CN101348791A - Populus euphratica Oliv hydrotropic gene PeXET and promoter thereof - Google Patents

Abstract

The invention relates to a hydrotropic gene, in particular to a root hydrotropic gene, namely a PeXET gene (SEQ ID No.1) for diversifolious poplar and proteins (SEQ ID No.2) translated by the gene. The invention also relates to a promoter (SEQ ID No.3) of the hydrotropic gene - the PeXET gene for the diversifolious poplar. The invention also relates to the PeXET gene and application of the promoter of the PeXET gene. Moreover, the invention also relates to a plant root hydrotropic reaction induction device.

Description

Populus euphratica Oliv hydrotropic gene PeXET and promotor thereof

Technical field

The present invention relates to a kind of hydrotropisms's gene, more specifically, relate to the root system hydrotropisms gene PeXET gene (SEQ ID No.1) of diversiform-leaved poplar, and the protein (SEQ ID No.2) of this gene translation.The invention still further relates to the promotor (SEQ ID No.3) of Populus euphratica Oliv hydrotropic gene PeXET gene.The invention still further relates to the application of PeXET gene and promotor thereof.In addition, the invention still further relates to the reaction induced device of root system of plant hydrotropisms.

Background technology

Can build the long high megaphanerophyte of all living creatures in the drought desert area and have diversiform-leaved poplar (Wang Shiji etc. only, the diversiform-leaved poplar woods, Beijing: China Forest press: 29,1995), therefore diversiform-leaved poplar is the very precious degeneration-resistant germ plasm resource of xylophyta, is popular to it in anti-contrary research aspect machine-processed always.

Diversiform-leaved poplar (Populus.euphratica Oliv.) is the most ancient in Salicaceae (Salicaeae) Populus (Populus), primary seeds, originates from fossil species and becomes the leaf poplar.Diversiform-leaved poplar originates from the ground that has abundant water resources as a kind of Relict Plant.Yet, along with its growth region changes of environment, the environmental compatibility of secular arid and Desertification makes the form of its assimilation organ that corresponding change take place, show as the leaf (Zheng Caixia etc. that variform on same strain tree body, occurred, the comparison of diversiform-leaved poplar multiform leaf stoma feature and photosynthesis characteristics, forest-science, 42 (8): 19-24,2006).And the diversiform-leaved poplar blade of these variforms is in growth course, variation (Qiu Jian etc. have taken place on its photosynthesis characteristics, the comparative studies of polymorphic leaf photosynthetic rate of diversiform-leaved poplar and fluorescent characteristic, the Jilin forestry science and technology, 3:19-21,2005), blade has the pore that heterogeneity opens and closes, stomatal frequency from lanceolar to the sawtooth ovaide leaf is changed to and waits characteristics (Zheng Caixia etc. from small to large, the comparison of diversiform-leaved poplar multiform leaf stoma feature and photosynthesis characteristics, forest-science, 8:19-24,2006), these characteristics of blade all are in order to adapt to the mal-condition under the arid and high light intensity environment in the habitat.

1. the drought-enduring research of diversiform-leaved poplar

Diversiform-leaved poplar is extremely drought-enduring, have " give birth to 3,000 years not dead, do not fall in dead 3,000 years, fall 3,000 years immortal " great fame.By diversiform-leaved poplar morphology and blade anatomical research result being shown (Wang Xilin etc., diversiform-leaved poplar seeding and seedling raising technology, forestry science and technology communication, 1:1-2,1985), diversiform-leaved poplar has some features of typical xeromorph seeds.

Morphologic feature:

(1) there is the cover of wax on the heteromorphism of leaf and keratin, blade face, and leaf is elongated, hard and thick, help setting the consumption that body reduces moisture;

(2) sprig drapes over one's shoulders wax and down, helps reflecting the radiation of high light and reduces water consumption;

(3) trunk is short and small and sturdy usually, helps wind resistance.

The anatomic characteristics of leaf (Luo Xiuying etc., the anatomical structure of several xerophyte leaves in Xinjiang (assimilation branch) is observed Xinjiang University's journal, 1:77-85,1986): diversiform-leaved poplar blade cutin bed thickness is big; The sagging degree of depth of pore is big; Palisade tissue is highly developed; It is flourishing to dredge tissue; The diversiform-leaved poplar leaf contains the druse cell, and moisture minimizing loss of water has special role in leaf for keeping for this; Mechanical tissue strengthen (Jiang advances, under the extreme weather conditions water regime of diversiform-leaved poplar and and environmental relation, arid zone research, 2:35-38,1991).

At occurring in nature, diversiform-leaved poplar has the characteristic of typical root system hydrotropisms growth.The drought-enduring growth mechanism of research diversiform-leaved poplar root system hydrotropisms has typical forestry characteristic and extreme important use is worth.

2. the hydrotropic progress of root system

Hydrotropic research concentrated on the aspects such as mensuration of hydrotropisms's apparatus for deivation and relevant physiological data mostly to root system in the past, Takano etc. are cloned into transglycosylase in the xyloglucan (Ps-EXGT1) gene from the ageotropum root system, obtain the partial cDNA Cloning of EXGT, be about 632bp, just begun research hydrotropic molecular level.This research also shows that this gene can be expressed in root and the stem that is extending and show very high transcriptional level in the stem of elongation fast, but does not express in sophisticated stem and spire.Simultaneously, Takano etc. think, the transcribing of Ps-EXGT1 relates to the cell growth and this transcriptional regulatory is played the part of important role (Takano M.et al. in the difference growth of root system hydrotropisms response, Calcium requirement for the induction of hydrotropism andenhancement of calcium-induced curvature by water stress in primary roots ofpea, Pisum sativum L, Plant and Cell Physiology, 2-3:157-174,1997).EXGT (endoxyloglucan transferase) is the xyloglucan transglucosidase with XET (xyloglucan endotransglycosylase) translator of Chinese, only there is difference nominally in both, both representatives all are structure gene (Rose Jocelyn K.et al. with XTH functional domain, The XTHFamily of Enzymes Involved in Xyloglucan Endotransglucosylation andEndohydrolysis:Current Perspectives and a New Unifying Nomenclature, Plant and Cell Physiology, 12:1421-1435,2002).This is an extended familys protein on the cell walls determined in more than 20 year in the past of a class.After first Plant Genome order-checking plan is finished, analyze demonstration by full sequence to this gene family, this only is difference name (the Rose Jocelyn K.et al. that same gene is carried out at the different times disparate databases, TheXTH Family of Enzymes Involved in Xyloglucan Endotransglucosylation andEndohydrolysis:Current Perspectives and a New Unifying Nomenclature, Plant and Cell Physiology, 12:1421-1435,2002).

