CN106148346A - A kind of isolated endosperm does not express promoter SAFES6 and application thereof - Google Patents
A kind of isolated endosperm does not express promoter SAFES6 and application thereof Download PDFInfo
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Abstract
The present invention relates to a kind of isolated endosperm and do not express promoter SAFES6 and application thereof.Specifically, the invention provides can be in promoter SAFES6 of endosperm transcription heterologous nucleic acid sequence and preparation and the method for this promoter of use in plant tissue.The method that the invention still further relates to the corresponding transgenic plant of the expression cassette containing this promoter, recombinant expression carrier and acquisition.Rice starter SAFES6 provided by the present invention can concentrate expression in controlling gene non-endosperm position in plant, and do not express in endosperm, the present invention utilizes this promoter improving plant growth characteristic in the case of not changing albumen character, improve the growth performance of Oryza sativa L., reduce the edible safety risk of Oryza sativa L. simultaneously, and then preferable transfer-gen plant can be obtained, help the business promotion of transgenic paddy rice.
Description
Technical field
The present invention relates to biotechnology and crop gene field of engineering technology.Specifically, the present invention relates to a kind of Oryza sativa L.
Endosperm does not express promoter SAFES6 and application thereof.
Background technology
One of target of crop gene engineering is to cultivate to have feature or the plant of character needed for agricultural.Engineered skill
Art development makes research worker be obtained in that required genes of interest and convert plant, it is thus achieved that have required character and feature turns base
Because of plant.On the one hand, gene can be promoted to express in the plant cell generally not expressing this genoid or plant tissue thus
Give the phenotype needed for these plants.On the other hand, gene or portion gene can stop or suppress transcribing of antisense orientation
Endogenous gene express thus reach required target.Utilize promoter directly to control transcribing of genes of interest, be regulation and control purposes
The most cost-effective means of gene expression, are also the feasible paths controlling to express risk.
From nineteen eighty-three the first Transgenic Tobacco crop since the U.S. is born, by the end of at present, genetically modified crops are entirely
Planting in the range of ball, the spendable product that goes through has kind more than 1000.The genetic improvement great majority of these crops concentrate at present
Improve yield, shorten breeding cycle, strengthen disease and insect resistance, improve agricultural product nutritional labeling, increase bio-diversity aspect.Many
Genetically modified crops and products thereof all enter Food circulation market, have come into the dining table of huge numbers of families.Oryza sativa L. is world's main food
One of crop, has more than 1/3rd populations in the world with rice as staple food.China is populous nation, and Oryza sativa L. is its people of China
Occupying considerable status in economy, for improving rice yield, resistance and quality, available technique for gene engineering is by external source mesh
In mark channel genes cultivated rice, create transgenic paddy rice.In order to solve, grain is in short supply and population is growing to a great extent for this
This contradiction opens up a new way.
Make a general survey of the research and development course of transgenic paddy rice, the character that Transgenic Rice engineering is improved relate generally to disease-resistant,
Antiweed, quality-improving and pest-resistant etc..
At disease-resistant aspect, mainly proceed to water resistant rice fungoid, bacillary, nematicide virus gene.Christou etc. will
After Xa21 channel genes Oryza sativa L., significantly improve Oryza sativa L. to bacterial leaf-blight and the resistance of rice blast.What winter jasmine etc. are by Nicotiana tabacum L. chitin
Matter enzyme channel genes Oryza sativa L. obtains high anti-banded sclerotial blight and rice blast transgenic plant.Sichuan Agricultural University is by disease-resistant and pest-resistant base
Because of in the restorer of introductive crossing rice, it is thus achieved that the dual anti-hybrid paddy rice of transgenic.
The eighties in 20th century, Monsanto Chemicals has wide spectrum with it, highy potent herbicide agriculture reaches the advantage of (glyphosate),
Take the lead in carrying out research and the exploitation of resistant variety of herbicide antibiotic property gene.China is main for the gene of rice herbicide resistant
It it is the bar gene of coding PAT.
In terms of quality-improving, rich proline gene, be the richest in methyllanthionine and lysine gene, octahydro Fructus Lycopersici esculenti synzyme and
Dehydrogenase gene, glycinin gene and high-lysine gene etc. are the most by successfully Introduced into Rice and obtain transgenic and plant
Strain, has very important significance for developing rice product.
From Bacillus thuringiensis insecticidal crystal protein gene (Bt gene), the insect protein inhibitor of Bacillus thuringiensis separation
(PI gene), lectin genes have been successfully applied in the pest-resistant improvement of Oryza sativa L. the most.
But anything all has dual character, genetically modified crops, while bring pleasantly surprised and Gospel to the mankind, also carry
Doubt and risk are come.The biosafety issues that transgenic paddy rice may bring has become as the focus of international concern.
From the perspective of microcosmic, the importing of promoter is likely to bring potential safety hazard to transgenic plant.Such as, start
Son is inserted into by the cryptovirogene being originally incorporated in Plant Genome, may reactivate virus；Promoter is inserted
To the upstream region of gene of a certain encoding toxin protein, the synthesis of this toxin may be strengthened；When transgenic plant is by animals or humans
Time edible, promoter may be horizontally inserted a certain oncogene upstream by gene, activates and cause the generation of cancer.
On the food-safety problem of macroscopic view, focus of concern is transgenic and product the most toxic or meeting when edible thereof
Cause allergic reaction；Next, when the selectable marker gene entrained by transgenic product is to encode some to be applied to clinic or intestinal
During the resistance thing of antibiotic, whether they may be transferred in microorganism, thus strengthen the Drug resistance of pathogenic microorganism, cause
Antibiotic inefficacy (Ebinuma etc., 2001；Hohn etc., 2001).
