CN104372005A - RhNAC3 promoter with series ABRE cis acting element and application thereof - Google Patents

Abstract

The invention provides an RhNAC3 promoter with series ABRE cis acting elements and application thereof. The promoter is an inducible promoter sensitive to abscisic acid, the contained ABRE cis acting elements have cumulative effect on the expression of downstream genes, so as to use the abscisic acid to conduct quantitative control on the expression of downstream genes. In some preferred embodiments, the downstream gene is a stress resistance gene such as RhNAC3; the abscisic acid produced by plants under adversity stress such as drought stress is used to induce the expression of the stress resistance gene, so that the plants can autonomously increase the expression of stress resistance gene in the face of adversity stress conditions to further enhance the stress resistance. Therefore, the promoter has very great application value in enhancing the stress resistance of plants.

Description

A kind of RhNAC3 promotor and application thereof with series connection ABRE cis-acting elements

Technical field

The present invention relates to field of plant molecular biology, provide inducible promoter, and utilize promotor to control the method for this promotor downstream gene expression of expression of plants by dormin.

Background technology

NAC transcription factor is a class transcription factor specific to plant, refers in particular to the class protein of the NAC structural domain with high conservative, and its title derives from the ATAF1 in the NAM gene of petunia, Arabidopis thaliana, the initial of 2 genes and CUC2 gene.Because the protein of these three kinds of genes encodings holds the NAC structural domain with high conservative at N-, found again that gene quantity in plant with this category feature was a lot of afterwards, just this genoid is called NAC (NAM, ATAF1,2, and CUC2) family gene, the albumen of its coding is called NAC albumen.

NAC proteinoid is extensively present in different plants, and in same plant One's name is legion.The structure of NAC class transcription factor is roughly divided into the conservative NAC structural domain of N-end and various C-terminal domains.NAC albumen also comprises N-and holds the nuclear localization signal of NAC structural domain and C-to hold well differentiated motif, and C-end is transcriptional regulatory territory, and majority has transcriptional activation function, and part NAC class transcription factor also has cross-film motif, for exercising cross-film function at C-end.

Due to the difference that NAC family member exists in sequence and other booster action factor etc. around structure, cis-acting elements, make it different in biological activity and physiological function, therefore NAC transcription factor extensively exists in Various Tissues, participates in the regulation and control of numerous metabolic process.The growing of NAC class transcription factor wide participation plant, hormone regulating and controlling and environment-stress response are the key factors of various physiological response in a class regulating plant body.NAC transcription factor in cytodifferentiation, embryo and flower development, the formation of side root, secondary wall formation, leaf senile, pathogenic bacteria defence, very important regulating and controlling effect should be played in poor environment etc. to external world.

Dormin (ABA, abscisic acid) as a stress signal plant hormone, can induce by Water deficit, in plant integral level to the adjustment of cell levels, play a very important effect.The diverse in function of ABA, many aspects of wide participation plant-growth and growth, as embryo maturation, seed development, seed germination, stomatal aperture regulate and activate many stress response genes, involved in plant is bloomed, ABA can also regulate transpiration efficiency to control to wilt, and then reduces plant moisture loss.In dehydration situation, a large amount of stress-related genes regulated by ABA.Although the diverse in function of ABA, in participation Water deficit, be mainly manifested in the moisture preserved in plant materials by promoting stomatal closure, it also regulates a large amount of Water deficit respond the expression of genes involved thus plant is responded to Water deficit simultaneously.At present, ABA mechanism of action is on a molecular scale unclear yet.

The present inventor's early-stage Study finds, RhNAC3 coerces relevant NAC (stress-associatedNAC in Chinese rose, SNAC) class transcription factor, it to be positioned in nucleus and to have transcripting activating characteristic, and the transcriptional level of RhNAC3 is induced by abiotic stress process such as Water deficit also can in conjunction with CAC [G/T] cis-acting elements.The tolerance to water deficit stress of petal is reduced after reticent RhNAC3 in Chinese rose petal.In Arabidopis thaliana, allos process LAN RhNAC3 improves the Water deficit tolerance of plant.Transcription factor RhNAC3 participates in tolerance to water deficit stress (Jiang X by regulating the expression of osmotic stress genoid, Zhang C, L ü P, Jiang G, Liu X, DaiF and Gao J.2014.RhNAC3, a stress-associated NAC transcription factor, has a rolein dehydration tolerance through regulating osmotic stress-related genes in rose petals.Plant biotechnology journal, 12 (1): 38-48).RhNAC3 is open by the present inventor, and its GenBank accession number is that JK617768.1 (can be see http:// www.ncbi.nlm.nih.gov/nucest/JK617768).Although existing work is studied RhNAC3 albumen, this area is still undistinct to the regulatory mechanism of RhNAC3 albumen, does not particularly know the relation of itself and plant hormone ABA.

Summary of the invention

The object of the invention is to make plant have favourable biological character by molecular biology method, the ability especially making plant have favourable stress tolerant to coerce, namely has favourable resistance.The present invention is achieved by the following technical solution:

1, a promotor for NAC protein transcription factor gene, wherein, described promotor and SEQ No.1 have at least 50%, the identity of such as 60%, 70%, 80%, 90%, 91%, 92%, 95%, 97%, 98%, 99% or 100%; Preferably, described nucleotide sequence comprises at least one in the group being selected from and being made up of acgtg, acgtgc, acgtgg, acgtgc and acgtgtc, at least two kinds, at least three kinds, at least four kinds or at least five kinds of cis-acting elements.

2, for cloning the promotor described in technical scheme 1, detecting, locate, the primer pair of process LAN or silence, wherein, described primer pair is selected from the group be made up of primer pair SEQ NO.2 and 15, SEQ NO.2 and 16, SEQ NO.2 and 17, SEQ NO.2 and 18, SEQ NO.3 and 19, SEQ NO.4 and 19, SEQ NO.5 and 6, primer pair SEQ NO.7 and 8, primer pair SEQ NO.9 and 12, primer pair SEQ NO.10 and 12, primer pair SEQ NO.11 and 12, primer pair SEQ NO.13 and 14.

The method of the promotor 3, described in a kind of clone technology scheme 1, wherein, described method uses at least one primer be selected from the group be made up of primer pair SEQ NO.2 and 15, SEQ NO.2 and 16, SEQ NO.2 and 17, SEQ NO.2 and 18, SEQ NO.3 and 19, SEQ NO.4 and 19 to carry out.

4, a method for clone or the promotor described in detection technique scheme 1, wherein, described method comprises use primer pair SEQ No.5 and 6 and/or primer pair SEQ NO.7 and 8 and carries out; Preferably, described detection is undertaken by GUS Activity determination, and uses primer pair SEQ NO.7 and 8 to carry out.

5, detect the method for plant to the susceptibility of ABA, described method comprises and detecting the promotor described in technical scheme 1; Preferably, described method comprises and detecting the existence of the ABA cis-acting elements of described promotor and/or quantity.

