CN105861506B - A rice endosperm non-expressing promoter SAFES2 and its isolation method and application - Google Patents
A rice endosperm non-expressing promoter SAFES2 and its isolation method and application Download PDFInfo
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Abstract
The present invention provides a kind of paddy endosperms not to express promoter SAFES2 and its separation method and application.The inventors found that, extract and the DNA sequence dna with transcriptional regulatory activity that identifies in OryzasativaLcv.Nipponbare rice one section, be named as promoter SAFES2 or SAFES2.The nucleotides sequence of the promoter is classified as sequence shown in SEQ ID No.1 or the filiation for the sequence in sequence table.The present invention utilizes crop binary expression vector pCAMBIA1391, promoter SAFES2 is implemented in gus reporter gene upstream, recombinant expression carrier is constructed, rice plant is regenerated by agrobacterium mediation converted Rice Callus, GUS Coloration experiment confirms that the promoter is that endosperm does not express promoter.It is used cooperatively using promoter of the invention with having the gene for improving plant strain growth characteristic, it can be while improving the metastomiums characters such as plant root, stem, leaf, it avoids bringing any adverse effect to plant endosperm fraction, there is significant application value and prospect at a specified future date.
Description
Technical field
The present invention relates to biotechnologys and crop gene field of engineering technology.Specifically, the present invention relates to a kind of rice
Endosperm does not express promoter SAFES2 and its application.
Background technique
In plant transgenic systems, imported foreign gene needs the transcription and translation and modified body by plant cell
System, generates active target protein, target gene is made to play a role, realize the genetic improvement of crop.Accurate control purpose base
It is extremely important to the specific aim and timeliness that improve target gene expression because of time, position or quantity that product generates, and mention
The importance of high genetically modified plants risk prevention system ability.In plant genetic engineering, target base is directly controlled by promoter
The transcription of cause is regulation destination gene expression means the most cost-effective, and the feasible path of control expression risk.Cause
This, fully understands the function and feature of different type plant promoter, is very heavy to suitable transgenic expression system is constructed
It wants.
Tissue-specific promoter is also known as organ specific promoter, only in specific histoorgan or cell type
With driving capability.The sphere of action of target gene is defined in genetic engineering, avoids constitutive promoter inessential
Matter energy caused by the expression of general formula wastes in tissue, particularly suitable for targetedly genetic improvement and bioreactor in a organized way
Using.It has been found that tissue-specific promoter specifically include that seed specific promoter, fruit differential type promoter, mesophyll
Cell-specific type promoter, root specific promoter.It has been applied to some tissue-specific promoters of Rice molecular breeding
It include: anther-specificpromoter BnBp10 (Cheng etc., 1998), anther specific promoter Pol (Peng etc., 2010), green
Tissue specific promoter OsrbcS (Ye etc., 2009), leaf specific promoter ZmPepc (Datta etc., 1998), endosperm specific opens
Mover Gt1 (Beyer etc., 2002), GluB4 (He etc., 2011), root-specific promoter OsAnt1 (Shrawat etc., 2008),
OsRCcs (Jeong etc., 2010) etc..
But the invention that endosperm does not express promoter is also rarely reported.The ready-to-serve polished rice position of the endosperm of rice, embryo
Cream, which does not express promoter, expresses that foreign gene only in non-edible part, guarantees that edible part does not contain the production of exogenous gene expression
Object reduces it to human health bring potential risk, is an effective way for promoting the commercialization process of transgenic paddy rice.
The research of Efforts To Develop functional genome is needed at present, and there is the endosperm of practical value not express starting for a large amount of excavations and separation
Son eliminates the public to the prejudice and misgivings of transgenic paddy rice, and does not express the characteristic of promoter using endosperm, gives full play to and turns base
Because of the advantage of breeding, more better new varieties are cultivated.