Transglycosylase belongs to transglycosylase lytic enzyme (Xyloglucanendotransglucosylase-hydrolase in the xyloglucan in the xyloglucan, XTH) gene family (Rose Jocelyn K.et al., TheXTH Family of Enzymes Involved in Xyloglucan Endotransglucosylation andEndohydrolysis:Current Perspectives and a New Unifying Nomenclature, Plant and Cell Physiology, 12:1421-1435,2002), main effect is the fracture by general acid catalysis effect catalysis glycosidic link.XTHs belong to a bigger enzyme family glycoside hydrolase (Glycoside hydrolases, GH).In recent years, from some plants, cloned the cDNAs of transglycosylase in the xyloglucan, as: from red bean (Vigna angularis), wheat (Triticum aestivum), Arabidopis thaliana (Arabidopsis thaliana), tomato (Lycopersicon esculentum) (AntosiewiczD.M.et al., Cellular localization of Arabidopsis xyloglucanendotransglycosylase-related proteins during development and after windstimulation., Plant Physiology, 4:1319,1997), paddy rice (Oryza sativa) etc.The ORF of 33 coding XTH protein family genes that from Arabidopis thaliana (Colombia's type), clone, relevant arabidopsis gene group database and cDNA sequence library (Initiative T.Ag have been submitted to, Analysis of the genome sequence of the flowering plant Arabidopsis thaliana, Nature, 796-815,2000).33 XTH genes involveds of this Arabidopis thaliana and proteic research will lay the foundation to other species XTH genes involved and proteic research.

The function of transglycosylase (XET) is to constitute the β-1 of cell walls micro-fibril xyloglucan in the xyloglucan, the 4-glycosidic link cuts off, and the oligosaccharide molecular transfer of downcutting laid equal stress on receive the congeneric elements end, this function is called again cuts chain transfer (Fry S.C., Polysaccharide-ModifyingEnzymes in the Plant Cell Wall, Annual Reviews in Plant Physiology and PlantMolecular Biology, 1:497-520,1995).Simultaneously XET can modified cellulose xyloglucan network system, and xyloglucan is one of main component of plant primary wall.In addition, XET can make changes the base effect in the xyloglucan generation, xyloglucan is cut, and form another xyloglucan molecule again, arranges little wooden Portugal again and gathers-fibrous reticulum, thereby cell walls is prolonged.Therefore, XETs reinvents the shape of cell walls in the cell expansion process.Under man-made environment, as: low moisture, high density material, endonuclease capable in several glycoside hydrolysis enzyme families has the glycosyl of commentaries on classics activity (Crout D.Hg et al., Glycosidases and glycosyl transferases in glycoside and oligosaccharidesynthesis, Current Opinion in Chemical Biology, 1:98-111,1998; YorkWilliam S.et al., Preparation of oligomeric{beta}-glycosides from celluloseand hemicellulosic polysaccharides via the glycosyl transferase activity of aTrichoderma reesei cellulase, Glycobiology, 2:193-201,2000).

The biological function of XET is the growth of its involved in plant, and make the wooden dextran of new synthetic polymkeric substance enter cell walls, or the reversible Mierocrystalline cellulose-wooden dextran network that loosens, the cell that allows turgescence to drive enlarges (De S.Ilva et al., Xyloglucan endotransglycosylase and plant growth, Journal of experimental botany, 280:1693-1701,1994).XET participates in the cell enlargement effect, the activity of various XET has shown relevant with elongation, maize root system (Pritchard J.Eremy et al. for example, Xyloglucan Endotransglycosylase Activity, MicrofibrilOrientation and the Profiles of Cell Wall Properties Along Growing Regions ofMaize Roots, J.Exp.Bot., 8:1281-1289,1993).GA (dormin) can significantly improve the activity of XET.The relevant gene (XTR) of 4 kinds of XET: OsXTR1 is arranged in the paddy rice, OsXTR2, OsXTR3 and OsXTR4, OsXTR1 wherein, OsXTR3 mainly expresses in the internode elongation district.OsXTR1 among the paddy rice mutant WaitoC of short stem, OsXTR3 mRNA level is than low in the wild plant, external source GA ₃Increase OsXTR1, the expression level of OsXTR3, show that GA promotes cell elongation (Cui D.et al. by the transcriptional level that improves the XET genes involved, Gibberellin-regulated XET isdifferentially induced by auxin in rice leaf sheath bases during gravitropicbending, Journal of Experimental Botany, 415:1327-1334,2005).

Purifying obtains transglycosylase (EXGT) in the xyloglucan first from red bean (Vigna angularis), this enzyme is acknowledged as and is playing an important role aspect the cell walls extensibility, can reconnect in another different xyloglucan chain by the sugar chain after also will cutting subsequently at inside cutting xyloglucan polymer, to regulate elongation growth (the Smith R.C.et al. of vegetable cell, Endotransglycosylation of xyloglucans in plant cell suspension cultures, Biochem.J, 529-535,1991; Nishitani K.et al., Endo-xyloglucan transferase, anovel class of glycosyltransferase that catalyzes transfer of a segment ofxyloglucan molecule to another xyloglucan molecule, Journal of BiologicalChemistry, 29:21058-21064,1992; Fry S.C.et al., Xyloglucanendotransglycosylase, a new wall-loosening enzyme activity from plants, Biochemical Journal, Pt 3:821,1992; Farkas V.et al., Cleavage of xyloglucanby nasturtium seed xyloglucanase and transglycosylation to xyloglucan subunitoligosaccharides., Arch Biochem Biophys, 2:365-70,1992; Fanutti C.et al., Action of a pure xyloglucan endo-transglycosylase (formerly calledxyloglucan-specific endo-(1 fi 4)-b-D-glucanase) from the cotyledons ofgerminated nasturtium seeds, Plant J, 691-700,1993; Nishitani K., Endo-xyloglucan transferase, a new class of transferase involved in cell wallconstruction, Journal of Plant Research, 1:137-148,1995; Nishitani K., Therole of endoxyloglucan transferase in the organization of plant cell walls., IntRev Cytol, 157-206,1997).Takano etc. are cloned into transglycosylase in the xyloglucan (Ps-EXGT1) gene (Takano M.et al. from the ageotropum root system, Endoxyloglucan transferasecDNA isolated from pea roots and its fluctuating expression in hydrotropicallyresponding roots, Plant and Cell Physiology, 2:135-42,1999a).Under the condition of water stress, obviously be obstructed near the zone that the root system stretching, extension is grown in the elongation zone, and cause this elongation zone (Sharp R.E.et al. that shortens, Growth of the Maize Primary Root at Low WaterPotentials:I.Spatial Distribution of Expansive Growth, Plant Physiology, 1:50-7,1988).But the growth velocity of elongation zone end is not subjected to influences of water stress, interior transglycosylase activity (the Wu Y.et al. of xyloglucan that is still keeping higher level, Root GrowthMaintenance at Low Water Potentials (Increased Activity of XyloglucanEndotransglycosylase and Its Possible Regulation by Abscisic Acid)., PlantPhysiology, 2:607,1994).Therefore, transglycosylase may play an important role aspect root elongation growth and the tropism's response in the xyloglucan, but the molecule mechanism of transglycosylase participation root system stretching, extension growth is still waited to be set forth in the xyloglucan.