6, improve a method for Plant Tolerance environment stress ability, described method comprises and utilizes the promotor described in technical scheme 1 to carry out; Preferably, described method realizes the susceptibility of ABA by changing plant; In addition preferably, described method realizes the susceptibility of ABA by improving plant; In addition preferably, described environment stress causes the ABA growing amount of described plant to increase; In addition preferably, described environment stress be selected from coerced by Chilling stress, high temperature stress, Drought Stress are coerced, waterlogging environment stress, high salt environment stress, biotic, mechanical wounding coerce the group formed.

7, the method as described in technical scheme 6, wherein, described method is realized by the amalgamation and expression of described promotor and stress tolerant being coerced relevant gene; Preferably, described stress tolerant coerces relevant gene is RhNAC3.

8, the method according to technical scheme 6 or 7, wherein, the endogenous ABA that described amalgamation and expression utilizes the exogenous ABA of outside applying or plant itself to produce induces; Further preferably, the endogenous ABA that described amalgamation and expression utilizes plant itself to produce induces.

9, RhNAC3 gene coerces the application in ability at raising Plant Tolerance; Preferably, described application is by realizing the amalgamation and expression of the promotor described in described RhNAC3 gene and technical scheme 1.

10, the carrier containing the promotor described in technical scheme 1; Preferably, described carrier is plasmid vector, phage vector or virus vector; Have choosing in addition, described carrier is cloning vector or expression vector.

The RhNAC3 promoter sequence comprising series connection ABRE cis-acting elements provided by the present invention participates in the regulation and control to plant drouhgt stress patience and ABA susceptibility.Such as, in plant, this promoter expression has popularity, shows accumulative effect to the response of ABA; In plant, this gene of process LAN shows ABA super quick in seed germination, primary root growth and stomatal aperture; The expression of ABA responsive genes can be regulated and controled in plant.This improves plant to ABA susceptibility having unique advantage by making NAC transcription factor albumen RhNAC3 at involved in plant abiotic stress tolerance.Described RhNAC3 promotor is inducible promoter, can merge mutually with the gene of difference in functionality type (drought resisting, cold-resistant, Salt And Alkali Tolerance), and regulate and control its expression in plant materials, can the expression of induced strong goal gene under ABA process, strengthen plant to the opposing of environment stress, ABRE cis-acting elements containing series connection in RhNAC3 promoter region, the deletion promoters that different deletion fragment comprises different number ABRE element can the expression of quantitatively regulating and controlling institute promotor gene.Described RhNAC3 promotor pair has great importance with the research of participation regulating plant to abiotic stress, can be the plant utilizing genetic engineering means to cultivate proterties excellent and provides genetic resources deposit.

Accompanying drawing explanation

Fig. 1 shows the important cis-acting elements signal of RhNAC3 promotor and expression characterization.Wherein, A figure: RhNAC3 promoter sequence schematic diagram.Different rectangle frame illustrates important stress response cis-acting elements such as W-box, ABRE, MYC, MYB, CBF etc. relevant to environment stress in RhNAC3 promotor.Scheme nucleotide sequence type and the particular location of ABRE element in the numeral promoter sequence in the form of A lower part, in RhNAC3 promoter region, be ABRE cis-acting elements position at-484 (Nucleotide acgtg) ,-516 (Nucleotide acgtgc) ,-600 (Nucleotide acgtgg) ,-914 (Nucleotide acgtgc) and-1134 (Nucleotide acgtgtc) place.Scheme by A the ABRE (ABA cis-acting elements) that known RhNAC3 has multiple series connection, the result of study that this RhNAC3 plays a role in plant reply stress response approach is coincide, especially in the regulatory pathway of ABA dependence.B schemes: the Histochemical localization of RhNAC3 in Arabidopis thaliana.By the expression of the gus gene of RhNAC3 promoters driven at 5d Arabidopis thaliana seedling (a), 14d seedling (b), young leaflet tablet (c), young leaflet tablet pore (d), flower (e), column cap (f), petal (g), inflorescence top stem (h), mellow fruit pod (i), the expression of immature seed (k), the scale on figure is 1mm.Schemed from B: RhNAC3 gene has expression in each stage of growth and development of plants, and in various piece wide expression, especially blade guard cell (Figure 1B-d) and some vascular tissues (Figure 1B-b and c) in have stronger expression.

Fig. 2 shows different fragments RhNAC3 promoter deletion and analyzes and ABA dosage effect.A schemes: the GUS activation analysis of RhNAC3 promoter deletion fragment in protoplasts of Arabidopsis thaliana broken by ultrasonic.A figure upper part schematically shows the distribution of ABRE cis-acting elements different in RhNAC3 promoter sequence, the bottom of A figure is divided into 3 different recombinant plasmid (N0, N1, N2) active with the relative GUS of unloaded contrast (NC), GUS activity detects after 24h after incubation, wherein, N0 (-1447 ~-160bp) includes the ABRE cis-acting elements of all 5 series connection shown in the A figure of Fig. 1, N1 (-707 ~-160bp) includes the ABRE cis-acting elements of wherein 3 series connection shown in the A figure of Fig. 1, ABRE cis-acting elements A figure shown in of N2 (-377 ~-160bp) not containing Fig. 1, NC position empty vector control, compared with NC, include N0 (-1447 ~-160bp) difference extremely significantly (double asterisk compared with NC of the ABRE cis-acting elements of 5 series connection, P < 0.01), include N1 (-707 ~-160bp) significant difference (single asterisk, P < 0.05) compared with NC of the ABRE cis-acting elements of 3 series connection, B schemes: Exogenous ABA is on the impact of GUS activity in the protoplasts of Arabidopsis thaliana broken by ultrasonic comprising RhNAC3 promoter deletion recombinant chou.RhNAC3 promotor (N0, N1, N2) and the unloaded contrast (NC) of different deletion fragment are transferred into protoplasts of Arabidopsis thaliana broken by ultrasonic, and wherein, N0, N1, N2 and NC are described above.Adopt 0 μM and 10 μMs of ABA processing primary plastid 2h; GUS activity is detected after hatching 24h.From figure B, compared with contrast NC, when not adding ABA, the active there was no significant difference of GUS between different fragments, when after applying 10 μMs of ABA process, the N0 (-1447 ~-160bp) of the ABRE cis-acting elements containing 5 series connection reaches pole significant difference (double asterisk compared with NC, P value < 0.01), the N1 (-707 ~-160bp) of the ABRE cis-acting elements containing 3 series connection reaches significant difference (single asterisk, P value < 0.05) compared with NC.C schemes: RhNAC3 promotor is to the response of ABA.The transgenosis seedling that MS substratum grows 9 days proceeds to the MS substratum growth 4d of 10 μMs of ABA, then carries out GUS dyeing.As seen from the figure, the expression induction of RhNAC3 transcriptional activity that ABA is strong.From the above: in RhNAC3 promotor, the transcriptional activity of ABREs element to RhNAC3 has a storage effect (Fig. 2 A), and ABA process more obviously can induce the transcriptional activity (Fig. 2 B) of RhNAC3 promotor.ABA process can strengthen the transcriptional activity (Fig. 2 C) of RhNAC3 promotor.

