CN102433338A - Plant stomata specific promoter and application thereof - Google Patents

Plant stomata specific promoter and application thereof Download PDF

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CN102433338A
CN102433338A CN2012100011172A CN201210001117A CN102433338A CN 102433338 A CN102433338 A CN 102433338A CN 2012100011172 A CN2012100011172 A CN 2012100011172A CN 201210001117 A CN201210001117 A CN 201210001117A CN 102433338 A CN102433338 A CN 102433338A
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plant
gbslsp
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CN102433338B (en
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肖兴国
韩蕾
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China Agricultural University
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Abstract

The invention discloses a plant stomata specific promoter and an application thereof. The promoter provided in the invention comprises the following nucleotide sequences: 1) a sequence 1 in a sequence table; 2) a nucleotide sequence still has the function of a stomata specific promoter after the 5' terminal and/or 3' terminal of the nucleotide sequence of the sequence 1 in the sequence table are deleted; and 3) a nucleotide sequence with the function of a stomata specific promoter when one or more deoxynucleotide residues of the nucleotide sequence in 1) or 2) is substituted, added or deleted. Experiments prove that: the promoter provided in the invention has excellent stomata specificity, and the specificity can be stably transferred to the next generation, which has important significance to molecular biological researches of stomata and guard cells, stomata motion and other theories, and has great application value and a wide market prospect in improving the photosynthetic efficiency of crops by culturing drought and disease resisting crops so as to improve yield, quality and other fields.

Description

A kind of plant stomata specificity promoter and application thereof
Technical field
The present invention relates to a kind of plant stomata specificity promoter and application thereof.
Background technology
Promotor (Promoter) is an important component part of genomic gene, and it is as " switch ", and whether the encoding sox under its control of basic decision on the transcriptional level is expressed, when expresses, where expressed and expression intensity.Generally it can be divided into three major types substantially: constitutive promoter, specificity promoter and inducible promoter (Wang Guanlin according to the promotor mode of action and function; Fang Hongjun, 2002, plant genetic engineering philosophy and technique (second edition); Beijing, science tech publishing house).But this classification is not absolute, and in some cases, one type promotor often has the characteristic of other type promotor concurrently, for example, and inducing reinforced composing type promoter (Xiao Xingguo etc., patent of invention number 200710177962.4).
Constitutive promoter (Constitutive promoter) is meant and can orders about the one type promotor of its control encoding sox down in Different Organs and/or the constant expression of tissue cardinal principle.Its feature is: receive the expression of the encoding sox of its control to have persistence, but do not have the space-time specificity; RNA and protein expression amount are constant relatively, do not receive inducing of extraneous factor.For example: cauliflower mosaic virus 35S promoter CaMV35S (Odell et al., Nature, 1985; 313:810-812), corn Ubiquitin promotor (Holtorf et al., Plant Mol.Biol., 1995; 29:637-646) with Actinl promotor (the McElroy et al of paddy rice; 1990, Plant Cell, 2:163-171).
Inducible promoter (Inducible promoter) is meant that the encoding sox that can respond some specific physics, chemistry and bio signal (being referred to as " elicitor " or " inducible factor ") and drive its control increases by one type of promotor of transcriptional level significantly.Being characterized as of it, under the condition that does not have inducible factor to exist, the encoding sox of its control is not expressed or is had only low-down expression (being also referred to as " background expressions "), but in case receive inducing of inducible factor, and the expression of gene amount is rapid and increase considerably.Inducible promoter is usually classified according to its inducement signal and is named, for example: fungal induction promotor, symbiotic bacterium evoked promoter, chemically inducible promoter, metals ion evoked promoter, photoinduction promoter, heat shock promoter and wound-induced promotor etc.
Organ and/or tissue-specific promoter (Organ-and/or Tissue-specific promoter); Can order about its control encoding sox down only at a certain of organism or organ and/or tissue that some is specific, perhaps only at a genoid of a certain or some specified phase expression of growing.Being characterized as of it receives the genetic expression of its control or adjusting to have temporal and spatial property, and often shows the characteristic of growing adjusting.For example, the root-specific promoter on the plant, blade specific promoter, flower specific promoter, pore specificity promoter, pore tapetum specific efficient promoter, pollen specific promoter, fruit-specific promoter, seed specific promoters, endosperm specificity promoter, cotton fiber specific property promotor and phloem specific promotor or the like.
Pore (Stomatal pore) is the door that plant and external environment are carried out gaseous interchange, also is the passage of moisture transpiration.Plant carries out the necessary CO of photosynthesis 2Rely on pore to get in the body, and the O that respiration produced 2It is external then to rely on pore to be rejected to.In addition, the moisture transpiration of plant also relies on pore as the principal passage.Therefore, the on-off control of pore the important physiological processes such as transpiration, photosynthesis and respiration of plant.
The pore of general plant is made up of a pair of stomata guard cell, also has subsidiary cell (Subsidiary cells) in some plant round this a pair of stomata guard cell, forms pore complex body (Stomatal complex) or claims stomatal apparatus (Stomaltal apparatus).Gas cell distribution mainly concentrates on blade at the over-ground part of plant.The stomata guard cell who forms pore is a kind of well differentiated cell, on most of Shuangzi plants and some monocotyledonss, gymnosperm, fern and mosses, is kidney shape, on grass and several kinds of sedges, then is dumb-bell shape.Stomata guard cell's the volume ratio leaf epidermal cell and the volume of mesophyll cell are much little.Its cell walls is named with the relative position of flanking cell by it: back of the body wall (Dorsal wal1) refers to the wall adjacent with epidermic cell; Stomach wall (Ventral wal1) is with the wall of carrying on the back place, relative, the near hole of wall; Outer side wall (Outer lateral wal1) is by the wall of blade outer surface; Inner side-wall (Inner lateral wal1) is by the wall of substomatic cavity.Stomata guard cell's wall is became uneven everywhere, and it is thin and stomach wall is thickeied especially generally to carry on the back wall.Like this, when cell turgor was big, the stomata guard cell was crescent moon, and pore is open; At cell turgor hour, the stomata guard cell is straight kidney shape or cylindrical, and stomatal closure (Wilmer and Fricker, 1996, Stomata, Ed Chapman and Hall, London, 1-375).The stomata guard cell changes the size of own vol and open and close that shape is regulated and control pore and stretching degree with the biology in the external environment of tackling its impression and abiotic stimulation (for example ABA, CO through self the change of turgescence size just 2With arid etc.) and the signal that transmits by self roots of plants (Bergmann and Sack FD, 2007, Annu Rev Plant Biol, 58:163-81).Cause this self the mechanism of change of change and own vol size that causes thus and shape of turgescence size; It is generally acknowledged; Mainly be the transportation of striding film ion and water (the Pandey et al. that some channel proteins through cytolemma and vacuole skin carry out; 2007, FEBS Lett, 581:2325-36; Schroeder et al., 2001, Annu Rev Plant Physiol Plant Mol Biol, 52:627-58).On C3 and C4 plant, can cause that the open main environment factor of pore has: blue light, ruddiness, high humidity and low CO 2(Marten et al, 2007, Plant J, 2007.50:129-139; Shimazaki et al, 2007, Annu Rev Plant Biol, 58:219-47); The main environment factor that can cause stomatal closure has: dark, arid, high CO 2With high ozone concentration (Sirichandra et al, 2009, J Exp Bot, 60:1439-63).In addition, endogenous hormones ABA can cause stomatal closure (Wilkinson and Davies, 2002, Plant Cell Env, 25:195-210; Israelsson M et al, 2006, Curr Opin Plant Biol; 9:654-63); Other hormone like growth hormone, kinetin, ethene, mustard acid and brassinolide, also all has certain influence (Acharya and Assmann to the switch of pore; 2009, Plant Mol Biol.69:4451-4462; Kim et al, 2010, Annu Rev Plant Biol, 61:561-591).
Stomatal movement (Kai Heguan) is a very complex physical process, and the someone thinks that it is a kind of active adjustment that plant conforms and changes, and also the someone thinks passive adjusting, and certain experimental result is all arranged as support.No matter be active adjustment or passive adjusting, people hope to start with from the motion of regulation and control air vent switch, improve saving water, resisting drought ability and the photosynthetic efficiency [Li Jun etc. of plant; Chinese science C collects life science; 2004,34 (4): 299-303], thus improve the yield and quality of plant, particularly crop.For this reason, evaluation, separation and functional study stomata guard cell specific gene and mainly the research of regulation and control person-promotor launch in the whole world.
Zhao etc. are separated to 3 * 10 from the Arabidopis thaliana of strain more than 22000 (Arabidopsis thaliana) 8Individual stomata guard cell's chloroplast(id) identifies 1734 kinds of unique albumen [Zhao et al.2008, Plant Cell, 20 (12): 3210-3226] thus.Some stomata guard cell's predominant expressions and/or specific expressed albumen and/or its encoding sox are like Arabidopis thaliana Akt1 (Sentenac et al, 1992; Science, 256:663-665), Kat1 (Anderson et al, 1992; PNAS, 89:3736-3740) and TPC1 (Rienmuller et al, 2010; Plant and Cell Physiol, 51:91548-91554) and yam Kst1 (Muller-Roeber et al, 1995; EMBO J 14:2409-2416) waits successively by evaluation and/or clone.Along with the clone of these genes, evaluation, separation, reorganization and functional study aspect that it mainly regulates and control zone-promotor have obtained tangible progress in recent years.For example, and the Kat1 promotor (Nakamura et al, 1995, Plant Physiol, 109:371-374), Kst1 (Plesch et al; 2000, Gene, 249:83～89), Abh1 promotor (Hugouvieux et al., Cell, 2001; 106:477-487), the Chl1 promotor (Guo et al., Plant Cell, 2001,13:1761-1777), Osm1 promotor (Zhu et al.; Plant Cell, 2002,14:3009-3028), Ost1 promotor (Mustilli et al., Plant Cell; 2002,14:3089-3099), Rac1 (Lemichez et al., Genes Dev.; 2001,15:1808-1816), SLAC1 promotor (Vahisalu et al, 2008, Nature; 452:487-491), CDeT6-19 promotor and Arabidopis thaliana monooxygenase gene CYP86A2 promotor (Taylor et al, 1995, Plant Journal, 7 (1): 129-134; Galbiati et al, 2008, Plant Journal, 53:750-762.Francia et al, 2008, Plant Signaling & Behavior, 3 (9): 684-686) etc.Yet,, also expression is in various degree arranged simultaneously at its tissue and organ though these promotors can drive the gene of its control in the surging expression of pore.That is to say that they also lack the specificity that pore is expressed.
