specific expression is as shown in Figure 5, in which: a is PTaEm2 driving
The positive transgenic plant of GUS；B is the positive transgenic plant of the GUS of PTaEm1 driving；C is the sun of the GUS of PTaEm3 driving
Property transgenic plant.As the result is shown: gus gene is in endosperm (asterisk institute in the positive transgenic plant of the GUS of PTaEm2 driving
Show) and aleurone (shown in arrow) in do not express；PTaEm1 promoter and PTaEm3 can drive gus gene in endosperm
Expression in (shown in asterisk) and aleurone (shown in arrow), it was demonstrated that PTaEm1 promoter and PTaEm3 can be specifically in the embryos of wheat
Destination gene expression is driven in laticiferous cell.
<110>Capital Normal University
<120>a kind of specifically expressed promoter of wheat endosperm and its application
<160> 11
<170> PatentIn version 3.5
<210> 1
<211> 1724
<212> DNA
<213>wheat (Triticum aestivum L.)
<400> 1
accccaacaa caccaacaaa gcagcaaagc gtgcccaacc cttctagtct aaataactag 60
cctccactaa cacatttctc tcgacatgca agcatgtcac ataagaaaat gcccacctca 120
atatgcaacc atgcatagaa gaaagctcag ctcagcggat gtggagtttc tacacccatg 180
agcacaaatg cttagtaaaa acaaaaaaaa caaaaaaagt tcaaaaaaat ctgaaattta 240
tttgtggcat actttcacaa atgtttgttg tgcatgcaaa atttcatcac aaaatcacat 300
tggtgaaagg cgtgataaaa aaacaaaatc gatgctctaa aaatgttact tacaaaagca 360
ttttggagtt tcgattttgt tttttttggc acgacttcca ccgtgatttt gcgatgaatt 420
tttgcgtgca cgataaacat ttgttaatgt cacaaaaaaa tttgattttt tattattttt 480
attttactgt tcacaccagg agcatttgct cctgggtgta taaaggcact ttcgcaactc 540
agcatgcaaa catgcaacat aatttacttt ttacttggct aattatattt gataaatatt 600
tcagaaatat acaataatca aacacaataa attatatgtg tttttagttt tagttctcat 660
atccaaatat acatgtttca tacaaccaaa tctcattcaa atatataaaa cattccagca 720
acaacttgtg gggtatatct agttacaagg gaatattagt ggtggcgtga gcaagcgata 780
aggccaagga gagaagaatt gtatcgtcta tggaggctag ggaaagacaa tggacatgca 840
gagaggcaag ggcggggaag aaacacttgg agatcataga agaagataag aggttaaaca 900
taggaggagg atataatgga caattaaatc tgcattaatt gaactcattt gggaagtaaa 960
caaattcttt attctgtgta aatcaaacta tttgacgcgg attttctctg aagatcctat 1020
attaatttta gacatggttt ggttagttca tttgtcacgg aaaggtgttt ccataagtcc 1080
aaaattctac caactttttt gtacggcgcg tcatagcata gatagatgtt gtgagtcact 1140
ggatagatat tgtgagtcat aggatggatt tgtgttgccg ggaaatccaa ctaacatgac 1200
aatcaacaaa acctgaaatg gcctttagga gttatccatt tacttgtttc atgcaggcta 1260
ccttccacta ctcgacatgc ttagaagcat tgagtggccg cagatttgca aaagcaatgg 1320
ctaacggaca catattctgc caaaccccaa gaaggataat cacttctctt agataaaaag 1380
gaacagacca atatacaaac atccacactt ctggaaacaa tacaccagaa ctaggattaa 1440
gccgattacg tggctttagc agaccgtcca aaaatctgtt ttacaaagct ccaattgctc 1500
cttgcttatc cagctttttt tgtgttggca aactacactt tttcaaccga ttttgttctt 1560
ctcacacttt cttcttaggc taaacaaacc ttaccgtgca cgcagccatg gtcctgaatc 1620
ttcacctcgt ccctataaaa gcctagccaa ccttcacaat ctcttcatca cccacaacac 1680
cgagcatcac aaactagaga tcaattcacc gacagtccac cgag 1724
<210> 2
<211> 36
<212> DNA
<213>artificial sequence
<400> 2
aaaaagcttc gcgtcatagc atagatagat gttgtg 36
<210> 3
<211> 32
<212> DNA
<213>artificial sequence
<400> 3
aaaggatccc tcggtggact gtcggtgaat tg 32
<210> 4
<211> 34
<212> DNA
<213>artificial sequence
<400> 4
aaaaagcttg aactaggatt aagccgatta cgtg 34
<210> 5
<211> 33
<212> DNA
<213>artificial sequence
<400> 5
aaaaagctta ccccaacaac accaacaaag cag 33
<210> 6
<211> 35
<212> DNA