Fig. 3 shows RhNAC3 transgenic line and improves susceptibility to ABA.A schemes: the germination rate of contrast (WT and VC) and RhNAC3 transfer-gen plant (OE#3,6,12) seed.3 RhNAC3 process LAN strains (OE#3,6,12) and contrast (WT, VC) seed are sowed respectively and are being contained in the MS substratum of 0,0.2,0.4 μM of ABA.Be the growing state after broadcast plant 5d shown in figure, germination rate is for adding up when growing 7d.B schemes: the seed germination rate statistics of RhNAC3 transgenic line (OE#3,6,12) and contrast (WT, VC).Double asterisk is shown as in 0.2 and 0.4 μM of ABA RhNAC3 transgenic line (OE#3,6,12) difference extremely remarkable (P value < 0.01) compared with contrast (VC).Error line is standard error (n=3).B schemes: in contrast and transfer-gen plant, ABA dosage affects main root length and side radical object.C schemes: contrast (WT and VC) and RhNAC3 transfer-gen plant (OE#3,6,12) root growth phenotype.Contrast seedling (the WT that 7d is large, VC) and 3 RhNAC3 transgenic line (OE#3, OE#6, OE#12) be transferred to respectively containing 0 respectively, 5, in the MS substratum of 10 and 30 μMs of ABA, root morphology phenotype is added up after 10d, as seen from the figure, when respectively with 0 and 5 μM of ABA process, RhNAC3 transgenic line (OE#3, 6, 12) and contrast (WT, VC) root phenotype is consistent, in the MS substratum of 10 μMs and 30 μMs ABA, compared with contrast (VC), RhNAC3 transgenic line (OE#3, 6, 12) primary root root is long shorter, side radical order is less, D schemes: RhNAC3 transgenic line (OE#3,6,12) and the long analysis of contrast (WT, VC) primary root main root.RhNAC3 transgenic line (OE#3,6,12) and contrast (WT, VC) primary root main root length statistics is carried out after plant strain growth 10d, hNAC3 transgenic line (OE#3,6,12) and contrast (WT, VC) plant add up with 15 young plants respectively; E schemes: RhNAC3 transgenic line (OE#3,6,12) and contrast (WT, VC) lateral root number analysis, RhNAC3 transgenic line (OE#3,6,12) and the rear side radical order statistical study of contrast (WT, VC) plant strain growth 10d, RhNAC3 transgenic line (OE#3,6,12) and contrast (WT, VC) plant add up with 15 young plants respectively.The above results shows: Seed Germination of Arabidopsis Pumila rate declines to some extent with the rising of ABA concentration, and under same level ABA (0.2 and 0.4 μM of ABA) process, compared with contrast (VC) plant, RhNAC3 transgenic line (OE#3,6,12) plant seed germination rate reduces more remarkable (Fig. 3 A and B), and illustrate in transgenic arabidopsis, RhNAC3 process LAN causes the ABA supersensitivity in seed germination stage.In seedling root phenotype, compared with contrast (VC) plant, RhNAC3 transgenic line (OE#3,6,12) all there is reduction trend in plant on length of root stalk and side radical order, and RhNAC3 process LAN also showed responsive (Fig. 3 C, D and E) ABA in the root growth stage.

Fig. 4 shows the stomatal aperture situation of RhNAC3 transgenic line in Arabidopis thaliana under ABA and Osmotic treatment.A schemes: the lower contrast (WT of ABA process, and RhNAC3 transgenic line (OE#3 VC), 6,12) Stoma of Leaves aperture, 2 contrast (WT of 3 weeks seedling ages, the mature leaf of the transgenic line (OE#3,6,12) of VC) and 3 RhNAC3 uses pore damping fluid (0 μM) and 10 μMs of ABA (on pore damping fluid basis interpolation 10 μMs of ABA) to process 2h respectively.Epidermic cell pore opticmicroscope (Olympus BX-51) observation choosing axle head far away is taken pictures.RhNAC3 transgenic line (OE#3,6,12) and contrast (WT, VC) plant, each strain sample at least measures stomatal aperture (width/height) with 20 guard cells.Double asterisk represents process LAN strain difference extremely remarkable (P < 0.01) compared with the control under 10 μMs of ABA process.Scale=10 μm.B schemes: contrast (WT, VC) and RhNAC3 transgenic line (OE#3,6,12) Stoma of Leaves aperture under Osmotic treatment.The contrast (WT, VC) in 3 Zhou Miao ridges and RhNAC3 transgenic line (OE#3,6,12) under normal condition growth and Osmotic treatment condition, 10d is grown respectively, the epidermic cell pore opticmicroscope (Olympus BX-51) getting axle head far away is respectively measured after taking pictures, and each strain measures stomatal aperture (width/height) with at least 20 guard cells.Single asterisk represents process LAN strain (OE#3) significant difference (P < 0.05) compared with contrast (VC) under 10d Osmotic treatment, double asterisk represents process LAN strain (OE#6,12) difference extremely remarkable (P < 0.01) compared with contrast (VC) under 10d Osmotic treatment.Scale=10 μm.The above results shows: ABA can promote stomatal closure, and greatly can promote the stomatal closure of RhNAC3 transgenic line, make RhNAC3 transgenic line (OE#3,6 under ABA process, 12) stomatal aperture is less, stomatal closure more rapid (Fig. 4 A); 10d Osmotic treatment can induce RhNAC3 Transgenic plant leaf stomatal aperture obviously to reduce (Fig. 4 B) relative to contrast (VC).RhNAC3 is to rely on the mode involved in plant of ABA to the response of water stress.

Fig. 5 shows the expression that RhNAC3 just can regulate and control ABA responsive genes.A schemes: the expression pattern of ABA responsive genes gene in RhNAC3 transgenic line (OE#3,6,12).Clear and definite, RD29A, RD29B, RD20, RD26, COR47, COR15A, KIN2, ABII, ABI3, ABF4, ABA3 are respectively ABA responsive genes in Arabidopis thaliana, under long day (16h illumination/8h is dark) condition, get 3 weeks large VC and and RhNAC3 transgenic line (OE#3,6,12) over-ground part grown is placed on (23-25 DEG C, 40-50% relative humidity) arid 3h on experiment table and samples.After Trizol method extracts total serum IgE, also reverse transcription becomes cDNA, and analyzes the relative fold induction of the gene expression amount after drought stress of 11 ABA responsive genes by qRT-PCR method, and fold induction is greater than 2 and is considered as up-regulated expression.In figure, numerical value represents arid with under collating condition, and the fold induction of each gene represents with the mean value that the biology of 3 strains repeats, and does stdn with internal standard gene Actin2.B schemes: respond genoid with the ABA of Arabidopis thaliana very high homology in Chinese rose.RU25535, RU07831, RU01455, RU06450, RU04740, RU22946, RU24499, RU03861, RU26868 are respectively the homologous gene with Arabidopis thaliana ABA responsive genes in Chinese rose.C schemes: ABA responsive genes expression of (TRV-RhNAC3) in normal plant (TRV) and RhNAC3 silence petal in Chinese rose.Heterogeneic data represent TRV-RhNAC3 and change relative to the multiple of each gene of TRV, and change multiple is less than 0.66 and is considered as expression and is suppressed, and RhUbil is that interior mark is as stdn.The above results shows: in Arabidopis thaliana, the RhNAC3 positive regulation expression of ABA responsive genes (Fig. 5 A), in the reticent Chinese rose petal of RhNAC3, the expression of ABA signal and downstream gene is subject to obvious suppression (Fig. 5 B and C), and these results show that RhNAC3 forward regulates the expression of ABA responsive genes to participate in ABA signal pathway.

