CN101565460B - Populus euphratica DREB2 transcription factor cDNA sequence, expression carrier containing same and application thereof - Google Patents

Abstract

The invention discloses a DREB transcription factor cDNA sequence (PeDREB2a gene) which is separated and cloned from Populus euphratica and is closely related to stress resistance, wherein the Populuseuphratica is a sand plant. The invention also discloses a high-efficiency plant expression carrier containing the cDNA sequence and an application thereof in improving the reverse resistance of plan ts. In the invention, the PeDREB2a gene is established into a plant high-efficiency expression carrier p2MDK-PeDREB2a; agrobacterium is utilized to carry out mediate transformation for tobaccos; positive seedlings are selected and identified by Kan resistance and genomes PCR and RT-PCR; the reverse resistance analysis of transgenic tobacco seedlings, such as drought, high salt, low temperature, and the like are carried out; and the correlative physiological-biochemical indexes of the transgenic tobacco seedlings are measured. A test result shows that the content of the dissoluble sugar, the cytoplast total protein and the praline of a PeDREB2a transgene plant strain is obviously higher than a contrast, so that the overexpression of the PeDREB2a gene can obviously improve the drought resistance and the infiltration stress resistance of transgenic tobaccos.

Description

Populus euphratica DREB 2 transcription factor cDNA sequence, the expression vector that contains this sequence and application thereof

Technical field

The present invention relates to the cDNA sequence of plant AP2/EREBP family transcription factor, relate in particular to psammophyte diversiform-leaved poplar (Populuseuphratica) DREB2 transcription factor cDNA sequence.The invention still further relates to plant expression vector, this cDNA sequence answering in improving stress resistance of plant that can in plant, efficiently express this cDNA sequence, belong to plant genetic engineering field.

Background technology

Arid, saline and alkaline and low temperature is three kinds of main abiotic stress factors that influence terrestrial plant growth and restriction crop yield.According to statistics, arid and semi-arid area, the world relates to more than 50 countries and regions, accounts for 34.9% of global land area.At present the whole world is used for 5,700,000,000 hectares of agricultural and can ploughs the nonirrigated farmland, and about 70% arable land is because factors such as arid cause soil property to be degenerated, and the land area that the Asia is influenced by this is the widest, nearly 1,400,000,000 hectares.And the arid of China, semiarid zone account for the over half of area, and a year area suffered from drought reaches ten thousand hectares of 200-270.Its harm is equivalent to other natural disaster sum.In addition, saltings, the world accounts for 7.6% of global land area, along with process of industrialization is accelerated, the continuous expansion of irrigated land and plastic greenhouse area, the soil salinization area of China constantly enlarges, the salinization soil area has 2,600 ten thousand hectares approximately at present, and wherein saline and alkaline arable land has 6,700,000 hectares approximately, has seriously restricted the utilization of agricultural to the soil.One of serious adverse circumstance factor as often taking place and endanger in low temperature, and it not only affects the seasonality growth of plant, also affects rational distribution the in ground of plant.According to statistics, China is annual because of damaging to plants caused by sudden drop in temperature the agricultural losses that cause up to billions of units, and these abiotic stress have direct or indirect damaging effect to agriculture production, ecological construction, the strategy of sustainable development.

Abiotic stresses such as arid, saline and alkaline and low temperature all can constitute the osmotic stress to plant, cause the environment osmotic potential to be lower than the vegetable cell osmotic potential and cause the vegetable cell dehydration, and serious caused cell turgor completely loses, even causes plant death.Certainly, plant is in order to conform, also formed complicated in the long-term evolution process and meticulous regulation mechanism, improved the opposing of self poor environment being coerced or restrained oneself ability, with the maladjustment environment.

Transcription factor (transcription factor, TF) also claim trans-acting factor, being meant can be conjugated protein with the interactional DNA of cis-acting elements generation specificity in the eukaryotic gene promoter region, by between them and and other associated protein between interaction activate or suppress some gene transcription.Since the clone of Paz-Ares reported first corn transcription factor gene in 1987, isolated regulation and control arid, high salt, low temperature, hormone, cause of disease are reacted and the transcription factor of related gene expression such as grow has reached hundreds of from higher plant in succession.The order-checking of arabidopsis gene group is finished, and by inference, has the gene of 1553 encoding transcription factors at least, accounts for it and estimates 5.9% of gene number.Transcription factor quantity is so big in the Arabidopis thaliana, more than the kind, has shown the complicacy of higher plant transcriptional control, also shows the importance of transcription factor research simultaneously.

AP2/EREBP class transcription factor is a peculiar huge transcription factor family in the plant, and its main involved in plant development, hormone, cause of disease reaction and arid, high salt and low temperature stress are replied.The member of this gene family is contained the very conservative DNA land of being made up of 60 left and right sides amino-acid residues (being that AP2/EREBP is in conjunction with the territory), and this proteinoid can fall into 5 types again according to the homology of DNA land: AP2 class, DREB class, ERF class, RAV class and other type.A lot of researchs are verified, and DREB class and ERF class transcription factor play an important role in stress resistance.

Quick, instantaneous, the early stage expression characteristic that the AP2/EREBP transcription factor is had participates in signal transduction pathway for it, bring into play its early stage gene regulating effect possibility is provided, in case have biology or abiotic stress to occur, this transcription factor family gene just can be expressed fast, by transcript and expression, and finally open the plant defense system and defend responsing reaction in conjunction with the cis-acting elements regulatory gene in the functional gene promotor of downstream.The main molecule responsing reaction that participates in ethene and cause of disease of EREBP subfamily transcription factor (as TINY, CBF1, Ptis, AtEBP, DREB1, DREB2).Wherein, the DREB transcription factor of this subfamily plays a crucial role in the plant stress-resistance response, it is discerned usually in conjunction with arid response element DRE/CRT, thereby regulate and control some and arid, high salt and low temperature patience gene expression related (Liu Q, Kasuga M, Sakuma Y, Abe H, MiuraS, Yamaguchi-Shinozaki K, Shinozaki K.Two transcription factors, DREB1 andDREB2, with an EREBP/AP2 DNA-binding domain separate two cellular signaltransduction pathway in drought and low-temperature-responsive gene expressionrespectively in Arabidopsis.Plant Cell, 1998,10:1391～1406).Yet the ERF transcription factor plays an important role in biological stress response, behind the overexpression, gray mold, sclerotium disease and Powdery Mildew is all had resistance as Arabidopis thaliana AtEFR1 gene in Arabidopis thaliana.ERF subfamily transcription factor is mainly regulated and control ethylene response and disease-resistant relevant (Pathogenesis-Related, PR) expression of gene.Usually contain in the promotor of PR gene with Ethylene Signal and reply relevant cis-acting elements GCC box, ERF subfamily transcription factor by with the interaction of GCC box, regulation and control PR expression of gene, thereby the resistance against diseases of regulation and control plant.

DREB transcription factor (Dehydration Responsive Element Binding Protein), it is arid response element binding protein, be that a class is reacted transcription factor relevant, that work with the resistance under the plant dehydration condition in signal transmission and regulate gene expression, energy specific combination DRE/CRT cis-acting elements, and activate transcribing of downstream goal gene.DRE/CRT (dehydration responsive element) cis-acting elements, core sequence is TACCGACAT, Yamaguchi Shinozaki in 1994 have found to contain the DRE core sequence of 9bpTACCGACAT sequence first in the promotor of analyzing Arabidopis thaliana rd29A gene, it can play an important role to adverse circumstance inducible gene expression under arid, high salt and the low temperature, and expression does not rely on the ABA signal transduction pathway.Meanwhile, be subjected to also to find in arid, high salt or the cryogenic promotor DRE element or DRE core sequence at some, as be subjected to find in the promotor of low temperature induction arabidopsis gene cor15a sequence TGGCCGAC similar in appearance to the DRE element, be called CRT (C-repeat) element, be subjected to also to find to have 5bp (CCGAC) the center sequence of DRE core sequence in the swede type rape gene BNIIS promotor of low temperature induction, be called as low temperature response element LTRE.The discovery of DREB is the breakthrough progress of tool in plant stress-resistance Journal of Sex Research aspect in recent years.(Liu Q such as Liu, Kasuga M, Saklam Y, et al.Two transcriptionfactors, DREB1 andDREB2, with an EREBP/AP2 DNA-binding domain separate two cellularsignal transduction pathway in drought and low temperature responsive geneexpression in Arabidopsis[J] .Plant Cell, 1998,10:1391-1406) utilize DRE cis-acting elements and yeast one-hybrid method in the rd29A gene promoter region, from the Arabidopis thaliana cDNA library of subzero treatment, be cloned into 3 and DRE combination of elements, the transcription factor that regulation and control reporter gene GUS expresses under low temperature stress, called after DREB1A, DREB1B, DREB1C.(Gihmur S J such as Gilmour, Zarka DG, Stockinger E J, et al.low temperatureregulation of the Arsbldopsis CBF family of AP2 transcriptional activator as anearly step in cold induced cor gene expression[J] .Plant J, 1998,16 (4): 433-442) also be separated to this three genes, respectively called after CBF1 (DREB1B), CBF2 (DREB1C) and CBF3 (DREB1A).Their abduction deliverings of catching a cold are cold response gene.With 4 ℃ of subzero treatment, in the 15min, DREB1A, DREB1B and DREB1C gene are by quick induced strong, but they are not subjected to inducing of Exogenous ABA.Liu etc. studies show that the composing type overexpression of DREB1B has improved plant to arid, high salt and cryogenic tolerance in the transgenic arabidopsis.Liu etc. also are cloned into 2 and DRE combination of elements from the Arabidopis thaliana cDNA library that arid is handled, regulate and control the gene that reporter gene GUS expresses under arid, high-salt stress, name to be DREB2A and DREB2B.They are subjected to arid and high salt abduction delivering, are arid and high salt response gene, as coercing processing with arid or high salt, in the 15min, DREB2A and DREB2B gene are also by quick induced strong, and especially at plant root, these two genes are not subjected to inducing of Exogenous ABA.In that homology is all very high separately with the DREB1 (CBF) of DRE combination of elements and DREB2 two class transcription factors, but except AP2 structural domain high conservative, there is not the similarity of sequence between two genoids substantially.The expression of DREB1 (CBF) is not but induced by arid and high-salt stress by low temperature induction, and DREB2 is induced by arid and high-salt stress, is not but induced by low temperature.Behind stress-inducing, the endogenous ABA level in the plant increases, but two kinds of DREB transcription factors all are not subjected to inducing of Exogenous ABA.The DREB transcription factor that produces can activate a series of goal gene with DRE cis-acting elements, as rd17, kin1, cor6.6, cor15a, erd10 and rd29A.The product of these genetic expressions is being brought into play different functions in the plant stress-resistance reaction, thereby the resistance of plant is improved.