The diversiform-leaved poplar root system has tangible hydrotropisms's growth characteristics, in Soil Moisture preferably under the situation, diversiform-leaved poplar root system main root is distributed in underground 50-80 centimetre, extremely pricking very deeply under the arid edaphic condition, can reach underground 13.5 meters (Wang Shiji etc., diversiform-leaved poplar woods, Beijing: China Forest press, 29,1995).Follow the tracks of, absorb at the desert area root system and utilize and undergroundly keep and continue the life of plant in the arid area.Compare with red bean, it is similar to red bean that the diversiform-leaved poplar root system has this specific character of hydrotropisms growth, and genes involved such as XET that the hydrotropisms who has in the red bean reacts also should exist in diversiform-leaved poplar; But the diversiform-leaved poplar root system can not appear in the newspapers in red bean to the underground this physiological phenomenon that extends to tens meters.No matter this ability is due to the qualitative character or the gene of quantitative character, before this as yet not relevant for the report of diversiform-leaved poplar XET gene.Therefore the XET gene of cloning and utilize diversiform-leaved poplar to contain is all had novelty, novelty and a practicality.Utilize particularly forest drought tolerance of this improvement of genes plant, be to satisfy pressing for of China's ecotope and the development of forestry products multiple-effect energy service society, meet National Program for Medium-to Long-term Scientific and Technological Development and " forest-science and technical development medium-term and long-term plans (2006-2020) ".

3. about the research of plant gene promoter

In plant genetic engineering, often efficiently express in the transgenic plant by highly active constitutive promoter driving foreign gene.But the continuous expression of foreign gene on time and space can increase the load of intracellular matter and energy, thereby influences growth and the growth of transgenic plant.Adopt tissue-specific promoter just can control foreign gene effectively and in specific tissue or organ, express, avoid gene product that receptor biological and environment are had side effects.CaMV (cauliflower mosaic virus) 35S promoter and NOS (rouge alkali synthetase) promotor have obtained a large amount of application in the research of transgenic plant.Because constitutive promoter can not be regulated and control the expression of goal gene on time and the space effectively, so in the genetic improvement of crop, there is certain defective (Gittins J.R.et al., Transgeneexpression driven by heterologous ribulose-1,5-bisphosphatecarboxylase/oxygenase small-subunit gene promoters in the vegetative tissuesof apple (Malus pumila Mill.), Planta, 2:232-240,2000).As utilize the constitutive promoter viral capsid proteins, just may cause that viral capsid shifts, thereby generation (the Robinson D.J. that causes the new strain of plant virus system, Environmental risk assessment of releases oftransgenic plants containing virus-derived inserts, Transgenic Research, 5:359-362,1996), therefore, seek the specificity promotor, make the key link that foreign gene is expressed has specifically become plant genetic engineering.The organizing specific promotor is as a class specificity promotor, can instruct foreign gene a certain specific spatial and temporal expression in the development of plants process, it can not only make the expression product of goal gene accumulate at certain space, increase regional expression amount, and avoid the unnecessary waste of plant nutrition, therefore, organizing specific promotor such as seed (Cahoon E.B.et al., Production ofFatty Acid Components of Meadowfoam Oil in Somatic Soybean Embryos, Plant Physiology, 1:243-252,2000), fruit (Chengappa S.et al., Transgenictomato plants with decreased sucrose synthase are unaltered in starch and sugaraccumulation in the fruit, Plant Molecular Biology, specific promoter such as 2:213-221,1999) has become the starting element of foreign gene the most promising in the transgenic research.

Summary of the invention

The present invention relates to a kind of hydrotropisms's gene, more specifically, relate to the root system hydrotropisms gene PeXET gene (SEQ ID No.1) of diversiform-leaved poplar, and the protein (SEQ ID No.2) of this gene translation.The invention still further relates to the promotor (SEQ ID No.3) of Populus euphratica Oliv hydrotropic gene PeXET gene.The invention still further relates to the application of PeXET gene and promotor thereof.In addition, the invention still further relates to the reaction induced device of root system of plant hydrotropisms.

According to embodiments more of the present invention, the present invention extracts RNA respectively from diversiform-leaved poplar blade, stem section, root system and the callus of drought stress.Then, according to the Ps-EXGT1 gene order on the GeneBank by BLAST at sequence of threads Populus sequence A F515607 and the EF194052 that search obtains that in the nucleic acid Non-redundant data storehouse nr that Polulus belongs to, compare.On the GenBank with AF515607 and EF194052 gene order by BLAST in the sequence of threads search of in the EST storehouse of diversiform-leaved poplar (P.euphratica), comparing, search obtains following two est sequence: AJ772752 and AJ778011.This two sequences is carried out sequence assembly by DNAMan software.Be used for from primer PeXET-722-1, PeXET-722-2 and the PeXET-999-3 of diversiform-leaved poplar according to above-mentioned splicing sequences Design by pcr amplification PeXET.The RNA that utilizes described primer and extract from the diversiform-leaved poplar tissue of drought stress carries out reverse transcription PCR, the dna sequence dna of the PeXET that obtains predicting.

According to another embodiment of the invention, the present invention has made up PeXET dna recombinant expression carrier, be further used for research about the PeXET gene function, for example can utilize the Agrobacterium transfection plant that has transformed described recombinant expression vector, obtain transgenic plant, for example transgenic plant of drought tolerance.

In addition, the present invention also provides the promoter sequence (pPeXET, SEQ IDNo.3) of PeXET gene, has made up the segmental expression vector of promoter deletion, the transient expression that drives GUS by pPeXET detects, and verifies the activity of described diversiform-leaved poplar root system hydrotropisms PeXET gene promoter.

At present, do not know also which effect the PeXET gene promoter specifically has, do not find as yet in the world this promotor to be done research.Find that by the result of study of describing among the present invention this gene is only expressed, and therefore, infers that this promotor has the function of tissue specific expression in callus and root system.After cloning this promotor, itself and gus reporter gene are merged, and by conversion to tobacco plant, after cultivating these tobaccos acquisition seeds, in the hope of passing through Eapen D.et al., Hydrotropism:root growth responses to water, Trends in Plant Science, 1:44-50, the method for describing in 2005 is carried out promoter function and is determined.