Summary of the invention
The purpose of this invention is to provide a kind of plant stomata specificity promoter and application thereof.
Plant stomata specificity promoter provided by the present invention derives from sea island cotton (Gossypium barbadense L.), be following a) or b) or dna fragmentation c):
A) 3 ' end contains the 529-993 position nucleotide sequence (GbSLSP-F5) of sequence 1 in the ordered list at least; And from the 529th beginning of sequence 1, prolong to 5 of sequence 1 ' end, obtain length and be any dna fragmentation of 465 to 993bp according to the nucleotide sequence of sequence 1; Said dna molecular has promoter function;
B) and a) nucleotide sequence that limits has 75% or 75% above homology, and has the dna fragmentation of promoter function;
C) under stringent condition and a) or b) nucleotide sequence hybridization that limits, and have the dna fragmentation of promoter function.
Said stringent condition is at 2 * SSC, in the solution of 0.1%SDS, under 68 ℃, hybridizes and washes film 2 times.Each 5min.0.5X SSC in the solution of 0.1%SDS, is hybridized under 68 ℃ and is washed film 2 times.Each 15min.
Last of said promotor Nucleotide is the 993rd of sequence 1.
Said promotor is following 1)-3) in any:
1) 3 of nucleotide sequence ' end contains the 511-993 position nucleotide sequence (GbSLSP-F4) of sequence 1 in the ordered list at least; And from the 511st beginning of sequence 1, prolong to 5 of sequence 1 ' end, obtain length and be any dna fragmentation of 483 to 993bp according to the nucleotide sequence of sequence 1;
2) 3 of nucleotide sequence ' end contains the 401-993 position nucleotide sequence (GbSLSP-F3) of sequence 1 in the ordered list at least; And from the 401st beginning of sequence 1, prolong to 5 of sequence 1 ' end, obtain length and be any dna fragmentation of 593 to 993bp according to the nucleotide sequence of sequence 1;
3) 3 of nucleotide sequence ' end contains the 346-993 position nucleotide sequence (GbSLSP-F2) of sequence 1 in the ordered list at least; And from the 346th beginning of sequence 1, prolong to 5 of sequence 1 ' end, obtain length and be any dna fragmentation of 648 to 993bp according to the nucleotide sequence of sequence 1.
In an embodiment of the present invention, said promotor is specially following a1)-a5) in any:
A1) nucleotides sequence is classified the dna molecular shown in the sequence 1 (GbSLSP) in the sequence table as;
A2) nucleotides sequence is classified dna molecular (GbSLSP-F2) shown in the 346-993 position Nucleotide of sequence 1 in the sequence table as;
A3) nucleotides sequence is classified dna molecular (GbSLSP-F3) shown in the 401-993 position Nucleotide of sequence 1 in the sequence table as;
A4) nucleotides sequence is classified dna molecular (GbSLSP-F4) shown in the 511-993 position Nucleotide of sequence 1 in the sequence table as;
A5) nucleotides sequence is classified dna molecular (GbSLSP-F5) shown in the 529-993 position Nucleotide of sequence 1 in the sequence table as.
Plant stomata specificity promoter provided by the invention can also be following d) or e) or f) dna fragmentation:
D) 3 ' end contains the 346-634 position nucleotide sequence (GbSLSP-SH) of sequence 1 in the ordered list at least; And from the 346th beginning of sequence 1, prolong to 5 of sequence 1 ' end, obtain length and be any dna fragmentation of 289 to 634bp according to the nucleotide sequence of sequence 1; Last of said promotor Nucleotide is the 634th of sequence 1;
E) and d) nucleotide sequence that limits has 75% or 75% above homology, and has the dna fragmentation of promoter function;
F) under stringent condition and d) or the nucleotide sequence hybridization that e) limits, and have the dna fragmentation of promoter function.
Said stringent condition is at 2 * SSC, in the solution of 0.1%SDS, under 68 ℃, hybridizes and washes film 2 times.Each 5min.0.5X SSC in the solution of 0.1%SDS, is hybridized under 68 ℃ and is washed film 2 times.Each 15min.
In an embodiment of the present invention, said promotor is specially following b1) or b2:
B1) nucleotides sequence is classified the dna molecular (GbSLSP-SH) shown in the 1-634 position Nucleotide of sequence 1 in the sequence table as;
B2) nucleotides sequence classify as sequence 1 in the sequence table from dna molecular (GbSLSPF2-SH) shown in the Nucleotide of 346-634 position.
GbSLSP promotor (sequence 1 in the sequence table) is carried out 5 ' end deletion analyze the promoter sequence that still has pore specificity driving force that obtains.Wherein, Upper reaches 96bp still has the small segment of pore specificity promoter vigor to GbSLSP-F5 (the 529-993 position Nucleotide of sequence 1 in the sequence table), is the core fragment of GbSLSP promotor up to transcription initiation site (sequence 1 is from the 625th Nucleotide of 5 ' end in the sequence table) for 5 ' the end deletion of GbSLSP promotor.
GbSLSP (sequence 1 in the sequence table) is carried out the deletion of 3 ' end analyze the promoter sequence that still has pore specificity driving force that obtains.Wherein, GbSLSPF2-SH (the 346-634 position nucleotide sequence of sequence 1 in the sequence table) is that GbSLSP promotor 3 ' end deletion is up to still having the small segment of pore specificity promoter vigor apart from transcription initiation site (sequence 1 is from the 625th Nucleotide of 5 ' end in the sequence table) 9bp.
The GbSLSP promotor is made up of 993 deoxynucleotides; Its nucleotides sequence is classified sequence 1 in the sequence table as; Wherein 594-599 position deoxynucleotide is TATA box (TATA box); The 625th deoxynucleotide is transcription initiation site, 626-993 position deoxynucleotide is 5 ' the end non-translational region (5 '-UTR).
The GbSLSP-F2 promotor is made up of 648 deoxynucleotides, and its nucleotides sequence is classified the 346-993 position of sequence 1 in the sequence table as.Wherein the 594-599 position deoxynucleotide of sequence 1 is TATA box (TATA box), and the 625th deoxynucleotide is transcription initiation site, 626-993 position deoxynucleotide is 5 ' the end non-translational region (5 '-UTR).
The GbSLSP-F3 promotor is made up of 593 deoxynucleotides, and its nucleotides sequence is classified the 401-993 position of sequence 1 in the sequence table as.Wherein the 594-599 position deoxynucleotide of sequence 1 is TATA box (TATA box), and the 625th deoxynucleotide is transcription initiation site, 626-993 position deoxynucleotide is 5 ' the end non-translational region (5 '-UTR).
The GbSLSP-F4 promotor is made up of 483 deoxynucleotides, and its nucleotides sequence is classified the 511-993 position of sequence 1 in the sequence table as.Wherein the 594-599 position deoxynucleotide of sequence 1 is TATA box (TATA box), and the 625th deoxynucleotide is transcription initiation site, 626-993 position deoxynucleotide is 5 ' the end non-translational region (5 '-UTR).
The GbSLSP-F5 promotor is made up of 465 deoxynucleotides, and its nucleotides sequence is classified the 529-993 position of sequence table sequence 1 as.Wherein the 594-599 position deoxynucleotide of sequence 1 is TATA box (TATAbox), and the 625th deoxynucleotide is transcription initiation site, 626-993 position deoxynucleotide is 5 ' the end non-translational region (5 '-UTR).
The GbSLSP-SH promotor is made up of 634 deoxynucleotides, and its nucleotides sequence is classified the 1-634 position of sequence table sequence 1 as.Wherein the 594-599 position deoxynucleotide of sequence 1 is TATA box (TATAbox), and the 625th deoxynucleotide is transcription initiation site, 626-634 position deoxynucleotide is 5 ' the end non-translational region (5 '-UTR).
The GbSLSPF2-SH promotor is made up of 289 deoxynucleotides, and its nucleotides sequence is classified the 346-634 position of sequence table sequence 1 as.Wherein the 594-599 position deoxynucleotide of sequence 1 is TATA box (TATA box), and the 625th deoxynucleotide is transcription initiation site, 626-634 position deoxynucleotide is 5 ' the end non-translational region (5 '-UTR).
The clone obtains GdSLSP total length promotor respectively from sea island cotton (Gossypium barbadense), and it is carried out functional analysis, has produced the present invention.
A primer that contains the genetic stocks of above-mentioned promotor or obtain said promotor is to also belonging to protection scope of the present invention.
Said genetic stocks is expression cassette, recombinant expression vector, reorganization bacterium, transgenic plant cells or tissue or organ.
In said expression cassette, the downstream syndeton gene of said pore specificity promoter, or regulatory gene; Or the inverted defined gene of structure gene or regulatory gene; Perhaps can disturb the coding DNA of the little RNA of native gene expression, be used for the Drive Structure gene, or regulatory gene; Or the inverted defined gene of structure gene or regulatory gene, the expression of the little RNA of perhaps natural little RNA or synthetic.