Oryza sativa L. is as one of main grain, the Oryza sativa L. i.e. endosperm of Leaf-feeding insects polished rice.Especially with external source in transgenic paddy rice
The albumen edible safety risk accumulating and being likely to result in endosperm, becomes the key factor of restriction commercialization process.Pin
To this problem, utilize promoter strategy i.e. to select induction type, tissue specificity and time-dependent promoter, make exogenous gene
Only express in non-edible part, it is ensured that edible part does not contains the product of exogenous gene expression, reduces it and brings human health
Potential risk, be the effective way of commercialization process promoting transgenic paddy rice.At present, it has been found that non-endosperm express
Promoter mainly include stem specificity promoter, mesophyll cell specificity promoter, root-specific promoter etc..Such as shi
Promoter RSs1 at phloem specific expressing Deng discovery；The work of research office of United States Department of Agriculture (USDA) Roger Thilmony leader
Thing improvement and research on utilization group find a kind of tissue-specific promoter LP2 recently, and it belongs to being subject in photosynthetic tissue
Body gene, the leaf camber at transgenic paddy rice enlivens, and is not detected in spending at seed；The root of hair that Varvara etc. find
The rolD promoter of Agrobacterium, only expresses in root and is transferred in purpose plant Fructus Lycopersici esculenti.But, the most at home in research,
Invention and the research of non-endosperm expression promoter rarely have report, and the rice non-endosperm being detected in the inventions such as Yang Jianbo expresses promoter
OsTSP I the most only expresses in rice non-endosperm tissue (root, stem, leaf etc.), does not expresses in endosperm.And Meng Cai etc. sends out
Existing promoter P only expressed at rice green tissue siteD540Deng.
Genetic engineering can inherently change the structure of organism, and this is the major progress in the history of science, the most also may be used
Hidden danger to a certain degree can be brought to human society.Although really there is potential food safety and environment peace in genetically modified crops
Congruence problem, but the reason hindering its development must not be become, and should pinpoint the problems and make great efforts solution problem.If transgenic
Oryza sativa L. can not finally realize Commercialization application in production, then the best achievement in research does not has any using value yet.Therefore, profit
Use promoter strategy, make exogenous gene only express in non-edible part, it is ensured that edible part does not contains the product of exogenous gene expression
Thing, the i.e. research of Efforts To Develop functional genome, excavate and separate the non-endosperm expression promoter with practical value in a large number, can
To reduce transgenic paddy rice potential risk that human health is brought, eliminate the public to the prejudice of transgenic paddy rice and misgivings, fill
The advantage of transgenic breeding is waved in distribution, cultivates more more preferable new varieties.Thus guide genetically modified crops in China's health, have
The development of sequence ground.
Summary of the invention
It is within the contemplation of the invention that provide a kind of promoter do not expressed at paddy endosperm, the present invention also provides for comprising this promoter
Expression cassette, recombinant expression carrier, transgenic plant or plant part.In an embodiment disclosed herein, promoter is even
It is connected to allos transcribable polynucleotide sequence.The method that manufacture and use disclosed herein promoter is also provided herein, including bag
Expression cassette containing promoter, recombinant expression carrier, and comprise be connected to allos transcribable polynucleotide sequence promoter turn
Gene plant or plant part.
Therefore, on the one hand, the present invention provides a kind of isolated endosperm not express promoter SAFES6, it is characterised in that
Described endosperm is not expressed promoter SAFES6 and is comprised at least one in following polynucleotide sequence:
A () comprises the polynucleotide sequence of nucleotide sequence shown in SEQ ID NO:1 in sequence table；
B () has at least 85% homology with nucleotide sequence shown in SEQ ID NO:1 in sequence table and has identical
The nucleotide sequence of function；
C one or more core is added, replaces or lacked to () in the nucleotide sequence shown in SEQ ID NO:1 in sequence table
In that obtained after thuja acid and sequence table, nucleotide sequence shown in SEQ ID NO:1 has the nucleotide sequence of identical function；With
And
(d) with comprise the nucleotide sequence of nucleotide sequence complementary shown in SEQ ID NO:1 in sequence table,
Wherein said endosperm does not express promoter SAFES6 can be connected to allos transcribable polynucleotide sequence and energy
Enough guide described allos transcribable polynucleotide sequence transcribing in non-endosperm tissue.
On the other hand, provided herein is a kind of expression cassette, it comprises described promoter and different with what described promoter was connected
The polynucleotide sequence that source is transcribed.Wherein said promoter can cause described allos transcribable polynucleotide sequence with institute
State the transcript and expression in the plant non-endosperm tissue that expression cassette converts.Described allos transcribable polynucleotide sequence is to have
Change the gene of crop character function.Wherein said allos transcribable polynucleotide sequence inserts this expression cassette with justice direction
In.Or, described allos transcribable polynucleotide sequence is inserted in this expression cassette with antisense orientation.In some embodiment
In, transcribable polynucleotide sequence transcribes anti-insect protein.
On the other hand, provided herein is a kind of recombinant expression carrier, it comprises described promoter or described expression cassette, institute
State promoter and be connectable to the transcribable polynucleotide sequence of allos.Wherein said allos transcribable polynucleotide sequence is
There is the gene changing crop character function.
On the other hand, the present invention provides a kind of side of improving plant growth characteristic in the case of not changing albumen character
Method, it is characterised in that described method includes:
(1), obtain the endosperm described in claim 1 and do not express promoter SAFES6；
(2) gene, by described endosperm not expressing promoter SAFES6 and have change crop character function is connected, and obtains
Obtain corresponding recombinant vector；
(3) described recombinant vector is proceeded in the cell of target plant；
(4) the corresponding transgenic plant of described cell culture is utilized；
(5) there is from the transfer-gen plant obtained the gene of change crop character function in non-endosperm portion described in selection
Position is expressed, and the unexpressed plant in endosperm position, this plant character at non-endosperm position obtains improvement, and endosperm position is not
Impacted.
Another further aspect, the present invention provides a kind of method converting plant or plant part, and described method includes with described
Described plant or plant part are stably converted by expression cassette or described recombinant expression carrier.Described plant part is
The group of choosing freely following part composition: cell, protoplast, cell tissue culture, callus, cell block, plumule,
Pollen, ovule, petal, style, stamen, leaf, root, the tip of a root and flower pesticide.Described plant is choosing freely following part composition
Group: cereal crops, vegetable crop, flower crop, energy crop.In a particular embodiment, plant part is rice callus
Tissue, plant is Oryza sativa L..
Another further aspect, the present invention provides a kind of side instructing transcribed polynucleotide sequence to express in plant cell
Method, it includes described promoter is connected to allos transcribable polynucleotide sequence construct recombinant expression carrier, and will recombinate
Expression vector converts plant cell and converts plant cell regenerating plants to produce.