The difference of the different subtribe protein functions of same family, often by the amino acid decision of a few key position, these amino acid are often conservative in subtribe, but different between subtribe.DREB/CBF and ERF belong to the AP2/EREBP transcription factor family together, but the function difference.ERF subfamily transcription factor identification bonded cis-acting elements GCC box, the main molecule responsing reaction that participates in ethene and cause of disease; And the identification of DREB/CBF subfamily transcription factor mainly participates in the molecule of low temperature, arid and high salt etc. is replied in conjunction with the CRT/DRE cis-acting elements.Aminoacid sequence is relatively found, the AP of ERF subfamily transcription factor ₂DNA is in conjunction with in the territory, and the 14th and 19 is conservative L-Ala (A) and aspartic acid (D); And the AP of DREB subfamily transcription factor ₂DNA is in conjunction with in the territory, and the 14th and 19 is conservative Xie Ansuan (V) and L-glutamic acid (E).

Equal intronless in the DREB transcription factor of hitherto reported, its gene.From structural analysis of protein, DREB albumen has the common configuration feature of transcription factor: be rich in acidic amino acid at the C-end, function is as transcriptional activation domain; The N-end is rich in basic aminoacids, is nuclear localization signal; The middle AP2/EREBP structural domain of forming by 58 amino-acid residues, this structural domain sequence and space conformation all have certain characteristics, comprise YRG district and RAYD district, the former is rich in alkalescence and the hydrophilic zone of being made up of 20 amino-acid residues for the N-end, proteinic three dimensional analysis shows, there are 3 antiparallel beta sheets in this district, to discerning all kinds of cis-acting elements and playing a crucial role with the element effect of mutually combining, the 14th of this district and the 19th 's amino acid is respectively the L-glutamic acid (E19) of conservative Xie Ansuan (V14), particularly the 14th Xie Ansuan (V) plays a crucial role to determining the DREB transcription factor to combine with the specificity of DRE cis-acting elements.Sakuma etc. and Qin etc. carry out point mutation to the amino acid in this two sites of the DREB transcription factor of Arabidopis thaliana and corn respectively, after 14 Xie Ansuans (V) become L-Ala (A), DREB does not combine with the DRE sequence, and the DREB transcription factor is almost lost its transcriptional activation ability; And after 19 L-glutamic acid (E) became aspartic acid (D), the DREB transcription factor can be in conjunction with the DRE sequence, but the transcriptional activation ability is a greater impact.In addition, the albumen of 56 DREB/CBF classes that retrieve in the Arabidopis thaliana database is Xie Ansuan all at the V14 place, is not L-glutamic acid and 19 genes are arranged at the E19 place, these show that all V14 plays a part even more important in the combining of DREB and cis-acting elements.The RAYD district is positioned at the C-end of AP2/EREBP structural domain, and the core area that wherein about 18 amino-acid residues are formed can form amphiphatic alpha-helix.DNA is in conjunction with depending on the YRG element, but the RAYD element is not participated in the specific recognition of same cis-acting elements directly, and by influence YRG district conformation or by with other albumen combining of interaction adjusting AP2/EREBP structural domain and DRE cis-acting elements being taken place.

The CBF/DREB transcription factor extensively exists in plant.More dicotyledonous and monocotyledons CBF/DREB1 transcription factor AP-1 2 is in conjunction with the 14th and the discovery of 19 amino acids in territory, and the 14th and 19 amino acids are V14 and E19 in the dicotyledons; The amino acid of corresponding position is except V14 and E19 in the monocotyledons, and great majority are V14 and V19.In sum,, only short 10 years from the clone of the evaluation of initial Arabidopis thaliana CRT/DRE element and CBF/DREB transcription factor apart from the present, but identified the CBF/DREB transcription factor from each kind of plant.DREB/DRE extensively exists in the plant and does not rely on the environment stress signal transduction pathway of ABA.CBF/DREB transcription factor specific recognition is regulated and control a series of arid low temperature response genes and is expressed in conjunction with the CRT/DRE element, and the product of these coded by said gene adapts to plant or resists adverse circumstance.In addition, the Arabidopis thaliana of overexpression CBF3/DREB1A is drought-enduring cold-resistant, and the raising of patience not only strengthens relevant, also relevant with the rising of proline(Pro) and sugared content with the CRT/DRE expression of gene.Therefore, the function of CBF/DREB transcription factor may the remarkable regulation and control that are confined to the CRT/DRE gene, may be also in some way, regulate and control the genetic expression of other and anti-retrocorrelation by certain approach.At present also very shallow to the understanding of CBF/DREB transcription factor, the accurate understanding and grasping to its function and the signal transduction path of regulation and control that participates in also needs unremitting effort.

Up to the present, from various plants clone and separate to hundreds of DREB transcription factor, comprise monocotyledons and dicotyledons, and the interactional regulation and control model of DRE/DREB cis-acting elements and trans-acting factor is found and is proved to be in the herbaceous plant kind, as Arabidopis thaliana, wheat, rye grass, tomato, corn, barley, paddy rice etc.But only in several xylophytas few in number, separate and obtained minority DREB transcription factor.

At present, the engineered research of plant stress-resistance has obtained certain progress, but its emphasis all improves certain resistance at importing discrete function gene, does not obtain improvement comprehensive, essence thereby reach the resistance that makes plant.The resistance of plant is not to be embodied by single or a few expression of gene, but by the quantitative character of controlled by multiple genes.Though clone a large amount of anti contravariance related genes successively at present from each kind of plant, these gene great majority can only increase certain single resistance of plant, can not comprehensively improve the resistance of plant on the whole.The by-pass cock that transcription factor is expressed as functional gene, can regulate accurately different genes, in plant adverse circumstance signal transduction process, bringing into play crucial effect, so by strengthening the effect of certain transcription factor, can impel a plurality of and degeneration-resistant function associated genetic expression, this is to make the plant stress-resistance proterties obtain a very effective approach of comprehensive improvement.DREB is the transcription factor of this class just, in coercing signal transduction process, play an important role, it can regulate and control a plurality of and plant arid, high salt and low temperature patience function associated expression of gene, strengthen the resistance of plant on the whole, improve its stability, resistance of reverse for genetically engineered improvement plant has huge potential using value, and great application prospect will be arranged.Therefore, clone new DREB class transcription factor gene with independent intellectual property right, study its basic biological characteristics and function, to provide fundamental basis for whole plants adversity gene regulated and control network and stress response reaction mechanism, for the improvement crop anti-adversity, create new degeneration-resistant material and provide material base.

Diversiform-leaved poplar (Populus euphratica) is the most ancient in the Salicaceae Populus, seeds of primary, and it is the Relict Plant that carries on the tertiary period, and deciduous tree is national three-level protective vulnerable species.Mainly be distributed in the lower areas, arid desert of height above sea level such as Xinjiang, Qinghai, the Inner Mongol, Gansu, Ningxia in China, distribution also arranged in Midwest, north African and the European south in Asia.Diversiform-leaved poplar can the anti-extreme highest temperature 45 ℃ and the extreme lowest temperature-40 ℃, have extremely strong drought resisting, salt tolerant alkali, windproof, ability such as fix the sand, improve the soil, can in the environment of extreme drought and water shortage, grow, except its morphologic part characteristics, main is because diversiform-leaved poplar has formed its distinctive gene formation and accurate expression regulation mode in the long term growth evolutionary process.Therefore, clone and the DREB transcription factor gene that resistance is closely related can not only provide the excellent genes source for genetic engineering breeding, and seem particularly important and urgent for the crop breeding for stress tolerance from the psammophyte diversiform-leaved poplar.

In sum, for a water scarcity, salinization soil area constantly enlarge, cultivated area is limited, populous large agricultural country, be cloned in the regulatory gene that plays an important role in the plant stress-resistance process, significant for the Sustainable development of agricultural.

Summary of the invention

One of the object of the invention provides that a class separates, clones from psammophyte diversiform-leaved poplar (Populus euphratica) the DREB transcription factor cDNA sequence that is closely related with resistance.

One of the object of the invention is achieved through the following technical solutions:

A kind of DREB transcription factor cDNA sequence that is closely related with resistance that from the psammophyte diversiform-leaved poplar, separate, clone, this cDNA sequence is following (a) or nucleotide sequence (b):

(a) nucleotide sequence shown in the SEQ ID NO:1; Or

(b) nucleotide sequence that obtains or/and insert is replaced, lacked to the one or more base of nucleotide sequence shown in the SEQ ID NO:1, the coded albumen of this nucleotide sequence still has the function of DREB transcription factor.

The present invention according to dreb gene DNA in the plant in conjunction with territory conserved regions design degenerated primer, utilize the RT-PCR method from the psammophyte diversiform-leaved poplar, to be separated to the homologous fragment of DREB2 gene, obtain new DREB2 transcription factor gene PeDREB2a (SEQ ID NO:1) by RACE-PCR technology clone then, and it has been carried out the prediction of sequential analysis and proteic three-dimensional structure, the result shows that this gene has peculiar three functional domains of DREB transcription factor, and promptly nuclear localization signal, DNA are in conjunction with territory and transcription activating domain.Sequence alignment shows this gene and other known dreb gene except having the very high homology at conservative AP2/EREBP structural domain place, and is not high at other regional sequence homology.Find that by the systematic evolution tree analysis that makes up plant DREB transcription factor the main and DREB2 class transcription factor of this PeDREB2a albumen gets together, and nearer with the sibship of the DREB2 class transcription factor of dicotyledons; Be that template is carried out pcr amplification to PeDREB2a with genomic dna and cDNA respectively, amplified production shows that through sequential analysis this gene does not contain intron; By sxemiquantitative RT-PCR it is coerced reaction detection under different stress conditions, the result shows: this expression of gene all is subjected to inducing of arid, high salt and low temperature environment adverse circumstance, and its expression does not rely on the regulation and control of ABA.