Said recombinant expression vector is to contain above-mentioned expression cassette, such as but not limited to plasmid and virus.Said recombinant expression vector also can be the recombinant plant expression vector.Said recombinant plant expression vector contain above-mentioned expression cassette and can with described expression cassette pass on get into plant host cell, tissue or organ and offspring thereof and can or convenient at least described expression cassette be incorporated in host's the genome, it includes but not limited to binary vector, closes carrier altogether.In an embodiment of the present invention, said recombinant expression vector is specially MCS at the pBI121 plasmid and inserts the recombinant plasmid that the nucleotide sequence of said promotor obtains.
Said recombinant expression vector can transform plant organ or tissue or cell by conventional biological methods such as Ti-plasmids, Ri plasmid, plant viral vector, directly DNA conversion, microinjection, electricity led, agriculture bacillus mediated or particle gun through using, and obtains transgenic plant cells or tissue or organ and differentiation, regenerated whole plant and clone thereof or generation thus thereafter.In an embodiment of the present invention, concrete use is agrobacterium-mediated transformation.
In an embodiment of the present invention, said primer is to being specially following c1)-arbitrary right in c5):
C1) primer that dna fragmentation shown in the 7-27 position Nucleotide of dna fragmentation shown in the 7-30 position Nucleotide of sequence 3 and sequence 2 is formed in the sequence table is right;
C2) primer that dna fragmentation shown in the 7-21 position Nucleotide of dna fragmentation shown in the 7-30 position Nucleotide of sequence 3 and sequence 5 is formed in the sequence table is right;
C3) primer that dna fragmentation shown in the 7-28 position Nucleotide of dna fragmentation shown in the 7-30 position Nucleotide of sequence 3 and sequence 6 is formed in the sequence table is right;
C4) primer that dna fragmentation shown in the 7-34 position Nucleotide of dna fragmentation shown in the 7-30 position Nucleotide of sequence 3 and sequence 7 is formed in the sequence table is right;
C5) primer that dna fragmentation shown in the 7-27 position Nucleotide of dna fragmentation shown in the 7-22 position Nucleotide of sequence 8 and sequence 2 is formed in the sequence table is right.
The application that said promotor starts in plant in the destination gene expression also belongs to protection scope of the present invention.
Said plant is a terrestrial plant, and preferably dicotyledons is more preferably Arabidopis thaliana or tobacco; It is said that to be expressed as pore specific expressed.
The experiment proof; Promotor provided by the present invention (comprising GbSLSP-F2, GbSLSP-F3, GbSLSP-F4) all can start reporter gene GUS and/or GFP (green fluorescent protein) specifically expressing in the pore of tobacco and/or Arabidopis thaliana, explains that promotor provided by the present invention has good pore specificity.Simultaneously prove also that through related experiment this specificity can stably pass to the offspring.
The coding DNA that promotor of the present invention downstream is connected the little RNA that native gene, foreign gene and inverted defined gene thereof maybe can disturb native gene to express (comprise natural with little RNA synthetic); The construction expression box also connects with different expression vector; Be transformed in the plant; Utilize its pore opposite sex expression activity, said native gene, foreign gene and the inverted defined gene thereof of specific expressed its control or guidance or little RNA in pore.The expression of the little RNA that in transfer-gen plant, makes its native gene, foreign gene and inverted defined gene thereof maybe can disturb native gene to express (comprise natural with little RNA synthetic) is confined in the pore, has got rid of these genes in the expression of planting other tissue of body or organ.
Utilize the specific expressed activity of pore of promotor of the present invention; With containing promotor of the present invention or this promotor and native gene and foreign gene and inverted defined gene thereof, can disturbing expression vector transformed host cell or the host tissue and the progeny cell thereof of little RNA (comprise natural with the little RNA synthetic) coding DNA of native gene expression; And the complete transfer-gen plant of regenerating thus, cultivate drought-enduring, anti-high or low concentration C O 2, resistance to fungal disease and high photosynthesis efficiency plant or crop varieties.Said plant is a polycarpeae; Include but not limited to terrestrial plant, angiosperm and gymnosperm, monocotyledons and dicotyledons, herbage, vine and xylophyta, gardens gardening plant and farm crop and forage grass, yearly plant and perennial plant and sexual and asexually propagated plant, be preferably terrestrial plant.
Pore specificity promoter of the present invention can be used for the gene that functional analysis, evaluation and separating plant pore formed and grew necessary gene and regulation and control stomatal movement, can be used to cultivate simultaneously drought-enduring, anti-high or low CO 2, resistance to fungal disease, high photosynthesis efficiency and high yield and high quality plant or crop varieties.Specifically can be connected allos or homologous gene or its inverted defined gene or little RNA coding DNA in pore specificity promoter of the present invention downstream; And be building up to plant expression vector; Transform host plant; Express target protein, inhibition or jamming target gene transcription or translation at its pore, to open or close the switch degree of pore or enhancing or reduction pore.The isolating pore specificity promoter of the present invention to the functional analysis of plant stomata g and D gene and evaluation, cultivate drought-enduring, anti-high or low CO 2, resistance to fungal disease, high photosynthesis efficiency and high yield and high quality plant or crop varieties etc. all have great application prospect.
This not only has great importance the present invention to the theoretical investigation of pore and stomata guard cell's molecular biology research and stomatal movement etc., and goes to improve the photosynthetic efficiency of crop cultivating drought resisting and disease-resistant crop and then aspect such as improve the yield and quality has great application value and vast market prospect.
Description of drawings
Fig. 1 is the PCR product electrophorogram of GbSLSP promotor full-length clone.Wherein swimming lane M is a molecular weight marker: λ DNA/EcoRI+Hind III; Swimming lane 1 is primer 1 and the two primer PCR amplified productions of primer 2; Swimming lane 2 is primer 1 single primer PCR amplified production; Swimming lane 3 is a primer 2 list primer PCR amplified production; Swimming lane 4 is two primer PCR amplification contrasts of no template DNA.
Fig. 2 is the GbSLSP promotor full length sequence figure that adds restriction enzyme site.Wherein, underscore partly for the restriction enzyme enzyme recognition site that adds (5 ' hold III into Hind, 3 ' hold I into BamH); The square frame place is the TATA box; " A " of overstriking band shading is transcription initiation site; The base that adds bold Italic is the Hind III restriction enzyme site of sequence self.
Fig. 3 is the plant expression vector collection of illustrative plates that pore specificity promoter GbSLSP drives reporter gene GUS or GFP.A is the plant expression vector that GbSLSP drives reporter gene GUS, and wherein pGbSLSP-GUS representes pBI-GbSLSP-GUS, and B is the plant expression vector that GbSLSP drives reporter gene GFP, and wherein pGbSLSP-GFP representes pBI-GbSLSP-GFP.
Fig. 4 is for changeing the tobacco T of pBI-GbSLSP-GUS 0Identify figure for plant PCR.Wherein, swimming lane M is DNA sized molecules mark λ DNA/EcoR I+Hind III; Swimming lane 1-8 is transgene tobacco T 0Generation 8 individual plants independently; Swimming lane 9 is vector plasmid (pBI-GbSLSP-GUS) contrast; Swimming lane 10 contrasts for not genetically modified tobacco NC89; Swimming lane 11 is the contrast of template for zero(ppm) water.
Fig. 5 is for changeing pBI-GbSLSP-GUS Arabidopis thaliana T 1PCR for plant identifies figure.Wherein, swimming lane M is DNA sized molecules mark λ DNA/EcoR I+Hind III; Swimming lane 1-8 is transgenic arabidopsis T 1In 8 of generations, are individual plant (the corresponding T of swimming lane 1,4,5,6,7 wherein independently 1For the Arabidopis thaliana plant is the GbSLSP positive plant); Swimming lane 9 is vector plasmid (pBI-GbSLSP-GUS) contrast; Swimming lane 10 contrasts for not genetically modified Arabidopis thaliana; Swimming lane 11 is the contrast of template for zero(ppm) water.
Fig. 6 is for changeing pBI-GbSLSP-GUS tobacco T 0GUS colored graph for the blade disk of plant.Wherein, A is for changeing pBI-GbSLSP-GUS tobacco T 0Leaf disc for plant; A is the local enlarged photograph of leaf disc shown in the A.
Fig. 7 is for changeing pBI-GbSLSP-GUS tobacco T 1For plant seedling GUS colored graph.Wherein A is pBI-GbSLSP-GUS tobacco T 1GUS dyeing photo for the whole strain of seedling; A is the painted local enlarged photograph of true leaf GUS of the corresponding strain of seedling shown in the A.
Fig. 8 is for changeing pBI-GbSLSP-GUS Arabidopis thaliana T 1GUS colored graph for the plant Different Organs.Wherein, A is an inflorescence; B is flower; C is a column cap; D is a flower pesticide; E is a stem; F is a bennet; G is a calyx; H is the fruit folder; I is a blade; J is the local enlarged photograph of I.
Fig. 9 is for changeing pBI-GbSLSP-GFP genetic tobacco T 0GFP confocal laser scanning microgram for plant leaf.
Figure 10 is that 5 of pore specificity promoter GbSLSP ' end deletion and 3 ' end is deleted the synoptic diagram that concerns between each fragment (GbSISP-F2, GbSISP-F3, GbSLSP-F4, GbSLSP-F5, GbSLSP-SH and GbSISPF2-SH) and the total length promotor (GbSLSP) behind the different lengths.Wherein, the data-X that is positioned at transcription initiation site (TSS) upper reaches (that is: left end) representes the length of GbSLSP through the residual nucleotide fragments in 5 ' end deletion back.
Each fragment behind the deletion of Figure 11 pore specificity promoter GbSLSP 5 ' end and the 3 ' end deletion different lengths is connected the restriction enzyme digestion and electrophoresis figure on the pBS-T carrier.Wherein, swimming lane M1 and M2 are respectively dna molecular amount mark λ DNA/EcoRI+HindIII and Marker I (100bp mark); Swimming lane 1 to 7 is respectively Hind III and the BamH I double digestion result of pBS-GbSLSP, pBS-GbSLSP-SH, pBS-GbSLSP-F2, pBS-GbSLSP-F3, pBS-GbSLSP-F4, pBS-GbSLSP-F5.