Embodiment

The present invention is providing a kind of promotor of NAC protein transcription factor gene, wherein, described promotor and SEQ No.1 have at least 50%, the identity of such as 60%, 70%, 80%, 90%, 91%, 92%, 95%, 97%, 98%, 99% or 100%; Preferably, described nucleotide sequence comprises at least one in the group being selected from and being made up of acgtg, acgtgc, acgtgg, acgtgc and acgtgtc, at least two kinds, at least three kinds, at least four kinds or at least five kinds of cis-acting elements.

The present inventor finds by carrying out further investigation to described promotor, described promotor is a kind of inducible promoter, there is the environment stress cis-acting elements of number of different types, particularly include the ABRE cis-acting elements of series connection, the effect of the trans-acting factors such as such as ABA can be subject to, thus induce the expression of its downstream gene such as ABA responsive genes.This makes this promotor have extremely important utility value in the genetically engineered of plant.

In some embodiments, what the present invention can utilize this promotor can inducing properties, by carrying out genetic modification to this promotor, thus promotes or suppresses this promotor to the induction of its downstream gene expression, promoting or suppress the expression of downstream gene thus.In some embodiments, by such as modifying with the internal promoter of this promotor homology in a certain noxious plant, thus the expression of the downstream gene of this promotor can be suppressed, affecting the normal growth of noxious plant thus.In other embodiment, can by carrying out genetic modification to the internal promoter same with this promotor in a certain plant, or by genetic engineering means such as transgenic technology, proceed to this promotor or proceed to the fusion gene of gene (i.e. downstream gene) of this promotor and target protein to be expressed, thus promote or process LAN downstream gene, controllably generate thus or promote to generate the useful proteins of being encoded by downstream gene.

In some embodiments, described downstream gene can be the gene of any useful proteins of encoding.Such as described useful proteins can be used in the albumen of the purposes such as medicine, nutrition, health care.Some preferred embodiment in, described downstream gene is the gene relevant with Plant Tolerance environment stress ability.Described environment stress be selected from coerced by Chilling stress, high temperature stress, Drought Stress are coerced, waterlogging environment stress, high salt environment stress, biotic, mechanical wounding coerce the group formed.

The present inventor also finds, described promotor is a kind of promotor of ABA susceptibility, can be subject to the effect of ABA and affect the expression of its downstream gene.

So in some embodiments, the present invention utilizes ABA to act on described promotor thus controls the expression of this promotor downstream gene.As described above, ABA is a kind of very important plant hormone, many aspects of wide participation plant-growth and growth, as embryo maturation, seed development, seed germination, stomatal aperture regulate and activate many stress response genes, involved in plant is bloomed, ABA can also regulate transpiration efficiency to control to wilt, and then reduces plant moisture loss.In addition, those skilled in the art will appreciate that plant usually can produce than ABA much more under non-adverse environmental factor under the effect of environment stress.These environment stresses comprise that such as Chilling stress is coerced, high temperature stress, Drought Stress are coerced, waterlogging environment stress, high salt environment stress, biotic, mechanical wounding are coerced.

So the invention provides a kind of method improving Plant Tolerance environment stress ability, described method is undertaken by promotor described in amalgamation and expression and degeneration-resistant protein gene.When plant runs into environment stress, can produce ABA, this ABA acts on the described promotor in plant materials, induces the expression of degeneration-resistant protein gene thus, thus improves the resistance of plant, and this is very favorable.Under plant is in non-adverse environmental factor, plant does not produce or just produces low-level ABA, so there is no or rarely ABA acts on described promotor, and therefore plant can not abduction delivering or few degeneration-resistant protein gene of abduction delivering.When plant is in the condition of environment stress, plant can produce than ABA more under non-environment stress condition, thus the degeneration-resistant protein gene of abduction delivering, thus enable plant can independently improve its resistance very cleverly when running into environment stress, without the need to adopting artificial measures to prevent plant cannot normal growth under environment stress condition.

Certainly, if the quantity not sufficient of the ABA that plant produces under adverse environmental factor is to act on described promotor fully, thus enough degeneration-resistant protein genes cannot be induced with the resistance enabling plant have expection level, can be also use the resistance that ABA improves plant by people.

In addition, because described promotor is responsive to ABA, therefore, ABA can be utilized to control with the internal promoter of described promotor homology or proceed to the expression being controlled downstream gene by the plant of described promotor or promotor and downstream gene, described downstream gene can be any useful gene, such as adversity gene, described adversity gene can be such as RhNAC gene.According to content disclosed in the present application, those skilled in the art are knowing that described promotor is inducible promoter, when inducible promoter especially to ABA sensitivity, have the ability useful gene merge as downstream gene and described promotor and be transferred in plant completely, thus utilize ABA to control the expression of useful gene in transgenic plant.Therefore, useful gene described herein is limited to absolutely not RhNAC gene.In addition, described ABA can be the endogenous ABA that plant itself produces, and also can be the outside exogenous ABA applied.Preferably, described ABA is endogenous ABA.

In addition, the present inventor also finds, in described promotor, in plant, this promoter expression has popularity, and significantly, described startup comprises multiple series connection ABRE cis-acting elements, and the response of these cis-acting elements to ABA shows accumulative effect.Especially can be controlled kind and the quantity of described cis-acting elements by genetic modification means, control the expression of described promotor downstream gene quantitatively or accurately.

Present invention also offers carry out cloning for described promotor, detect, locate, the primer pair of process LAN or silence, wherein, described primer pair is selected from the group be made up of primer pair SEQ NO.2 and 15, SEQ NO.2 and 16, SEQ NO.2 and 17, SEQ NO.2 and 18, SEQ NO.3 and 19, SEQ NO.4 and 19, SEQ NO.5 and 6, primer pair SEQ NO.7 and 8, primer pair SEQ NO.9 and 12, primer pair SEQ NO.10 and 12, primer pair SEQ NO.11 and 12, primer pair SEQ NO.13 and 14.