Two of the object of the invention provides a class by the coded diversiform-leaved poplar DREB transcription factor of above-mentioned cDNA sequence.

Two of the object of the invention is achieved through the following technical solutions:

One class is by the coded diversiform-leaved poplar DREB transcription factor of above-mentioned cDNA sequence, for following (a) or the aminoacid sequence (b):

(a) aminoacid sequence shown in the SEQ ID NO:2; Or

(b) with the replacement of the aminoacid sequence shown in the SEQ ID NO:2 by one or more amino-acid residues, disappearance or/and insert the aminoacid sequence that still has the DREB functional transcription factor that obtains.

Preferably, diversiform-leaved poplar DREB transcription factor of the present invention is the aminoacid sequence shown in the SEQID NO:2.

Herein, described " a plurality of " mean 2～8 usually, are preferably 2～4, and these depend on the position or the amino acid whose kind of amino-acid residue in the DREB transcription factor three-dimensional structure; Described " replacement " is meant respectively and replaces one or more amino-acid residues with different amino-acid residues; Described " disappearance " is meant the change of aminoacid sequence, wherein lacks one or more amino-acid residues respectively; Described " insertion " is meant the change of aminoacid sequence, relative natural molecule, and described change causes adding one or more amino-acid residues.

The invention still further relates to the plant expression vector that can in plant, efficiently express described DREB transcription factor cDNA sequence (SEQ ID NO:1).

Described DREB transcription factor cDNA sequence is inserted between the suitable restriction enzyme site of plant expression vector, exercisablely is connected, can obtain in plant, to express the expression vector of this cDNA sequence with expression regulation sequence.This expression vector can 5 ' non-coding region, cDNA sequence and the 3 ' non-coding region shown in the SEQ ID NO:1, and wherein, described 5 ' non-coding region can comprise that promoter sequence, enhancer sequence are or/and the translation enhancement sequences; Described promoter sequence can be composing type, induction type, tissue or organ specificity elicitor.As an embodiment preferred, described plant expression vector is p2MDK.

Described 3 ' non-coding region can comprise terminator sequence, mRNA cutting sequence etc., cDNA sequence shown in the SEQ ID NO:1 can replace, lack the nucleotide sequence that obtains or/and insert by the base that it is one or more and be replaced, and the coded albumen of this nucleotide sequence still has the function of DREB transcription factor.As preferably, described plant expression vector also can contain reporter gene, and is preferred, and described reporter gene is GFP.As the most preferred embodiment of the present invention, described high-efficiency plant expression vector is p2MDK-PeDREB2a.

The invention still further relates to the vegetable cell that comprises above-mentioned plant expression vector.

In addition, the application of plant expression vector in improving stress resistance of plant that the invention still further relates to a DREB transcription factor cDNA sequence that separates, clones, contains this cDNA sequence.For example, the plant expression vector that contains this cDNA sequence can be imported in the vegetable cell, cultivate screening and obtain the transfer-gen plant that resistance to environment-stress has improved, wherein, described environment-stress can be adverse circumstances such as arid, high salt or low temperature.

Transcription factor combines by the cis element with the downstream gene promoter region can regulate and control a series of correlation function expression of gene, therefore, in the plant resistance to environment stress molecular breeding, imports transcription factor gene and can improve the resistance of transgenic plant effectively.In order further to analyze the function of the PeDREB2 transcription factor gene of being cloned into, the present invention at first makes up the high efficiency plant expression vector p2MDK-PeDREB2a that obtains containing the PeDREB2a gene, utilizes agriculture bacillus mediated it to be transformed in the tobacco; By the Kan resistance, the positive seedling of genome PCR and RT-PCR Screening and Identification, the transgene tobacco seedling is carried out arid, resistance analyses such as high salt and low temperature and its relevant physiological biochemical indicator measured, draw by measuring physiological and biochemical index, the soluble sugar of transfer-gen plant, kytoplasm total protein and proline content are higher than contrast, a large amount of accumulation of these osmoregulation materials help to reduce the injury to plant of osmotic stress and drought stress, become the important biochemical evidence that transgenic plant impermeabilisation ability and drought-resistant ability improve, also further specified and expressed drought resistance and the osmotic stress resistance that the PeDREB2a gene may improve transgene tobacco by the content of regulating the osmoregulation material, to keep the normal biochemical functions of plant.The DREB transcription factor being transcribed and the promoter action of proline(Pro) and the sugared content explanation DREB factor is played an important role in the plant stress-resistance reaction a series of degeneration-resistant functional genes.

Description of drawings

Fig. 1 is the degenerate pcr amplification;

Fig. 2 is 3 ' RACE-PCR amplification.

Fig. 3 is 5 ' RACE-PCR amplification.

Fig. 4 is the amplification of PeDREB2a cDNA full length sequence.

Fig. 5 is the proteic three-D space structure prediction of PeDREB2a.

Fig. 6 is the genomic dna structural analysis of Populus euphratica DREB 2 a gene.

Fig. 7 coerces for difference and handles PeDREB2a expression of gene pattern analysis down.

Fig. 8 is the structure schema of expression vector p2MGK-PeDREB2a

The collection of illustrative plates of Fig. 9 plant expression vector p2MDK.

Figure 10 recombinant plasmid p2MGK-PeDREB2a colony PCR amplification result

The enzyme of Figure 11 recombinant plasmid p2MGK-PeDREB2a is cut the result; 1, Sal I single endonuclease digestion, 2, XbaI I and SalI double digestion.

Figure 12 contains the bacterium colony PCR of the Agrobacterium LBA4404 of expression vector p2MGK-PeDREB2a; PCK: be connected with the pMD19-T (positive control) of goal gene, nCK: Agrobacterium LBA4404 (negative control).

The PCR of Figure 13 transgene tobacco dna level detects.

The RT-PCR of Figure 14 transgene tobacco detects.

The drought resistance analysis of Figure 15 transgene tobacco; A: change p2MGK-PeDREB2a gene masculine tobacco strain system; The B:p2MGK contrast.

The salt tolerance analysis of Figure 16 transgene tobacco; A: change p2MGK-PeDREB2a gene masculine tobacco strain system; The B:p2MGK contrast.

Transgenosis and adjoining tree blade behind Figure 17 leaf-cutting 7h; A.p2MGK-PeDREB2a B.p2MGK contrast.

Figure 18 unit leaf area accumulative total fluid loss.

The influence of the high salt pair kytoplasm of Figure 19 total protein content.

Figure 20 arid is to the influence of kytoplasm total protein content.

The influence of the high salt pair proline content of Figure 21.

Figure 22 arid is to the influence of proline content.

The influence of the high salt pair leaf soluble of Figure 23.

Figure 24 arid is to the influence of leaf soluble.

Embodiment

Further describe preparation method of the present invention and beneficial effect by the following examples, it should be understood that these embodiment only are used for the purpose of illustration, never limit protection scope of the present invention.

Illustrate: make the experimental methods of molecular biology specify in following examples, all carry out, perhaps carry out according to test kit and product description with reference to listed concrete grammar in " molecular cloning experiment guide " (third edition, J. Sa nurse Brooker) book.

Separation, the clone of embodiment 1 diversiform-leaved poplar DREB transcription factor (PeDREB2a) gene

1 material and method

1.1 material

1.1.1 processing is cultivated and coerced to vegetable material

Diversiform-leaved poplar (Populus euphratica) seedling is collected in area, Lop Nur, Xinjiang, now be colonizated in experiment base, Langfang, Hebei, clip diversiform-leaved poplar brachyplast is inserted into and carries out simulating drought in the 20%PEG6000 solution and coerce and handle after 10 hours during sampling, gather be stored in the Ultralow Temperature Freezer after the vanes liquid nitrogen is handled standby.

Difference is coerced the processing of material: clip diversiform-leaved poplar brachyplast, be inserted into respectively GA3 solution, the 100 μ M of ABA solution, the 100 μ M of sodium-chlor, 20%PEG6000, the 100 μ M of 250mM BA solution, 100 μ M NAA solution and place 4 ℃ of refrigerators to handle respectively 0.5,3,6,12 and 24 hour.Material after handling is freezing rapidly with liquid nitrogen, be positioned in the Ultralow Temperature Freezer standby.

1.1.2 bacterial strain

Intestinal bacteria (Escherichia coli): DH5 α

Agrobacterium (Agrobacterium tumefaciens): LBA4404

1.1.3 carrier

The pMD19-T cloning vector is available from TaKaRa biotech firm

1.1.4 enzyme and reagent

DNA reclaims test kit available from sky, Beijing root Science and Technology Ltd.; SuperScript ^TMIII Reverse Transcriptase and Gene RacerTM kit are available from Invitrogen company; Enzymes commonly used such as pMD19-T carrier and restriction enzyme, Taq enzyme are available from precious biotech firm; Easy Pure Mini Plasmid Purification Kit is available from the Beijing Quanshijin Biotechnology Co., Ltd.

1.1.5 other reagent

Peptone, yeast extract, chloroform, Virahol, ethanol, primary isoamyl alcohol, NaCl, MgCl ₂, SODIUM PHOSPHATE, MONOBASIC, water-saturated phenol, the saturated phenol of Tris etc. be homemade analytical reagent; 5-bromo-4-chloro-3-indoles--D-galactoside (X-gal), own propyl group-β-D-thiogalactoside (IPTG), penbritin etc. be available from Beijing ancient cooking vessel state biotech firm; Agarose is the Spain product, and diethylpyrocarbonate (DEPC) is a U.S. Genview company product; Bromohexadecane base Trimethylamine 99 (CTAB) is available from Beijing Baeyer enlightening biotech firm; Polyvinylpyrrolidone (PVPK-25) is glad through Bioisystech Co., Ltd of section available from Beijing; Quadrol tetrem disodium salt (EDTA Na ₂) available from Beijing Pu Boxin biotechnology limited liability company.

1.1.6 substratum

LB liquid and solid medium are according to the disclosed method preparation of document (molecular cloning experiment guide (third edition) J. Sa nurse Brooker).The preparation of B5 medium and stock solution is prepared (Chinese agriculture press publishes for " plant tissue culture ", Wang Di chief editor) according to the disclosed method of relevant teaching material.