Figure 12 is that each fragment behind the deletion of pore specificity promoter GbSLSP 5 ' end and the 3 ' end deletion different lengths is connected to the PCR evaluation electrophorogram on the pBI121 carrier.Wherein, swimming lane M1 and M2 are respectively dna molecular amount mark λ DNA/EcoRI+HindIII and Marker 1 (100bp mark); Swimming lane 1 to 7 is respectively the PCR product electrophorogram of pBI-GbSLSP, pBI-GbSLSPF2, pBI-GbSLSP-SH, pBI-GbSLSPF3, pBI-GbSLSPF4, pBI-GbSLSPF5, pBI-GbSLSPF2-SH.
Figure 13 is for changeing GbSLSPX-GUS (F=2,3,4,5 ,-SH or F2-SH) genetic tobacco T 0For the GUS colored graph of plant leaf with flower.Wherein, F2 representes that GbSLSPF2-GUS is at transgene tobacco T 0Drive the GUS colored graph that gus gene is expressed for plant; F3 representes that GbSLSPF3-GUS is at transgene tobacco T 0Drive the GUS colored graph that gus gene is expressed for plant; F4 representes that GbSLSPF4-GUS is at transgene tobacco T 0Drive the GUS colored graph that gus gene is expressed for plant; F5 representes that GbSLSPF5-GUS is at transgene tobacco T 0Drive the GUS colored graph that gus gene is expressed for plant; F-SH representes that GbSLSP-SH-GUS is at transgene tobacco T 0Drive the GUS colored graph that gus gene is expressed for plant; F2-SH representes that GbSLSPF2-SH-GUS is at transgene tobacco T 0Plant drives the GUS colored graph that gus gene is expressed.F2 to F2-SH in totally 6 series of drawing, all is that left side (A) be the GUS colored graph of leaf dish, and middle (a) be the partial enlarged drawing of left side (A) picture, and right side (B) is colored GUS colored graph.
Figure 14 is for changeing GbSLSPX-GUS (F=2,4,5 ,-SH or F2-SH) genetic tobacco T 1GUS colored graph for seedling.Wherein, F2 representes that GbSLSPF2-GUS is at transgene tobacco T 1Drive the GUS colored graph that gus gene is expressed for plant; F4 representes that GbSLSPF4-GUS is at transgene tobacco T 1Drive the GUS colored graph that gus gene is expressed for plant; F5 representes that GbSLSPF5-GUS is at transgene tobacco T 1Drive the GUS colored graph that gus gene is expressed for plant; F-SH representes that GbSLSP-SH-GUS is at transgene tobacco T 1Drive the GUS colored graph that gus gene is expressed for plant; F2-SH representes that GbSLSPF2-SH-GUS is at transgene tobacco T 1Drive the GUS colored graph that gus gene is expressed for plant.In 5 series of drawing of above-mentioned F2 to F2-SH, all be that top (A) is transgene tobacco T 1The GUS colored graph of the whole strain of plant seedling, below (B) is the true leaf partial enlarged drawing of top (A).
Figure 15 is for changeing GbSLSPX-GUS (X=2,5) gene Arabidopis thaliana T 1For the specific expressed GUS colored graph of the pore of plant Different Organs.Wherein, F2 representes that GbSLSPF2-GUS is at transgenic arabidopsis T 1Drive the GUS colored graph that gus gene is expressed for plant; F5 representes that GbSLSPF5-GUS is at transgenic arabidopsis T 1Drive the GUS colored graph that gus gene is expressed for plant.A is a titbit; B is flower; C is a column cap; D is a flower pesticide; E is a stem; F is a bennet; G is a calyx; H is the fruit folder; I is a blade.
Figure 16 is for changeing GbSLSPF2-GUS gene Arabidopis thaliana T 2For seedling GUS colored graph.Wherein, A is the whole strain of seedling; B is a cotyledon; B is the partial enlarged drawing of B; C is a true leaf; C is the partial enlarged drawing of C.
Figure 17 changes GbSLSPX-GFP (F=2,3,4,5 ,-SH or F2-SH) genetic tobacco T 0For the specific expressed GFP confocal laser scanning microgram of plant leaf pore.Wherein, A representes that GbSLSPF2-GFP is at transgene tobacco T 0Drive the confocal laser scanning microgram of GFP genetic expression for plant; B representes that GbSLSPF3-GFP is at transgene tobacco T 0Drive the confocal laser scanning microgram of GFP genetic expression for plant; C representes that GbSLSPF4-GFP is at transgene tobacco T 0Drive the confocal laser scanning microgram of GFP genetic expression for plant; D representes that GbSLSPF5-GFP is at transgene tobacco T 0Drive the confocal laser scanning microgram of GFP genetic expression for plant; E representes that GbSLSP-SH-GFP is at transgene tobacco T 0Drive the confocal laser scanning microgram of GFP genetic expression for plant; F representes that GbSLSPF2-SH-GFP is at transgene tobacco T 0Drive the confocal laser scanning microgram of GFP genetic expression for plant.
Figure 18 is for being used to make up the nucleotide sequence of the used GFP gene of GbSLSPX-GFP (F=2,3,4,5 ,-SH or F2-SH) serial carrier.
Embodiment
Promotor nucleotide sequence described in the present invention can be that wherein one or more Nucleotide replace, the nucleotide sequence of disappearance, insertion or inversion; Promptly artificial mutant or " natural " two mutants of isolating nucleotide sequence, it keeps its promoter function; It can also be the fusion sequence of the conservative regulating and controlling sequence (" motif " or " box ") of described nucleotide sequence and other promoter sequence or promoter region." promotor " described in the present invention is for having the promotor of TATA box (TATAbox); TATA box or zone similarity are positioned transcription initiation site (TSS is "+1 " on the Nucleotide counting) upper reaches and have the guide RNA polysaccharase on the tram, to start the function of transcribing, but be not limited near the part this zone; In addition, it also can contain being used to of being associated with protein except that RNA polymerase regulate another of expression must the zone." promoter region " described in the present invention is defined as the zone of at least 10 Nucleotide in zone and transcription initiation site downstream that contain the promotor that defines in the preceding text." promoter activity " described in the present invention but be meant when be connected to the downstream of promotor with the phraseology of certain gene; And import among the host; This host shows to have the ability during with function of producing this gene product in the host or outside the host, and this promotor has the activity of startup.Usually; Be will encode easily qualitative or detection by quantitative proteinic gene (for example; Reporter gene) is connected to the downstream of this promotor; This gene is imported in the host, and detect expressed protein, can confirm the active of specific promotor or not have the perhaps effectiveness of this promotor of this promotor.Structure gene described in the present invention is meant one section nucleotide sequence of can encode certain protein or other active substance function, comprises RNA or dna sequence dna.Described regulatory gene is meant one section nucleotide sequence that certain RNA or protein or other active substance of its coding can be regulated or regulate and control the expression of other structure gene, comprises RNA or dna sequence dna.Described just gene comprises above-mentioned structure gene and regulatory gene.Described inverted defined gene is meant RNA complementary RNA or the dna sequence dna with said structure gene or regulatory gene coding.Described native gene is meant the gene from host self, comprises RNA or dna sequence dna.Said foreign gene is meant any one section nucleotide sequence, and has coding certain protein or other active substance function, comprises natural and RNA synthetic or dna sequence dna, and this sequence does not combine in normal circumstances with the pore specificity promoter.Described little RNA is meant that separation is from RNA sequence fragment organism or synthetic; Its length is generally 20-26 deoxynucleotide or after importing the dormitory cell, can be sheared into the RNA fragment of 20-26 deoxynucleotide, and itself is nontoxic or toxicity is extremely low to organism.
That conversion described in the present invention is meant is well known in the prior art, can be with any methods for plant transformation of exogenous gene transfered plant cell or plant tissue, like agrobacterium-mediated transformation and particle gun etc.
Host cell described in the present invention or host tissue or host's organ and progeny cell thereof be meant all vegetable cells or plant tissue or plant organ or by these cells, tissue or organ through tissue differentiation or asexual embryo regeneration and the whole plant (comprising seed) of fully-developed.
Term " nucleotide sequence " or " nucleotide sequence " refer to contain the sequence of naturally occurring Nucleotide or nucleoside monomers.This sequence also comprises the monomer of the non-natural existence with identity function or modification or the substituted sequence of its part.
Term " pore specificity promoter " be meant can instruct its control gene down only pore express and other of planting body organize do not express or according to the ordinary method detection less than expression promoter.
Term " sequence with 75% or 75% above homology " is meant compares same or analogous those nucleotide sequences of the nucleotide sequence that has more than 75% or 75% or nucleotide sequence with the nucleotide sequence of above-mentioned promotor; These sequences play a role with essentially identical mode and can to drive the pore of its downstream gene specific expressed; The difference of sequence possibly be because modification or the sudden change on the local structure comprises artificial mutation and unartificial sudden change in they and the above-mentioned promotor.
Term " rigorous hybridization conditions " is meant well known by persons skilled in the art, perhaps can be at molecular biology or genetically engineered experiment guide, and " Molecular Cloning " (3 for example RdEd) (Sambrook etc., Cold Spring Harbor Laboratory, New York; 2001) find in the general scheme in (hereinafter to be referred as " " molecular cloning " third edition "); Be specially at 2 * SSC, in the solution of 0.1%SDS, under 68 ℃, hybridize and wash film 2 times.Each 5min.0.5X SSC in the solution of 0.1%SDS, is hybridized under 68 ℃ and is washed film 2 times.Each 15min.
Employed experimental technique is ordinary method like no specified otherwise among the following embodiment.
Used material, reagent etc. like no specified otherwise, all can obtain from commercial sources among the following embodiment.
