CN101698854A - Application of transcription thellungiella halophila CBF1 gene in improving drought resistance and salt tolerance of corn and wheat - Google Patents

Abstract

The invention discloses an application of a transcription factor CBF1 gene. The CBF1 gene cloned from the thellungiella halophila is transcribed into cells of corn and wheat in a sense form to obtain transgenic plants which can have obvious improved stress resistance. The invention can improve the salt tolerance and the drought resistance of corn and wheat in a way of transcribing TsCBF1 gene, thereby being used for creating stress-resistant new germ plasm.

Description

Change the application that salt mustard CBF1 gene improves corn, Drought-resistance in Wheat salt tolerance

Technical field

The invention belongs to the biotechnology breeding field of farm crop, specifically, relate to a kind of by changeing the scheme and the purposes of salt mustard CBF1 gene alteration corn, wheat resistance.

Background technology

CBFs (CRT/DRE-binding factor) is and DNA cis element CCGAC bonded transcription factor.Transcription factor be a class can with the cis-acting elements generation specificity bonded protein molecule in the eukaryotic gene promoter region, thereby guarantee that goal gene is with specific intensity, express in the specific time and space.When changes in environmental conditions, plant is by a series of signal transmission, excite transcription factor, transcription factor is with after corresponding cis-acting elements combines, activator RNA polymerase II transcription complex, thereby start transcribing of specific gene, make conditioned reaction by the internal outer signals of the effect of gene product at last.Be used to improve the engineered transcription factor of plant stress-resistance and see Table 1-1.

The transcriptional control of genetic expression

The regulation and control of osmotic stress response gene be divided into the regulation and control of transcriptional level and transcribe after regulation and control.The regulatory factor of transcriptional level mainly is cis-acting elements and the trans-acting albumen relevant with osmotic stress.It is generally acknowledged that the being adjusted in genetic expression on the transcriptional level occupies an leading position in regulating, transcription factor plays a crucial role in transcriptional regulatory.Many stress response genes encode transcription factors, thereby in the regulation and control of genetic expression, work.So far isolating salt stress and the drought-induced transcription factor gene of being subjected to mainly contain: myb transcription factor, contain alkaline whorl one spiral (bHLH) and leucine zipper the MYC transcription factor, contain alkaline zone and leucine zipper bZIP transcription factor, zinc and refer to the class transcription factor and have a DRE of AP2 structural domain conjugated protein as DREB1 and DREB2 etc.

Table 1-1 is used to improve the transcription factor (Taishi et al., 2006) of stress tolerance in plants

The strategy that the plant stress-resistance genetically engineered is adopted

In the plant long-term evolution process, aspects such as its habit, morphological structure and Physiology and biochemistry have formed a cover to various adverse circumstance adaptive strategies.Wherein comparatively generally metabolic change, i.e. the metabolism adjustment of stress-inducing.Under the environment stress condition, plant itself can perception and conduction adverse circumstance signal, starts Expression of Related Genes, and then activates the corresponding protection pathways metabolism.Plant stress-resistance border research has in recent years obtained remarkable progress, has cloned in succession that some important salt tolerants, water-fast branch are coerced, cold-resistant genes involved.Along with plant salt tolerance anti-morning of Study on Molecular Mechanism progressively deeply and the becoming better and approaching perfection day by day of biotechnology research, among the research by genetic engineering technique cultivation salt tolerant drought-enduring plant is in and develops rapidly.

Transcription factor also claims trans-acting factor, be 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 related proteins between interaction, activate or the transcribing of suppressor gene.Transcription factor generally is made up of DNA land, transcription regulatory region (comprising active region or inhibitory area), oligomerization site and 4 functional zone of nuclear localization signal, and transcription factor interacts transcribing of regulatory gene by these functional zone and the effect of promotor cis element or with the functional zone of other transcription factor.Have a large amount of transcription factors in the plant materials, the Arabidopis thaliana that only contains 27000 genes is the encoding transcription factor (Riechmannand Ratcliffe, 2000) with regard to 5.6% gene is arranged.In recent years, isolate from higher plant in succession that a series of regulation and control are grown, metabolism and transcription factor that the factors such as arid, high salt, low temperature, hormone, cause of disease are reacted.These transcription factors can be divided into several families according to the characteristics with the DNA calmodulin binding domain CaM, and the transcription factor relevant with the plant stress resistance mainly contains 4 classes: bZIP class, WRKY class, AP2/EREBP class and MYB class.