1.2 method

1.2.1 the extraction of the total RNA of diversiform-leaved poplar (hot CTAB method)

According to " the disclosed hot CTAB method of plant molecular biotechnology applications handbook (Peng Xuexian chief editor, a Chemical Industry Press) book is extracted the total RNA of diversiform-leaved poplar;

1.2.2 synthetic (being undertaken) of the first chain cDNA by Invitrogen company reverse transcription test kit specification sheets

1. pre-sex change: total RNA is a template with diversiform-leaved poplar, is primer (working concentration is 10pmol/ μ l) with Oligo-dT.Reaction system such as table 1, behind the mixing, 65 ℃ of sex change 5min place 1-2min on ice immediately.

The pre-sex change system of table 1 RNA

The composition consumption

Total?RNA 10ng-1μg

Oligo-dT?Primer(10pmol/μl) 1.0μl

dNTPs(10mM?each) 1.0μl

DEPC?H ₂O X

Total 13μl

2. reverse transcription: in above-mentioned centrifuge tube, add in the table 2 mixing after the ingredients listed, 42 ℃ of 1h; 70 ℃ of following 15min deactivation ThermoScript II;

Table 2 reverse transcription system

The composition consumption

5×Buffer 4μl

DTT(0.1M) 1.0μl

RNase?OUT(10mM?each) 1.0μl

Reverse?Transcriptase(200U/μl) 1.0μl

Total 7μl

3.RNaseH digestion: in reaction solution, add the RNaseH of 1 μ l (2U/ μ l), 37 ℃ of 20min, digestion and cDNA bonded single stranded RNA ,-20 ℃ of refrigerators are preserved reverse transcription product.

1.2.3 the clone of dreb gene homologous fragment

1.2.3.1 primer design is with synthetic

Utilize DNAMAN software that the amino acid and the nucleotide sequence of the relevant platymiscium DREB2 of the section genoid announced among the GENBANK are carried out the homology analysis, then, according to dreb gene DNA in conjunction with the synthetic pair of degenerate primers (table 3) of territory conserved regions design.

The primer that uses in table 3 degenerate pcr

Primer numbering primer sequence

DREBP1?5’-AA(G/A)CT(T/C)TA(T/C)AGAGGAGTGAGGCAG-3’

DREBP2?5’-(A/G)AG(A/G)T(T/G)(T/A)GG(A/G)AA(G/A)TTGAG(C/A)C(T/G)(G/A)GC-3’

QT 5’-GCTGTCAACGATACGCTACGTAACGGCATGACGG(T) ₂₄-3’

1.2.3.2?RT-PCR

In conjunction with territory conserved regions design pair of degenerate primers S and AS, is that material extraction total RNA carry out RT-PCR with the blade according to DREB2 gene DNA in the plant, and the PCR condition is: 94 ℃ of 5min; 94 ℃ of 45sec, 50 ℃ of 45sec, 72 ℃ of 30sec, 30 circulations; 72 ℃ of 10min.Reaction finishes the back and with 0.8% agarose gel electrophoresis the PCR product is identified.

1.2.3.3 the recovery of PCR product

Adopt the PCR product of sky, Beijing root Science and Technology Ltd. to reclaim test kit, operate according to the method for test kit specification sheets:

1.2.3.4 the purpose fragment is connected with cloning vector

The PCR product that reclaims is connected on the pMD19-T carrier, ligation system such as table 4,16 ℃ of connections are spent the night.

Table 4 ligation system

The composition consumption

PCR product (200-300ng/ μ l) the 4 μ l that reclaim

PMD19-T carrier (50ng/ μ l) 1.0 μ l

Ligation?Mix 5.0μl

Total 10μl

1.2.3.5 the competent preparation of intestinal bacteria

Carry out with reference to listed concrete grammar in " molecular cloning experiment guide " (third edition, J. Sa nurse Brooker) book.

1.2.3.6 the conversion of recombinant plasmid

Carry out with reference to listed concrete grammar in " molecular cloning experiment guide " (third edition, J. Sa nurse Brooker) book.

1.2.3.7 bacterium colony PCR identifies

White single bacterium colony with picking on the LB flat board that contains Amp, change in 1mlLB (Amp+) liquid nutrient medium, 37 ℃, 200r/min shakes bacterium 2h, draws 1 μ l bacterium liquid respectively as template, carrying out bacterium colony PCR identifies, primer is S and AS, and amplification condition is: 94 ℃ of 5min:94 ℃ of 45sec, 50 ℃ of 45sec, 72 ℃ of 30sec, 30 circulations; 72 ℃ of 10min.Reaction finishes the back and with 0.8% agarose gel electrophoresis the PCR product is identified.

1.2.3.8 the nucleotide sequencing of recombinant plasmid

Change bacterium colony PCR male bacterium colony over to contain Amp LB liquid nutrient medium, 37 ℃ of following 200r/min incubated overnight are prepared into glycerol stock, send the order-checking of order-checking portion of the Chinese Academy of Agricultural Sciences.

1.2.4 the clone of 3 ' cDNA of diversiform-leaved poplar dreb gene

1.2.4.1 primer design is with synthetic

According to the homologous fragment of acquired dreb gene, the nido gene specific primer of 1 couple of 3`RACE of design sees Table 5.

The primer that uses in table 53 ' the RACE reaction

Primer numbering primer sequence

3HY1-GSP1 5’-CAGAGGAGTGAGGCAGAGACATTG-3’

3HY1-GSP2 5’-GGAGTGAGGCAGAGACATTGGGG-3’

QT 5’-GCTGTCAACGATACGCTACGTAACGGCATGACAGTG(T) ₂₄-3’

Q1 5’-GCTGTCAACGATACGCTACGTAACG-3’

Q2 5’-CGCTACGTAACGGCATGACAGTG-3’

1.2.4.2 the extraction of the total RNA of diversiform-leaved poplar

With the diversiform-leaved poplar blade is the total RNA of material extraction, the same 1.2.1 of extracting method.

1.2.4.3 the first chain cDNA's is synthetic

The same 1.2.2 of method.

1.2.4.4 nest-type PRC amplification 3 '-end cDNA

1. obtaining cDNA with the reverse transcription of QT primer is template, carries out first round PCR, reaction parameter with gene specific primer 3HY1-GSP1 and universal primer Q1: 94 ℃ of 5min; 94 ℃ of 4sec, 60 ℃ of 45sec, 72 ℃ of 1min, 30 circulations; 72 ℃ of 10min; 4 ℃ of preservations.

2. first round PCR product is diluted 100 times as template, carry out second with gene specific primer 3HY1-GSP2 and universal primer Q2 and take turns nest-type PRC, reaction parameter: 94 ℃ of 5min; 94 ℃ of 45sec, 63 ℃ of 45sec, 72 ℃ of 1min, 30 circulations; 72 ℃ of 10min; 4 ℃ of preservations.

3. 0.8% agarose gel electrophoresis reclaims the PCR product and is connected to the pMD19-T carrier, and transformed into escherichia coli DH-5 α utilizes α complementation and bacterium colony PCR screening positive clone, and send the order-checking of order-checking company, the same 1.2.3 of method.

1.2.5 nest-type PRC amplification 5 ' end cDNA

1.2.5.1 primer design is with synthetic

According to 1 pair of nido gene specific primer of acquired 3 '-end cDNA sequences Design, see Table 6.

The primer that uses in table 65 ' the RACE reaction

Primer numbering primer sequence

5HY1-GSP1 5’-TTCACCAGACACATGAGCTCCTTGG-3’

5HY1-GSP2 5’-GGTGACGAAGATGTGGAAAGTTGAG-3’

GeneRacer5’primer 5’-CGACTGGAGCACGAGGACACTGA-3’

GeneRacer?5’Nested 5’-GGACACTGACATGGACTGAAGGAGTA-3’

primer

1.2.5.2 synthetic (the carrying out) of the first chain cDNA according to the explanation of the GeneRACER of Invitrogen company test kit

1. dephosphorization acid: in the PCR pipe, add table 7 ingredients listed mixing, centrifugal, behind the processing 1hr, centrifugal under 50 ℃ of conditions, place 1-2min on ice.

Table 7 dephosphorylation reaction system

The composition consumption

Total?RNA(1-5μg) 7μl

10×CIP?Buffer 1.0μl

RNaseOut(40U/μl) 1.0μl

CIP(10U/μl) 1.0μl

Total 10μl

2.RNA purifying

Carry out with reference to listed concrete grammar in " molecular cloning experiment guide " (third edition) J. Sa nurse Brooker one book.

3. remove cap: in the PCR pipe, add following table ingredients listed mixing, centrifugal, behind the processing 1hr, centrifugal under 37 ℃ of conditions, place 1-2min on ice.

Table 8 goes the cap reaction system

The composition consumption

dephosphorylated?RNA 7μl

10×TAP?Buffer 1.0μl

RNaseOut(40U/μl) 1.0μl

TAP(0.5U/μl) 1.0μl

Total 10μl

4.RNA purifying

Method is with step 2.

5.RNA the connection of joint sequence

1) will change in another PCR pipe through dephosphorylation and the RNA that goes cap to handle, and add the RNA linker fragment, mixing is centrifugal gently;

2) 65 ℃, 5min, ice bath 2min is centrifugal;

3) in the PCR pipe, add following table ingredients listed mixing, centrifugal, behind the processing 1hr, centrifugal under 37 ℃ of conditions, place 1-2min on ice.

6.RNA purifying

Method is with step 2

7. reverse transcription

The same 1.2.2 of method.

Table 9 RNA fragment ligation system

The composition consumption

Dephosphorylated?decapped?RNA?and?RNA?Oligo 7.0μl

10×Ligase?Buffer 1.0μl

RNaseOut(40U/μl) 1.0μl

ATP(10mmol/L) 1.0μl

TAP(0.5U/μl) 1.0μl

Total 11μl

1.2.5.3 nest-type PRC amplification 5 '-end cDNA

1. obtaining cDNA with the reverse transcription of QT primer is template, carries out first round PCR, reaction parameter with gene specific primer 5HY1-GSP1 and GeneRacer5 ' primer: 94 ℃ of 5min; 94 ℃ of 45sec, 60 ℃ of 45sec, 72 ℃ of 1min, 30 circulations; 72 ℃ of 10min; 4 ℃ of preservations.