Description of drawings

From the detailed description below in conjunction with accompanying drawing, above-mentioned feature and advantage of the present invention will be more obvious, wherein:

Fig. 1. root system hydrotropisms induced reaction device.A. the culture apparatus of horizontal positioned, have only a flank side surface have the infiltration hole (among Figure 1A the infiltration hole in the left side,

Be represented by dotted lines), rotary axle is arranged in the middle of the bottom; B. behind the plant normal growth that the culture apparatus of axis top is downward-sloping, the angle of inclination determines at the actual grade of field growing that according to plant the infiltration hole in culture apparatus left side can draining towards ground.

Fig. 2. the prediction of diversiform-leaved poplar PeXET gene order.2A.AJ772752 and the sequence assembly of AJ778011; 2B. the splicing sequence of AJ772752 that obtains and AJ778011; 2C.PeXET splicing sequence and AF515607 and EF194052 compare.

Fig. 3. diversiform-leaved poplar PeXET Gene RT-PCR.A.722bp big or small fragment; B.999bp big or small fragment.M:100bp Ladder; 1: leaf cDNA; 2: root system cDNA; 3: stem section cDNA; 4: callus cDNA.

The gene order of Fig. 4 .PeXET and protein prediction sequence.

Fig. 5. diagram PeXET expression vector construction strategy.

Fig. 6. the structure of diagram PeXET expression vector.The enzyme-added site PCR that cuts of A.PeXET detects; The B.PeXET-pMD18-T double digestion detects and reclaims; The double digestion of C.pBin438 carrier; The PCR of D.pBin438-PeXET carrier detects; The double digestion of E.pBin438-PeXET carrier detects; F. changeing pBin438-PeXET Agrobacterium PCR detects.

Fig. 7. diagram iPCR design of primers.7A.iPCR the principle of work synoptic diagram; 7B.PeXET the gene structure synoptic diagram, intron, exon and restriction enzyme site that diagram is wherein comprised; 7C. diagram iPCR design of primers.

The total length promoter sequence of Fig. 8 .PeXET gene.

Fig. 9 .pCAMBIA1301 carrier collection of illustrative plates.

Figure 10. the big logotype and the PCR result of disappearance promotor.

Figure 11. the promotor transient expression detects.Ck-is unconverted tobacco leaf disc, and the p1301 representative has transformed the tobacco leaf disc of pCAMBIA1301 empty carrier, and 1831,1425,993,717 and 588 representatives have transformed the tobacco leaf disc of the promoter deletion carrier of different lengths respectively.

Embodiment

Come further to illustrate the present invention by the following examples.But should be appreciated that described embodiment is illustrational purpose, and be not intended to limit the scope of the invention and spirit.

The drought stress of embodiment 1. diversiform-leaved poplars

Design is applicable to the experimental installation (referring to Fig. 1) of Populus euphratica Oliv hydrotropic induced reaction, and the physiological response of inducing plant that is grown in is wherein measured.With reference to the hydrotropic research report of maize root system (Takahashi H.et al., Hydrotropism and its interaction with gravitropism inmaize roots, Plant Physiology, 558-64,1991), design xylophyta root system hydrotropisms induced reaction device, as shown in Figure 1, it comprises: (1) culture apparatus, have only a side side that the infiltration hole is arranged; (2) rotary axle is arranged in the middle of the bottom of described culture apparatus, described axle is fixed by A-frame, and the rotation of described axle can be so that described culture apparatus tilts, and a side side that makes the infiltration hole is towards ground.In addition, above described culture apparatus, be equipped with demountable at any time fluorescent light source and Controlling System thereof, plant is obtained and the similar illumination condition of physical environment.Referring to Fig. 1, Figure 1A: the device during horizontal positioned, have only side, the left side have the infiltration hole (

Be represented by dotted lines), rotary axle is arranged in the middle of the bottom; Figure 1B: behind the plant normal growth, with described axle rotating certain angle, with oblique the putting down of culture apparatus of axis top, the infiltration hole in left side is towards ground, the simulating drought stress conditions.In the present embodiment, the long 170cm of described root system hydrotropisms induced reaction device, wide 110cm, high 50cm; Support: end 60cm, hypotenuse 65cm, high 63.5cm.Yet, it should be appreciated by those skilled in the art that the size of device can be adjusted according to actual needs.

Described root system hydrotropisms induced reaction apparatus features is horizontal positioned to plant nursery stock, and the drought condition on simulating nature level land is handled nursery stock; Also can the simulating nature hillside, the angle of inclination of adjusting described culture apparatus by the rotation of axle forms the drought condition processing nursery stock consistent with the natural hill gradient, and the simulation massif gradient of this device is in 0 ° of-45 ° of scope; Above nursery stock, be equipped with demountable at any time fluorescent light source and Controlling System thereof, plant is obtained and the similar illumination condition of physical environment.Described device can also be made the proterties of different sizes according to actual place.When the simulating drought stress conditions, described culture apparatus has a side in infiltration hole towards ground.

In the present embodiment, described root system hydrotropisms reacts analogue experiment installation can simulate the diversiform-leaved poplar regional physical environment of growing, and produces the water potential gradient on the slope, induces the hydrotropic growth response of root system.

2 years living seeds to fetching from field, nursery, diversiform-leaved poplar breeding base, Ejina Banner are implanted in the described culture apparatus, the river sand that the culture medium of filling in the culture apparatus is to use 8 mesh sieves to sieve.The plant of plantation is 3 on 6 row * 6 row * every piers.The delegation of next-door neighbour's infiltration hole side is set to guard rows (that is the 6th of Figure 1B the row).This culture apparatus is placed in the greenhouse, the biological center of Beijing Forestry University cultivates.Room temperature maintains 28 ± 2 ℃, relative air humidity 60-80%, and the natural lighting on daytime, behind the 17:30, light filling 2000Lux to 21:00 keeps more than the whole day illumination 15h.

Root system hydrotropisms after the diversiform-leaved poplar nursery stock normal growth in the culture apparatus can tilt when living spray mean length is about 15-20cm then induces experiment.Through being cultured to label is that wilt (temporary wilting) has just appearred in the diversiform-leaved poplar blade of 1 plant row.Collection is numbered delicate white root root system of 1～5 plant and young leaflet tablet, and numbering is placed in the liquid nitrogen quick-frozen and continues to employ rapidly.