Summary of the invention
The first purpose of this invention is that separation clones a kind of paddy endosperm and do not express promoter SAFES2, utilizes the starting
Sub on the one hand driving target gene is expressed in rice plant, improves its production performance；On the other hand mesh is not expressed in endosperm
Gene is marked, the edible safety risk of transgenic rice is reduced with this.
A second object of the present invention is to provide promoter SAFES2 to cultivate the application in genetically modified crops.It is related
" crop " refers to cereal crop, such as rice, wheat, sorghum, barley, oat, rye etc., preferably rice.
To achieve the goals above, on the one hand, present inventor is from OryzasativaLcv.Nipponbare rice (Oryza sativa L
Cv.Nipponbare separation clone obtains one section of DNA sequence dna with transcriptional regulatory activity in), is named as SAFES2 or starting
Sub- SAFES2.Promoter SAFES2 includes one of one of following sequences or complementary series of following sequences:
(a) nucleotide sequence shown in SEQ ID NO:1 in sequence table；
(b) it is obtained after adding one or more nucleotide in nucleotide sequence shown in SEQ ID NO:1 in sequence table
The nucleotide sequence obtained；
(c) there is the nucleotides sequence of at least 90% homology with nucleotide sequence shown in SEQ ID NO:1 in sequence table
Column；
(d) it is obtained after replacing one or more nucleotide in nucleotide sequence shown in SEQ ID NO:1 in sequence table
The nucleotide sequence obtained；
(e) obtained after sequential nucleotide deletion one or more nucleotide shown in SEQ ID NO:1 in sequence table
Nucleotide sequence.
Preferably, the paddy endosperm does not express promoter SAFES2 DNA sequence as shown in SEQ ID No:1 in sequence table
Column are constituted.
It should be understood that DNA sequence dna shown in SEQ ID No:1 in sequence table, the 22bp that sequence 5 ' is held, i.e.,
" gcggtaatag aaatgtggag gg " its be obtain promoter process used in forward primer retention sequence；Sequence 3 '
The 22bp at end, i.e. " cactttaaac acataattaa cc ", to obtain staying for reverse primer used in promoter process
Deposit sequence (the retention sequence is complementary with the corresponding sequence of reverse primer).It is emphasized that promoter mentioned herein
Both it can refer to above-mentioned entire DNA sequence dna, and can also refer to that removing above-mentioned primer retained the DNA sequence dna after sequence.Even if this field skill
Art personnel obtain similar sequence on the basis of the present invention, using other primers, also fall into protection scope of the present invention it
It is interior.
On the other hand, the present invention also provides a kind of expression cassettes for not expressing promoter SAFES2 comprising above-mentioned paddy endosperm.
Another aspect, the present invention also provides a kind of recombinant expression carrier, the recombinant expression carrier includes above-mentioned rice
Endosperm does not express promoter SAFES2.In the recombinant expression carrier, the paddy endosperm does not express promoter SAFES2 company
It is connected to the upstream of gene order to be expressed, gene to be expressed there can be improvement ability for the growth characteristics of any pair of rice
Gene, promoter through the invention concentrate expression with driving tissue specificity of the gene other than endosperm, change to realize
Function of the kind paddy growth performance without influencing endosperm growth performance；Preferably, the gene to be expressed is Gus gene.
On the other hand, promoter SAFES2 acquisition is not expressed accordingly using the paddy endosperm the present invention also provides a kind of
The method of host strain, the method includes by the paddy endosperm do not express promoter SAFES2, the expression cassette or
The recombinant expression carrier is transferred to Agrobacterium tumefaciems using freeze-thaw method.
On the other hand, the present invention, which provides, a kind of do not express promoter SAFES2 using the paddy endosperm and obtains corresponding turn
The method of beggar, the method includes host strain obtained above is passed through mediated by agriculture bacillus method to target cell, callus
Tissue or plant are converted, and corresponding transformant is obtained.Wherein, the transformant is preferably transgenic cell line, callus
Or plant.