2. first round PCR product is diluted 100 times as template, carry out second with gene specific primer 5HY1-GSP2 and GeneRacer5 ' Nested primer and take turns nest-type PRC, reaction parameter: 94 ℃ of 5min; 94 ℃ of 45sec, 62 ℃ of 45sec, 72 ℃ of 1min, 30 circulations; 72 ℃ of 10min; 40 ℃ of preservations.

3.0.8% agarose gel electrophoresis reclaims the PCR product and is connected to the pMD19-T carrier, and transformed into escherichia coli DH-5 α utilizes α complementation and bacterium colony PCR screening positive clone, and send the order-checking of order-checking company, the same 1.2.3 of method.

1.2.6 dreb gene cDNA total length amplification

1.2.6.1 primer design is with synthetic

Splice known 3 '-end and 5 '-end cDNA sequence, obtain the cDNA full length sequence, in view of the above, the design gene specific primer, the cDNA full length sequence of amplification dreb gene, the primer sees Table 10.

The table 10 cDNA full length sequence employed primer that increases

Primer numbering primer sequence

HY1-F1 5′-CAACTTGAAGGGTTTGTTTTTGGTCC-3′

HY1-F2 5′-GGTTTGTTTTTGGTCCCTCAAGGTT-3′

1.2.6.2 the first chain cDNA's is synthetic

The same 1.2.2 of method.

1.2.6.3 the clone and the bioinformatic analysis of dreb gene cDNA full length sequence

1. obtaining cDNA with the reverse transcription of QT primer is template, utilizes gene specific primer HY1-F1 and universal primer Q1, carries out first round PCR, reaction parameter: 94 ℃ of 5min; 94 ℃ of 45sec, 64 ℃ of 45sec, 72 ℃ of 2min, 30 circulations; 72 ℃ of 10min; 4 ℃ of preservations

2. first round PCR product is diluted 100 times as template, utilize gene specific primer HY1-F2 and universal primer Q2, carry out second and take turns nest-type PRC, reaction parameter: 94 ℃ of 5min; 94 ℃ of 45sec, 60 ℃ of 45sec, 72 ℃ of 2min, 30 circulations; 72 ℃ of 10min; 4 ℃ of preservations.

3.0.8% agarose gel electrophoresis reclaims the PCR product and is connected to the pMD19-T carrier, and transformed into escherichia coli DH-5 α utilizes α complementation and bacterium colony PCR screening positive clone, and send the order-checking of order-checking company, the same 1.2.3 of method.

4. utilize DNAMAN software to carry out sequence alignment and analysis; Utilize the SMART server ( Http:// coot.embl-heidelberg.de/SMART/) analyze the structural domain of PeDREB2a gene; Utilize the proteic three-D space structure of CPHmodels-2.0 server analyses and prediction PeDREB2a ( Http:// genome.cbs.dtu.dk/services/CPHmodels-2.0 Server-3D.htm); Utilize the ScanProsite server ( Http:// www.expasy.org/cgi-bin/prosire) the structure function site of analysis PeDREB2a gene.

1.2.7 the amplification of dna sequence dna and analysis

1.2.7.1 primer design is with synthetic

According to the cDNA sequence of acquired PeDREB2a gene, at gene 5 '-end and 3 '-end design gene specific primer, the primer sees Table 11 respectively.

The employed primer of genome amplification of table 11 PeDREB2a gene

Primer numbering primer sequence

Gn-HY1(F) 5′-CGTAATTTTGCCACCTCTTTAGTTT-3′

Gn-HY1(R) 5′-AGAATCTAAAACTGACTGACCCCTT-3′

1.2.7.2 the extraction of diversiform-leaved poplar genomic dna

Extract the diversiform-leaved poplar genomic dna with reference to listed DNA extraction method in " molecular cloning experiment guide " (third edition) J. Sa nurse Brooker one book.

1.2.7.3 pcr amplification

Genomic dna with diversiform-leaved poplar is a template, carries out pcr amplification with gene specific primer Gn-HY1 (F) and Gn-HY1 (R); 0.8% agarose gel electrophoresis reclaims the PCR product and is connected to the pMD19-T carrier, and transformed into escherichia coli DH-5 α utilizes α complementation and bacterium colony PCR screening positive clone, and order-checking, the same 1.2.3 of method.

1.2.9 the expression pattern analysis of dreb gene under different stress conditions

1.2.9.1 primer design is with synthetic

With diversiform-leaved poplar actin gene Actin (EF148840) is internal standard gene, design diversiform-leaved poplar actin gene special primer HY Actin-F and HY Actin-R; According to the PeDREB2a gene cDNA sequence that oneself obtains, at the non-conserved regions design gene specific primer of gene, the primer sees Table 13.

The employed primer of the expression analysis of table 13 PeDREB2a gene under abiotic stress

Primer numbering primer sequence

HY1-RT(F) 5′-GTTTGTTTTTGGTCCCTCAAGGT-3′

HY1-RT(R) 5′-AAGCTAAGGTCCAAGTTGGGATATG-3′

HY?Actin-F 5′-AAGTCCTCTTCCAGCCATCTCTCAT-3′

HY?Actin-R 5′-GTATTTTCTCTCTGGTGGTGCAACC-3′

1.2.9.2 the DREB transcription factor gene is replied arid, high salt and low temperature stress condition

The fresh brachyplast of the diversiform-leaved poplar of clip is inserted NaCl solution, the 20%PEG6000 solution that contains 250mM respectively and place 4 ℃ of refrigerators to carry out high-salt stress, drought stress and low temperature stress processing.Extract the total RNA of blade that handled 0,0.5,3,6,12 and 24 hour respectively, the cDNA that reverse transcription obtains is as the pcr amplification template, with the diversiform-leaved poplar actin gene is internal standard gene, utilize gene specific primer HY1-RT (F) and HY1-RT (R) to carry out the RT-PCR amplification, detect the PeDREB2a expression of gene and change.

1.2.9.3 the DREB transcription factor gene is to replying that different hormone conditions are coerced

The fresh brachyplast of the diversiform-leaved poplar of clip is inserted respectively carry out different hormones in dormin (ABA) solution of the Plant hormones regulators,gibberellins GA3 solution of phytokinin 6-benzylaminopurine (BA) solution, 100 μ M of the growth hormone naphthylacetic acid NAA solution that contains 100 μ M, 100 μ M and 100 μ M and coerce processing; Extract the total RNA of blade that handled 0,0.5,3,6,12 and 24 hour respectively, the cDNA that reverse transcription obtains is as the pcr amplification template, with the diversiform-leaved poplar actin gene is internal standard gene, utilize the special primer HY1-RT (F) and the HY1-RT (R) of gene to carry out the RT-PCR amplification, detect the PeDREB2a expression of gene and change.

2 test results and analysis

2.1 the acquisition of dreb gene homologous fragment

The DREB2 protein sequence that diversiform-leaved poplar and Arabidopis thaliana, paper mulberry, officinalis and white poplar have been reported has carried out the homology compare of analysis, find that the proteic DNA of DREB2 presents the characteristic of high conservative in conjunction with the aminoacid sequence in territory in different plants, different DREB2 family, therefore utilize this conservative region to carry out the design of degenerated primer, the cDNA that obtains with the total RNA reverse transcription of blade is a template, amplify the object tape (Fig. 1) of about 200bp by degenerate pcr, consistent with the expection size.The PCR product that reclaims is connected on the pMD19-T carrier, and transformed into escherichia coli DH-5 α utilizes α complementation and bacterium colony PCR to filter out 4 positive colonies, obtains the sequence of 1 183bp after the order-checking.Sequence retrieval result shows, with soybean, cotton, and the dreb gene nucleotide sequence of Arabidopis thaliana have higher consistence, judge that tentatively this fragment is a diversiform-leaved poplar dreb gene conserved regions.

2.2 the acquisition of dreb gene cDNA3 ' sequence and 5 ' sequence

Through sequence alignment, utilize acquired dreb gene homologous fragment, the design gene specific primer; Utilize gene specific primer 3HY1-GSP1 and 3HY1-GSP2 to carry out 3 ' RACE amplification with 3 ' universal primer Q1, Q2 respectively, electrophoresis detection, obtain the amplified production (Fig. 2) of 1 treaty 1000bp, sequencing result shows, this fragment is 969bp, and 5 ' terminal sequence and former homologous fragment are overlapped, and are judged as 3 ' terminal sequence of target dreb gene.

Utilize gene specific primer 5HY1-GSP1 and 5HY1-GSP2 to carry out 5 ' RACE amplification with GeneRacer 5 ' Primer and 5 ' NestedPrimer respectively, electrophoresis detection obtains the amplified production (Fig. 3) of 1 treaty 900bp, sequencing result shows, this fragment is 893bp, and 3 ' terminal sequence and former homologous fragment are overlapped, and are judged as 5 ' terminal sequence of target dreb gene.

2.3 the acquisition of dreb gene cDNA full length sequence

Utilize full length gene special primer HY1-F1 and HY1-F2 respectively with 3 ' universal primer Q1, Q2, nest-type PRC obtains the amplified production (Fig. 4) of 1 treaty 1.7KB.Sequencing result shows that its full length sequence is 1601bp, and the sequence alignment result shows, with the gene of various plants DREB family higher similarity is arranged, and is inferred as new dreb gene, called after PeDREB2a.

2.4 the bioinformatic analysis of diversiform-leaved poplar dreb gene

2.4.1 the sequence signature analysis of dreb gene and the prediction of the structure function of proteins encoded

Utilize DNAMAN software that the full length cDNA sequence of PeDREB2a gene is analyzed, the result shows: this gene order total length is 1601bp, complete open reading frame 1125bp, and 375 amino acid of encoding, 5`-UTR and 3`-UTR comprise 151bp and 322bp respectively.By utilizing its molecular weight of Compute pI/Mw tool software prediction to be 41.7kDa, iso-electric point is 6.16.Functional analysis shows SMART software to the PeDREB2a encoded protein, this albumen contains a typical A P2/EREBP structural domain, this structural domain is made up of 58 amino acid, and at a front end existence nuclear localization signal (NLS) of AP2/EREBP structural domain, it guides this albumen to be positioned nucleus; And find to have two Serine enrichment regions and a glutamine enrichment region in its sequence, the function that strengthens transcriptional activation may be played in this zone; End sequence (PSXEIDW) conserved regions that acidic activated territory is arranged at C-terminal.