In some embodiments, present invention also offers a kind of method of cloning described promotor, wherein, described method uses at least one primer be selected from the group be made up of primer pair SEQ NO.2 and 15, SEQ NO.2 and 16, SEQ NO.2 and 17, SEQ NO.2 and 18, SEQ NO.4 and 19 to carry out.Described method comprises the steps: to take DNA of plants as template, the first product obtained by any pair primer in primer pair SEQ NO.2 and 15, SEQ NO.2 and 16, SEQ NO.2 and 17, SEQ NO.2 and 18 by PCR; With described first product for template, obtain the second product by PCR by described primer pair SEQ NO.3 and 19; And with described second product for template, by PCR by the described promotor of described primer pair SEQ NO.4 and 19 clone.In other embodiment, described method obtains described promotor using DNA of plants as template by described primer pair SEQ NO.5 and 6 Direct Clonings.

In some embodiments, the invention provides a kind of method of clone or the promotor described in detection technique scheme 1, wherein, described method comprises use primer pair SEQ NO.5 and 6 and/or primer pair SEQ NO.7 and 8 and carries out; Preferably, described detection is undertaken by GUS Activity determination, and uses primer pair SEQNO.7 and 8 to carry out.

In some embodiments, present invention also offers and a kind ofly detect the method for plant to the susceptibility of ABA, described method comprises and detecting the promotor described in technical scheme 1; Preferably, described method comprises and detecting the existence of the ABA cis-acting elements of described promotor and/or quantity.

In some embodiments, the invention provides a kind of method improving Plant Tolerance environment stress ability, described method comprises and utilizes the promotor described in technical scheme 1 to carry out; Preferably, described method realizes the susceptibility of ABA by changing plant; In addition preferably, described method realizes the susceptibility of ABA by improving plant; In addition preferably, described environment stress causes the ABA growing amount of described plant to increase; In addition preferably, described environment stress be selected from coerced by Chilling stress, high temperature stress, Drought Stress are coerced, waterlogging environment stress, high salt environment stress, biotic, mechanical wounding coerce the group formed.In some embodiments, described method is realized by the amalgamation and expression of described promotor and stress tolerant being coerced relevant gene; Preferably, described stress tolerant coerces relevant gene is RhNAC3.In some embodiments, the endogenous ABA that described amalgamation and expression utilizes the exogenous ABA of outside applying or plant itself to produce induces; Further preferably, the endogenous ABA that described amalgamation and expression utilizes plant itself to produce induces.

The present inventor also surprisingly finds, RhNAC3 transgenic line improves the susceptibility to ABA.Under ABA and Osmotic treatment, the stomatal closure of RhNAC3 transgenic line is rapider, thus the dehydrating speed reduced when plant is subject to drought stress and fluid loss, which thereby enhance the ability of Plant Tolerance Water deficit.Plant all can produce ABA in numerous environment stress, and the above-mentioned promotor of RhNAC3 is responsive to ABA.Therefore, present invention also offers RhNAC3 and improving the application in Plant Tolerance environment stress ability, especially improve the application in Plant Tolerance Water deficit ability.Preferably, described application is by realizing the amalgamation and expression of described RhNAC3 gene and described promotor.

In addition, present invention also offers the carrier containing the promotor described in technical scheme 1; Preferably, described carrier is plasmid vector, phage vector or virus vector; Have choosing in addition, described carrier is cloning vector or expression vector.

Hereafter will be further detailed the present invention by embodiment.Should be understood that, these embodiments only for purpose of explanation, should not be understood to be the restriction of the scope to request protection of the present invention.

Embodiment

In order to study the regulatory mechanism of RhNAC3, RhNAC3 upstream regulatory sequence is separated, be extracted arge gene group DNA, utilize genomic dna, with High-tail PCR (Liu Y, Chen Y (2007) High-efficiency thermal asymmetric interlaced PCR for amplification of unknownflanking sequences.BioTechniques43:649-656.) method takes turns inverse PCR through 3 and obtains the promoter fragment of RhNAC3, and adopt the method for information biology to analyze the promoter fragment obtained, with clear and definite wherein comprised important cis-acting elements.

1.RhNAC3 promotor is cloned

Test materials cut rose flower (Rosa hybrid L.) kind ' Sa Mansha ' (' Samantha '), in vigorous vegetative growth phase gather healthy Chinese rose young young stem and leaf and be in business and gather rank the be 2 grades spray of (be in bud period), insert in immediately in water.Be put in plastics bag, for the extraction of DNA after bud point and spire are removed petiole.

The extraction of Chinese rose leaves genomic DNA adopts CTAB method, and concrete operations are as follows:

A. the young leaflet tablet sample of 2g cut rose flower is got, the mortar being placed in Liquid nitrogen precooler is fully ground to sample and presents Powdered, proceed to rapidly in 50mL centrifuge tube, add 2 × CTAB solution (1M Tris-HCl of 10mL65 DEG C of preheating, pH8.0,0.5M EDTA, 5M NaCl, 2%CTAB).Be placed in trash ice ice bath.

B. add 1mL B-mercaptoethanol, stir with smooth glass stick.Be placed in water-bath 45min under shaking bath 56 DEG C, rotating speed 100rpm, be carefully uniformly mixed for several times with glass stick in water-bath process.

C. add 10mL CIA (chloroform: primary isoamyl alcohol=24: 1, volume ratio) extract, gentle inversion mixes, and puts into water-bath 20min under 37 DEG C of shaking baths, rotating speed 120rpm.

D. centrifugal 20min under normal temperature, 3000rpm rotating speed.With the 1mL rifle head that excision is most advanced and sophisticated, Aspirate supernatant (containing genomic dna), proceeds in new 50mL centrifuge tube.

E. add the CTAB solution of supernatant volume 1/10, softly mix, and then add 10ml CIA (chloroform: primary isoamyl alcohol=24: 1, volume ratio) and to insert in 37 DEG C of shaking baths water-bath 20min under, rotating speed 120rpm.

F. centrifugal 20min under normal temperature, 3000rpm rotating speed.Careful Aspirate supernatant is in new 50mL centrifuge tube.

G. slowly add DNA precipitation buffering liquid (1M Tris-HCl, pH8.0,0.5M EDTA, 1%CTAB) along test tube wall, gentle inversion mixing 15-20 time, precipitates completely to DNA.

H. use glass crotch (alcohol blast burner drawing by high temperature is made) picking to go out DNA flocks, proceed in the test tube of the 1M NaCl containing 5mL, 56 DEG C of water-baths shake 2h gently.

I. use 70% and 100% ethanol purge DNA twice respectively, dry in air or vacuum.

J. the TE solution (10mM Tris-HCl, pH8.0,1mM EDTA) adding 1mL makes DNA dissolve, and spends the night at 37 DEG C.

After obtaining Chinese rose DNA, utilize general degenerated primer (SEQ NO.15 to 19) and the reverse special primer (SEQ NO.2, SEQ NO.3 and SEQNO.4) of RhNAC3 gene 3 to carry out 3 according to the method for High-Tail PCR and take turns PCR reaction and increase.