Thering is provided defined below is to preferably define the present invention and instructing those skilled in the art to put into practice this with method
Bright.Unless otherwise noted, otherwise term should understand according to the common usage of the those of ordinary skill of correlative technology field.
Term " DNA sequence ", " nucleotide sequence " and " nucleic acid molecules " refers to genome or the double chain DNA molecule in synthesis source.
Term " is expressed " and is referred to that generation corresponding mRNA, this mRNA translation of transcribing of gene produces corresponding gene outcome (i.e.
Peptide, polypeptide or protein).
Term " expression cassette " refers to comprise and is connected to one or more controlling element, the DNA molecular of transcribed DNA molecular.
Term " recombinant expression carrier " refers to that a class, by particular/special requirement design construction, has the regulation correct table of exogenous gene
Reach signal, and clone gene can be made to obtain the proprietary vector of effective expression in the host cell converted.Carrier generally includes one
Individual or multiple expression cassettes.It is included in and does not has in the case of human intervention will not to divide the DNA of combination DNA molecule of Lock-in completely
Son.Term " recombinant vector " refer to such as plasmid, cosmid, virus, self replication sequence, phage or linear strand, cyclic single strand,
Linear double-strand or circular double stranded DNA or RNA nucleotide sequence.Recombinant vector any source, and can producer group
Integrate or self replication.
Term " expression of antisense RNA " refers to the RNA molecule with certain natural mRNA reverse complemental.It is by double-stranded DNA
Nonsense strand transcribes generation, can be used to stop the translation activity of mRNA complementary therewith present in the cell that converted by it.
Using the gene of encoding antisense RNA, the mRNA blocking genes of interest (i.e. with antisense RNA complementation) is translated into the table of protein
Reach.
Term " sequence homology " refers to the degree that two optimal comparison DNA sequence are identical.Such as, reference sequences and another
Individual DNA sequence, improves the number of nucleotides match to greatest extent in sequence alignment.As used herein, term " reference sequences "
Refer to the DNA sequence of SEQ ID NO:1.
Term " allos " " external source " refers to the mutual relation between nucleic acid or the protein sequence of two or more separate sources.
Such as, the combination between promoter and coded sequence there is usually no at nature, then both allos each other.Additionally, specific sequence
Cell or organism that row can be inserted relative to it are that (it is generally not present in certain detail to external source the most under field conditions (factors)
In born of the same parents or organism).
Term " connects " functional spatial arrangement referring to two or more nucleotide region or nucleotide sequence.As, start
The position relationship of sub-district and nucleotide sequence makes nucleotide sequence can be regulated and controled by this promoter region and guides and transcribe.Now, promoter
District belongs to " connection " with nucleotide sequence.
Term " transcribable polynucleotide sequence " refers to be transcribed into any DNA molecular of RNA molecule, including but do not limit
In, there are those molecules of protein coding sequence and produce those points with the RNA molecule that sequence is applicable to gene inhibition
Son.DNA molecular type can include, but are not limited to, and from the DNA molecular of identical plant, the DNA from another kind of plant divides
Son, from the DNA molecular of different organisms, or synthetic dna molecule, such as the DNA molecular of the antisense information containing gene, or coding
Manually, synthesis or the DNA molecular of the other transgenic modifying pattern.Exemplary in recombinant expression carrier incorporated herein can
Transcription DNA molecule includes, Reporter gene GUS, Bt (the Bacillus thuringiensis Su Yun gold of codon optimized synthesis
Bacillus cereus) protein coding gene mCry1Ab.
Term " promoter " or " promoter region " refer to be positioned at gene 5 '-end upstream outer, next-door neighbour's transcriptional start site
The noncoding nucleotide sequence of one section of tool specific function.RNA polymerase by with the transcribing of its combination and promotor gene.
Promoter or promoter region provide RNA polymerase or other be the recognition site of transcription initiation factor.The intensity of promoter or work
Property can be transcribed the amount of the RNA of generation by it and be weighed, or weighed by albumen accumulated amount in cell or tissue, or
It is determined relative to the transcriptional activity of other promoter compared with it.
Term " converts " process referring to exogenous DNA into cells, will import in receptor host by nucleic acid.Term " host "
Refer to bacterial cell, fungus, animal or zooblast, plant or seed or any plant part or tissue, including plant cell,
Protoplast, callus, root, tuber, seed, stem, leaf, Seedling, embryo and pollen.
Term " transgenic plant " instruct into nucleic acid stability be incorporated into the plant of its genome.Such as it is incorporated into core base
Because of in group.
Term " has the gene changing crop character function " and refers to be expressed in concrete plant tissue, cell or cell type
In, give the transcribed DNA molecular of plant desirable characteristics.The product with the gene changing crop character function can be in plant
Function such that plant morphology, physiology change.In one embodiment of the invention, the promoter of the present invention is incorporated to
Recombinant expression carrier is so that promoter is connected to have in the transcribed DNA molecular of the gene changing crop character function.?
In transgenic plant containing this kind of recombinant expression carrier, the expression with the gene changing crop character function can give plant
Favourable character.Beneficial traits can include, such as but be not limited to, herbicide tolerance, insect resistace, disease resistance, resistance, improvement
Plant growing and growth, improvement yield, improved protein content, improvement fruit maturation, biopolymer produce, improve local flavor,
Hybrid seed breeds effectiveness.
In the present invention, inventors herein have recognized that Japanese fine Oryza sativa L. (Oryza sativa L cv.Nipponbare) base
Because of the DNA sequence with transcriptional regulatory activity of one section of 1931bp in group, there is driving target gene at rice non-endosperm tissue
The effect expressed.And separating clone has obtained the DNA sequence shown in SEQ ID No:1 in sequence table.Named SAFES6 or
Promoter SAFES6.
