CN1624129A - Specific promoter of root and its application - Google Patents
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Abstract
A plant root specific expression promotor sequence, and the recombinant nucleic acid sequence, configurator and expression system, which containing said promotor are disclosed. It can drive the functional gene to be specifically expressed and preferably expressed in plant root.
Description
Technical field
The present invention relates to the genetically engineered plant field.More specifically, the present invention relates to the specific expressed promoter sequence of a kind of roots of plants, contain the recombinant nucleic acid sequence of this promotor, construct and expression system, and drive the purposes that functional gene is expressed and preferentially expressed at the roots of plants internal specific.
Background technology
The molecular nature of higher plant cell differentiation is the result of different genes differential expression, and most of developmental regulation expression of gene all have timeliness and spatiality.Timeliness is meant that the expression of specific gene is discontinuous, of short duration, and spatiality to be specific gene have in vivo privileged site to express be the characteristic of tissue specific expression (tissue-specific expression), tissue specific expression comprises that tissue specificity expression (tissue-unique expression) and tissue strengthen property expression (tissue-enhanced expression), the former is the genetic expression that only exists in particular organization, as glycinin gene etc., and the latter is meant its hetero-organization relatively, has the genetic expression of high expression level in particular organization.
Adopt molecular biology that farm crop are carried out in the genetic improvement process, usually the foreign gene of expectation insertion can be limited in the specific tissue and express, and this tissue should be the most significant position of exogenous gene expression.This has two important reasons: at first, for whole and persistence were expressed, restricted expression (restricted expression) was minimum for the metabolic influence of plant self; Secondly, when it doesn't matter at the edible position of human or animal on heterogenous expression albumen and the plant, foreign gene preferably directly is expressed in other positions in addition, these edible positions, particularly the human or animal absorb the proteic consequence of heterogenous expression also not fully aware of in, the second reason seems even more important.
Simple in structure, the composition of higher plant root are stablized, be that the research organ takes place and the good model system of growing, effectively the root specific expression promoter is to utilize disease-resistant worm gene, resistance gene, promote the gene that mineral substance absorbs and promote useful goal gene such as the metabolic gene of specific secondary substance to import farm crop and drive the prerequisite of expressing in roots of plants.
At present, the tissue-specific promotor of having reported has at blade, root, the pollen tapetum, the embryo, endodermis, specific expressed polytype promotor in the organ or tissue such as aleurone layer and phloem, the root-specific promoter of wherein having found is also very few, at present in the United States Patent Office (USPO) registration tobacco RB7 root promotor (U.S.Pat.No.5459252) arranged, corn MR7 (U.S.Pat.No.5837848) etc. also have Brassica plants Gent recessive allele regulating and controlling sequence (U.S.Pat.No.5401836) to wait and the root-specific promoter related DNA sequence in addition.
Summary of the invention
Summary of the invention
At above-mentioned needs of the prior art, the inventor is through long-term exploration and a large amount of research, finally successfully having obtained a kind of functional gene that can drive expresses or preferential promoter sequence of expressing at the root internal specific, and its sequence and function have been carried out deep research, thereby finished the present invention.
Therefore, in one aspect of the invention, the specific expressed promoter sequence of a kind of roots of plants or its function equivalent are provided, variant, perhaps fragment.
In another aspect of the present invention, provide a kind of recombinant nucleic acid sequence that contains above-mentioned promotor.
In another aspect of the present invention, provide a kind of construct that contains above-mentioned recombinant nucleic acid sequence.
In another aspect of the present invention, provide a kind of above-mentioned construct expression system that contains.
Aspect another, provide above-mentioned promotor of the present invention, contained the recombinant nucleic acid sequence of this promotor, construct or expression system are expressed and the preferential purposes aspect the expression at the roots of plants internal specific driving functional gene.
But on " detailed Description Of The Invention " below having read, the especially basis of the disclosure of " embodiment " part, other aspects and advantages of the present invention are conspicuous to those skilled in the art.
Description of drawings
Accompanying drawing 1:DcRB7 promoter sequence.
Accompanying drawing 2: plant conversion carrier synoptic diagram.