Embodiment 2. extracts the RNA of the diversiform-leaved poplar of drought stress

Select diversiform-leaved poplar blade, stem section, root system and the callus of drought stress, extract RNA respectively according to the CTAB-LiClRNA extracting method.Concrete extracting method is as follows:

1) will be used for CTAB that RNA extracts extracts damping fluid (prescription: 2%CTAB (ten

Six alkyl trimethyl ammonia bromides); 2%PVP (polyvinylpyrrolidone K30); 100mMTris-HCl (pH8.0); 25mM EDTA; 2.0M NaCl; 0.5g/L spermidine (Spermidine) (mixing and sterilization); 2% beta-mercaptoethanol (adding during use)) 65 ℃ of preheating;

2) liquid nitrogen grinding diversiform-leaved poplar sample keeps freezing state to put into 2mL EP pipe, and adds 800 μ L CTAB rapidly and extract damping fluid, uses the concussion of vortex vibrator, fully mixing;

3) 65 ℃ of water-bath incubations are 5 minutes, put upside down counter-rotating EP pipe therebetween 3-4 time, and the assurance damping fluid fully contacts with sample powder;

4) above-mentioned EP pipe is transferred to ice bath, makes content temperature decline in the pipe.Use isopyknic 24: 1 chloroform/primary isoamyl alcohol (available from Beijing chemical reagents corporation) extracting subsequently, and centrifugal under 4 ℃ with 10000rpm, draw supernatant;

5) the centrifugal back absorption supernatant that finishes is transferred to new EP pipe, and repeats the 4th) go on foot twice;

6) draw supernatant, add 1/4 volume 10mol/L LiCl ( -20 ℃, LiCl is available from Beijing chemical reagents corporation) and to the top layer, and mixing;

7) precipitation is spent the night in the ice bath, next day centrifugal 20 minutes of 10000rpm under 4 ℃ of conditions;

8) abandon supernatant, the gained resolution of precipitate is in 0.5%SDS;

9) with twice of isopyknic 24: 1 chloroform/primary isoamyl alcohol extracting.Method such as step 4);

10) behind the absorption supernatant, add 2.5 times of cold (20 ℃) dehydrated alcohols of volume and 1/10 volume 3mol/L NaAc (pH5.2) in-80 ℃ of precipitations 30 minutes or-20 ℃ of precipitation 2h;

11) under 4 ℃ of 12000rpm conditions after centrifugal 15 minutes, abandon supernatant;

12) add 70% ethanol washing and precipitating, and under 4 ℃ of 12000rpm conditions centrifugal 5 minutes, supernatant abandoned;

13) with the water dissolution precipitation that does not contain DNase-RNase of 20-30ul, get RNA solution;

14) usefulness DNase I treatment step 13 under 37 ℃ of conditions) gained RNA solution is 30 minutes, dna digestion.

Embodiment 3. clone PeXET genes

Based on document (Takano M.et al., Endoxyloglucan transferase cDNA isolatedfrom pea roots and its fluctuating expression in hydrotropically respondingroots, plant and cell physiology, 1:135-142,1999) and (Bourquin Veronica et al., Xyloglucan Endotransglycosylases Have a Function during the Formation ofSecondary Cell Walls of Vascular Tissues, Plant Cell, 12:3073-3088,2002) report is studied diversiform-leaved poplar XET gene emphatically.On the GenBank with Ps-EXGT1 gene order (GenBank Acc.AB015428) by BLAST in the sequence of threads search of in the nucleic acid Non-redundant data storehouse nr that Polulus belongs to, comparing, search for and obtain following two Populus sequences:

AF515607：Populus tremula x Populus tremuloidesxyloglucan endotransglycosylase precursor(XET16A)mRNA

EF194052：Populus trichocarpa xyloglucanendotransglycosylase/hydrolase precursor XTH-27mRNA

On the GenBank with AF515607 and EF194052 gene order by BLAST in the sequence of threads search of in the EST storehouse of diversiform-leaved poplar (P.euphratica), comparing, search obtains following two est sequence: AJ772752 and AJ778011.This two sequences is downloaded to local hard drive, carries out sequence assembly by DNAMan software, see Fig. 2 A, splicing obtains the sequence as Fig. 2 B.

Analysis chart 2B, this sequence has the transcription initiation codon (showing with yellow background) of prediction, but does not find suitable Transcription Termination codon according to the triplet rule of codon.Therefore this splicing sequence is incomplete, needs with the prediction of comparing of other poplars again.Utilize VectorNTI software that the splicing sequence of Fig. 2 B and AF515607 and EF194052 are compared result such as Fig. 2 C.

In considering the diversiform-leaved poplar EST splicing sequence of Fig. 2 B, do not comprise terminator codon, and comparison result shows also is like this among Fig. 2 C.Design during primer at first the diversiform-leaved poplar EST splicing sequence with Fig. 2 B design, as the PeXET-722-1 that marks among Fig. 2 C and the position of PeXET-722-2, this guarantees to obtain the fragment of a PeXET to primer from diversiform-leaved poplar cDNA, prove with this, comprises PeXET among this cDNA.Again owing to find that when BLAST the characteristics that this gene is guarded relatively are according to consequence devised three primer (the comprise terminator codon) PeXET-999-3 (position of this sequence among Fig. 2 Cs mark) of Fig. 2 C by two Populus XET genes comparisons on sequence.Designed primer sequence sees the following form 1.

Table 1. is used for the primer of pcr amplification PeXET

The primer title	Sequence
		PeXET-722-1	5’-TCGAGATATGGCTGCTTCT-3’
PeXET-722-2	5’-GTGGCACAGAATTTCGCTT-3’
		PeXET-999-3	5’-AAACAGGATGGAAGCAGCT-3’

Carry out reverse transcription experiment back PCR by the RNA that embodiment 2 is extracted, obtain result as Fig. 3.As can be seen, the 2nd swimming lane (root system cDNA is a template) and the 4th swimming lane (callus cDNA is a template) RT-PCR clearly obtain the band that a size is about 722bp from Fig. 3 A, and this proof all comprises this gene of PeXET in these two cDNA.In Fig. 3 B, find out, when using the primer PeXET-999-3 of comparison prediction, also obtained the RT-PCR result of root system and callus, show the band of an about 999bp.Size is about 722bp carries out the sepharose recovery with the band that size is about 999bp, transformed into escherichia coli TOP10 competent cell behind the connection T carrier send company's order-checking after the detection.Sequencing result shows: in this PCR result, sequence and reality that the sequence of so-called 999bp comprises 722bp are the 1000bp size, and wherein the sequence of PeXET gene is SEQ ID No.1.