It should be understood that in the DNA sequence of the promoter of SEQ ID No:1, the sequence " gcattcaaga of sequence beginning
Aagcaatcag cc " for obtaining the retention sequence of the forward primer used during promoter, 22bp altogether；Sequence end
Sequence " ggcgaccgat cgattagcta gc " is to obtain the retention sequence of the reverse primer used during promoter (this stays
Deposit sequence complementary with the corresponding sequence of reverse primer), 22bp altogether.Both it is emphasized that promoter mentioned herein
Above-mentioned whole DNA sequence can be referred to, it is also possible to refer to remove the DNA sequence after above-mentioned primer retains sequence.Even if art technology
Personnel, on the basis of the present invention, use other primers to obtain similar sequence, within it also falls into protection scope of the present invention.
The recombinant expression carrier of the present invention is referred to as pCAMBIA1391-SAFES6, and this recombinant expression carrier is by SEQ ID
Sequence shown in No:1 i.e. SAFES6 or promoter SAFES6 are implemented in the recombinant expression carrier obtained in pCAMBIA1391, this
Referred to herein as pCAMBIA1391-SAFES6.Promoter SAFES6 sequence is connected to crop double base after enzyme action and expresses by inventor
On carrier pCAMBIA1391, it is thus achieved that corresponding recombiant plasmid (i.e. recombinant expression carrier), SAFES6 drives gus gene to express.Profit
With this recombinant plasmid transformed Agrobacterium tumefaciens strain EHA105, then carry out the conversion of Oryza sativa L. by agriculture bacillus mediated method,
To transgenic rice plant.The transgenic paddy rice obtained carrying out histochemistry detection find, transfer-gen plant is at root, stem, leaf
Sheath, leaf, embryo have GUS strongly expressed, endosperm is not expressed.Thus prove the sequence of this 1931bp have drive gene specific
The activity expressed in non-endosperm tissue.
Gus gene quantitative analysis shows, Oryza sativa L. SAFES6 promoter is expressed in leaf and sheath tissue and is significantly stronger than gene
Rice constitutive type promoter ACTIN promoter conventional in engineering；In root and stem tissue, two promoter activities are the most suitable；
In flower tissue, SAFES6 promoter activity is slightly weaker than ACTIN promoter；And in endosperm, SAFES6 promoter is inactive.
In pCAMBIA1391-SAFES6, with mCry1Ab (bacillus thuringiensis Bacillus thuringiensis
I.e. Bt protein coding gene, Academy of Agri-Science and Technology Anhui Province autonomous Design, ZL 2,009 1 0185774.5) substitute gus gene, obtain
The expression vector of Bt, named SAFES6-BT must be driven by SAFES6.In related transgenic strain, start relative to ACTIN
Son drives the strain of Bt, and in leaf and root tissue, Bt expression is slightly strong or is on close level for SAFES6-BT plant, and at endosperm
In, there is expression in ACTIN-BT, SAFES6-BT then can't detect (less than detection line).
Technique effect
The endosperm that the present invention is cloned does not expresses promoter SAFES6 can controlling gene non-endosperm position collection in plant
Middle expression, has notable value in actual applications.As by this promoter regulation target gene table in chlorenchyma and root
Reach activity, and do not express in endosperm, improve the growth performance of Oryza sativa L., reduce the edible safety risk of Oryza sativa L. simultaneously, help
The business promotion of transgenic paddy rice.
Sequence table is sketched
SEQ ID NO:1 represents SAFES6 promoter.
SEQ ID NO:2-11 represents primer sequence.
Accompanying drawing explanation
Fig. 1 is SAFES6 promoter and pcr amplification primer thing sequence thereof, illustrated therein is and has the SAFES6 of PCR primer and open
Mover, 1943 base pairs, further it is shown that HindIII site: 1-6, EcoRI site: 1938-1943；
Fig. 2 is that SAFES6 promoter is implemented in the schematic diagram in pCAMBIA1391 vector plasmid, and in figure, A is
PCAMBIA1391 schematic diagram, B is pCAMBIA1391-SAFES6 schematic diagram, illustrated therein is and utilizes SAFES6 promoters driven
Gus gene expression downstream.
Fig. 3 is the result schematic diagram that the promoter to the present invention carries out digestion verification.
Fig. 4 is the result schematic diagram utilizing SAFES6 promoters driven Gus gene expression, shows each position of Oryza sativa L. in figure
Gus coloration result, Gus gene is strongly expressed in root (a), stem (b), sheath (c), leaf (d), embryo (e), does not expresses in endosperm,
Scale=2.5mm in figure.
Fig. 5 is pCAMBIA1391-SAFES6 Yu pCAMBIA1391-ACTIN plant GUS quantitative analysis results figure.
Fig. 6 is SAFES6-BT expression cassette schematic diagram.This expression cassette comprises two expression cassettes, i.e. by CaMV35S promoter,
Hyg gene, CaMV terminate molecular expression cassette, and the expression of SAFES6 promoter, mCry1Ab gene, NOS terminator composition
Frame.Wherein, LB: left margin；CaMV terminator:CaMV terminator, long 0.22kb；Hyg: hygromycin gene
Hygromycin, long 1.86Kb；CaMV35S promoter:CaMV35S promoter, long 0.53Kb；SAFES6promoter:
SAFES6 promoter, long 1.96Kb；MCry1Ab:Bacillus thuringiensis (Bt) protein coding gene, long
1.84Kb；NOS terminator:NOS terminator, long 0.25Kb；RB: right margin.
Fig. 7 is ACTIN-BT expression cassette schematic diagram.This expression cassette comprises two expression cassettes, i.e. by CaMV35S promoter,
Hyg gene, CaMV terminate molecular expression cassette, and the expression of ACTIN promoter, mCry1Ab gene, NOS terminator composition
Frame.Wherein, LB: left margin；CaMV terminator:CaMV terminator, long 0.22kb；Hyg: hygromycin gene
Hygromycin, long 1.86Kb；CaMV35S promoter:CaMV35S promoter, long 0.53Kb；ACTIN promoter:
ACTIN promoter, long 1.3Kb；MCry1Ab:Bacillus thuringiensis (Bt) protein coding gene, long 1.84Kb；
NOS terminator:NOS terminator, long 0.25Kb；RB: right margin.