2.4.2 the proteic three-D space structure prediction of DREB

Utilize CHPmodels-2.0 ( Http:// genome.cbs.dtu.dk/services/CPHmodels-2.0server) server is predicted the proteic three-D space structure of DREB2a, the result shows: have 1 alpha-helix in the proteic space structure of DREB2a, 3 beta sheets are formed, this is a space structure that typical DREB transcription factor is had, and this angle from the space structure that albumen had has proved that the albumen of this coded by said gene may be DREB class transcription factor (Fig. 5).

2.4.3PeDREB2a comparison of the homology of albumen and other DREB2 proteinoid and systematic evolution tree analysis

DREB genoid in the plant can be divided into two big classes, and a class is the DREB1 class transcription factor by low temperature induction; Another kind of is by DREB2 class transcription factor arid, the hypersaline environment stress-inducing.By DNAMAN software Populus diversifolia PeCBL 2 a gene and other dreb gene of having cloned are carried out the systematic evolution tree analysis, the result shows: the main and DREB2 class transcription factor of PeDREB2a gene is got together, and nearer with the sibship of the DREB2 class transcription factor of dicotyledons.

Therefore, the DREB2 class transcription factor in the main and plant is carried out the homology compare of analysis with the DREB transcription factor gene PeDREB2a of being cloned into.The sequence alignment result shows: this transcription factor gene complete sequence homology with other plant DREB2 proteinoid on protein level is not high, and consistence is about 33.42%, only high conservative is arranged at AP2/EREBP in conjunction with the territory.And the Xie Ansuan (V14) of finding the 14th of DREB class transcription factor is very conservative, and the 19th amino acid is conservative, and what have is L-glutamic acid (E19), and what have is leucine (L19).Further aspect the decision DNA binding specificity, the effect of V14 is obviously important than E19 and L19 in the DREB associated protein for proof.

2.5 the genomic dna structural analysis of Populus euphratica DREB 2 a gene

With diversiform-leaved poplar genomic dna and cDNA is template, is that primer carries out pcr amplification to PeDREB2a with Gn-HY1 (F), Gn-HY1 (R), obtains the amplified production of 1 treaty 1.5Kb; Show that through reclaiming order-checking this band is 1303bp.The sequencing result analysis shows, PeDREB2a gene intronless (Fig. 6).

2.6 the expression pattern analysis of dreb gene under different stress conditions

The diversiform-leaved poplar brachyplast is carried out low temperature, high salt and arid (PEG) etc. respectively coerce processing, in the different treatment time, extract the total RNA of its blade respectively, reverse transcription obtains cDNA.Sxemiquantitative RT-PCR detected result shows that after handling with 250mM NaCl high level salt solution, the PeDREB2a gene is induced by high-salt stress obviously at transcriptional level, expression amount just began to improve after salt was handled 0.5h, along with the prolongation in treatment time, expression amount increases sharply, and reaches maximum value to 3h.And brachyplast is when being in the simulating drought condition (20%PEG solution), and the PeDREB2a gene can be by its abduction delivering, and higher salt handles and reach peak value earlier, and gene expression amount reaches maximum after handling 6h, descends rapidly again then.After with 4 ℃ of subzero treatment, the result shows that this gene also is subjected to cryogenic abduction delivering, and change also apparent in view, at 0.5h abduction delivering just, the 3h expression amount reaches the highest, in processing subsequently, weaken gradually again, show that this gene is subjected to the abduction delivering of arid, high salt and low temperature abiotic stress environment.Basically do not change with the expression of PeDREB2a on transcriptional level in the dormin ABA processing, this illustrates that also it does not rely on the Signal Regulation approach of ABA.With shifting to an earlier date of hormone NAA abduction delivering than GA3 and BA abduction delivering time, 0.5h in time, just obviously express, and the abduction delivering amount obviously reduces again in the whole process thereafter, and in handling with GA3 and BA all at 12h and 24h abduction delivering, these illustrate that all these three kinds of hormones also induce the PeDREB2a expression of gene.Find out that thus PeDREB2a has significantly abiotic stress and replys (Fig. 7).In sum, the PeDREB2a gene can be by high salt, arid and low temperature institute abduction delivering, and this inducing action and do not rely on ABA Signal Regulation approach.

Utilize biological software that the PeDREB2a gene is analyzed, find that in the AP2/EREBP structural domain of this gene the 14th exists to be proved the functional amino that has the specificity binding mechanism with the DRE cis-acting elements, i.e. Xie Ansuan (14V).And the 19th be leucine, is not glutamine, and this also proves absolutely the non-conservation of 19 amino acids, and further aspect the decision DNA binding specificity, the effect of V14 is obviously important than E19 and L19 in the DREB associated protein for proof.In addition, be subjected to the abduction delivering of different condition, the DREB class transcription factor in the plant can be divided into two types of DREB1 and DREB2 according to plant DREB.To the analysis revealed of different plant DREB transcription factor sibships, PeDREB2a is sorted in the DREB2 transcription factor class of dicotyledons, and is nearest with the GmDREB relation of soybean.

Studies show that at transcription activating domain of the general existence of the C-of DREB transcription factor end, they are feature to be rich in acidic amino acid, glutamine, proline(Pro) or Serine and Threonine, this structural domain is to activate target gene to transcribe necessary.Have tangible transcription activating domain at PeDREB2a transcription factor gene C-end, infer that this transcription factor protein should have transcriptional activation function, its biological function remains further to be furtherd investigate.Infer simultaneously phosphorylation that Serine enrichment region in the PeDREB2a albumen may be by the site respectively the DNA of modulin in conjunction with the territory to the binding ability of DRE element and the transcriptional activation ability in acid activatable territory.

The correlation function checking in tobacco of the structure of test example 1 high efficiency plant expression vector and Populus diversifolia PeCBL 2 a gene

2. materials and methods

2.1 material

2.1.1 vegetable material

Tobacco NC89 is preserved by this laboratory.

Bacterial strain and carrier

Intestinal bacteria (E.coli): DH5 α preserves this chamber

Agrobacterium (Agrobacterium tumefaciens) bacterial strain LBA4404 preserves this chamber

PUC-ST4A preserves this chamber

PCAMBIA2301 preserves this chamber

PMD19-T cloning vector TaKaRa

PUC19 TaKaRa

Illustrate: the complete sequence of pCAMBIA2301 has been disclosed in AF234316 Binary vector pCAMBIA-2301 among the NCBI.

2.1.3 agents useful for same and medicine in the experiment

Various restriction enzymes, rTaq enzyme, ExTaq enzyme are available from Takara company, and the T4 dna ligase is available from New EnglandBiolabs, and the Taq enzyme is available from Beijing Pu Boxin biotech company; SuperScript ^TMIII Reverse Transcriptase is available from Invitrogen company; The TRNzol total RNA extraction reagent is available from sky, Beijing root Science and Technology Ltd.; Sepharose DNA reclaims test kit available from sky, Beijing root Science and Technology Ltd.; Easy Pure Mini Plasmid Purification Kit is available from the Beijing Quanshijin Biotechnology Co., Ltd.

2.1.4 substratum

LB bacteria culture medium .YEB substratum and MS substratum are prepared according to the disclosed method of " molecular cloning experiment guide " (third edition, J. Sa nurse Brooker);

MS selects substratum: add 6-BA (3.0mg/L) in the MS minimum medium, NAA (0.2mg/L), Cephradine (500mg/L) and kantlex (100mg/L).

MS root media: add Cephradine (500mg/L) and kantlex (200mg/L) in the MS minimum medium.

2.2 method

2.2.1 design of primers is with synthetic

Synthetic primer p35SGKNOS-PeDREB2a (F) and the p35SGKNOS-PeDREB2a (R) that contains restriction enzyme site Sal I and Xba I of design, primer sequence sees Table 14.

Table 14 makes up the plant expression vector the primer

Primer numbering primer sequence

p35SGKNOS-PeDREB2a(F) 5’-GGAGTCGACACCATGGCAGCAGCT-3’

p35SGKNOS-PeDREB2a(R) 5’-GGATCTAGACTGACTGACCCCTTAA-3’

2.2.2 the structure of plant expression vector

2.2.2.1 the structure of high efficiency plant expression vector p2MDK-PeDREB2a

Utilize primer p35SGKNOS-PeDREB2a (F) and p35SGKNOS-PeDREB2a (R) to amplify complete open reading frame from the pMD19-T carrier that is connected with total length PeDREB2acDNA.Amplified production and carrier are after double digestion, agarose gel electrophoresis, recovery, and the T4 ligase enzyme makes up high efficiency plant expression vector p2MGK-PeDREB2a.

2.2.2.2 recombinant plasmid transformed bacillus coli DH 5 alpha and bacterium colony PCR identify

Method is with 2.2.3.6 and the 2.2.3.7 of embodiment 1

2.2.2.3 a small amount of of recombinant plasmid is extracted and enzyme is cut evaluation

2.2.3 the genetic transformation of tobacco

2.2.3.1 the preparation of Agrobacterium competent cell and conversion

Carry out the preparation and the conversion operation of Agrobacterium competent cell according to the disclosed method of " plant genetic engineering philosophy and technique " (Wang Guanlin chief editor, Dalian press of normal university).

2.2.3.2 containing the PCR of the Agrobacterium of recombinant plasmid identifies

Single bacterium colony on the picking YEB flat board is inoculated in the 5ml YEB liquid nutrient medium (containing 50mg/L Rif, 100mg/L Kan), cultivates 1-2d for 28 ℃, compares with unconverted Agrobacterium, carries out bacterium colony PCR and identifies.

2.2.3.3 the genetic transformation of tobacco

1. the Agrobacterium bacterium liquid that will contain plant expression vector evenly is applied to YEB flat board (containing 50mg/L Rif, 100mg/L Kan), 1ml/ plate, 28 ℃ of overnight incubation;

2. scrape gently with the rifle head and get thalline, and change in the MS liquid nutrient medium, make suspension OD ₆₀₀Be about 0.5;

3. get the blade of aseptic seedling, cut edge and main vein, be cut into the square of 0.5cm size;

With the explant that cuts at Agrobacterium bacterium liquid (OD ₆₀₀=0.5) soaks 5-10min in;

5. blot the bacterium liquid on vegetable material surface with aseptic filter paper, change the MS solid-based basal culture medium that the surface is covered with one deck filter paper over to, 28 ℃, secretly cultivate 3d;

6. material is forwarded to 25 ℃ of cultivations (photoperiod is 16h illumination/8h dark) to resistant buds breaks up in the division culture medium that contains Kan (100mg/L).