First round PCR degenerated primer (SEQ NO.15 to 18) reacts with primer SEQ NO.2 respectively, and condition is as follows: 94 DEG C of 5min, 95 DEG C 10min1 circulation; 95 DEG C of 15s, 61 DEG C of 15s, 72 DEG C 30s5 circulation; 95 DEG C of 15s, 25 DEG C of 3min, 0.5 DEG C/s rise to 72 DEG C of 3min, 72 DEG C 2min1 circulation;

95 DEG C of 15s, 44 DEG C of 15s, 72 DEG C of 30s3min; 95 DEG C of 15s, 61 DEG C of 15s, 72 DEG C of 2min, 95 DEG C of 15s, 61 DEG C of 15s, 72 DEG C of 2min, 95 DEG C of 15s, 44 DEG C of 15s, 72 DEG C 2min15 circulation; 72 DEG C 10min1 circulation; 10 DEG C of end;

Be used for second after four first round products that each pair of primer obtains are diluted 50 times respectively and take turns PCR reaction template, second takes turns PCR degenerated primer (SEQ NO.19) and Auele Specific Primer (SEQ NO.3) reacts, and condition is as follows:

95 DEG C of 15s, 63 DEG C of 15s, 72 DEG C of 2min, 95 DEG C of 15s, 63 DEG C of 15s, 72 DEG C of 2min, 95 DEG C of 15s, 44 DEG C of 15s, 72 DEG C 2min15 circulation; 72 DEG C 10min1 circulation; 10 DEG C of end.

Four second are taken turns after product dilutes 100 times respectively and is used for third round PCR reaction template, third round PCR degenerated primer (SEQ NO.19) and Auele Specific Primer (SEQ NO.4) react, condition is as follows: 95 DEG C of 15s, 64 DEG C of 15s, 72 DEG C of 2min, 95 DEG C of 15s, 64 DEG C of 15s, 72 DEG C of 2min, 95 DEG C of 15s, 44 DEG C of 15s, 72 DEG C 2min15 circulation; 72 DEG C 10min1 circulation; 10 DEG C of end.

3 take turns after reaction terminates, take turns second and be separated with third round PCR reaction product agarose gel electrophoresis, take turns according to second and to determine fragment for the purpose of gained fragment whether with the size of third round reaction product and the position that differs from of design SEQ NO.4 and SEQ NO.3, gained object fragment is adopted the DNA fast purifying/recovery test kit (AP-GX-50 of Axygen, Axygen) reclaim target DNA, be connected to pGEM-T Easy Vector Systems (Promega) of Promega company.To connect product conversion intestinal bacteria Dh5 α, getting 1 μ l bacterium liquid is template, determines positive colony with PCR detection and agarose gel electrophoresis analysis.Utilize positive colony to check order and preserve positive colony for future use.According to result design primer pair SEQ NO.5 and SEQ NO.6 after order-checking.

Using genomic dna as masterplate, SEQ NO.5 and SEQ NO.6 primer amplification is utilized to obtain the sequence (see SEQNO.1) that length is 1447bp.Be RhNAC3 promoter sequence by this sequence designations.

2. promoter sequence analysis

Plant cis-regulating element online database PLACE (http://www.dna.affrc.go.jp/PLACE/signalscan.html) is submitted to and PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) carries out cis-acting elements analysis by being separated the RhNAC3 promotor that obtains, analytical results shows, in the promoter sequence of 1447bp, except containing except the basal promoter elements such as CAAT and TATA, also include W-box, MYC binding member, CBF binding member, the cis-acting elements that different types of environment stresses such as MYB binding member are relevant.And more surprisingly, the ABRE binding member of the 5 kind different nucleotide type of RhNAC3 promotor also containing series connection.

Through database retrieval, there are no about the separation of RhNAC3 promotor and the report of functional study.

The Histochemical localization that 3.GUS expresses and fluorescence activity detect

After obtaining RhNAC3 promotor, in order to study the fundamental characteristics of RhNAC3 promotor, its Histochemical localization is studied, simultaneously owing to including the ABRE cis-acting elements of 5 series connection in RhNAC3 promotor, the GUS activity of its inducing properties under ABA process and different deletion fragment is probed into, to the regulatory mechanism of clear and definite RhNAC3.

Histochemical localization: using GUS as report, proceed in Arabidopis thaliana after RhNAC3 promotor is merged GUS, utilize primer pair SEQ NO.7 and SEQ NO.8 to detect the transfer-gen plant (adopt inflorescence dip method RhNAC3 promotor fusion GUS carrier proceeded in Arabidopis thaliana) of acquisition with RhNAC3 promotor.The T3 of different seedling age is put into 2ml test tube for organs such as the blade of Arabidopis thaliana transfer-gen plant, root and flowers, add 200 μ l histochemical stain liquid (75.5mM sodium phosphate pH7.0,0.1%Triton X-100,0.05mM K3/K4FeCN, 10mM EDTA, 20% methyl alcohol (v/v), 50 μ g ml ^-1the chloro-3-indyl-glucuronic acid of the bromo-4-of 5-).Place 5h in 37 DEG C of incubators, then decolour to manifesting white with the alcohol of 70%.GUS expressive site is observed and Taking Pictures recording under Stereo microscope (Olympus SEX16).

Fluorescence activity detects: utilize the N0 of primer pair SEQ NO.9 and SEQ NO.12 acquisition containing 5 series connection ABRE cis-acting elements, utilize the N1 of primer pair SEQ NO.10 and SEQ NO.12 acquisition containing 3 series connection ABRE cis-acting elements, primer pair SEQ NO.11 and SEQ NO.12 is utilized to obtain the N2 not containing ABRE cis-acting elements, will containing different number ABRE cis-acting elements deletion fragment (N0, N1, N2 and NC) proceed to protoplasts of Arabidopsis thaliana broken by ultrasonic after measure fluorescence activity (method is the same), RhNAC3 promotor is merged the transgenic line of GUS with after 10 μMs of process, measure the GUS fluorescence activity before and after ABA process respectively, Arabidopsis plant is put into 1.5ml Eppendorf test tube, add 300 μ l Arabidopis thaliana GUS extracting solution (50mM sodium phosphates (pH7.0), 10mM Na ₂-ethylenediamine tetraacetic acid (EDTA) (EDTA, pH8.0), 0.1% sodium lauroyl sareosine, 0.1%Triton X-100,10mM beta-mercaptoethanol), tissue is ground with refiner on ice.4 DEG C of centrifugal 10min of 8000rmp, proceed to new pipe by supernatant liquor.Prepare the MUG solution (1mM) of 160 μ L in advance in 1.5ml Eppendorf test tube, water-bath 5min at 37 DEG C, add the supernatant liquor of 40 μ l, rapidly fully mixing, and take out 100 μ l reaction mixtures at once and join (the Na of 0.2mol/L in the stop buffer of 900 μ l ₂cO ₃), using 0 point of this pipe as enzymatic reaction.After 15min, take out remaining 100 μ l reaction mixtures, join in the stop buffer of 900 μ l.With spectrophotometer under excitation wavelength 365nm, emission wavelength 455nm, the fluorescent value of different treatment sample before and after assaying reaction.Get the supernatant liquor of 100 μ l, Coomassie Brilliant Blue working sample protein content.The GUS calculated in each sample unit time is active.