Detailed description of the invention
The present invention provide following example so that the present invention is preferably illustrated, unless there are specified otherwise, following enforcement
Example is merely to illustrate and the unrestricted present invention.Those skilled in the art are it is understood that disclosed technology generation in the following embodiments
What table the inventors have found that plays the technology of preferably effect in practice.But, it will be understood by a person skilled in the art that based on this
Bright design, those skilled in the art can carry out many on the basis of disclosed specific embodiments and change, and still
Obtain same or like result, without departing from the spirit and scope of the present invention, therefore, accompanying drawing proposes or the institute that illustrates is busy
Item is interpreted as exemplary, and the most in a limiting sense.
Experimental technique in following embodiment, if no special instructions, is conventional method.Life used in following embodiment
Change reagent, carrier consumptive material etc., if no special instructions, be commercially available purchase product.
Embodiment 1, the acquisition of SAFES6 promoter
Step 1, the design of primer
According to rice varieties Japan fine (the Oryza sativa L cv.Nipponbare) full-length genome provided in NCBI
Sequence, according to the sequential design amplimer of rice starter SAFES6 (i.e. sequence shown in SEQ ID No:1 in sequence table), and
According to the carrier selected and the feature of target gene, the restriction enzyme site of design primer.Rice varieties day used in the present invention
This fine seed is obtained from Paddy Rice Inst., Anhui Agriculture Science Academy's seed bank.
In the present embodiment, with Oryza sativa L. binary expression vector pCAMBIA1391, (in Fig. 2, part A, comes from CAMBIA, for public affairs
Opening use carrier, genetically modified organism product composition supervision and inspection center of Academy of Agri-Science and Technology Anhui Province Ministry of Agriculture Oryza sativa L. group is protected
Deposit) as a example by, genes of interest is Gus gene, and the primer of specific design is: forward primer (SEQ ID No:2) 5 ' holds band enzyme action position
Point HindIII
(AAGCTT), reverse primer (SEQ ID No:3) 5 ' end band restriction enzyme site EcoRI (GAATTC), primer sequence is such as
Under:
Forward primer: 5 '-AAGCTTGCATTCAAGAAAGCAATCAGCC-3 '
Reverse primer: 5 '-GAATTCGGCGACCGATCGATTAGCTAGC-3 '
Synthesized by Shenzhen Hua Da genome company.
Step 2, the acquisition of promoter SAFES6
With the fine DNA of rice varieties Japan as template, forward primer, reverse primer is utilized to expand promoter SAFES6, by often
(using conventional PCR amplification method, the detail of this amplification method is referred to document to rule PCR system: Bachmann H
S,Siffert W,Frey U H.Successful amplification of extremely GC-rich promoter
regions using a novel'slowdown PCR'technique.Pharmacogenetics,2003,13(12):
759-66.) use following amplification program:
When 95 DEG C, template DNA is carried out denaturation 5min.Subsequently enter degeneration-annealing-extension to circulate i.e.: 95 DEG C of changes
Property 30s, template DNA double-strand solution is split into strand；58 DEG C of annealing 30s, primer hybridizes with DNA single-stranded template, formation DNA profiling-draw
Thing complex；72 DEG C extend 2min30s, and DNA profiling-primer complex, under the effect of Taq archaeal dna polymerase, is anti-with dNTP
Answering raw material, target sequence is template, by base pairing and semiconservative replication principle, synthesizes a new chain complementary with template DNA chain.
Totally 35 circulations.Last extension 10min at 72 DEG C.
The purpose fragment reclaiming PCR amplification (see Fig. 1, shows the sequence of this promoter, 5 ' end and the 3 ' enzymes held in Fig. 1
Cut site), it is connected to PGEM-T-Easy vector plasmid and (purchased from Promega company, mixes in the ratio in carrier description
Close) on, by heat shock method, the PGEM-T-Easy vector with purpose fragment is thin to escherichia coli XL-Blue competence
In born of the same parents.Escherichia coli bacteria liquid is uniformly coated on LB plating medium, cultivates 24 hours for 37 DEG C.Utilize cloning promoter just
Reverse primer, carries out bacterium colony PCR checking to the single Bacillus coli cells clone obtained, filters out positive colony therein.With
After, 2 positive cell clones of picking, carry out liquid culture overnight, collect thalline, for extracting containing promoter SAFES6 fragment
PGEM-T-Easy vector plasmid.Double digestion checking is carried out, as shown in Figure 3 with HindIII and the EcoRI plasmid to obtaining.Will
Positive colony through bacterium colony PCR checking and the dual qualification of digestion verification delivers the order-checking of Invitrogen company.Verify correct
Clone is promoter SAFES6 to be obtained, and its nucleotide sequence is as shown in SEQ ID No:1.
Embodiment 2, the structure of crop expression vector and the conversion of Agrobacterium
The escherichia coli positive colony obtained during " acquisition of promoter SAFES6 " from above, extract containing starting
The PGEM-T-Easy vector plasmid of sub-SAFES6 fragment, and carry out double digestion with HindIII and EcoRI, reclaim promoter
SAFES6 fragment.After utilizing HindIII and EcoRI that crop expression vector pCAMBIA1391 carries out enzyme action, recovery enzyme action simultaneously
PCAMBIA1391 fragment.(will in the present invention containing the reporter gene Gus with or without promoters driven on pCAMBIA1391 carrier
The promoter of research is inserted in Gus upstream region of gene, for driving the expression of Gus gene, reflects quilt with the expression characteristic of Gus gene
Research the character of promoter) SAFES6 fragment and the pCAMBIA1391 fragment T4DNA ligase of above-mentioned recovery (are purchased
In TaKaRa company) it is attached, obtain the crop expression vector pCAMBIA1391-of promoter SAFES6 and Gus gene fusion
SAFES6 (Fig. 2 B).With reference to (Hofgen R, Willmitzer L.Storage of competent cells such as Hofgen
For Agrobacterium transformation.1988.Nucleic Acids Res 16:9877.) freeze thawing that proposes
Method, proceeds to Agrobacterium tumefaciems (Agrobacterium tumefaciens) EHA105 (Anhui agricultural section by crop expression vector
Genetically modified organism product composition supervision and inspection center of the Ministry of Agriculture of institute Oryza sativa L. group preserves).