Accompanying drawing 3: the enzyme of plant conversion carrier is cut evaluation, wherein: M: λ DNA/EcoRI+Hind III; 1:pCAMBIA-C10/SspI; 2:pCAMBIA-C10/Ncol+Hind III; 3:pCAMBIA-CE/Ssp I.
Accompanying drawing 4: the histochemical stain that the transgenic tobacco plant gus gene is expressed.
Accompanying drawing 5: transgene tobacco GUS expression activity is at the quantitative fluorescence analysis of each tissue site, wherein: 1.DcRB7 promotor-GUS recombinant expression vector pCAMBIA-C10; 2. the promotor of the DcRB7 under the drying treatment-GUS recombinant expression vector pCAMBIA-C10; 3.CaMV35S-GUS recombinant expression vector pCAMBIA1305.1; 4. promoterless GUS expression vector pCAMBIA-CE; 5. the adjoining tree that does not have expression vector.
Detailed Description Of The Invention
In the context of the present specification, unless specialize, otherwise the used any skill of this specification The art term has the implication that those of ordinary skills understand in the art usually, and unreceipted tool The experimental technique of concrete conditions in the establishment of a specific crime is according to normal experiment method or the operational manual of advising according to supplier Carry out.
Particularly, the term below using in the application's context and claims has following implication:
" function equivalent " refers under strict hybridization conditions assorted with a target DNA molecular sequences Any dna molecular of handing over, this dna molecular have the biology that is similar to described target DNA molecule and live The property.
" strict hybridization conditions " refers to that experiment condition is: 1, and hybridization is at 65 ℃ and 5 * SSPE (0.75 M NaCl, 50mM NaH2PO4,5mM EDTA), 0.2%SDS, 5 * Denhardt (0.1% Polyvenylpyrrolidon, 0.1%BSA, 0.1% ficoll 400) and 0.2mg/ml salmon Carried out 12 hours in the smart DNA hybridization solution; 2, washing the film condition is 68 ℃, film washing liquid I (5 * SSC, 0.1% SDS) 20 minutes, film washing liquid II (2 * SSC, 0.1%SDS) washed 20 minutes, film washing liquid III (1 * SSC, 0.1%SDS) 20 minutes.
" variant " refers to a target DNA sequence is carried out any replacement of one or more base, Variation is modified, replaces, and the sequence that disappearance or interpolation produce, this sequence still shows similar Activity in described target DNA sequence.
" fragment " refers to one or more zone of basic dna sequence dna, and it still has and is similar to The activity of basic dna sequence dna.
" heterologous gene " refers in the present invention Resistant Gene, resistance gene, promotes mineral matter The gene that absorbs and the desirable genes such as gene that promote specific secondary substance metabolism.
The inventor screens from carrot radicle cDNA library and obtains a global cDNA segment, its Dna sequence dna and tobacco root-specific expressing gene TobRB7 (Yamamoto, YT.et al., 1991) The dna sequence dna homology reach 72%, the amino acid sequence of its supposition and TobRB7 amino acid sequence Homology reach 80%, protein structure prediction contains 6 TMDs, and contains aquaporin Significant NPA motif. Therefore, think that this gene is that to separate of obtaining from carrot new Water channel protein gene, its called after DcRB7.
DcRB7 is carried out the Northern hybridization analysis, and the result shows that the DcRB7 gene is the gene of a different expression of Gent.Utilize the method for inverse PCR, obtained one section DcRB7 promoter sequence of DcRB7 upstream region of gene from the Radix Dauci Sativae genome, it has comprised the scope from translation initiation codon ATG upstream 66bp to-601bp.With this section sequence (+48bp to-601bp) be incorporated into plant expression vector, and transformation of tobacco (Nicotiana tabacum L.) NC89 by vitro recombination.After transgene tobacco carried out that gus reporter gene is active and detect, find that this promoter sequence can drive functional gene and express or preferential the expression at the root internal specific, have certain root-specific and drive active.
Below in conjunction with specific embodiment, further illustrate the present invention.Should be understood that these embodiment just in order to demonstrate the invention, but not limit the scope of the invention by any way.In the following example, the experimental technique of unreceipted actual conditions carries out according to the normal experiment method usually, or carries out according to the operation instructions that manufacturer advises.