In another aspect, the present invention, which provides above-mentioned paddy endosperm, does not express promoter SAFES2 in cultivating genetically modified crops
Using.The application includes:
A), the paddy endosperm upstream that promoter SAFES2 is connected to target gene is not expressed into；
B), the recombinant expression carrier for not expressing promoter SAFES2 and target gene containing the paddy endosperm is transferred to
In Agrobacterium tumefaciems；
C), Agrobacterium is carried out to target plant callus using the Agrobacterium tumefaciems for being transferred to the recombinant expression carrier
Conversion；
D), corresponding plant is cultivated using the callus after conversion, in the plant, the paddy endosperm is not expressed
Promoter SAFES2 can drive destination gene expression, and destination gene expression is not driven in endosperm.
In addition, the present invention provides the separation method that a kind of paddy endosperm does not express promoter SAFES2, feature exists
In, which comprises
Step (1), the sequence design amplimer for not expressing promoter SAFES2 according to the paddy endosperm；
Step (2), using the DNA of rice varieties OryzasativaLcv.Nipponbare as template, institute is expanded by PCR amplification program using the primer
It states paddy endosperm and does not express promoter SAFES2；
Step (3), the target fragment for recycling PCR amplification, and be connected on PGEM-T-Easy carrier；
Step (4) converts Escherichia coli XL-Blue competent cell according to heat shock method using connection product；
Step (5) carries out PCR screening to converted product, obtains positive colony, and picking monoclonal shakes bacterium solution upgrading grain, uses
Corresponding restriction enzyme carries out double digestion verifying；
Step (6) will be sequenced by the positive colony of identification, obtains and correct nucleotide sequence is sequenced, as mesh
Mark sequence.
In conclusion the inventors found that, extract and identify OryzasativaLcv.Nipponbare rice (Oryza sativa L
Cv.Nipponbare one section of DNA sequence dna with transcriptional regulatory activity in), and SAFES2 is named as (in sequence table
SEQ ID No:1).Specifically, inventor has found that there is the sequence driving gene to express in rice plant without in endosperm
The ability of middle expression extracts the sequence and is identified aforementioned capabilities.The sequence is connected to by inventor after digestion
On crop binary expression vector pCAMBIA1391, corresponding recombinant plasmid (i.e. recombinant expression carrier) is obtained, utilizes the recombination matter
Grain conversion Agrobacterium tumefaciens strain EHA105, the conversion of rice is then carried out with the method for mediated by agriculture bacillus, obtains transgenosis water
Rice plants.Histochemistry's detection discovery is carried out to the transgenic paddy rice of acquisition, gus gene is in root, stem, leaf, leaf sheath, embryo and kind skin
In have more strongly active, but do not expressed in albuminous cell.To prove that the sequence has planting in rice for driving gene specific
The activity expressed in strain without being expressed in endosperm.
Technical effect
Promoter SAFES2 of the present invention can be connect with crop binary expression vector, for replacing composing type to start
Son.Also, the promoter can be connect with required target gene, constructed recombinant expression carrier, after inverted, can be planted in rice
In strain expression improves the production performance of rice, also ensures the edible safety of rice without expressing in endosperm, has weight
The theory and practical significance wanted.
Detailed description of the invention
Hereinafter, carrying out the embodiment that the present invention will be described in detail in conjunction with attached drawing, in which:
Fig. 1 is that SAFES2 promoter is implemented in the schematic diagram in pCAMBIA1391 vector plasmid, and wherein A is in Fig. 1
PCAMBIA1391 schematic diagram, B are pCAMBIA1391-SAFES2 schematic diagram, are shown and are driven using SAFES2 promoter
The gus gene expression locateding downstream；
Fig. 2 is the result schematic diagram that digestion verification is carried out to promoter of the invention.
Fig. 3 is root (a), stem (b), leaf sheath (c), leaf (d), GUS coloration result in seed (e).Blue is GUS histochemistry
Dyeing, representing in tissue has GUS expression.Scale=2.5mm
Specific embodiment
The present invention is described below with reference to specific embodiments.It will be appreciated by those skilled in the art that these embodiments are only
For illustrating the present invention, do not limit the scope of the invention in any way.