Tobacco T 0Transfer-gen plant and the clone thereof that is obtained by callus, T are shown in representative 1Expression T 0The seed that produces for selfing reaches plant and clone by it grew up to; Arabidopis thaliana T 1Plant and the clone thereof that forms through the seed germination that produces after directly " dipping in flower " infects shown in representative.
The clone of embodiment 1, sea island cotton pore specific promoter SLSP (GbSLSP) with separate
1, the extraction of the total DNA of sea island cotton
Material is taken from sea island cotton (Gossypium barbadense L.) kind " SHZ2-214 " (Shihezi Univ buys from Xinjiang), and leaflet tablet 1-2g extracts total DNA with CTAB method (" molecular cloning " third edition).Get 1-5 μ l DNA sample, measure its concentration and purity with ultraviolet spectrophotometer, the agarose gel electrophoresis with 0.8% detects DNA purity and integrity.With the DNA that extracts in-20 ℃ of preservations.
2, the recovery of pcr amplification and amplified fragments
Design and synthesize following two primers (primer 1 and primer 2) amplification sea island cotton pore specificity promoter; Later for ease clone and structure have added the recognition sequence site (the underscore sequence in the following primer sequence) of restriction enzyme Hind III and BamH I respectively at 5 of two primers ' end
Primer 1:5 '- AAGCTTACAACTTTTCTCTACCAATCA-3 ' ( Hind III) (sequence 2); Primer 2: 5 '- GGATCCGCTAGAGAAAAATGGGAAGGTGAG-3 ' ( BamH I) (sequence 3).
The cotton genomic dna that extracts with step 1 is a template, carries out the PCR reaction:
Reaction system is: template DNA 800ng; 10 * Ex buffer, 2 μ l; DNTP mixture (2.5mM) 2 μ l; ExTaq DNA Polymerase (5U/ μ l) 0.5 μ l; Primer 1 (10 μ M) 2 μ l; Primer 2 (10 μ M) 2 μ l; Aseptic double distilled water (sddH 2O) complement to 20 μ l.
PCR response procedures: 94 ℃ of preparatory sex change 5min of elder generation; 94 ℃ of sex change 30s then, 60 ℃ of annealing 30s, 72 ℃ are extended 1min, 30 circulations.
PCR result is as shown in Figure 1; After the PCR reaction is accomplished; Get 15 μ l amplified productions through 1.0% agarose gel electrophoresis; Uv lamp is positioned at the specific fragment about dna marker 1000bp with careful cutting-out of disposable blade down fast, reclaims test kit with dna fragmentation and reclaims and purifying (Beijing ancient cooking vessel state Company products), is dissolved in 50 μ l ddH 2Among the O ,-20 ℃ of preservations are subsequent use.
3, reclaim segmental subclone and order-checking
Adopt two step connection methods that the fragment that above-mentioned steps 2 reclaims is inserted into carrier pBS-T carrier (sky, Beijing root Company products).Concrete operations are: with the fragment that Hind III and BamH I double digestion above-mentioned steps 2 reclaim, obtain two bands (containing a HindIII restriction enzyme site owing to reclaim fragment (sequence is as shown in Figure 2) inside) that size is about 650bp and 350bp.Return the fragment (fragment between Hind III and the BamH I) that size is about 650bp earlier, insert pBS-T carrier, form pBS-Gb650 through the HindIII-BamHI double digestion; Reclaim the fragment that two sizes between the Hind III are about 350bp then, insert pBS-Gb650, form pBS-Gb650+350, identify direction of insertion through order-checking through Hind III single endonuclease digestion.
The recombinant plasmid that obtains (pBS-Gb650+350) is transformed into coli strain DH5 α competent cell through " freeze-thaw method ".The DH5 α that transforms scribbles on the surface on the LB solid medium that contains penbritin 50mg/L of IPTG and X-gal in 37 ℃ of incubated overnight; The white colony of growing on the picking flat board inserts in the LB liquid nutrient medium of penbritin 50mg/L in 37 ℃ of incubated overnight.When bacterial concentration reaches OD 600It is 0.8 o'clock; Centrifugal collection thalline; Extract plasmid by a small amount of alkaline lysis (" molecular cloning " third edition); Behind Restriction Enzyme Hind III and BamH I double digestion, use 1.0% agarose gel electrophoresis, visible molecular size approximately is respectively 3000bp (carrier band), 650bp (object tape 1) and 350bp (object tape 2) three bands under uv lamp.Enzyme is cut plasmid that the correct clone of checking and clone thus extract to be delivered commercial order-checking company and checks order.The carrier called after pBS-GbSLSP that order-checking is correct wherein inserts fragment (object tape 1+ object tape 2=1000bp) called after GbSLSP.The sequencing result of GbSLSP is as shown in Figure 2, contains the sequence of primer 1 and primer 2 respectively at 5 ' end and 3 ' end, shows to contain above-mentioned dna fragmentation GbSLSP by the sea island cotton amplification among the pBS-GbSLSP really.The nucleotides sequence of GbSLSP is classified sequence 1 in the sequence table as.
Embodiment 2, sea island cotton pore specificity promoter (GbSLSP) drive the structure of gus gene or GFP gene plant expression vector
Two the step connection methods: with pBS-GbSLSP with restriction enzyme Hind III and BamH I double digestion after; Use 1.0% agarose gel electrophoresis; Cut two object tapes of about 650bp and about 350bp, reclaim test kit (Beijing ancient cooking vessel state Company products) with dna fragmentation and reclaim and purifying.The object tape (object tape 1) of about 650bp behind the purifying is linked to each other the recombinant plasmid called after pBI-Gb650 that obtains with the big fragment (being approximately 13.9kb) of the plant expression vector pBI121 plasmid (former Clontech Company products) of process Hind III and BamH I double digestion; Object tape (object tape 2) with about 350bp behind the purifying links to each other with the recombinant plasmid pBI-Gb650 of process Hind III single endonuclease digestion again, obtains transforming and uses recombinant plasmid pBI-Gb650+350.
The above-mentioned conversion that obtains is transformed into coli strain DH5 α with recombinant plasmid (pBI-Gb650+350) with " freeze-thaw method " (" molecular cloning " third edition).The DH5 α that transforms scribbles on the surface on the LB solid medium that contains kantlex 50mg/L of IPTG and X-gal in 37 ℃ of incubated overnight; Single bacterium colony of growing on the picking flat board inserts in the LB liquid nutrient medium that contains kantlex 50mg/L and cultivates in 37 ℃ of shaken overnight.When bacterial concentration reaches OD 600During value 0.6-0.8; Centrifugal collection thalline; By above-mentioned a small amount of alkaline lysis method of extracting plasmid, behind restriction enzyme Hind III and BamH I double digestion, at 1.0% agarose gel electrophoresis; Visible molecular size is about three bands of 13.9kb (pBI121 carrier band), 650bp (object tape 1) and 350bp (object tape 2) respectively under uv lamp, and carries out sequence verification.Enzyme is cut and order-checking shows and contains pore specificity promoter GbSLSP fragment (sequence 1 in the sequence table) and the pBI121 enzyme is cut the big segmental recombinant expression vector called after pBI-GbSLSP-GUS that obtains.Equally, substitute the GUS (restriction enzyme BamH I and Sac I double digestion) among the pBI-GbSLSP-GUS, obtain plant expression vector pBI-GbSLSP-GFP with GFP.Concrete grammar is following: GFP gene (sequence is shown in figure 18) the PCR product that two ends is had BamH I and a Sac I be connected with the big fragment that Sac I double digestion pBI-GbSLSP-GUS obtains with BamH I, obtain pBI-GbSLSP-GFP.
Gus gene in recombinant expression vector pBI-GbSLSP-GUS and the GFP gene in recombinant expression vector pBI-GbSLSP-GFP all need pore specific promoter GbSLSP to drive expression.Constructed plant binary expression vector pBI-GbSLSP-GUS and the collection of illustrative plates of pBI-GbSLSP-GFP are seen Fig. 3.
The acquisition and the evaluation of embodiment 3, commentaries on classics GbSLSP tobacco and Arabidopis thaliana
1, the evaluation of conversion of Agrobacterium and transformant
Competent cell with CaCl2 method (" molecular cloning " third edition) preparation agrobacterium tumefaciens bacterial strain LBA4404 and GV3101 (U.S. Life Technology Company products).Utilize freeze-thaw method (" molecular cloning " third edition) two recombinant expression vectors (pBI-GbSLSP-GUS and pBI-GbSLSP-GFP) of embodiment 2 gained to be changed over to the LBA4404 competent cell of preparation; Simultaneously, change the pBI-GbSLSP-GUS carrier over to the GV3101 competent cell.With the LBA4404 inoculation that transforms to the YEB solid medium that contains Streptomycin sulphate (Str) 100mg/L and kantlex (Kan) 50mg/L; With the GV3101 inoculation that transforms to the YEB solid medium that contains Rifampin (Rif) 25mg/L and kantlex (Kan) 50mg/L; Place 28 ℃, in the dark cultivate 48-72h; Single bacterium colony on two flat boards of picking inserts separately and contains corresponding antibiotic YEB liquid nutrient medium respectively, cultivates 28 ℃ of shaken overnight.When the concentration of culture reaches OD600 value 0.4-0.6; Bacterium liquid (1.5-2ml) takes a morsel; By above-mentioned a small amount of alkaline lysis method of extracting plasmid; Identify that with restriction enzyme Hind III and BamH I double digestion at 1.0% agarose gel electrophoresis, visible molecular size approximately is respectively three bands of 13.9kb (carrier band), 650bp (object tape 1) and 350bp (object tape 2) under uv lamp.
According to contained goal gene (GUS or GFP) and transform the different of bacterial strain (LBA4404 and GV3101), enzyme cut and check order identify and show the LBA4404 positive colony called after LBA4404/pBI-GbSLSP-GUS that contains the segmental pBI-GbSLSP-GUS of changing over to of GbSLSP, enzyme cut and checked order identify and show the LBA4404 positive colony called after LBA4404/pBI-GbSLSP-GFP that contains the segmental pBI-GbSLSP-GFP of changing over to of GbSLSP, enzyme cut and checked order identify and show the GV3101 positive colony called after GV3101/pBI-GbSLSP-GUS that contains the segmental pBI-GbSLSP-GUS of changing over to of GbSLSP.