Process DNAMAN software obtains the result as Fig. 4 after carrying out ORF prediction and translation with universal code, these ORF translation 292 amino-acid residues (SEQ ID No.2), and predicted protein matter size is 33.8KDa.Utilize the online comparison of BLAST comparison instrument as can be seen, this predicted amino acid sequence and other plant (for example pea, comospore poplar, Arabidopis thaliana etc.) XET gene has the similarity more than 80%.

Embodiment 4. makes up the PeXET expression vector

The process that the PeXET expression vector makes up as shown in Figure 5, according to the result of this route as shown in Figure 6.The final pBin438-PeXET plant binary expression vector (Fig. 6 E) that obtains, and transform in agrobacterium strains GV3101 (Fig. 6 F).Pass through standard molecular biological technique, the PeXET gene clone is arrived the pBin438 plant binary expression vector (referring to the patent No.: ZL00103561.4, but reference: Li Taiyuan also, Yingchuan, field, Qin Xiaofeng, big gram is strong. the research of efficient insect-resistant transgenic tobacco. and Chinese science (B), 1994,24 (3): 276-282), this recombinant expression vector is by repetition CaMV35S promotor (Kay R.et al., the Duplication of CaMV 35SPromoter Sequences Creates a Strong Enhancer for Plant Genes of composing type, Science, 4806:1299-1302,1987) driving, the effectively expression of enhancing gene (Yingchuan, field etc., express the insect-resistance of the transgene tobacco of Bacillus thuringiensis delta-endotoxin genes, the biotechnology journal, 1:1-10,1991; Li Taiyuan etc., the research of efficient insect-resistant transgenic tobacco, Chinese science (B collects), 3:276-282,1994; Tang W.et al., Transgenic loblolly pine (Pinus taeda L.) plantsexpressing a modified δ-endotoxin gene of Bacillus thuringiensis withenhanced resistance to Dendrolimus punctatus Walker and Crypyotheleaformosicola Staud, Journal of Experimental Botany, 383:835-844,2003).

PeXET expression vector construction strategy is to introduce two restriction enzyme sites at the gene two ends by PCR method, the upstream is SalI for the BamHI downstream, connect on the T carrier and be transformed in the intestinal bacteria, extract plasmid and carry out double digestion, obtain notched gene fragment; Simultaneously pBin438 is carried out same double digestion, reclaim big fragment; Utilize the T4DNA ligase enzyme to be connected said gene fragment and breach expression vector, obtain expression vector.

The research of embodiment 5.PeXET gene promoter

5.1PeXET the prediction of gene promoter

Result based on Fig. 3, the PeXET gene is only expressed in diversiform-leaved poplar and root system and callus, therefore this research is according to document (Li Shanshan etc., promotor cloning process progress, Chinese biological engineering magazine, 7:9-16,2005) inverse PCR of Jie Shaoing (Inverse PCR, iPCR) method, and use for reference the way of nest-type PRC, cloned the promoter sequence (SEQ ID No.3) of diversiform-leaved poplar PeXET gene, and promoter sequence has been merged mutually with reporter gene GUS, by examining report genetic expression, the adjusting activity of promotor is carried out qualitative analysis.The promoter sequence of the PeXET gene that the present embodiment utilization is cloned into is replaced expression vector pCAMBIA1301, and (can order from network: the CaMV 35S promoter http://www.cambia.org/daisy/cambia/585.html) merges mutually with reporter gene GUS sets up plant expression vector, and importing Agrobacterium GV3101 (available from the excellent bok gene in Beijing Science and Technology Ltd.), contaminate tobacco and cultivate transfer-gen plant, provide condition to identify its promoter function.

Because the differential expression (as Fig. 3) of this gene in tissue, whether the promotor of this gene has tissue specific expression and can drive foreign gene effectively becomes a good problem to study at plant interior expression.

The dna sequence dna of analyzing the PeXET gene as can be seen, this gene fragment is bigger, and should consider that as the design of primers of iPCR restriction endonuclease sites should not occur in that known array is inner.Therefore, choose the sequence of front, EcoRI site (Fig. 7 B) at PeXET gene 748bp place as the target area of design of primers.Principle (figure mistake according to iPCR! Do not find Reference source.), design primer such as Fig. 7 C, the primer sites design has a benefit at this, can use the EcoRI site that does not have in this section zone exactly.And fully take into account the not intellectual of gene 5 ' flanking sequence, and known amplification region is dwindled as far as possible, it is wise therefore choosing this section sequence.

Referring to Fig. 8, place PlantCARE to search for described promoter sequence, be positioned at-464 position as can be seen, prediction has ARE (Anaerobic Response Element, the anaerobic reaction element) element, this element finds that in corn (Zea mays L.) this is the cis-acting elements to anaerobic condition.And in the reality investigator in other species under anaerobic the clone obtain XET gene (Wang Wenquan etc., the expression of anaerobic induction xyloglucan transglucosidase (XET) gene in sesame and wheat root, Journal of Agricultural Biotechnology, 3:258-263,2004).And the GC motif at-72 places is considered to the enhancing element of gene at the anoxia condition abduction delivering.

Be positioned at-46 position, it is a class ethylene response element of finding in carnation (carnation, Dianthus caryophyllus L.) that prediction has ERE (Ethylene-Responsive Element, ethylene reaction element) element, this element.And in root system of plant hydrotropisms response process, the investigator thinks that ethene may participate in this process (Eapen D.et al. as plant hormone, Hydrotropism:root growth responses to water, Trends in Plant Science, 1:44-50,2005), in this case, may the playing a role of PeXET gene at root system hydrotropisms response process.

The CCGTCC-box at-65 places is considered to relate to the active especially cis-acting elements of meristematic tissue, this may with relevant (the Takano M.et al. of the anisopleual growth of meristematic zone that causes in the root system hydrotropisms BENDING PROCESS, Endoxyloglucan transferase cDNA isolated from pea rootsand its fluctuating expression in hydrotropically responding roots, Plant andCell Physiology, 2:135-42,1999a).

Also comprise many other controlling elements in the described promoter sequence, but the function of these cis-acting elements also not fully aware of.Find out in the prediction that a part of element is relevant with photoresponse, another part element is relevant with materials such as jasmonic, Whitfield's ointments, between these factors and material and PeXET expression what interactively is arranged so, remains further to be excavated.

5.2 make up the segmental expression vector of promoter deletion

By above-mentioned analysis, infer that SEQ ID No.3 has promoter function, in order to prove this point, simultaneously for study among the total RNA in diversiform-leaved poplar root, stem, blade and the callus that in embodiment 2, extracts motif in which has decisive role to tissue specific expression, test design is the promoter deletion experiment.Therefore at the pCAMBIA1301 vector construction plant expression vector of five different lengthss (be used to replace " the replacement section " shown in Fig. 9, this section was 35S promoter originally), in order to the research promoter function.