Embodiment 3, promoter SAFES6 drive the expression of Gus reporter gene
Step 1: agriculture bacillus mediated rice transformation
After Japanese for maturation fine rice paddy seed is removed grain husk shell, with 70% alcohol-pickled seed 1min, outwell ethanol.With containing
50% sodium hypochlorite (stock solution effective chlorine density is more than 4%) the solution soaking seed 40min of 1 Tween 20, and on shaking table
Rock uniformly (150r/min).Outwell sodium hypochlorite, clarify to solution, without sodium hypochlorite taste for 5 times with aseptic washing.Sterilized water
Soak seed overnight.Along the aleurone of seed, embryo is peeled with dissecting knife, embryo is inoculated on calli induction media.At 30 DEG C
Light culture after 11 days by wound healing and endosperm and germ separation, by go bud in good condition, divide vigorous primary callus and enter
Row preculture 3～after 5 days for Agrobacterium-mediated Transformation.
Recombinant expression carrier has been proceeded to during using above-mentioned " building and the conversion of Agrobacterium of crop expression vector "
Agrobacterium tumefaciems carries out Agrobacterium-mediated genetic transformation, and this genetic transformation, transformant screening and transgenic plant regeneration etc. are joined
According to Yongbo Duan (Yongbo Duan, Chenguang Zhai, et al.An efficient and high-
throughput protocol for Agrobacterium mediated transformation based on
phosphomannose isomerase positive selection in Japonica rice(Oryza sativa L.)
[J] .Plant Cell Report, 2012.DOI 10.1007/s00299-012-1275-3.) etc. proposition method.The method
It is roughly divided into following step: (1) callus induction: the seed sterilized water after sterilization soaks under 30 DEG C of dark conditions
Overnight, with dissecting knife embryo peeled and be placed on inducing culture.Every ware (specification is the disposable plastic culture dish of 100 × 25mm,
Include 50mL inducing culture) uniformly place 12 embryos, under 30 DEG C of dark conditions, place 2～3 weeks callus induction, to long
Go out faint yellow graininess wound healing.(inducing culture based component is: N6majors, MS iron salts, B5minors,
B5vitamins,500mg/L proline,500mg/L glutamine,300mg/L casein enzymatic
hydrolysate,30g/L sucrose,2mg/L 2,4-D,3g/L phytagel,pH 5.8)
(2) preculture: select granular callus from inducing culture and be placed on new inducing culture, in 30
Cultivate 3～5 days under DEG C dark condition.
(3) infect and co-culture: pre-incubated callus is transferred in 50mL sterile tube, adding Agrobacterium bacterium solution
(OD600=0.2) soak 20min, pour out bacterium solution, and with aseptic filter paper, the remaining bacterium solution in wound healing is blotted.Wound healing is uniform
It is sprinkling upon and co-cultures in culture medium, cultivate 2～3 days under 23 DEG C of dark.(co-culturing based component is: N6majors, MS iron
salts,B5minors,B5vitamins,500mg/L proline,500mg/L casein enzymatic
hydrolysate,30g/L sucrose,2mg/L2,4-D,3g/L phytag-el,100μL/L acetosyingone,4g/
L phytagel,pH 5.2)
(4) recover: the wound healing co-cultured is transferred on recovery media, 30 DEG C of dark culturing 3～5 days.(renewal cultivation
Based component is: N6majors, MS iron salts, B5minors, B5vitamins, 500mg/L proline, 500mg/L
glutamine,500mg/L casein enzymatic hydrolysate,30g/L sucrose,2mg/L 2,4-D,
250mg/L carbenicillin, 3g/L phytagel, pH 5.8)
(5) screening: select without bacterial plaque color vivid in faint yellow granular embryo callus subculture group from screening culture medium
Knit, be inoculated in screening culture medium, 30, every ware.30 DEG C of dark culturing 2～3 weeks, to the long resistance graininess wound healing made new advances.
(screening and culturing based component is: N6majors, MS iron salts, B5 minors, B5vitamins, 500mg/L
proline,500mg/L glutamine,500mg/L casein enzymatic hydrolysate,10g/L sucrose,
20g/L Mannose, 2mg/L 2,4-D, 3g/Lphytagel, 250mg/L carbenicillin, 25mg/L
hygromycin,pH 5.8)
(6) differentiation: each transformation event selects three independent embryo callus subcultures to a certain region of division culture medium, 30 DEG C of light
Cultivate 3～4 weeks according under the conditions of culturing room (16h illumination/8h is dark), treat that seedling grows.(differentiation culture based component is:
N6majors,MS iron salts,B5minors,B5vitamins,1g/Lcasein enzymatic hydrolysate,
30g/L sucrose,30g/L sorbitol,500mg/L MES,2.5mg/L CuSO4,0.5mg/L KT,AA amino
acids,2g/L phytagel,pH 5.8)
(7) take root: two healthy and strong seedling replantings are selected to root media in each region, 30 DEG C of tissue culture room light weeks
Phase (16h illumination/8h is dark) cultivates about three weeks, carries out identifying and transplanting to field.(root culture based component is: 1/2MS
basal salts(2.165g/L),B5vitamins,1.0g/Lcasein enzymatic hydrolysate,20g/L
sucrose,0.2mg/L NAA,125mg/Lcarbenicillin,3.5g/L phytagel,25mg/L hygromycin,
pH5.8)
Finally give the rice plant containing pCAMBIA1391-SAFES6 expression vector, we term it
PCAMBIA1391-SAFES6 plant.
Step 2, GUS histochemical stain
With reference to Jefferson (Jefferson RA et al.GUS fusion: β-Glucuronidase as a
Sensitive and versatile gene fusion marker in higher plant [J] .EMBO J., 1987,6:
3901-3907) etc. the method for proposition, carries out GUS histochemical stain to the tissue after Agrobacterium-mediated Transformation.The group of dyeing will be needed
Knitting evacuation, be then immersed in dyeing liquor, 37 DEG C are dyeed 24 hours.Then decolour, during decolouring, tissue is immersed in 95%
In ethanol, white to chlorenchyma material, coloration result is shown in Fig. 4, and as can be seen from the figure GUS is at root (a), stem (b), sheath
C strongly expressed in (), leaf (d), embryo (e), does not expresses in endosperm (e).