2, the acquisition of transgene tobacco
1) preparation of Agrobacterium
Picking carries the single bacterium colony of Agrobacterium LBA4404 of plant expression vector pBI-GbSLSP-GUS or pBI-GbSLSP-GFP respectively; Being inoculated in 5ml contains in the YEB liquid nutrient medium of Streptomycin sulphate (Str) 100mg/L and kantlex (Kan) 50mg/L; 28 ℃ of shaking culture incubated overnight; Get the activatory agrobacterium liquid, join in the YEB liquid nutrient medium that contains Streptomycin sulphate (Str) 100mg/L and kantlex (Kan) 50mg/L, continue to be cultured to OD in 1: 100 ratio 600Value is 0.4-0.6; The centrifugal 5min of 5000rpm collects thalline; (MS inorganic salt+B5 VITAMINs+inositol 0.1g/L+ sucrose 30g/L pH5.80) wash thalline once, and it is diluted in the 1/2MS liquid nutrient medium of centrifugal preceding 3 times of volumes of bacterium liquid, prepare to infect usefulness with the 1/2MS0 liquid nutrient medium.
2) regeneration of conversion of tobacco and transformed plant
The conversion of tobacco according to leaf dish method (Horsch RB etc., 1985, Science 227:1229-1231) carries out.Concrete operations are: choose tobacco (kind " NC89 ", seed is available from the Chinese Academy of Agricultural Sciences) aseptic seedling of about 30 days seedling ages, downcut fresh and tender dark green blade, produce leaf dish explant with the punch tool of diameter 9mm; Freshly prepd explant is dropped into above-mentioned steps 1) in the off-the-shelf Agrobacterium bacterium liquid, infect 15min; Take out the leaf dish; Absorb the remaining Agrobacterium bacterium liquid of leaf panel surface with autoclaved thieving paper; (MS inorganic salt+B5 VITAMINs+inositol 0.1g/L+BA 2.0mg/L+IAA 0.5mg/L+ sucrose 30g/L+ agar powder 0.7% pH5.8) are gone up the dark place and were cultivated altogether 2 days to place solid regenerated substratum; Forward the leaf dish of cultivating altogether to contain Pyocianil (Carb) 500mg/L and Kan 50mg/L solid regenerated substratum and carry out screening and culturing, every separated 2-3 week is changed a subculture, and reduces Carb to 200mg/L gradually.When treating Kan resistant buds length to 1-1.5cm; Its cutting-out is changed to solid root media (the MS inorganic salt+B5 VITAMINs+inositol 0.1g/L+IAA0.5mg/L+ sucrose 30g/L+ agar powder 0.7% that contain Carb 200mg/L and Kan 50mg/L; PH5.80) go up root induction, obtain complete transgene tobacco T with kalamycin resistance 0For plant.
3) conversion of Arabidopis thaliana and screening
The conversion of Arabidopis thaliana is dipped in method (Clough and Bent, 1998, Plant Journal, 16 (6): 735-743) carry out through flower.Picking carries the single bacterium colony of Agrobacterium GV3101 of plant expression vector pBI-GbSLSP-GUS, is inoculated in 5ml and contains in the YEB liquid nutrient medium of Rifampin (Rif) 25mg/L and Kan 50mg/L, and 28 ℃ of shaking culture are spent the night.Get the Agrobacterium that activation is spent the night, join in the fresh YEB liquid nutrient medium that contains Rif 25mg/L and Kan 50mg/L, continue to be cultured to OD in 1: 100 ratio 600Value is approximately 1.2-1.6, and the centrifugal 5min of 5000rpm collects the about 30mL of thalline; Permeate the resuspended thalline of damping fluid (1 * MS macroelement, 5% sucrose) with 50mL, add the Selwet L-77 of 10 μ L 0.02%; Mixing divides in 5mL Eppendorf (EP) pipe of packing into every pipe 3mL.When infecting, Arabidopis thaliana inflorescence to be infected is inserted in the bacterium liquid, infect 5min.After infecting, on the pallet that the Arabidopis thaliana plant is kept flat, cover preservative film and preserve moisture, recover normal illumination behind 16 ℃ of dark condition held 24h and cultivate, results seed (transgenic T 1For seed).The seed of keeping well is transferred in the 1.5mL EP pipe, adds the ethanol of 1mL 75%, mixing, surface sterilization 1min.Then, outwell ethanol, add 0.5% Youxiaolin 1mL, surface sterilization 10min, during constantly shake up.Outwell thimerosal, add the 1mL sterilized water, mixing is placed 5min.Repeat above-mentioned steps and give seed disinfection 2-3 time, the seed that disinfects evenly is layered on the flat board of (MS+Kan 50mg/L) on the screening culture medium, put 4 ℃ of vernalization 2-4d; Transfer to again in the incubator and cultivate (22 ℃, 16h illumination/8h is dark), behind the cultivation 10-14d; Select and grow true leaf and take root normal plant; Tentatively be decided to be transfer-gen plant, move into (nutrition soil: grow two to three months results T in soil vermiculite=1: 1) 2For seed.
4) transgene tobacco T 0For plant and Arabidopis thaliana T 1Molecular Identification for plant
To step 2) and 3) the tobacco T that obtains 0Generation and Arabidopis thaliana T 1Carrying out PCR for plant identifies.Extract the above-mentioned tobacco T that obtains according to SDS method and CTAB (" molecular cloning " third edition) with Kan resistant gene 0For plant, Arabidopis thaliana T 1For plant and not genetically modified contrast strain (tobacco bred " NC89 "; Arabidopis thaliana " Colombia " wild-type) blade genomic dna; With pore specificity promoter GbSLSP two ends Auele Specific Primer (primer 1 and primer 2) carried out pcr amplification, the result shows, transgene tobacco T 0For (Fig. 4) and Arabidopis thaliana T 1In generation (Fig. 5), all increases and obtains the object tape that size is about 1000bp, and unconverted adjoining tree does not then amplify this target fragment in the corresponding position.This result shows that tentatively pore specificity promoter GbSLSP and target gene thereof have been incorporated in tobacco and the arabidopsis gene group.
Embodiment 4, utilize histological chemistry to detect the expression of gus gene in transgene tobacco and the Arabidopis thaliana, confirm GbSLSP promoter activity and specificity
The histological chemistry that transgene tobacco and Arabidopis thaliana gus gene are expressed detects and carries out according to the method for Jefferson RA [1987, Plant Mol Biol Rep, 5 (4): 387-405].With transgene tobacco T 0For the leaf disc of plant, T 1For the plant seedling, and transgenic arabidopsis T 1For the fresh titbit of plant, flower, column cap, flower pesticide, stem, bennet, calyx, fruit folder, leaf (leaf disc) in GUS staining fluid (X-GLuc solution) in 37 ℃ of dyeing 12-16h; Then through destainer (ethanol: Glacial acetic acid min. 99.5=3: 1) after the decolouring, at the microscopically observing samples and take pictures.Not genetically modified acceptor kind plant is dyeed as contrast equally.The tobacco leaf disk is taken from nearly petiole place.Change the tobacco T of each expression vector over to 0Generation, T 1For plant and Arabidopis thaliana T 1Plant is all got the above independent individual plant that transforms of 10 strains.
Transgenic T 0Show that for the painted result of the GUS of tobacco each tissue and the organ of not genetically modified adjoining tree (CK) all can not dye, but the blade stomata guard cell of commentaries on classics GbSLSP dyeed by the degree of depth, a little less than the dyeing of mesophyll cell, vein and epidermal hair (Fig. 6).
Get T at random 0The seed that transgene tobacco is tied; With being seeded in the solid MS media surface that contains Kan100mg/L after the 5% Youxiaolin surface sterilization, germinate 24 ± 1 ℃ of dark places, it is dark that the back illumination condition that germinates changes 16h illumination/8h into; Temperature is 24 ± 1 ℃, screens transgenic progeny with this understanding.At Kan resistance seedling (T 1Generation) when presenting 3-4 sheet true leaf, gets at random and put in order strain GUS dyeing more than 10 strains.T 1GUS dyeing for tobacco seedling shows that the pore of cotyledon and true leaf is all dyed the GUS mazarine, and the contiguous mesophyll of pore also is colored, and the young root axis presents weak blueness (Fig. 7).And each tissue of not genetically modified adjoining tree (CK) and organ all can not dye.
Change GbSLSP-GUS gene Arabidopis thaliana T 1Show for the painted result of the GUS of plant Different Organs; Each tissue and the organ of not genetically modified adjoining tree (CK) all can not dye; And the pore of the common peduncle of transfer-gen plant, bennet, calyx, petal, flower pesticide, style, fruit pod and blade is all dyed the GUS mazarine, and other organizes do not dye (Fig. 8).
Embodiment 5, utilize confocal laser scanning microscope, CLSM to observe GFP expression of gene in the transgene tobacco, confirm the activity and the specificity of GbSLSP promotor
At potted plant commentaries on classics GbSLSP-GFP tobacco T 0Long during for Kan resistance seedling to 3-5 sheet leaf, get the 2nd tender leaf in its top, in confocal laser scanning microscope, CLSM (model: Carl Zeiss LSM510) down with the 488nm wavelength excites, 550nm detection GFP sends green fluorescence.Tear the then lower epidermis of blade, green fluorescence and taking a picture under similarity condition.Microscopic inspection shows that the GFP expression of gene product GFP that is driven by GbSLSP has not only sent green fluorescence, and green fluorescence only appears at pore (Fig. 9).