The pCAMBIA1301 carrier (Fig. 9, can order from network: Http:// www.cambia.org/daisy/cambia/585.html) have HindIII and NcoI mono-clonal site in the expression promoter 35S upstream and downstream that drives gus gene.Therefore, utilize the method for PCR, on purpose promoter deletion fragment, introduce above-mentioned two sites, PCR product size and electrophoretogram such as Figure 10.Reclaim above-mentioned PCR product, add and connect pMD19-T-Sample carrier (available from the precious biotech firm in Dalian) behind the A, and transformed into escherichia coli.

After extracting above-mentioned connection plasmid, carry out double digestion with HindIII and NcoI, and while double digestion pCAMBIA 1301 carriers, and each fragment (result does not show) of recovery big fragment of pCAMBIA 1301 carriers and promoter deletion.PCAMBIA 1301 plant binary expression vectors that the dna fragmentation that enzyme is cut back to close cuts back to close with enzyme respectively according to the segmental mol ratio of carrier and purpose be 1: 3～1: 8 ratio under the effect of T4DNA ligase enzyme 16 ℃ spend the night and be connected transformed into escherichia coli.Extract plasmid subsequently, utilize PCR, enzyme to cut and the means that check order are carried out connectivity verification.The checking result shows that the promotor of different lengths is all with in the correct insertion pCAMBIA1301 expression vector.Next step needs these expression vectors of verifying are transformed into Agrobacterium in order to study the function of promotor, is used to infect tobacco.

5.3pPeXET drive the transient expression of GUS

With the CaMV35S among the promoter sequence replacement plant binary expression vector pCAMBIA1301 of diversiform-leaved poplar PeXET gene, be transformed in the tobacco through Agrobacterium tumefaciens mediated leaf disc transformation method, detect the activity of the promotor of diversiform-leaved poplar PeXET gene by the expression of gus gene, this experiment is with the negative contrast of empty bacterial strain.

(β-glucuronidase, GUS) gene are called for short gus gene to β-glucuronidase, are present in some bacterial body coding β-Pu Taotanggansuanmei (EC 3.2.1.31, a kind of lytic enzyme, the hydrolysis of the many poly glucoside Esters of catalysis).Most of plants lack endogenous glucuronidase, and the importing of gus gene can not influence it grows normally, is widely used a kind of reporter gene.The expression product of gus gene is easy to detect.GUS is very stable in transformant and extracting solution, and heat and stain remover during to extraction have certain tolerance; Transformation period in mesophyll protoplast is 50 hours.The suitableeest pH of this enzyme reaction is 5.2～8.0, does not need coenzyme and special ion, and can adapt to the ionic strength scope of broad; But some divalent-metal ion can suppress its activity.This enzyme specificity is very low, but catalysis kinds of artificial substrate generation color reaction and detected easily.Detecting the most frequently used method of gus gene expression is the histochemical stain localization method, indoles-β-D-glucoside acid esters (X-Gluc) is a substrate with 5-bromo-4-chloro-3-, give expression to GUS if change tissue, cell, the protoplastis of gus gene, this enzyme can generate blue material 5 with the X-Gluc hydrolysis under optimum conditions, 5 '-two bromo-4,4 '-dichloro bipseudoindoxyl dye can be with the naked eye, microscope, laser confocal microscope observations such as (Confocal).Present embodiment transfer GUS and adjoining tree do not have significant difference on phenotype.

Result such as Figure 11.From the figure mistake! Do not find Reference source.The promoter sequence of the diversiform-leaved poplar PeXET gene of different lengths all can expression activity in tobacco as can be seen, and this provides the basis for identifying the tissue specific expression of promotor in tobacco.

Should be appreciated that, although with reference to its exemplary embodiment, the present invention is shown particularly and describe, but will be understood by those skilled in the art that, can carry out the variation of various forms and details therein, and not deviate from by the defined the spirit and scope of the present invention of accompanying Claim.

Reference:

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SEQUENCE LISTING

＜110〉Beijing Forestry University

＜120〉Populus euphratica Oliv hydrotropic gene PeXET gene and promotor thereof

<130>IB085344

<160>3

<170>PatentIn version 3.1

<210>1

<211>882

<212>DNA

＜213〉diversiform-leaved poplar (Populus.euphratica Oliv.)

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atggctgctt ctctatggac tttttttctt ggcatgctgt ttatggtatc tgggacaatg 60

ggagctgccc caaggaagcc agtggatgtg ccttttggaa ggaactatgt tcctacatgg 120

gcttttgacc acattaagta cttcagagga ggctccgaga ttcagctcca gttggataaa 180

tacaccggta ctggtttcca atcaaaaggg tcatacttat ttggccattt cagtatgcaa 240

gtgaagttgg ttcctggtga ttcagctgga acagttactg ctttctatct atcttcacaa 300

aactcagagc atgatgagat agactttgag ttcttaggaa acaggactgg ccagccttat 360

attttgcaga caaatgtttt cacaggaggc aagggagaca gagaacagag gatttacctt 420

tggtttgacc caaccataag ataccactct tactccgtcc tatggaattc gtatctggta 480

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aaatttcctt tcaaccagcc aatgaagata tactcaagcc tatggaacgc cgatgattgg 600

gctaccagag gtggacttga gaagacagac tggtccaagg cgccctttat agcttcctac 660

aagagcttcc acatagatgg ctgtgaggcc tccgtggaag cgaaattctg tgccacacag 720

ggtaccagat ggtgggccca gaaggagttc caggatcttg atgccttgca gtacaggagg 780

ctcagatggg tacgccagaa atacaccatc tacaactact gcactgatag atcaagatac 840

ccttcactgc caccagaatg caagagagac agagacatat aa 882

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＜213〉infer

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Met Ala Ala Ser Leu Trp Thr Phe Phe Leu Gly Met Leu Phe Met Val

1 5 10 15

Ser Gly Thr Met Gly Ala Ala Pro Arg Lys Pro Val Asp Val Pro Phe

20 25 30

Gly Arg Asn Tyr Val Pro Thr Trp Ala Phe Asp His Ile Lys Tyr Phe

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Gly Phe Gln Ser Lys Gly Ser Tyr Leu Phe Gly His Phe Ser Met Gln

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Val Lys Leu Val Pro Gly Asp Ser Ala Gly Thr Val Thr Ala Phe Tyr

85 90 95

Leu Ser Ser Gln Asn Ser Glu His Asp Glu Ile Asp Phe Glu Phe Leu

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Gly Asn Arg Thr Gly Gln Pro Tyr Ile Leu Gln Thr Asn Val Phe Thr