The quantitative analysis of embodiment 4, promoter SAFES6 activity
Take pCAMBIA1391-SAFES6 plant and the pCAMBIA1391-ACTIN plant as comparison
(pCAMBIA1391-ACTIN plant is obtained by directly plantation respective seed, the seed containing pCAMBIA1391-ACTIN
Come from Cornell Univ USA, constitutive promoter Oryza sativa L. ACTIN conventional in current genetic engineering drive Gus
Gene, genetically modified organism product composition supervision and inspection center of the Academy of Agri-Science and Technology Anhui Province Ministry of Agriculture preserve) leaf, sheath, stem,
Root, flower, endosperm sample, use the plant total RNA extraction reagent box of Tian Gen biochemical technology company limited to extract in every kind of sample
Total serum IgE, the total serum IgE reverse transcription extracted is by the FastQuant RT test kit re-using Tian Gen biochemical technology company limited
CDNA, with the cDNA that obtains from every kind of sample as template, respectively with ACTIN gene as reference gene, has with sky root biochemical technology
The SuperReal PreMixPlus real-time fluorescence quantitative PCR premixed liquid of limit company is reaction reagent, at the PRISM of ABI company
On 7500 fluorescent PCR instrument, by qRT-PCR reaction detection pCAMBIA1391-SAFES6, pCAMBIA1391-ACTIN transgenic
The expression intensity of reporter gene Gus in each tissue site in plant, this expression intensity reflects corresponding promoter activity.Wherein,
It is (SEQ ID No:4-5) for demarcating the quantitative qRT-PCR primer of reference gene ACTIN:
ACTIN forward primer: 5'-CCTTCAACACCCCTGCTATG-3 '
ACTIN downstream primer: 5'-CAATGCCAGGGAACATAGTG-3 '
It is (SEQ ID No:6-7) for detecting the qRT-PCR primer of Gus gene expression:
Gus forward primer: 5'-TACGGCAAAGTGTGGGTCAATAATCA-3 '
Gus downstream primer: 5'-CAGGTGTTCGGCGTGGTGTAGAG-3 '
Quantitative analysis results shows as it is shown in figure 5, it can be seen that Oryza sativa L. SAFES6 promoter is in leaf and sheath group
Knit expression activity and be significantly stronger than in genetic engineering conventional Rice constitutive type promoter ACTIN promoter；At root and stem tissue
In, two promoter activities are the most suitable；In flower tissue, Oryza sativa L. SAFES6 promoter is slightly weaker than ACTIN promoter；And at endosperm
In, Oryza sativa L. SAFES6 promoter is inactive.
Embodiment 5, promoter SAFES6 drive the activity analysis of BT
1, the structure of SAFES6-BT and ACTIN-BT expression vector
According to carrier pCAMBIA1391-SAFES6, pCAMBIA1391-ACTIN and gene mCry1Ab (Bacillus
Thuringiensis (Bt) protein coding gene, Academy of Agri-Science and Technology Anhui Province autonomous Design, in patent ZL 2,009 1
Disclosed in 0185774.5) the primer of sequential design band restriction enzyme site, forward primer 5 ' holds band restriction enzyme site EcoRI
(GAATTC), reverse primer 5 ' end band restriction enzyme site SpeI (ACTAGT), primer sequence is following (SEQ ID No:8-9):
Primer sequence is as follows:
BT FP:GAATTCATGGACAACAACCCGAACATCA
BT RP:ACTAGTTCAGTACTCGGCCTCGAAGGTC
With this to primer amplification gene mCry1Ab, with EcoRI and SpeI to amplified fragments and carrier pCAMBIA1391-
SAFES6, pCAMBIA1391-ACTIN carry out double digestion, are separately recovered the mCry1Ab fragment after enzyme action, pCAMBIA1391-
SAFES6 carrier large fragment and pCAMBIA1391-ACTIN carrier large fragment, with T4DNA ligase respectively to mCry1Ab fragment
With pCAMBIA1391-SAFES6 carrier large fragment, and mCry1Ab fragment and pCAMBIA1391-ACTIN carrier large fragment carry out
Connect, it is thus achieved that substitute Gus gene with mCry1Ab, driven the expression vector of Bt, named SAFES6-BT (Fig. 6) by SAFES6；
The named ACTIN-BT of the expression vector (Fig. 7) of Bt is driven by ACTIN.
2, the quantitative analysis of BT
Utilize freeze-thaw method that SAFES6-BT carrier and ACTIN-BT carrier are proceeded to Agrobacterium tumefaciems (Agrobacterium
Tumefaciens) in EHA105, using agriculture bacillus mediated rice transformation method, the transgenic obtaining respective carrier is planted
Strain, referred to as SAFES6-BT plant and ACTIN-BT plant.Concrete grammar is with reference to " step 1 in embodiment 3 ".Extract
SAFES6-BT plant and the leaf of ACTIN-BT plant, root, endosperm sample rna, and reverse transcription become cDNA.Reacted by qRT-PCR
Detect the relative expression quantity of BT gene in the different parts cDNA of two plants.For demarcating the quantitative of reference gene ACTIN
QRT-PCR primer (SEQ ID No:4-5) as previously mentioned.It is (SEQ ID for detecting the qRT-PCR primer of BT gene expression
No:10-11):
BT forward primer: 5 '-ATCCCGCCGCAGAACA-3 '
BT downstream primer: 5 '-GAGCGGAACATGGACACG-3 '
Quantitative analysis the results are shown in Table 1, in SAFES6-BT plant and ACTIN-BT plant, relative to ACTIN promoter
Driving the strain of Bt, in leaf and root tissue, Bt expression is slightly strong or is on close level for SAFES6-BT plant, and in endosperm,
There is expression in ACTIN-BT, SAFES6-BT then can't detect (less than detection line).Illustrate that the endosperm of the present invention does not express startup
Sub-SAFES6 does not drive Bt to express in endosperm or expression is the lowest, is negligible.
Table 1
N.D., non-detected (less than detection line)
Although the principle of the present invention being described in detail above in conjunction with the preferred embodiments of the present invention, this area skill
Art personnel are it should be understood that above-described embodiment is only the explanation of the exemplary implementation to the present invention, not to bag of the present invention
Restriction containing scope.Details in embodiment is not intended that limitation of the scope of the invention, without departing substantially from the present invention spirit and
In the case of scope, any equivalent transformation based on technical solution of the present invention, simple replacement etc. obviously change, and all fall within
Within scope.