The analysis of short active fragments of embodiment 6, pore specificity promoter
What the analysis of the shortest active fragments of pore specificity promoter was adopted is that GbSLSP promoter fragment 5 ' end and/or 3 ' end is deleted analytical method.In order to resolve the shortest active fragments of pore specificity promoter GbSLSP, we have designed 45 ' end deletion fragment (GbSLSP-F2, GbSLSP-F3, GbSLSP-F4, GbSLSP-F5) and 23 ' end deletion fragment (GbSLSP-SH and GbSLSPF2-SH).Relation that these 6 part sheets are intersegmental and size are shown in (Figure 10).
1, pore specificity promoter 5 ' end and/or 3 ' end is deleted segmental clone and is separated
Design and synthesize following 5 primers according to the GbSLSP full length sequence, 3 of upstream primers (also claim 5 ' end primer) (F3-F5) wherein, downstream primer (also claiming 3 ' end primer) 1 (R2).Later for ease clone and structure; Added restriction enzyme Hind III recognition sequence site at 5 of 3 upstream primers (F3-F5) ' end, added the recognition sequence site (the underscore sequence in the following primer sequence) of restriction enzyme BamH I at 3 of 1 downstream primer (R2) ' end.These primers are following:
F3:5 '- AAGCTTATTTTGGAAGATGAC-3 ' (sequence 5);
F4:5 '- AAGCTTCTTTACATGCATCATGTGATCG-3 ' (sequence 6);
F5:5 '- AAGCTTATCGTGGGGGACCCGAAACTTGGCATAC-3 '; (sequence 7)
R2:5 '- GGATCCGTGG TTGGATGAGA CT-3 '; (sequence 8).
According to embodiment 1 said method; Identify that with embodiment 1 the correct recombinant plasmid pBS-GbSLSP that contains the GbSLSP full length sequence is a template; With above-mentioned 3 upstream primers (F3-F5) respectively with primer 2 (sequence 3 in the sequence table) combination as primer to carrying out pcr amplification, the two ends that obtain 3 different lengthss have 5 of restriction enzyme site ' end and delete promoter fragment (GbSLSP-F3, GbSLSP-F4 and GbSLSP-F5); With total length upstream primer (primer 1, i.e. sequence 2 in the sequence table) and R2 (sequence 8) combination as primer to carrying out pcr amplification, obtain 1 two ends and have 3 of restriction enzyme site ' end and delete promoter fragment GbSLSP-SH; The fragment that the size of using Hind III and BamH I double digestion pBS-GbSLSP carrier to obtain is about 650bp is promotor GbSLSP-F2; The small segment (about 280bp) that obtains with Hind III and BamH I double digestion pBS-GbSLSP-SH carrier is promotor GbSLSPF2-SH.
The two ends that above-mentioned pcr amplification is obtained have 4 promoter fragments (3 5 ' end deletion promoter fragment of restriction enzyme site; 13 ' end deletion promoter fragment GbSLSP-SH) and through GbSLSPF2-SH promoter fragment and GbSLSP-F2 promoter fragment that enzyme is cut acquisition reclaim according to ordinary method, and insertion pBS-T carrier.Afterwards again with 6 above-mentioned recombinant vectorss according to the described method of embodiment 1 step 3 transform, the picking mono-clonal, cut evaluations (Figure 11) through Hind III and BamH I are two, identify that correct person is again through the order-checking affirmation.
The gained recombinant vectors is according to the difference difference called after pBS-GbSLSPF2, pBS-GbSLSPF3, pBS-GbSLSPF4, pBS-GbSLSPF5, pBS-GbSLSP-SH, the pBS-GbSLSPF2-SH that contain promoter fragment.Sequencing result shows the fragment that all contains in above 6 parts of plasmids after GbSLSP promotor 5 ' end or 3 ' end is deleted the back different lengths.The nucleotides sequence of GbSLSP-F2 is classified the 346-993 position of sequence 1 in the sequence table as, called after GbSLSP-F2 promotor; The nucleotides sequence of GbSLSP-F3 is classified the 401-993 position of sequence 1 in the sequence table as, called after GbSLSP-F3 promotor; The nucleotides sequence of GbSLSP-F4 is classified the 511-993 position of sequence 1 in the sequence table as, called after GbSLSP-F4 promotor; The nucleotides sequence of GbSLSP-F5 is classified the 529-993 position of sequence 1 in the sequence table as, called after GbSLSP-F5 promotor; The nucleotides sequence of GbSLSP-SH is classified the 1-634 position of sequence 1 in the sequence table as, called after GbSLSP-SH promotor; The nucleotides sequence of GbSLSPF2-SH is classified the 346-634 position of sequence 1 in the sequence table as, called after GbSLSPF2-SH promotor.
2, each fragment of pore specific promoter drives the structure of the plant expression vector of gus gene or GFP genetic expression
The structure of 6 pore specific promoter fragment driving gus gene plant expression vectors adopts a conventional step enzyme to cut connection and gets final product method, makes the GbSLSP total length promoter fragment in the specific promoter fragment alternate embodiment 2pBI-GbSLSP-GUS carrier.Transform according to embodiment 2 said methods afterwards; Shake bacterium; A small amount of alkaline lysis method of extracting plasmid; Again respectively when cloning separately in the step 1 used Auele Specific Primer right; And sequence 4 is identified carrying out PCR with two pairs of primers that sequence 3, sequence 4 and sequence 8 are formed in the sequence table, and the corresponding size that amplifies is about 650,600,480,460,640 or the specificity band (Figure 12) of 280bp, should be GbSLSP-F2, GbSLSP-F3, GbSLSP-F4, GbSLSP-F5, GbSLSP-SH and GbSLSPF2-SH mutually.These fragments and pBI121 enzyme are cut the big segmental recombinant expression vector that obtains, and the name life (abbreviates GbSLSPX-GUS as for pBI-GbSLSPF2-GUS, pBI-GbSLSPF3-GUS, pBI-GbSLSPF4-GUS, pBI-GbSLSPF5-GUS, pBI-GbSLSP-SH-GUS, pBI-GbSLSPF2-SH-GUS successively; X=F2, F3, F4, F5 ,-SH or F2-SH).
Then; Substitute the GUS (restriction enzyme BamH I and Sac I double digestion) in above-mentioned 6 expression vectors with GFP, corresponding 6 recombinant expression vector pBI-GbSLSPF2-GFP, pBI-GbSLSPF3-GFP, pBI-GbSLSPF4-GFP, pBI-GbSLSPF5-GFP, pBI-GbSLSP-SH-GFP, pBI-GbSLSPF2-SH-GFP that obtain containing pore specificity promoter fragment driving GFP gene (abbreviate GbSLSPX-GFP as; X=F2, F3, F4, F5 ,-SH or F2-SH).Concrete grammar is said with embodiment 1.
In above-mentioned 12 plant expression vectors, gus gene or GFP gene are driven by each pore specific promoter fragment and express.
3, change the acquisition of pore specificity promoter fragment tobacco and Arabidopis thaliana
Method according to embodiment 3 changes above-mentioned GbSLSPX-GUS and 12 plant recombination expression vector of GbSLSPX-GFP series over to tobacco (kind " NC89 " respectively; Seed is available from the Chinese Academy of Agricultural Sciences) in; 6 respectively that above-mentioned GbSLSPX-GUS is serial simultaneously recombinant expression vectors change in the Arabidopis thaliana (Colombia is environmental), thereby screening obtains genetically modified tobacco T 0For plant and Arabidopis thaliana T 1Plant.
Extract the tobacco T that changes each expression vector respectively 0For plant and Arabidopis thaliana T 1The plant genomic dna; Form primer to carrying out pcr amplification with any and primer 2 (sequence 3) among the primers F 2-F5 (sequence is respectively sequence 4, sequence 5, sequence 6, sequence 7); Perhaps use primer 1 (sequence 2), F2 (sequence 4) and primer R2 (sequence 8) to form primer to carrying out pcr amplification, thereby detect whether changed corresponding promotor in genetically modified tobacco and the Arabidopis thaliana over to respectively.Then, carry out histological chemistry's detection (according to the method for embodiment 4) of gus gene expression and the confocal laser scanning microscope, CLSM observation (according to the method for embodiment 5) that GFP expresses, see step 4 and 5 for details detecting male transgene tobacco and Arabidopis thaliana.
4, change the tobacco of GbSLSPX-GUS series recombinant vectors and histological chemistry's detection that the Arabidopis thaliana gus gene is expressed
Method according to embodiment 4 detects the tobacco T that male changes GbSLSPX-GUS series recombinant expression vector to above-mentioned PCR respectively 0Generation, T 1For plant and Arabidopis thaliana T 1Generation, T 2Carry out the histochemical stain that gus gene is expressed for plant, the equal random sampling 3-5 of each promoter fragment GbSLSPX-GUS male plant independent individual plant is contrast with not genetically modified tobacco or Arabidopis thaliana simultaneously.
Transgene tobacco T 0For the coloration result of blade shown in A among Figure 13 and a.The result shows, GbSLSP-F2 (Figure 13 F2-A and a), GbSLSP-F3 as if (Figure 13 F3-A and a) can both drive gus gene strongly expressed specifically in the stomata guard cell is although there is the expression of trace in mesophyll cell; GbSLSP-F4 can drive gus gene the stomata guard cell specific expressed fully (Figure 13 F4-A and a), just expression intensity than GbSLSP-F3 a little less than some; GbSLSP-F5 can drive gus gene at stomata guard cell's predominant expression, at relatively weak (Figure 13 F5-A and a) of the expression of mesophyll cell; GbSLSP-SH driven GUS gene all has expression in stomata guard cell, mesophyll and vein, although in the stomata guard cell, express strong slightly (Figure 13 F-SH-A and a); GbSLSPF2-SH driven GUS gene has trace expression in the stomata guard cell, and painted (Figure 13 F2-SH-A and a) of GUS can only be arranged in the part.Transgene tobacco T 0The GUS coloration result of seville orange flower (B among Figure 13) shows that GbSLSP-F2 can drive gus gene all has certain expression (Figure 13 F2-B) at ovary, sepal, petal, column cap, the flower pesticide of flower; GbSLSP-F4 only can drive gus gene has faint expression (Figure 13 F4-B) in ovary; GbSLSP-F5 can drive gus gene has faint expression (Figure 13 F5-B) in the ovary of spending, petal, flower pesticide; GbSLSP-SH only can drive gus gene has stronger expression (Figure 13 F-SH-B) in ovary; GbSLSPF2-SH only can drive gus gene has very faint expression (Figure 13 F2-SH-B) in flower pesticide.Transgene tobacco T 1GUS coloration result (Figure 14) for seedling shows that each makes up the heredity that the promoters driven gus gene all can be stable.And each tissue of not genetically modified adjoining tree (CK) and organ all can not dye.