<213>artificial sequence
<400> 6
aaaggatcca tgttacgtcc tgtagaaacc ccaac 35
<210> 7
<211> 30
<212> DNA
<213>artificial sequence
<400> 7
aaagagctct cattgtttgc ctccctgctg 30
<210> 8
<211> 1812
<212> DNA
<213>artificial sequence
<400> 8
atgttacgtc ctgtagaaac cccaacccgt gaaatcaaaa aactcgacgg cctgtgggca 60
ttcagtctgg atcgcgaaaa ctgtggaatt gatcagcgtt ggtgggaaag cgcgttacaa 120
gaaagccggg caattgctgt gccaggcagt tttaacgatc agttcgccga tgcagatatt 180
cgtaattatg cgggcaacgt ctggtatcag cgcgaagtct ttataccgaa aggttgggca 240
ggccagcgta tcgtgctgcg tttcgatgcg gtcactcatt acggcaaagt gtgggtcaat 300
aatcaggaag tgatggagca tcagggcggc tatacgccat ttgaagccga tgtcacgccg 360
tatgttattg ccgggaaaag tgtacgtatc accgtttgtg tgaacaacga actgaactgg 420
cagactatcc cgccgggaat ggtgattacc gacgaaaacg gcaagaaaaa gcagtcttac 480
ttccatgatt tctttaacta tgccggaatc catcgcagcg taatgctcta caccacgccg 540
aacacctggg tggacgatat caccgtggtg acgcatgtcg cgcaagactg taaccacgcg 600
tctgttgact ggcaggtggt ggccaatggt gatgtcagcg ttgaactgcg tgatgcggat 660
caacaggtgg ttgcaactgg acaaggcact agcgggactt tgcaagtggt gaatccgcac 720
ctctggcaac cgggtgaagg ttatctctat gaactgtgcg tcacagccaa aagccagaca 780
gagtgtgata tctacccgct tcgcgtcggc atccggtcag tggcagtgaa gggcgaacag 840
ttcctgatta accacaaacc gttctacttt actggctttg gtcgtcatga agatgcggac 900
ttgcgtggca aaggattcga taacgtgctg atggtgcacg accacgcatt aatggactgg 960
attggggcca actcctaccg tacctcgcat tacccttacg ctgaagagat gctcgactgg 1020
gcagatgaac atggcatcgt ggtgattgat gaaactgctg ctgtcggctt taacctctct 1080
ttaggcattg gtttcgaagc gggcaacaag ccgaaagaac tgtacagcga agaggcagtc 1140
aacggggaaa ctcagcaagc gcacttacag gcgattaaag agctgatagc gcgtgacaaa 1200
aaccacccaa gcgtggtgat gtggagtatt gccaacgaac cggatacccg tccgcaaggt 1260
gcacgggaat atttcgcgcc actggcggaa gcaacgcgta aactcgaccc gacgcgtccg 1320
atcacctgcg tcaatgtaat gttctgcgac gctcacaccg ataccatcag cgatctcttt 1380
gatgtgctgt gcctgaaccg ttattacgga tggtatgtcc aaagcggcga tttggaaacg 1440
gcagagaagg tactggaaaa agaacttctg gcctggcagg agaaactgca tcagccgatt 1500
atcatcaccg aatacggcgt ggatacgtta gccgggctgc actcaatgta caccgacatg 1560
tggagtgaag agtatcagtg tgcatggctg gatatgtatc accgcgtctt tgatcgcgtc 1620
agcgccgtcg tcggtgaaca ggtatggaat ttcgccgatt ttgcgacctc gcaaggcata 1680
ttgcgcgttg gcggtaacaa gaaagggatc ttcactcgcg accgcaaacc gaagtcggcg 1740
gcttttctgc tgcaaaaacg ctggactggc atgaacttcg gtgaaaaacc gcagcaggga 1800
ggcaaacaat ga 1812
<210> 9
<211> 23
<212> DNA
<213>artificial sequence
<400> 9
agccgattac gtggctttag cag 23
<210> 10
<211> 25
<212> DNA
<213>artificial sequence
<400> 10
gcatcggcga actgatcgtt aaaac 25
<210> 11
<211> 503
<212> DNA
<213>artificial sequence
<400> 11
agccgattac gtggctttag cagaccgtcc aaaaatctgt tttacaaagc tccaattgct 60
ccttgcttat ccagcttttt ttgtgttggc aaactacact ttttcaaccg attttgttct 120
tctcacactt tcttcttagg ctaaacaaac cttaccgtgc acgcagccat ggtcctgaat 180
cttcacctcg tccctataaa agcctagcca accttcacaa tctcttcatc acccacaaca 240
ccgagcatca caaactagag atcaattcac cgacagtcca ccgaggtcga cgatatctct 300
agaggatccc cgggtaccga gctcgaattc atgttacgtc ctgtagaaac cccaacccgt 360
gaaatcaaaa aactcgacgg cctgtgggca ttcagtctgg atcgcgaaaa ctgtggaatt 420
gatcagcgtt ggtgggaaag cgcgttacaa gaaagccggg caattgctgt gccaggcagt 480
tttaacgatc agttcgccga tgc 503
Claims (10)
1.DNA molecule, for it is following a) or b) or c):
A) 3 ' 1107-1724 nucleotide sequences of the end at least containing sequence 1 in ordered list, and from the 1107th of sequence 1 the