2.2.4 the screening of transgene tobacco

2.2.4.1 the screening of Kan resistance seedling

Treat that resistant buds grows to 2-3cm when high, downcut budlet and change in the root media that Kan concentration is reduced to 50mg/L and is beneficial to root induction in the substratum.After treating that seedling takes root, it is changed in the new root media, be increased to 200mg/L with the concentration of Kan this moment, in order to reducing the false positive rate of transformed plant, thereby reduces the workload of follow-up screening.

2.2.4.2 the dna level of transgene tobacco is identified

Utilize the CTAB method to be extracted in a small amount to contain total DNA of the tobacco seedling of taking root of growing in the Kan substratum, detect the further positive transformation of tobacco plant of screening by PCR.If amplified target fragment, and do not amplify fragment in the negative control, prove that tentatively goal gene is incorporated in the tobacco gene group.

2.2.4.3 the RT-PCR of transgene tobacco detects

The positive transformant of selecting PCR to determine, clip blade, TRNzol extract total RNA, and reverse transcription obtains cDNA as template, and RT-PCR detects the positive seedling that transforms of screening.Method is with reference to the 2.2.1 and the 2.2.8.4 of chapter 2

2.2.5 transgene tobacco anti-coerced analysis

2.2.5.1 the drought resistance analysis of transgene tobacco

The commentaries on classics PeDREB2a gene masculine tobacco seedling that the size growth is consistent takes out with the contrast tobacco seedling band root that changes the p2MGK empty plasmid over to, it is transferred in the MS substratum that contains 20%PEG, in 28 ℃ illumination box, cultivate, after 4 days, after root salt solution cleaned with sterilized water, move into the MS substratum again, recover growth 14 days, observe the plant strain growth changing conditions, and the Taking Pictures recording experimental result.3 repetitions are established in test.

2.2.5.2 the salt tolerance analysis of transgene tobacco

The commentaries on classics PeDREB2a gene masculine tobacco seedling that the size growth is consistent takes out with the contrast tobacco seedling band root that changes the p2MGK empty plasmid over to, its transfer is contained in the MS substratum of 300mMNaCl, in 28 ℃ illumination box, cultivate, observe the plant strain growth changing conditions after 7-10 days, and the Taking Pictures recording experimental result.3 repetitions are established in test.

2.2.5.3 the low temperature resistant analysis of transgene tobacco

The commentaries on classics PeDREB2a gene masculine tobacco seedling that size growth is consistent and the contrast tobacco seedling that changes the p2MGK empty plasmid over to place-4 ℃ of refrigerators to carry out being transferred under the normal condition behind the subzero treatment 20h and cultivate, observe seedling and whether can restore normal growth, and the Taking Pictures recording experimental result.3 repetitions are established in test.

2.2.6 the physiological and biochemical index of transgene tobacco under adverse environmental factor measured

2.2.6.1 water-retentivity experiment

The 3rd expansion leaf from top to bottom of the contrast cigarette strain of the cigarette strain of the commentaries on classics PeDREB2a of clip PCR tests positive and commentaries on classics p2MGK empty plasmid, C1-202AREA METER type leaf area instrument is measured leaf area, put on the filter paper then, under the room temperature condition, nature dehydration 7h, heavy every leaf of weighing half an hour, record blade dehydration situation, Units of Account leaf area accumulative total fluid loss.

2.2.6.2 the mensuration of kytoplasm total protein content

The transgenic positive tobacco seedling of flush away root matrix and the contrast cigarette strain of changeing the p2MGK empty plasmid be put in the MS solution that contains 20%PEG6000 respectively and contain in the MS solution of 300mmol/L NaCl and carry out arid and the processing of high salt respectively, after handling 7h and 12h, get the 3rd and 4 leaf and measure kytoplasm total protein content, proline content and soluble sugar content.

1. experimental principle

Xylene Brilliant Cyanine G G-250 measures protein content and belongs to a kind of of dye binding method, is a kind of reasonable quantification of protein method.

2. the protein content typical curve is drawn

The disclosed method of reference literature is drawn protein content typical curve (high refined plum .TERF1 gets functional analysis [D] .2006 in transgenic paddy rice)

3. the mensuration of protein concn in the sample extracting solution

Proteinic concentration in the disclosed method working sample of the reference literature extracting solution (high refined plum .TERF1 gets functional analysis [D] .2006 in transgenic paddy rice).

2.2.6.3 the mensuration of proline content

1. experimental principle

Under acidic conditions, triketohydrindene hydrate and proline(Pro) reaction generate stable red compound, this product has maximum absorption band under the 520nm wavelength, the depth of color is promptly represented the height of proline content, acidic amino acid and neutral amino acids can not react with acid ninhydrine, basic aminoacids content is very little, can ignore.

2. the proline content typical curve is drawn

The disclosed method of reference literature is drawn the typical curve (high refined plum .TERF1 gets functional analysis [D] .2006 in transgenic paddy rice) of proline content.

3. the mensuration of proline content in the sample

The content of proline(Pro) in the disclosed method working sample of reference literature (high refined plum .TERF1 gets functional analysis [D] .2006 in transgenic paddy rice).

2.2.6.4 the mensuration of soluble sugar content

1. experimental principle

Phynol method is measured the soluble sugar principle: sugar is under vitriol oil effect, furfural or hydroxymethylfurfural that dehydration generates can be condensed into a kind of orange red compound with phenol, its shade is directly proportional with the content of sugar in the 10-100mg scope, and under the 485nm wavelength maximum absorption band is arranged, so available colorimetry is measured under this wavelength.

2. the typical curve of soluble sugar content is drawn

The disclosed method of reference literature is drawn the typical curve (high refined plum .TERF1 gets functional analysis [D] .2006 in transgenic paddy rice) of soluble sugar content.

3. the mensuration of soluble sugar content in the sample

The content of soluble sugar in the disclosed method working sample of reference literature (high refined plum .TERF1 gets functional analysis [D] .2006 in transgenic paddy rice).

3. experimental result and analysis

3.1 the structure of plant expression vector

3.1.1 the structure of efficient expression vector p2MGK-PeDREB2a

3.1.1.1 pass through pcr amplification, obtain having the CaMV 35S promoter sequence of 2 enhansers, two ends have HindIII and Pst I restriction enzyme site respectively, and HindIII and this fragment of Pst I double digestion and cloning vector PUC19 obtain carrier PUC-35S. through the connection of T4 ligase enzyme

3.1.1.2 pcr amplification obtains PolyA, the NOS sequence that two ends have EcoR I and Kpn I restriction enzyme site respectively from the plant expression vector pT Ω 4A, EcoR I and Kpn I enzyme are cut this fragment and carrier PUC-35S, connect through the T4 ligase enzyme to obtain carrier PUC-35SNOS.

3.1.1.3 design two long-chain primers, two ends have Pst I enzyme point of contact respectively, two primer annealings obtain the Ω sequence with the restriction enzyme site sticky end, and Pst I enzyme is cut PUC-35SNOS, are connected with Ω through the T4 ligase enzyme to obtain carrier PUC-Ω 35SNOS.

3.1.1.4 pcr amplification obtains the GFP+KDEL sequence that two ends have BamH I and Kpn I restriction enzyme site respectively from the carrier 163, BamH I and Kpn I enzyme are cut this fragment and carrier PUC-Ω 35SNOS, connect through the T4 ligase enzyme to obtain carrier PUC-Ω 35SGKNOS.

3.1.1.5 the PeDREB2a and the PUC-Ω 35SGKNOS that will be connected on the cloning vector PMD-19 distinguish Sal I and Xba I double digestion, are connected with Ω through the T4 ligase enzyme to obtain carrier PUC-Ω 35SNOS-PeDREB2a.

3.1.1.6HindHI and EcoR I double digestion PUC-Ω 35SNOS-PeDREB2a, reclaim small segment, be connected on the pCAMBIA2301 plant expression vector behind HindIII and the EcoR I double digestion and obtain carrier pGK.

Obtain the MAR sequence that two ends have EcoR I restriction enzyme site respectively 3.1.1.7 go up pcr amplification from carrier PMD-MAR, the EcoRI enzyme is cut this fragment and carrier pGK, connects through the T4 ligase enzyme to obtain carrier pMGK.

Obtain the MAR sequence that two ends have the HindIII restriction enzyme site respectively 3.1.1.8 go up pcr amplification from carrier PMD-MAR, the HindIII enzyme is cut this fragment and carrier pMGK, connects through the T4 ligase enzyme to obtain carrier p2MGK-PeDREB2a.Tool makes up flow process and sees Fig. 8.

Fig. 9 is the collection of illustrative plates of p2MGK.

3.1.2 the bacterium colony PCR of recombinant plasmid identifies

On the conversion LB of three recombinant plasmids (Kan+) flat board, two single bacterium colonies of white of picking carry out bacterium colony PCR and identify that the result is all positive, sees Figure 10 respectively.

3.1.3 the enzyme of recombinant plasmid is cut evaluation

The positive bacterium colony that PCR is detected changes LB (Kan+) liquid nutrient medium over to, and 37 ℃ are shaken bacterium and spend the night, and alkaline process extracts recombinant plasmid in a small amount, carries out enzyme and cuts evaluation.The p2MGK-PeDREB2a recombinant plasmid is cut out object tape, and it is correct that enzyme is cut the result, sees Figure 11.

3.2 containing the Agrobacterium PCR of expression vector detects

Recombinant plasmid p2MGK-PeDREB2a is transformed Agrobacterium LBA4404 make template with the bacterium liquid of the Agrobacterium after transforming, unconverted Agrobacterium compares, and carries out bacterium colony PCR and identifies.Detected result shows that recombinant plasmid successfully changes in the Agrobacterium, sees Figure 12.

3.3 the screening of transgene tobacco and detection

3.3.1 the dna level of transgene tobacco detects

Adopt classical agrobacterium-mediated transformation that tobacco is carried out genetic transformation.The Kan resistance seedling that selection has been taken root, its blade of clip, adopt the CTAB method to extract tobacco DNA, with tobacco DNA is template, the p35SGKNOS-PeDREB2a (F) and p35SGKNOS-PeDREB2a (R) primer that design during with the structure conversion carrier are primer, adopt round pcr, Kan resistance seedling is carried out the detection of dna level.The result shows, detects Kan resistance seedling 36 strains that p2MGK-PeDREB2a transforms, and it is positive that PCR detects 24 strains, and positive rate 67% proves that PeDREB2a has been incorporated in the tobacco gene group.See Figure 13.