Experimental method in following embodiments is unless otherwise specified conventional method.Life as used in the following examples
Change reagent, carrier consumptive material etc. is commercially available products unless otherwise specified.
Embodiment 1 separates cloning promoter SAFES2
Step 1, the design of primer
According to rice varieties OryzasativaLcv.Nipponbare (the Oryza sativa L cv.Nipponbare) full-length genome provided in NCBI
Sequence, according to the sequence design amplimer of rice SAFES2 gene, and the characteristics of according to the carrier and target gene of selection, if
Count the restriction enzyme site of primer.
With rice binary expression vector pCAMBIA1391, (part A, open from CAMBIA in Fig. 1 in the present embodiment
Using carrier, Academy of Agri-Science and Technology Anhui Province Ministry of Agriculture genetically modified organism product composition supervision and inspection center rice group is saved)
For, target gene is Gus gene, the primer specifically designed are as follows: the end of forward primer (SEQ ID No:2) 5 ' band HindIII, enzyme
Enzyme site (AAGCTT), the end of reverse primer (SEQ ID No:3) 5 ' band EcoRI, restriction enzyme site (GAATTC), primer sequence is such as
Under:
Forward primer: AAGCTTGCGGTAATAGAAATGTGGAGGG HindIII
Reverse primer: GAATTCGGTTAATTATGTGTTTAAAGTG EcoRI
It is synthesized by Shenzhen Huada gene company.
The separation clone of step 2, promoter SAFES2
Using rice varieties OryzasativaLcv.Nipponbare DNA as template, promoter SAFES2 is expanded using forward primer, reverse primer, by normal
PCR system is advised, using following amplification program:
95 DEG C of initial denaturation 5min；95 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C of extension 2min30s, 35 recycle；Finally
72 DEG C of extension 10min.
The target fragment for recycling PCR amplification is connected to PGEM-T-Easy carrier (purchased from Promega company, by load
Ratio mixing in body specification) on, after converting Escherichia coli XL-Blue competent cell according to heat shock method, sieved through bacterium colony PCR
Choosing obtains positive colony, and picking monoclonal shakes bacterium solution upgrading grain, carries out double digestion verifying, target fragment with HindIII and EcoRI
Length 2018bp, as shown in Figure 2.The sequencing of Invitrogen company will be delivered by the positive colony of identification.Verify correct gram
Grand is the promoter SAFES2 to be obtained, and nucleic acid sequence is as shown in SEQ ID No:1.
The functional verification and application of 2 promoter SAFES2 of embodiment
1. the building of genetic transformation carrier and the conversion of Agrobacterium
Used carrier is crop binary expression vector pCAMBIA1391, carries out double digestion with HindIII and EcoRI, is recycled
The pCAMBIA1391 of linearization process.Digestion is carried out with the promoter SAFES2 that HindIII and EcoRI obtains clone simultaneously,
Recycle target fragment.By above-mentioned pCAMBIA1391 segment and SAFES2 segment with T4DNA ligase (being purchased from TaKaRa company)
It is attached, obtains the recombinant expression carrier pCAMBIA1391-SAFES2 (Figure 1B) of promoter SAFES2 Yu Gus Gene Fusion,
Recombinant expression carrier is transferred to the (Anhui EHA105 Agrobacterium tumefaciems (Agrobacterium tumefaciens) using freeze-thaw method
The Ministry of Agriculture, Shanxi Academy of Agricultural Sciences genetically modified organism product composition supervision and inspection center rice group saves).
The functional verification and application of 2 promoter SAFES2
Step 1: the rice transformation of mediated by agriculture bacillus
After mature rice paddy seed removes glume, seed 1min is impregnated with 70% alcohol, outwells alcohol.Tween is dripped with containing 1
20 50% sodium hypochlorite (stoste effective chlorine density is greater than 4%) solution impregnates seed 40min (150r/min).Outwell hypochlorous acid
Sodium, it is sterile to wash 5 times to solution clarification, no sodium hypochlorite taste.Sterile water impregnates seed and stays overnight.With scalpel along the paste of seed
Bisque peels embryo, and embryo is inoculated on calli induction media.By callus and endosperm and plumule after dark culture 11 days at 30 DEG C
Separation is used for Agrobacterium-mediated Transformation after carrying out the primary callus in good condition, that division is vigorous for removing bud preculture 3~5 days.