GUS activity=(after reaction the front fluorescent value of fluorescent value-reaction)/protein content/reaction times

Result after GUS measures as shown in Figure 2, A figure shows the GUS activation analysis of RhNAC3 promoter deletion fragment in protoplasts of Arabidopsis thaliana broken by ultrasonic, compared with NC, include N0 (-1447 ~-160bp) difference extremely significantly (double asterisk compared with NC of the ABRE cis-acting elements of 5 series connection, P < 0.01), include N1 (-707 ~-160bp) significant difference (single asterisk, P < 0.05) compared with NC of the ABRE cis-acting elements of 3 series connection.B figure shows the impact of Exogenous ABA on GUS activity in the protoplasts of Arabidopsis thaliana broken by ultrasonic comprising RhNAC3 promoter deletion recombinant chou.Compared with contrast NC, when not adding ABA, the active there was no significant difference of GUS between different fragments, when after applying 10 μMs of ABA process, the N0 (-1447 ~-160bp) of the ABRE cis-acting elements containing 5 series connection reaches pole significant difference (double asterisk compared with NC, P value < 0.01), the N1 (-707 ~-160bp) of the ABRE cis-acting elements containing 3 series connection reaches significant difference (single asterisk, P value < 0.05) compared with NC.C figure shows the response of RhNAC3 promotor to ABA.The expression induction of RhNAC3 transcriptional activity that ABA is strong.

4.RhNAC3 transfer-gen plant phenotype is observed

The transcriptional activity of existence to RhNAC3 of ABRE cis-acting elements has very important effect, in order to the function of further clear and definite RhNAC3, the particularly phenotype of RhNAC3 transfer-gen plant after ABA process, measures the seed germination rate of RhNAC3 transfer-gen plant and Seedling Stage root growth phenotype and ABA process air holes aperture.

1) germination rate statistics

The RhNAC3 transgenosis T2 filtered out is sowed at containing ABA (0 for homozygote plant, 0.2 μM, 0.4 μM) MS substratum in (containing 3% sucrose and 0.7% agar), after 4 DEG C of low temperature vernalization 4d, long day (16h illumination, 8h is dark) after CMC model 7d, take pictures and add up germination rate.

Germination rate as shown in Figure 3, Seed Germination of Arabidopsis Pumila rate declines to some extent with the rising of ABA concentration, and under same level ABA (0.2 and 0.4 μM of ABA) process, compared with contrast (VC) plant, RhNAC3 transgenic line (OE#3,6,12) plant seed germination rate reduces more remarkable (Fig. 3 A and B)

2) primary root root length and side root statistics

T2 sprouts on the MS substratum adding 3% sucrose and 0.6% agar for seed, after 4 DEG C of low temperature vernalization 4d, after long-day conditions cultivates 7d, transfer on new MS substratum, comprise in substratum ABA (0,5 μMs, 10 μMs, 30 μMs), under long-day conditions, culture dish is stood to taking pictures after growth 10d and adding up the long and side radical order of plant root.

In seedling root phenotype, compared with contrast (VC) plant, RhNAC3 transgenic line (OE#3,6,12) all there is reduction trend in plant on length of root stalk and side radical order, and RhNAC3 process LAN also showed responsive (Fig. 3 C, D and E) ABA in the root growth stage.

3) transfer-gen plant stomatal movement observation

Select the Arabidopis thaliana of 10 Leaf-Age-Periods (transplanting about 11-13d later, the young plant of about 3 weeks sizes), select time new round size, the blade of state consistency.If blade there are the unsound states such as fold all do not select.

A. choose the Arabidopsis plant of neat and consistent, choose size in same impeller blade, the blade of state consistency, band petiole is cut.

B. vacuum side of blade upward, puts into epidermis bar damping fluid (2.0 centrifuge tube), immerses below damping fluid liquid level.Leaf in each pipe, and under the liquid of different pipe Leaf, depth requirements is consistent.

C., under different strains being evenly placed in cold light source, 3h is irradiated.

D. tear the lower epidermis getting blade Zhong Mai side to observe, see whether pore is opened completely.

If E. pore is opened completely, then add ABA and carry out process 3h.

F. tear the lower epidermis getting arteries and veins opposite side in blade to observe.

ABA dehydrated alcohol is configured to 100mM mother liquor, and working fluid is 1mM, dilute with water.Be stored in-20 DEG C at ordinary times.

The measurement result of stomatal aperture as shown in Figure 4, ABA can promote stomatal closure, and greatly can promote the stomatal closure of RhNAC3 transgenic line, make RhNAC3 transgenic line (OE#3 under ABA process, 6,12) stomatal aperture is less, stomatal closure more rapid (Fig. 4 A); 10d Osmotic treatment can induce RhNAC3 Transgenic plant leaf stomatal aperture obviously to reduce (Fig. 4 B) relative to contrast (VC).RhNAC3 is to rely on the mode involved in plant of ABA to the response of water stress.

RhNAC3 transgenic line shows as ABA more responsive under ABA process, in order to explain the control methods of high ABA susceptibility further, choose ABA responsive genes in Arabidopis thaliana and Chinese rose, real-time fluorescence quantitative PCR is adopted to detect its expression amount, to the relation of clear and definite ABA responsive genes and ABA susceptibility.

5. real-time fluorescence quantitative PCR (qRT-PCR)

A. adopt Trizol method to extract plant tissue materials RNA, use spectrophotometer detectable level, denaturing formaldehyde glue Detection job.

B. DNase I (Takara company, D2215,5U/ μ L) is utilized to process RNA sample.

Adopt the 10 μ L reaction systems that breast is following: total serum IgE 5 μ g (2 ~ 5 μ g can be processed); DNase I1 μ L; DEPC H2O polishing is to 10 μ L;

C. gently centrifugal after system mixing, at 37 DEG C, process 20min.

D. reacted rear 65 DEG C of process 10min and made DNaseI sex change (completing in PCR instrument), the complete sample of sex change is-80 DEG C of preservations.

E. get 1 μ L sample and run glue detection RNA quality, get 1 μ L in addition and dilute 100 times, measure the content of RNA.

F. adopt SuperScriptTM II RNase H Reverse Transcriptase (Invitrogen) and Oligo dT18 (Invitrogen) to carry out reverse transcription, adopt 25 following μ L reaction systems: oligodT primer 1 μ L; Total serum IgE 2 μ g; Moisturizing to 18 μ L, 70 DEG C of sex change 5min, cool rapidly at least 1min, centrifugal; 5 × the first chain damping fluid 5 μ L; DNTP1 μ L; UperScriptTM II RNase H reversed transcriptive enzyme 1 μ L.