Embodiment
The acquisition of embodiment 1:DcRB7 promoter fragment
One. extract the Radix Dauci Sativae genomic dna with reference to CTAB method (Wang Guanlin etc., 1998).
(1) in centrifuge tube, adds 10ml CTAB and extract damping fluid (2%CTAB, 1.4M NaCl, 20mMEDTA, 100mM TrisHCL pH8.0,0.2% β-mercaptoethanol), 65 ℃ of preheatings;
(2) take by weighing 1-2 and restrain fresh blade, become powder with liquid nitrogen grinding in mortar, change in the centrifuge tube, the limit edged stirs, and makes it to mix;
(3) 65 ℃ water bath heat preservation 0.5-1 hour, middle gentle mixing is several times;
(4) be cooled to room temperature after, the equal-volume of adding (10ml) chloroform: primary isoamyl alcohol (24: 1), light and slowly put upside down mixing 10 minutes;
(5) the centrifugal 10000rpm of room temperature, 10 minutes, go precipitation, supernatant changes in the new pipe;
(6) Virahol of adding 2/3 volume in the supernatant, the light and slow mixing of putting upside down, room temperature was placed 10-20 minute;
(7) the centrifugal 10000rpm of room temperature 10 minutes, abandons supernatant;
(8) precipitate twice with desalination with 2ml 75% washing with alcohol;
(9) remove ethanol, 37 ℃ of dryings, (about 20 minutes) are dissolved in 60 μ l TE damping fluids;
(10) add 5 μ l RNaseA (10mg/ml), 37 ℃ are incubated 30 minutes;
(11) equal-volume chloroform: primary isoamyl alcohol (24: 1) extracting 1-3 time;
(12) room temperature centrifugal (10000rpm, 10 minutes) is got supernatant, changes new pipe over to;
(13) the 4 M NaCl (final concentration 0.1-4M) of adding 1/10 volume add the two volumes dehydrated alcohol, and room temperature was placed after 20 minutes, and centrifugal 10000rpm 5 minutes, abandons supernatant;
(14) DNA precipitation is used 75% washing with alcohol, and 2-3 time, it is standby to be dissolved in an amount of TE damping fluid-20 ℃ of preservations in 37 ℃ of insulation cans after the drying.
Two. the inverse PCR method obtains the DcRB7 promoter fragment
The method that the enzyme of DNA is cut, the extraction conversion of electrophoresis, recovery, connection, subclone and plasmid etc. all described with reference to " molecular cloning experiment guide (third edition) " (work such as Joseph Sambrook, Huang Peitang etc. translate).Template preparation: the Radix Dauci Sativae genomic dna with the complete digestion of Hind III enzyme, is lower than in DNA concentration and carries out recirculation under the 2g/ml condition and connect.Adopt phenol: the chloroform extracting connects product, is dissolved in TE solution behind the ethanol sedimentation.
The PCR reaction system is (reagent is all purchased the company in Promega):
Primer (10 μ M) 1 μ l
Primer (10 μ M) 1 μ l
dNTP(10mM) 0.4μl
Mg
++(25mM) 1.6μl
10×Buffer 2μl
Taq enzyme 2unit
ddH
2
O 11.5μl
Cumulative volume 20 μ l
According to the sequences Design of DcRB7 four nested primerss (hundred victory companies are synthetic by match):
P1: the outside, upstream 5 '-gaggcctaacaaattgaagaaga-3 ' (SEQ ID NO:2)
P2: inboard, upstream 5 '-ggctgccaattgatatcttc-3 ' (SEQ ID NO:3)
P3: downstream interior side 5 '-ctgagtttattgccaccctt-3 ' (SEQ ID NO:4)
P4: the outside, downstream 5 '-ccgaaggtgtagtgtttgagat-3 ' (SEQ ID NO:5)
The Radix Dauci Sativae genomic dna of cutting the back recirculation with Hind III enzyme is a template, is that primer carries out the pcr amplification first time with P2, P3.Reaction conditions is:
1 circulation of 95 ℃ of 5min
1 circulation of 72 ℃ of 10min
With the first time PCR product be template, be that primer carries out the pcr amplification second time with P1, P4.Reaction conditions is:
1 circulation of 95 ℃ of 5min
1 circulation of 72 ℃ of 10min
Pcr amplification product confirms that through agarose gel electrophoresis size is about the single fragment of 1.3kb.This fragment and pGEM-Teasy (promega company) carrier are carried out vitro recombination, obtain to contain the clone pGEM-RP of DcRB7 promoter region.Extract the pGEM-RP plasmid DNA, carry out nucleotide sequencing.