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Gly Gly Lys Gly Asp Arg Glu Gln Arg Ile Tyr Leu Trp Phe Asp Pro

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Val Phe Phe Val Asp Asp Val Pro Ile Arg Val Phe Lys Asn Cys Lys

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Asp Leu Gly Val Lys Phe Pro Phe Asn Gln Pro Met Lys Ile Tyr Ser

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Ser Leu Trp Asn Ala Asp Asp Trp Ala Thr Arg Gly Gly Leu Glu Lys

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Thr Asp Trp Ser Lys Ala Pro Phe Ile Ala Ser Tyr Lys Ser Phe His

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Ile Asp Gly Cys Glu Ala Ser Val Glu Ala Lys Phe Cys Ala Thr Gln

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Gly Thr Arg Trp Trp Ala Gln Lys Glu Phe Gln Asp Leu Asp Ala Leu

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Gln Tyr Arg Arg Leu Arg Trp Val Arg Gln Lys Tyr Thr Ile Tyr Asn

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Tyr Cys Thr Asp Arg Ser Arg Tyr Pro Ser Leu Pro Pro Glu Cys Lys

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Arg Asp Arg Asp Ile

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<210>3

<211>2015

<212>DNA

＜213〉diversiform-leaved poplar (Populus.euphratica Oliv.)

<400>3

aaacacagga acatggaaag tggtgatcgg tgagaactga taagaaagac atggatcgtg 60

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ttcttcttgt ctctctcgtt ctgaaactat ttttttcact atgtttggca cgtttggaaa 180

taaaattgag tggatgcggg ggggagtcta acagcgttcc tgcaagtccc cagccctaac 240

tcgcaacgac aggagcacat ctggatttca agggttgtct tctgtacata tctcttcact 300

agtgacaaag tgtcattgtt cactgtttcc tctaaaaatc catgcatcgc tcatttaaaa 360

ccaaaaaaat taatattggt cacaaaactg atctaattcc ttgatggtaa gttatgatat 420

cagttgatgg gtcggggttt aatttctcaa gttgcttata cgcggcatgt acgtagacta 480

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ttttaatact tcaccttttc ttttcttttc ctaatgttat gtttgacaag tggttataaa 600

ttatctttgt aaaatctaac tttattcgtt ttttttcttt taaaaaaaga ggtgaattat 660

taaggagata atgaccggta gattgttttt agatgaacta atcaccaaca aaactaaaat 720

aaaaataatg gttattttac tatccattgc actgtttaca taaacagtgc aacttttttt 780

tttaattttt tctctttttt tattttttta aaaattggtt cattgcacta ttcatgtgag 840

tattagtttt ttttccggtt cattgcacca cccatgcaat tttcttaatt ttttttctgt 900

tcatgaatag tgttgttttt gcttttctcg tcgaatttga tttatttata attttatcct 960

ttcaatatac aataacatga ttttatatat caaaatcttt ttttctctct caatattctc 1020

acatactgtg aaagtttcgt ctgtatcaaa acgaagacac atgcctttgt gattaacaat 1080

gtaagaacga atcgtgggtt tcctaggaag gaaaattaga aagtattcga cccattcact 1140

tgcttgcttg cttctctcaa atttgcttct ctgcacttgc aattagccta tcaaaacgaa 1200

gacacatgcc ttttgaattt taccttgacg gtagaaatct ccttccttca cgccaaacgt 1260

caccgtagta gagtcggtgg gcttgaagtc atcattagca aaagagcccg agacaaaagc 1320

agaaaagtgc agtcctcgaa aagagaaaaa accagggaca aaagccgaat atgtgaaggt 1380

ggcccagata tcttaggtga gagatgcagc tagaaagaaa gagagaagga aatttcaaga 1440

ttggtcagct gtgctcatat ttttcctttc ccatcaaccc ccccagaaaa ataaacaaat 1500

accacacgag tctcagcgtt tccaactttc aagatgtttt caaaacacat tcttctcctg 1560

tcgtcgagca tgccctccta gtccatccac tttttcaaca tccagaagtt aaataattgc 1620

acttttctcc tctacccact atcacataaa aattaaaata ggctgttgtg gtagctacta 1680

gctagctgta ttaccactgc ccattattat agttttttta atcaaatcaa atagtttgcc 1740

cccgcatacc gtccaataat ttaattttag ttttgaaata accactattt aaagggactc 1800

tcctcctcca ttctcctcgt ccaattagca ccccttcagg agtactctct acgctcaggt 1860

aatgctagct ctagaacctt gaatatgctt gcatcttgat tcctctaatg ctcaacatct 1920

ctagtccctt taaggtctta tattgtgtct caagaactca catttctctt gaaatgtgca 1980

gagatatggc tgcttcttta tggacttttt ttctt 2015

Claims (10)

1. the root system hydrotropisms gene PeXET of diversiform-leaved poplar (Populus.euphratica Oliv.), its nucleotides sequence is classified SEQ ID No.1 as.

2. the protein of the PeXET genes encoding of claim 1, its aminoacid sequence is SEQ IDNo.2.

3. the promotor of the PeXET gene of claim 1, its nucleotides sequence is classified SEQ ID No.3 as.

4. the application of the promotor of claim 3, it is used for making foreign gene to carry out tissue specific expression at root system or callus.

5. the recombinant expression vector of the PeXET gene of claim 1, it prepares by described diversiform-leaved poplar root system hydrotropisms gene PeXET is cloned in the expression vector.

6. the described recombinant expression vector of claim 5, wherein said expression vector comprises the pBin438 plant binary expression vector.

7. the application of claim 5 or 6 described recombinant expression vectors, its by utilize described recombinant expression vector transform Agrobacterium again the transfection plant be used to prepare transgenic plant, or prepare transgenic plant by other physics and chemical process with described recombinant expression vector.

8. root system of plant hydrotropisms induced reaction device, it comprises:

(1) culture apparatus has only a side side that the infiltration hole is arranged;

(2) rotary axle is arranged in the middle of the bottom of described culture apparatus, described axle is fixed by A-frame, and the rotation of described axle can be so that described culture apparatus tilts, and a side side that makes the infiltration hole is towards ground.

9. the described root system of plant hydrotropisms of claim 8 induced reaction device, it also is included in the dismountable light source and the Controlling System thereof of described culture apparatus top.

10. the application of claim 8 or 9 described root system of plant hydrotropisms induced reaction devices, the pitch angle of described culture apparatus is regulated in its rotation by axle, and the natural hillside drought condition of angle of inclination in 0 ° of-45 ° of scope simulated towards ground in a side side that makes the infiltration hole.

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