Position starts, extends according to the nucleotide sequence of sequence 1 to 5 ' ends of sequence 1, obtains any one that length is 618 to 1724bp
DNA fragmentation；The DNA molecular has promoter function；
B) nucleotide sequence and a) limited has 75% or 75% or more identity, 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.
2. DNA molecular according to claim 1, it is characterised in that: last nucleotide that the DNA molecular 3 ' is held is
The 1724th of sequence 1.
3. DNA molecular according to claim 1 or 2, it is characterised in that: the DNA molecular be it is following 1) or 2):
1) DNA molecular shown in 1107-1724 of sequence 1 in sequence table；
2) DNA molecular shown in sequence 1 in sequence table.
4. it is following B1 the biomaterial containing the DNA molecular any in claim 1-3) any one of to B19):
B1 the expression cassette) containing the DNA molecular any in claim 1-3；
B2 the recombinant vector) containing the DNA molecular any in claim 1-3；
B3) contain B1) recombinant vector of the expression cassette；
B4 the recombinant microorganism) containing the DNA molecular any in claim 1-3；
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 the DNA molecular any in claim 1-3；
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 the DNA molecular any in claim 1-3；
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 the DNA molecular any in claim 1-3；
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.
5. DNA molecular described in claim 1 is as the application in promoter.
6. application according to claim 5, it is characterised in that: the promoter is plant tissue specificity promoter.
7. application according to claim 6, it is characterised in that: the tissue is albumen.
8. biomaterial described in any DNA molecular or claim 4 is any in following (a) to (c) in claim 1-3
Application in:
(a) plant variety or strain are cultivated；
(b) expression of target gene is driven in plant；
(c) driving target gene is expressed in albumen.
9. target gene method specific expressed in albumen, comprising: will contain as claimed in any one of claims 1-3
The expression cassette of DNA molecular and target gene imports in plant, realizes specifically expressing of the target gene in albumen.
10. expanding any DNA molecular overall length or the primer pair of Partial Fragment in claim 1-3.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109355291A (en) * | 2018-11-22 | 2019-02-19 | 深圳市作物分子设计育种研究院 | A kind of identification and application of plant endosperm specificity expression promoter pOsEnS93 |
-
2018
- 2018-11-13 CN CN201811343855.9A patent/CN109337906B/en active Active
Non-Patent Citations (1)
| Title |
|---|
| JIHU LI等: "Dissecting conserved cis-regulatory modules of Glu-1 promoters which confer the highly active endosperm-specific expression via stable wheat transformation", 《THE CROP JOURNAL》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109355291A (en) * | 2018-11-22 | 2019-02-19 | 深圳市作物分子设计育种研究院 | A kind of identification and application of plant endosperm specificity expression promoter pOsEnS93 |
| CN109355291B (en) * | 2018-11-22 | 2022-01-18 | 深圳市作物分子设计育种研究院 | Identification and application of plant endosperm specific expression promoter pOsEnS93 |
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| CN109337906B (en) | 2021-06-01 |
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