With Hind III and Nco I digestion with restriction enzyme pGEM-RP, the fragment of the about 650bp that obtains is the promoter fragment of being identified among the embodiment 2, and it has comprised that NcoI is to the sequence (referring to Promega pGEM-T and pGEM-Teasy Vector Systems specification sheets) of the 25bp of EcoRI on the upstream promoter fragment of DcRB7 gene and the pGEM-Teasy carrier multiple clone site.
The promoter sequence that is obtained is through order-checking, and its sequence (SEQ ID NO:1) as shown in Figure 1.
The transgenosis Function Identification of embodiment 2 promotors
One. the structure of plant conversion carrier
With Hind III and NcoI digestion with restriction enzyme pGEM-RP, reclaim the purpose fragment of about 650bp, use Hind III and NcoI restriction enzyme to binary expression vector pCAMBIA1305.1 (CAMBIA simultaneously, Australia) digest, reclaim carrier segments, then purpose fragment and carrier are carried out in-vitro directed reorganization acquisition clone called after pCAMBIA-C10.PCAMBIA-C10 replaces the plant expression vector that obtains after the original CaMV35S promotor of pCAMBIA1305.1 with the DcRB7 promotor.
Digest pCAMBIA-C10 with EcoRI, reclaim carrier part, and carrier segments is carried out obtaining cloning pCAMBIA-CE from connecting, this carrier is the negative control carrier (Fig. 2) that is not activated son before the gus reporter gene.With SspI, NcoI, Hind III these two positive colonies are carried out enzyme and cut evaluation, NcoI and HindIII digestion pCAMBIA-C10, the insertion fragment of the about 650bp of acquisition; Cut the fragment (Fig. 3) that pCAMBIA-CE can obtain about 7.3kb and about 4.7kb with the SspI enzyme.
Two. Agrobacterium is infected the cultivation of tobacco leaf explant and transgene tobacco
Transform Agrobacterium EHA105 (Hood et al.1993) with reference to Bevan method (Bevan, 1984) respectively with pCAMBIA-C10, pCAMBIA-CE, three kinds of carriers of pCAMBIA1305.1.The Agrobacterium that will have pCAMBIA-C10, pCAMBIA-CE, pCAMBIA1305.1 is by leaf dish method transformation of tobacco (Nicotiana tabacum L.) NC89 (available from the Chinese Academy of Agricultural Sciences), and regeneration obtains the transgenosis plantlet.
Three .GUS genetic expressions detect
Histochemical stain detects: carry out with reference to the method that Jefferson et al (1987) describes, plant is positioned over GUS dye liquor (0.1M sodium phosphate buffer; The 5mM yellow prussiate of potash; The 5mM Tripotassium iron hexacyanide; 10mM Na
2EDTA; 0.5mg/ml X-Gluc), be incubated a few hours to spending the night at 37 ℃.Change in 70% ethanol and decolour 2-3 time, the part of the blueness on the white background is the (see figure 4) as a result that gus gene is expressed.
Fluorescent quantitative measurement GUS enzymic activity:, measure the GUS activity in plant root to be measured, stem, the leaf texture respectively with reference to the method for Jefferson et al (1987).With reference to Bradford (1976) albuminometry the plant total protein is measured.Measurement result is seen Fig. 5.
The result shows, the DcRB7 gene upstream sequence (+48bp to-601bp) be incorporated into plant expression vector by vitro recombination, and find behind the transformation of tobacco that this promoter sequence can drive functional gene and express or preferential the expression at the root internal specific, have certain root-specific and drive active.