G. flick mixing at the bottom of pipe after adding sample centrifugal, 42 DEG C of reactions 90min, rear ice bath 10min, dilute-20 DEG C, 5 times of acquisition cDNA first chains and save backup.

H. real-time fluorescence quantitative PCR: adopt instrument ABI PRISM Step ONE PlusReal-time PCR System (Applied Biosystems, Foster City, CA, and test kit KAPA SYBR FAST qPCR KIT (KAPA Biosystems USA), Boston, Massachusetts, USA) carry out quantitative PCR reaction.

I., after having added reaction system, sample is added in real-time PCR (AppliedBiosystems, Foster City, CA, USA), it is as follows that reaction conditions is set:

95 DEG C of 10min; 95 DEG C of 15s; 60 DEG C of 1min, 40 circulations.

J. reaction terminates laggard row data processing, adopts two Δ Δ T method to calculate the relative expression quantity of each gene.

In Arabidopis thaliana and Chinese rose to the detection of expression result of ABA responsive genes as shown in Figure 5.In Arabidopis thaliana, the RhNAC3 positive regulation expression of ABA responsive genes (Fig. 5 A), in reticent RhNAC3 Chinese rose petal, ABA signal and downstream gene must be expressed and be subject to obvious suppression (Fig. 5 B and C), and these results show that RhNAC3 forward regulates the expression of ABA responsive genes to participate in ABA signal pathway.

Utilize Arabidopis thaliana database (http://www.arabidopsis.org) TAIR Loci Upstream Seq-1000bp method, further analysis has been carried out to the upstream regulatory sequence of ABA responsive genes in Arabidopis thaliana, to determine these genes upstream regulatory domains in whether containing NAC protein binding element, acquired results is as shown in table 1.Wherein obtain the regulating and controlling sequence of the upstream 1000bp of 11 ABA responsive genes, by the analysis to upstream regulatory sequence.In analyzed 11 ABA responsive genes, all find the NAC protein binding site containing different number and type, show that said gene is the direct target gene of RhNAC3 regulation and control.

Table 1ABA responsive genes promoter sequence NAC binding member is analyzed

This clone upstream promoter sequence of RhNAC3 gene 1447bp, and bioinformatic analysis has been carried out to acquisition sequence, containing the cis-acting elements that the stress response that ABRE, W-BOX, MYC, MYB, CBF etc. are dissimilar is relevant in RhNAC3 promotor, using GUS as report, Histochemical localization has been carried out in Arabidopis thaliana, the expression of RhNAC3 promotor has popularity, especially with strong expression in Stoma of Leaves and in vascular bundle.Transcriptional activity containing 5 series connection ABRE cis-acting elements in RhNAC3 promotor is higher than the transcriptional activity of 3 series connection ABRE cis-acting elements, the existence of ABRE element has accumulative effect to RhNAC3 promoter activity, and ABA process can strengthen the raising of RhNAC3 promoter activity.Analyzed by the seed germination rate in RhNAC3 transgenic line, primary root growth and side radical order, and the stomatal conductance under ABA condition and under Osmotic treatment is measured, prove that RhNAC3 can improve the susceptibility of transgenic plant to ABA.ABA responsive genes in Arabidopis thaliana and Chinese rose is detected, finds that RhNAC3 just can regulate and control the expression of ABA responsive genes, and in the promoter region of said gene, containing numerous NAC protein binding site.

Claims (10)

1. a promotor for NAC protein transcription factor gene, wherein, described promotor and SEQ No.1 have at least 50%, the identity of such as 60%, 70%, 80%, 90%, 91%, 92%, 95%, 97%, 98%, 99% or 100%;

Preferably, described nucleotide sequence comprises at least one in the group being selected from and being made up of acgtg, acgtgc, acgtgg, acgtgc and acgtgtc, at least two kinds, at least three kinds, at least four kinds or at least five kinds of cis-acting elements.

2. for cloning promotor according to claim 1, detecting, locate, the primer pair of process LAN or silence, wherein, described primer pair is selected from the group be made up of primer pair SEQ NO.2 and 15, SEQ NO.2 and 16, SEQ NO.2 and 17, SEQ NO.2 and 18, SEQ NO.3 and 19, SEQ NO.4 and 19, SEQ NO.5 and 6, primer pair SEQ NO.7 and 8, primer pair SEQ NO.9 and 12, primer pair SEQ NO.10 and 12, primer pair SEQ NO.11 and 12, primer pair SEQ NO.13 and 14.

3. the method for clone's promotor according to claim 1, wherein, described method uses at least one primer be selected from the group be made up of primer pair SEQ NO.2 and 15, SEQ NO.2 and 16, SEQ NO.2 and 17, SEQ NO.2 and 18, SEQ NO.3 and 19, SEQ NO.4 and 19 to carry out.

4. clone or test right require a method for the promotor described in 1, and wherein, described method comprises use primer pair SEQ No.5 and 6 and/or primer pair SEQ NO.7 and 8 and carries out;

Preferably, described detection is undertaken by GUS Activity determination, and uses primer pair SEQ NO.7 and 8 to carry out.

5. detect the method for plant to the susceptibility of ABA, described method comprises and detecting promotor according to claim 1;

Preferably, described method comprises and detecting the existence of the ABA cis-acting elements of described promotor and/or quantity.

6. improve a method for Plant Tolerance environment stress ability, described method comprises and utilizes the promotor described in claim 1 to carry out;

Preferably, described method realizes the susceptibility of dormin by changing plant;

In addition preferably, described method realizes the susceptibility of dormin by improving plant;

In addition preferably, described environment stress causes the dormin growing amount of described plant to increase;

In addition preferably, described environment stress be selected from coerced by Chilling stress, high temperature stress, Drought Stress are coerced, waterlogging environment stress, high salt environment stress, biotic, mechanical wounding coerce the group formed.

7. method as claimed in claim 6, wherein, described method is realized by the amalgamation and expression of described promotor and stress tolerant being coerced relevant gene;

Preferably, described stress tolerant coerces relevant gene is RhNAC3.

8. the method according to claim 6 or 7, wherein, the endogenous dormin that described amalgamation and expression utilizes the exogenous abscisic acid of outside applying or plant itself to produce is induced;

Further preferably, the endogenous dormin that described amalgamation and expression utilizes plant itself to produce is induced.

9.RhNAC3 gene coerces the application in ability at raising Plant Tolerance;

Preferably, described application is by realizing the amalgamation and expression of described RhNAC3 gene and promotor according to claim 1.

10. the carrier containing promotor according to claim 1;

Preferably, described carrier is plasmid vector, phage vector or virus vector;

Have choosing in addition, described carrier is cloning vector or expression vector.