Transgenic arabidopsis T 1The GUS coloration result in generation is shown in figure 15.Change GbSLSP-F2 and drive gus gene and not only express specifically at the blade pore, and all have organizing of pore and all have specifically and express stem, anthocaulus, flower pesticide, sepal and anthocaulus, the pore of these organs is all dyed GUS blueness (Figure 15 F2).In addition, very strong GUS dyeing is arranged also on filigree, but can't see GUS dyeing on petal and the fruit folder.Change GUS coloration result (Figure 16) demonstration of the T2 of GbSLSPF2-GUS for seedling; GUS dyeing (Figure 16 A) can both be dyed in the whole leachy position of strain of seedling; Its cotyledon (Figure 16 B and b) and true leaf (Figure 16 C and c) only pore demonstrate the GUS blueness, and organizing of other is not colored.Change the T of GbSLSPF5-GUS 1The GUS coloration result in generation (Figure 15 F5) is approximate with commentaries on classics GbSLSPF2-GUS's, but intensity obviously weakens.In addition, present sheet GUS dyeing (Figure 15 F5C) in the part of style except that neck.And not genetically modified T 1Generation and T 2Each tissue and organ for adjoining tree (CK) all can not dye.
5, change GbSLSPX-GFP series tobacco T 0The confocal laser scanning microscope, CLSM of the GFP that representative reaches is observed
Picked at random is changeed the tobacco T of GbSLSPFX-GFP series recombinant expression vector 0For Kan resistance seedling; The equal random sampling 3-5 of each promoter fragment GbSLSPX-GFP male plant independent individual plant; Get its spire and under confocal laser scanning microscope, CLSM is observed, observe expression and the photograph of GFP, photo is shown in figure 17, and 6 deletion GFP genes that fragment drove of GbSLSP are all only expressed at pore specifically; Green fluorescence only occurs at pore, does not detect and organize at other.
The above embodiments result shows and proves conclusively; Cotton promotor GbSLSP provided by the present invention has stronger predominant expression in pore; Promoter fragment GbSLSP-F2, GbSLSP-F3, GbSLSP-F4 after 5 ' the end deletion have good pore specificity, especially GbSLSP-F2 and have very strong promoter activity.GbSLSP-F5, GbSLSP-F-SH, GbSLSPF2-SH have more weak predominant expression in pore, and the promoter fragment that obtains all can stably pass to the offspring.
Figure IDA0000128543820000011
Figure IDA0000128543820000021
Figure IDA0000128543820000031
Figure IDA0000128543820000041

Claims (10)

1.DNA molecule, be following a) or b) or dna fragmentation c):
A) 3 ' end contains the 529-993 position nucleotide sequence of sequence 1 in the ordered list at least, and from the 529th beginning of sequence 1, according to the nucleotide sequence of sequence 1 to 5 of sequence 1 ' end prolongation, obtain length and be any dna fragmentation of 465 to 993bp; Said dna molecular has promoter function;
B) and a) nucleotide sequence that limits has 75% or 75% above homology, and has the dna fragmentation of promoter function;
C) under stringent condition and a) or b) nucleotide sequence hybridization that limits, and have the dna fragmentation of promoter function.
2. dna molecular according to claim 1 is characterized in that: last of said dna molecular Nucleotide is the 993rd of sequence 1.
3. dna molecular according to claim 1 and 2 is characterized in that: said dna molecular is following 1)-3) in any:
1) 3 of nucleotide sequence ' end contains the 511-993 position nucleotide sequence of sequence 1 in the ordered list at least; And from the 511st beginning of sequence 1, prolong to 5 of sequence 1 ' end, obtain length and be any dna fragmentation of 483 to 993bp according to the nucleotide sequence of sequence 1;
2) 3 of nucleotide sequence ' end contains the 401-993 position nucleotide sequence of sequence 1 in the ordered list at least; And from the 401st beginning of sequence 1, prolong to 5 of sequence 1 ' end, obtain length and be any dna fragmentation of 593 to 993bp according to the nucleotide sequence of sequence 1;
3) 3 of nucleotide sequence ' end contains the 346-993 position nucleotide sequence of sequence 1 in the ordered list at least; And from the 346th beginning of sequence 1, prolong to 5 of sequence 1 ' end, obtain length and be any dna fragmentation of 648 to 993bp according to the nucleotide sequence of sequence 1.
4. according to arbitrary described dna molecular among the claim 1-3, it is characterized in that: said dna molecular is following a1)-a5) in any:
A1) nucleotides sequence is classified the dna molecular shown in the sequence 1 in the sequence table as;
A2) nucleotides sequence is classified dna molecular shown in the 346-993 position Nucleotide of sequence 1 in the sequence table as;
A3) nucleotides sequence is classified dna molecular shown in the 401-993 position Nucleotide of sequence 1 in the sequence table as;
A4) nucleotides sequence is classified dna molecular shown in the 511-993 position Nucleotide of sequence 1 in the sequence table as;
A5) nucleotides sequence is classified dna molecular shown in the 529-993 position Nucleotide of sequence 1 in the sequence table as.
5.DNA molecule is following d) or e) or f) dna fragmentation:
D) 3 ' end contains the 346-634 position nucleotide sequence of sequence 1 in the ordered list at least, and from the 346th beginning of sequence 1, according to the nucleotide sequence of sequence 1 to 5 of sequence 1 ' end prolongation, obtain length and be any dna fragmentation of 289 to 634bp; Said dna molecular has promoter function; Last of said dna molecular Nucleotide is the 634th of sequence 1;
E) and d) nucleotide sequence that limits has 75% or 75% above homology, and has the dna fragmentation of promoter function;
F) under stringent condition and d) or the nucleotide sequence hybridization that e) limits, and have the dna fragmentation of promoter function.
6. dna molecular according to claim 5 is characterized in that: said dna molecular is following b1) or b2):
B1) nucleotides sequence is classified the dna molecular shown in the 1-634 position Nucleotide of sequence 1 in the sequence table as;
B2) nucleotides sequence is classified dna molecular shown in the 346-634 position Nucleotide of sequence 1 in the sequence table as.
7. the genetic stocks that contains arbitrary said dna molecular among the claim 1-6, or a primer of arbitrary said dna molecular is right among the acquisition claim 1-6; Said genetic stocks is expression cassette, recombinant vectors, reorganization bacterium or transgenic plant cells or tissue or organ.
8. primer according to claim 7 is right, it is characterized in that: said primer is to for following c1)-arbitrary right in c5):
C1) primer that dna fragmentation shown in the 7-27 position Nucleotide of dna fragmentation shown in the 7-30 position Nucleotide of sequence 3 and sequence 2 is formed in the sequence table is right;
C2) primer that dna fragmentation shown in the 7-21 position Nucleotide of dna fragmentation shown in the 7-30 position Nucleotide of sequence 3 and sequence 5 is formed in the sequence table is right;
C3) primer that dna fragmentation shown in the 7-28 position Nucleotide of dna fragmentation shown in the 7-30 position Nucleotide of sequence 3 and sequence 6 is formed in the sequence table is right;
C4) primer that dna fragmentation shown in the 7-34 position Nucleotide of dna fragmentation shown in the 7-30 position Nucleotide of sequence 3 and sequence 7 is formed in the sequence table is right;
C5) primer that dna fragmentation shown in the 7-27 position Nucleotide of dna fragmentation shown in the 7-22 position Nucleotide of sequence 8 and sequence 2 is formed in the sequence table is right.
9. arbitrary said dna molecular starts the application in the destination gene expression among the claim 1-6 in plant.
10. application according to claim 9 is characterized in that: said plant is a terrestrial plant, and preferably dicotyledons is more preferably Arabidopis thaliana or tobacco; It is said that to be expressed as pore specific expressed.
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107287195A (en) * 2017-05-18 2017-10-24 青岛农业大学 One kind is in the single-minded expression promoter of guard cell and its creation method
CN116463349A (en) * 2023-06-07 2023-07-21 河北科技大学 Promoter OsP04g0617800 for rice stomata tissue specific expression and application thereof

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CN101245349A (en) * 2007-02-16 2008-08-20 中国科学院上海生命科学研究院 Plants impeller vane all-level vein and tillering base section special expression promoter and application
CN101792761A (en) * 2009-10-23 2010-08-04 武汉大学 Induction-enhanced tissue specific promoter and application thereof

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CN101082047A (en) * 2006-05-31 2007-12-05 中国农业大学 Beta-glucosidase gene promoter and uses thereof
CN101245349A (en) * 2007-02-16 2008-08-20 中国科学院上海生命科学研究院 Plants impeller vane all-level vein and tillering base section special expression promoter and application
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Publication number Priority date Publication date Assignee Title
CN107287195A (en) * 2017-05-18 2017-10-24 青岛农业大学 One kind is in the single-minded expression promoter of guard cell and its creation method
CN116463349A (en) * 2023-06-07 2023-07-21 河北科技大学 Promoter OsP04g0617800 for rice stomata tissue specific expression and application thereof
CN116463349B (en) * 2023-06-07 2023-12-15 河北科技大学 Promoter OsP04g0617800 for rice stomata tissue specific expression and application thereof

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