Reference:
Wang Guanlin etc., " plant genetic engineering philosophy and technique " (first version), ISBN 7-03-006452-6, Science Press, Beijing, 600-601 (1998).
Hood, E.E., S.B.Gelvin, L.S.Melchers, new Agrobacterium helper plasmid (the New Agrobacterium helper plasmids forgene transfer to plants) .Transgen.Res.2:208-218 (1993) of and A.Hoekema. plant transgene operation.
Bevan M., the double base agrobacterium vector of Plant Transformation (Binary Agrobacterium vectorsfor plant transformation) .Nucleic Acids Res., 12,8711-21 (1984).Bradford MM, the proteic method of measurement Gamma Magnitude (A rapid and sensitive method for the quantitation ofmicrogram quantities of protein utilizing the principle ofprotein-dye binding) the .Anal Biochem 7 of a kind of rapid sensitive of utilization dye-bond protein principle; 72:248-54 (1976).
Jefferson, RA., Kavanagh, TA., Bevan, MW., GUS fusions: β-Pu Taotanggansuanmei, the sensitivity of a kind of higher plant and the general warm mark of gene (GUS fusions:beta-glucuronidase as a sensitive and versatile gene fusionmarker in higher plants) .EMBO J, 6,3901-7 (1987).
Sambrook, J., the yellow training hall of Russell DW. waits translates molecular cloning experiment guide (third edition) .ISBN 7-03-010338-6, Science Press, Beijing, (2002).
Yamamoto, instruct description (Characterization of cis-Acting Sequences Regulating Root-specific Gene Expression in Tobacco) the .The plant cell. of the cis-acting elements of the different expression of gene Gent in the YT.et al. tobacco, 3:371-82 (1991).
Sequence table
＜110〉Inst. of Genetics and Development Biology, CAS
＜120〉root-specific promoter and uses thereof
<130>I030697
<160>5
<170>PatentIn?version?3.1
<210>1
<211>701
<212>DNA
＜213〉Radix Dauci Sativae
<220>
＜221〉promotor
<222>(1)..(701)
<223>
<400>1
aagcttatcc?gctcataacc?tactcaatta?ttcaaaaact?tgtaggggtg?tccctaatac 60
caatcggtgt?gtagggtggt?tagattactt?tattcttaaa?ctgacataag?aaaccacaat 120
tttacatatt?gttcatttca?tgattattac?aaaccaaaac?ttagttcttt?tcaatcgatt 180
atcaaattat?ggtcggcacc?ggtgattagt?cttaaaaaag?tccaaaatag?accatataat 240
ttagtgggta?acatcagtga?gataaaatat?taggggacag?tcggtccaga?aaagtcgaat 300
gtcagcagca?tacatttgac?gggaaggaaa?aaagtgttgg?acaagaagaa?ttccaggatg 360
agttaatttt?cgttggatga?cacagtaata?tccttgcata?ttgtttaaaa?acttgaattt 420
aaggtgttga?gagatttttt?ccgagtatac?ttgacattcg?tatgtcagtc?cctgacctat 480
acttaaatgt?atcattatat?gagtatatgt?ttcggtttgt?tcattaactg?aattcctaaa 540
ctttctagta?tataaacaac?attgctgctc?tgtagaattc?gcaaaagaag?cccttaatca 600
atacttggaa?ttttccaagg?cttttatctt?cttcaatttg?ttaggcctct?ctagctagct 660
ttaaaaatgg?tgaagatatc?aattggcagc?cttggtgact?c 701
<210>2
<211>23
<212>DNA
<213>Artificial
<220>
＜221〉outside, upstream primer
<222>(1)..(23)
<223>
<400>2
gaggcctaac?aaattgaaga?aga 23
<210>3
<211>20
<212>DNA
<213>Artificial
<220>
＜221〉primer
<222>(1)..(20)
<223>
<220>
＜221〉the inboard primer in upstream
<222>(1)..(20)
<223>
<400>3
ggctgccaat?tgatatcttc 20
<210>4
<211>20
<212>DNA
<213>Artificial
<220>
＜221〉downstream interior side primer
<222>(1)..(20)
<223>
<400>4
ctgagtttat?tgccaccctt 20
<210>5
<211>22
<212>DNA
<213>Artificial
<220>
＜221〉outside, downstream primer
<222>(1)..(22)
<223>
<400>5
ccgaaggtgt?agtgtttgag?at 22
Claims (8)
1. promotor and its function equivalent thereof that roots of plants is specific expressed, variant, perhaps fragment, it has the part of the dna sequence dna shown in the SEQ ID NO:1 at least.