Claims (10)
1. an isolated endosperm does not express promoter SAFES6, it is characterised in that described endosperm does not express promoter
SAFES6 comprises at least one in following polynucleotide sequence:
A () comprises the polynucleotide sequence of nucleotide sequence shown in SEQ ID NO:1 in sequence table；
B () has at least 85% homology with nucleotide sequence shown in SEQ ID NO:1 in sequence table and has identical function
Nucleotide sequence；
C one or more nucleotide is added, replaces or lacked to () in the nucleotide sequence shown in SEQ ID NO:1 in sequence table
Rear obtained and in sequence table nucleotide sequence shown in SEQ ID NO:1 there is the nucleotide sequence of identical function；And
(d) with comprise the nucleotide sequence of nucleotide sequence complementary shown in SEQ ID NO:1 in sequence table,
Wherein said endosperm is not expressed promoter SAFES6 and can be connected to allos transcribable polynucleotide sequence and can draw
Lead described allos transcribable polynucleotide sequence transcribing in non-endosperm tissue.
2. an expression cassette, it is characterised in that described expression cassette comprise the promoter described in claim 1 and with described promoter
The transcribed polynucleotide sequence of allos connected, wherein, described promoter can cause described allos transcribable polynucleotide
Sequence transcript and expression in the plant non-endosperm tissue converted with described expression cassette.
3. the expression cassette of claim 2, it is characterised in that described allos transcribable polynucleotide sequence is to have crop to express
The gene of characteristic, described allos transcribable polynucleotide sequence with justice direction insert in this expression cassette, or, described is different
Source transcribable polynucleotide sequence is inserted in this expression cassette with antisense orientation.
4. a recombinant expression carrier, it is characterised in that comprise described in the promoter described in claim 1 or claim 2
Expression cassette.
5. the recombinant expression carrier described in claim 4, it is characterised in that described allos transcribable polynucleotide sequence is tool
The gene of the crop character function that changes.
6. the method for improving plant growth characteristic in the case of not changing albumen character, it is characterised in that described side
Method includes:
(1), obtain the endosperm described in claim 1 and do not express promoter SAFES6；
(2), described endosperm is not expressed promoter SAFES6 to be connected with the gene with change crop character function, it is thus achieved that phase
Answer recombinant vector；
(3) described recombinant vector is proceeded in the cell of target plant；
(4) the corresponding transfer-gen plant of described cell culture is utilized；
(5) there is from the transfer-gen plant obtained the gene of change crop character function at non-endosperm position table described in selection
Reach, and the unexpressed plant in endosperm position, this plant character at non-endosperm position obtains improvement, and endosperm position is not by shadow
Ring.
7. the method converting plant or plant part, it is characterised in that described method includes with the table described in claim 2
Reach the recombinant expression carrier described in box or claim 4 described plant or plant part are converted.
Method the most according to claim 7, it is characterised in that the group of described plant part choosing freely following part composition
Group: cell, protoplast, cell tissue culture, callus, cell block, plumule, pollen, ovule, petal, style, hero
Stamen, leaf, root, the tip of a root and flower pesticide；The group of described plant choosing freely following part composition: cereal crops, vegetable crop, flowers
Crop, energy crop.
9. one kind is instructed the method that transcribed polynucleotide sequence is expressed in plant cell, it is characterised in that described method
Including the promoter described in claim 1 being connected to allos transcribable polynucleotide sequence construct recombinant expression carrier, and will
Recombinant expression carrier is transformed in plant cell.
Method the most according to claim 9, it is characterised in that described transcribable polynucleotide sequence is used for transcribing following
Protein: anti-insect protein, antibacterium albumen, antifungal protein, antiviral protein, nematicide albumen, herbicide resistant protein, anti-
Stress protein, the labelled protein that can screen, described method also includes the Plant cell regeneration plant after converting.
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| JP2005185101A (en) * | 2002-05-30 | 2005-07-14 | National Institute Of Agrobiological Sciences | VEGETABLE FULL-LENGTH cDNA AND UTILIZATION THEREOF |
| CN101230348A (en) * | 2007-11-19 | 2008-07-30 | 安徽省农业科学院水稻研究所 | Rice non-endosperm tissue expression promoter (OsTSP I) and uses thereof |
| CN103074342A (en) * | 2013-01-24 | 2013-05-01 | 山东农业大学 | Inducible promoter for pathogenic bacteria of rice |
| CN104342441A (en) * | 2014-11-04 | 2015-02-11 | 安徽省农业科学院水稻研究所 | Plant non-endosperm expression promoter SAFE S1 and acquiring method thereof |
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| CN105567694B (en) * | 2016-03-08 | 2019-08-13 | 安徽省农业科学院水稻研究所 | A kind of non-seed expression promoter SAFES4 of the active rice of strongly expressed and its application |
| CN106148346B (en) * | 2016-07-15 | 2019-10-08 | 安徽省农业科学院水稻研究所 | A kind of endosperm isolated does not express promoter SAFES6 and its application |
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|---|---|---|---|---|
| JP2005185101A (en) * | 2002-05-30 | 2005-07-14 | National Institute Of Agrobiological Sciences | VEGETABLE FULL-LENGTH cDNA AND UTILIZATION THEREOF |
| CN101230348A (en) * | 2007-11-19 | 2008-07-30 | 安徽省农业科学院水稻研究所 | Rice non-endosperm tissue expression promoter (OsTSP I) and uses thereof |
| CN103074342A (en) * | 2013-01-24 | 2013-05-01 | 山东农业大学 | Inducible promoter for pathogenic bacteria of rice |
| CN104342441A (en) * | 2014-11-04 | 2015-02-11 | 安徽省农业科学院水稻研究所 | Plant non-endosperm expression promoter SAFE S1 and acquiring method thereof |
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| WO2018010459A1 (en) * | 2016-07-15 | 2018-01-18 | 安徽省农业科学院水稻研究所 | Isolated promoter safes6 not expressed in endosperm and application of promoter safes6 |
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