Recombinant expression carrier has been transferred to using above-mentioned " building of crop expression vector and the conversion of Agrobacterium " in the process
Agrobacterium tumefaciems carries out Agrobacterium-mediated genetic transformation, the ginseng such as the genetic transformation, transformant screening and transgenic plant regeneration
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. propositions method.Turned
Gene regeneration plant.
Step 2, GUS histochemical stain
Referring to Jefferson (fusion: β-Glucuronidase as a of Jefferson RA et al. .GUS
Sensitive and versatile gene fusion marker in higher plant [J] .EMBO J., 1987,6:
The method of propositions such as 3901-3907), the tissue that the needs separated in transgenic regenerated plant obtained from the above are dyed immerse
It in dyeing liquor, is placed 24-36 hours in 37 DEG C of incubators, soaked in absolute ethyl alcohol is added until decoloration completely, is clapped under the microscope
According to record.As a result see Fig. 3 (it should be noted that in view of color image, therefore, present invention is not received in patent text
Colored Fig. 3 is revised as gray level image by people, still, even if in this way, based on the colour darkness in Fig. 3, it can also be clearly
Tell whether each position is dyed), root (a), stem (b), leaf sheath (c), leaf (d), GUS dye in seed (e) are shown in figure
For color as a result, blue is GUS histochemical stain, representing in tissue has GUS expression.Coloration result shows that SAFES2 promoter exists
Root, stem, leaf, leaf sheath, embryo and kind skin in have it is more strongly active, Gus gene expression can be guided, but the not table in albuminous cell
It reaches.
Therefore, can clearly be judged based on the coloration result, endosperm of the invention does not express promoter not table in endosperm
It reaches, and is expressed in other Main Tissues except endosperm.
For the repeatability of confirmatory experiment, the present invention has carried out three repeated experiments, experimental result with implement above
Example is similar, is described again here.
Although the present invention is described by taking rice as an example, it is applicant's understanding that the application of the obtained promoter of the present invention
Scene is not limited to rice, and promoter of the present invention can be used for various gramineous crops, such as rice, wheat, corn, barley, height
Beam or oat, preferably rice.
Specific description of embodiments of the present invention above is not intended to limit the present invention, and those skilled in the art can be according to this
Invention is variously modified or deforms, and as long as it does not depart from the spirit of the invention, should belong to the model of appended claims of the present invention
It encloses.
Claims (3)
1. a kind of paddy endosperm does not express promoter SAFES2 and is cultivating the application in genetically modified crops, which is characterized in that described
As shown in SEQ ID NO:1 in sequence table, the application include: by paddy endosperm shown in SEQ ID NO:1 in sequence table not
Expression promoter SAFES2 is connected to gene sequence upstream to be expressed in carrier, to construct recombinant expression carrier；It will be described
Recombinant expression carrier is transformed into crop cell, tissue or organ and is cultivated, and then obtains corresponding transgenic plant, sequence table
Sequence shown in middle SEQ ID NO:1 are as follows:
2. application according to claim 1, which is characterized in that the application is used for Crop Improvement growth characteristics, the work
Object rice.
3. application according to claim 1, which is characterized in that the gene to be expressed is Crop Improvement growth characteristics
Structural gene, the antisense gene for adjusting gene or structural gene.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| US7790958B2 (en) * | 1999-07-20 | 2010-09-07 | Monsanto Technology Llc | Genomic plant sequences 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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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7790958B2 (en) * | 1999-07-20 | 2010-09-07 | Monsanto Technology Llc | Genomic plant sequences and uses 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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