3.3.2 the RT-PCR of transgene tobacco detects

Choosing 24 positive tobacco strains that dna level identifies is material, extracts total RNA, and the cDNA that obtains with reverse transcription is a template, and RT-PCR does further to detect to positive seedling.The result shows, in the tobacco strain that 24 strain p2MGK-PeDREB2a transform, and 18 strain test positive, positive rate 75% is seen Figure 14.

3.4 transgene tobacco is to the tolerance analysis of abiotic stress

3.4.1 the drought resistance analysis of transgene tobacco

Tobacco conversion seedling with the p2MGK empty plasmid compares, and transgenic positive tobacco seedling and contrast seedling band root are taken out, and it is transferred in the MS substratum that contains 20%PEG, carries out the drought resistance analysis.Handle after 6 days, can observe contrast tobacco plant blade wilts serious than transgene tobacco, the lobus cardiacus of transgene tobacco blade and last several second leaf still keep certain turgescence, and the lobus cardiacus of contrast tobacco leaf has just begun to wilt, phenomenons such as leaf margin curls, blade is sagging, the contrast tobacco seedling above 95% is containing substantially all death on the substratum of PEG.After this tobacco recovered to cultivate 20 days again, can see that the recovery extent of transgenic tobacco plant blade is better than contrast (Figure 15).

3.4.2 the salt tolerance analysis of transgene tobacco

Tobacco conversion seedling with the p2MGK empty plasmid compares, transgenosis and contrast tobacco seedling are changed in the MS substratum that contains 300mmol/L NaCl, after handling 7d continuously, contrast tobacco seedling leaf takes place obviously to wilt, and part jaundice, the wilting degree is more serious, the degree that wilting takes place transfer-gen plant is lighter, recover to cultivate after 20 days, the recovery extent that can see the transgenic tobacco plant blade is significantly better than contrast (Figure 16), and above-mentioned test-results shows that the overexpression of Populus diversifolia PeCBL 2 A gene in tobacco can improve the resistance of transfer-gen plant to salt stress really.

3.4.3 the low temperature resistant analysis of transgene tobacco

Tobacco conversion seedling with the p2MGK empty plasmid compares, to contrast with transfer-gen plant and be placed in-6 ℃ of low temperature and irradiance incubators, handled 8 hours, observe the phenotype of contrast and transfer-gen plant, find the blade frostbite wilting of adjoining tree, transfer-gen plant also has the minority seedling to wilt but the overwhelming majority is still normal, puts then to normal condition to grow, and the equal yellow of plant that takes place after 3 days to wilt is die.Survival rate after contrast is handled with the transfer-gen plant freeze injury is respectively 43.3% and 83.4%.This shows, change the winter resistance that the PeDREB2a gene makes plant over to and obtained certain raising.

3.5 the physiological and biochemical index of transgene tobacco under adverse environmental factor measured

3.5.1 transgene tobacco blade water-retentivity is analyzed

Can be found out obviously that by Figure 17 behind the leaf-cutting 7h, the tobacco contrast seedling leaf fluid loss that changes the p2MGK empty plasmid is big, the wilting degree is serious, and leaf area is obviously dwindled, and dehydration becomes dry around the blade, is irregular shape; And it is lighter to change the positive seedling leaf wilting of PeDREB2a, and only the limb edge curls, and the leaf look brighter, and leaf area is dwindled degree, and contrast is not serious, and has certain turgescence, maintenance blade basic configuration.

As shown in Figure 18, in half an hour, the blade dehydration is all very fast after leaf-cutting for contrast and transgenic positive plant, and fluid loss is big; After half an hour, rate-of-loss of coolant slows down, but the accumulative total dehydration constantly increases.Comparatively speaking, contrast is obviously fast than transgenosis dehydration generally, and this illustrates that also Pe DREB2a gene has improved the blade water retention property of tobacco.By two sample average difference t test, its average significance of difference test t=4.3998, conspicuous level p=0.0064, difference is extremely remarkable.

3.5.2 the mensuration of transgenic tobacco plant kytoplasm total protein content

Kytoplasm total protein content difference under table 15 transfer-gen plant and the contrast high-salt stress

Kytoplasm total protein content difference under table 16 transfer-gen plant and the contrast drought stress

According to BSA concentration and corresponding OD595nm value production standard curve thereof, gained regression equation y=0.0715X-0.0412, R ²=0.9417, thus calculate the content of sample kytoplasm total protein.By Figure 19 and Figure 20 as can be seen, after salt is handled 7h and arid processing 12h, protein content in contrast and the transgene tobacco blade all raises to some extent, comparatively speaking, protein content after the processing finds out that by table 15 transgenic line 6,7,10 high salt front and back kytoplasm total protein contents increase per-cent and reach 48.13%, 40.95%, 37.36% respectively all than the height before handling.And contrast high salt front and back content increase per-cent is 19.83%, 11.71%.Proof transfer-gen plant salt tolerance is higher than adjoining tree.Can obtain conclusion by table 16: contrasting arid cross-reference kytoplasm total protein content increases per-cent far away from transfer-gen plant, differs significantly, has proved that the transfer-gen plant drought resistance is apparently higher than adjoining tree.

3.5.3 the mensuration of transgenic tobacco plant proline content

Proline content difference under table 17 transfer-gen plant and the contrast high-salt stress

Proline content difference under table 18 transfer-gen plant and the contrast drought stress

Proline(Pro) is as a kind of effective organic osmoregulation material, plays an important role in abiotic stresses such as arid, low temperature, high temperature, freezing, salt marsh are coerced.Almost desired adverse circumstance all can cause the accumulation of proline(Pro) in the plant materials, and the index of accumulation is relevant with the resistance of plant.Therefore, proline(Pro) Chang Zuowei plant drought physical signs.OD520nm value production standard curve according to concentration of proline is measured obtains regression equation Y=0.3167X-0.0595, R ²=0.9492, thus calculate proline content in the sample.Result of study is seen Figure 20 and 21 and table 17,18, find out intuitively that by Figure 21 and 22 high salt and arid are handled cross-reference and the intravital proline(Pro) of transfer-gen plant has all improved, analysis through table 17 and 18, data presentation on the whole, the increase per-cent of transfer-gen plant all is higher than contrast, thereby the drought resisting, salt tolerance of releasing transfer-gen plant are apparently higher than contrast.

3.5.4 the mensuration of transgenic tobacco plant soluble sugar content

Soluble sugar content difference under table 19 transfer-gen plant and the contrast high-salt stress

Soluble sugar content difference under table 20 transfer-gen plant and the contrast drought stress

Usually accumulate a large amount of soluble sugars under the adverse environmental factor in the plant materials, it is a class osmoregulation material, and plant can be improved its resistivity to adverse circumstance by the osmoregulation effect.There are some researches prove: in several osmoregulation materials of cell, be K the Relative Contribution size of steady seepage regulating power ⁺＞soluble sugar＞other total free aminoacids＞Ca ²⁺＞Mg ²⁺＞proline(Pro).Therefore, under drought condition, the interior osmoregulation thing of cell is K particularly ⁺It with soluble sugars one of level of signification of reflection drought resistance power.Find out that by histogram (Figure 23 and Figure 24) all there is more significantly raising high salt and the arid back of handling than the intravital soluble sugar content of plant before the processing, by calculating comparison transfer-gen plant and to impinging upon the difference of soluble sugar content under high salt and the drought stress, the result shows: transfer-gen plant is being coerced processing back soluble sugar increase per-cent all apparently higher than contrast, and some transgenic lines increases per-cent and has reached 155.68%, and the salt tolerant, drought-resistant ability that can prove transfer-gen plant thus are apparently higher than contrast.

Plant shows morphological specificity under the adverse circumstance on the one hand to the resistance of osmotic stress and arid, and on the other hand, the variation of plant physiology and biochemistry also highly adapts to stress resistance.The content of proline(Pro) is as the plant drought index, become and weigh the whether biochemistry foundation of drought resisting of plant, soluble sugar, kytoplasm total protein are as the osmoregulation material, accumulation under osmotic stress, help vegetable cell to reduce the osmotic potential and the flow of water, reduce the harm of osmotic stress to plant, the content of these osmoregulation materials also becomes the important indicator that the plant impermeabilisation is coerced.Draw by measuring physiological and biochemical index, the soluble sugar of transfer-gen plant, kytoplasm total protein and proline content are higher than contrast, a large amount of accumulation of these osmoregulation materials help to reduce the injury to plant of osmotic stress and drought stress, become the important biochemical evidence that transgenic plant impermeabilisation ability and drought-resistant ability improve, also further specified and expressed drought resistance and the osmotic stress resistance that the PeDREB2a gene may improve transgene tobacco by the content of regulating the osmoregulation material, to keep the normal biochemical functions of plant.The DREB transcription factor being transcribed and the promoter action of proline(Pro) and the sugared content explanation DREB factor is played an important role in the plant stress-resistance reaction a series of degeneration-resistant functional genes.

Sequence table

Claims (6)

1. the arid response element binding protein of diversiform-leaved poplar (Populus euphratica) is characterized in that: it is the aminoacid sequence shown in the SEQ ID NO:2.

2. the cDNA of the coding described diversiform-leaved poplar of claim 1 (Populus euphratica) arid response element binding protein, it is characterized in that: described cDNA is the nucleotide sequence shown in the SEQ ID NO:1.

3. plant expression vector is characterized in that: the cDNA that contains claim 2.

4. the arid response element binding protein of the described diversiform-leaved poplar of claim 1 (Populus euphratica) is improving plant to the application in the tolerance to environmental stress.

5. the described cDNA of claim 2 is improving plant to the application in the tolerance to environmental stress, comprising: described cDNA can be operated with the expression regulation sequence of plant expression vector be connected, obtain the recombinant plant expression vector; The recombinant plant expression vector is imported to vegetable cell, and screening obtains transgenic plant.

6. the described plant expression vector of claim 3 is improving plant to the application in the tolerance to environmental stress, comprising: described plant expression vector is imported to vegetable cell, and screening obtains transgenic plant.

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