2. the promotor of claim 1 and its function equivalent thereof, variant, perhaps fragment, it has the dna sequence dna shown in the SEQ ID NO:1.
3. recombinant nucleic acid sequence, it contains promotor or its function equivalent of claim 1 or 2, variant, perhaps fragment.
4. the recombinant nucleic acid sequence of claim 3, it also contains at least a functional gene.
5. the recombinant nucleic acid sequence of claim 3, promotes the gene that mineral substance absorbs and promotes the metabolic gene of specific secondary substance the disease-resistant worm gene of functional gene wherein, resistance gene.
6. expression system, it contains among the claim 3-5 each recombinant nucleic acid sequence.
7. claim 1 or 2 promotor or its function equivalent, variant, perhaps fragment is expressed or the preferential purposes of expressing at the roots of plants internal specific driving functional gene.
8. the purposes of claim 7, promotes the gene that mineral substance absorbs and promotes the metabolic gene of specific secondary substance the disease-resistant worm gene of functional gene wherein, resistance gene.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200310119747 CN1624129A (en) | 2003-12-03 | 2003-12-03 | Specific promoter of root and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200310119747 CN1624129A (en) | 2003-12-03 | 2003-12-03 | Specific promoter of root and its application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1624129A true CN1624129A (en) | 2005-06-08 |
Family
ID=34761415
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200310119747 Pending CN1624129A (en) | 2003-12-03 | 2003-12-03 | Specific promoter of root and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1624129A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100345973C (en) * | 2004-11-18 | 2007-10-31 | 中国农业大学 | Process for expressing gene of superoxide nutase and its special carrier |
| CN101875937A (en) * | 2010-07-15 | 2010-11-03 | 福建农林大学 | Cloning of tobacco root-specific promoter and application thereof to transgenic plant |
| CN101831424B (en) * | 2009-09-28 | 2011-12-14 | 江苏省农业科学院 | Promoter for expressing specificity of plant tissue and later development and application thereof |
| CN102618543A (en) * | 2012-03-13 | 2012-08-01 | 青岛农业大学 | Root-specific and harm inducible promoter from glycine max |
| CN104673793A (en) * | 2013-11-27 | 2015-06-03 | 中国科学院上海生命科学研究院 | Specific promoter in leguminous plant root tissue and application thereof |
-
2003
- 2003-12-03 CN CN 200310119747 patent/CN1624129A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100345973C (en) * | 2004-11-18 | 2007-10-31 | 中国农业大学 | Process for expressing gene of superoxide nutase and its special carrier |
| CN101831424B (en) * | 2009-09-28 | 2011-12-14 | 江苏省农业科学院 | Promoter for expressing specificity of plant tissue and later development and application thereof |
| CN101875937A (en) * | 2010-07-15 | 2010-11-03 | 福建农林大学 | Cloning of tobacco root-specific promoter and application thereof to transgenic plant |
| CN101875937B (en) * | 2010-07-15 | 2012-05-09 | 福建农林大学 | Cloning of tobacco root-specific promoter and application thereof to transgenic plant |
| CN102618543A (en) * | 2012-03-13 | 2012-08-01 | 青岛农业大学 | Root-specific and harm inducible promoter from glycine max |
| CN104673793A (en) * | 2013-11-27 | 2015-06-03 | 中国科学院上海生命科学研究院 | Specific promoter in leguminous plant root tissue and application thereof |
| CN104673793B (en) * | 2013-11-27 | 2017-12-08 | 中国科学院上海生命科学研究院 | Legume root system tissue-specific promoter and its application |
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