The transcription factor relevant with the plant stress resistance can be divided into two classes according to its expression characteristic, one class is the composing type transcription factor, as barley (Hordeum vulgare) HvCBF2 (C-repeat/DRE binding factor2) (Xue, 2003), gene is all expressed under adverse circumstance still is normal condition; One class is the induction type transcription factor, and only gene just can be expressed under adverse circumstance is induced, and the transcription factor of the overwhelming majority belongs to back one class.The genetic expression of stress-inducing is regulated and control by multiple cis-acting elements in proper order.

Though every kind of transcription factor has major function separately, same stress response can have the participation of multiple transcription factor, and same transcription factor also can participate in a plurality of stress response.When plant is in adverse circumstances, the common factor by more than one of coercing that suffers causes jointly, as the osmotic stress that causes with regard to inevitably loss of moist being arranged under low temperature or the high temperature, so any one coerces expression and regulation and control that reaction may relate to a plurality of transcription factors.Drought-induced DREB class, ABRE class and MYB class transcription factor are expressed simultaneously, regulate and control the expression of multiple downstream gene and the synergy of their products.

In research, find plant adverse circumstance signal transduction, transcription factor plays a crucial role in plant reacts adverse circumstance, some transcription factors are constantly synthetic coercing under the stimulation, by starting or suppress target gene expression with signal transmission and amplification, thereby cause the change of plant physiology and biochemistry.In the research in early days, approach by the screening difference expression gene, identify a series of and arid, salt and the cold genes involved of replying, as rd (responsive to dehydration), erd (early responsive to dehydration), cor (cold-regulated), cin (cold-inducible) and lti (low-temperature induced) (Steponkus et al., 1998; Thomashow, 1998; Shinozaki and Yamaguchi-Shinozaki, 2000) etc.Promotor to these genes is carried out detailed analysis, has therefrom determined several important cis-acting elements.At present to the understanding of the molecular regulation of plant drought stress, mainly from based on big quantity research to these cis elements and corresponding transcription factor thereof.

Because transcription factor energy regulatory function expression of gene and signal transduction under adverse environmental factor that some adverse circumstances are relevant, their overexpressions in transgenic plant can activate the expression of many anti contravariance related genes, improve the expression intensity of a transcription factor, just can impel a plurality of functional genes to play a role by it, thereby make the plant resistance trait obtain comprehensive improvement (Liu Qiang, 2000).Therefore, utilizing transcription factor gene to improve crop anti-adversity research is quite paid attention to.

The discovery of transcription factor CBF/DREB and analysis

The promoter Analysis of Baker etc. (1994) by typically being subjected to the gene C OR15 of low temperature induction to one identifies an important cis-acting elements called after C-repeat (CRT), and its core sequence is CCGAC.Shinozaki etc. (1994) are by to the analysis of arid response gene RD29 promotor, have found that the cis-acting elements that a control drought stress is replied is DRE element (dehydration-responsive element).DRE contains the core sequence (TACCGACAT) of conservative 9bp, and is very similar to CRT, and they all participate in the genetic expression of arid and low temperature induction.At the deficient mutants (aba) of ABA with to ABA in insensitive mutant (abi), rd29A still can hard to bear arid and cold inducing and high strength is expressed, and illustrates that this process does not rely on ABA.Further experiment proves, DRE can make rd29A because of arid and the cold up-regulated expression of inducing not relying under the condition of ABA.

After the relevant cis-acting elements of drought stress is identified out, determine can and the research of the trans-acting factor (transcription factor) of these combination of elements in, the yeast one-hybrid technique functions keying action.Stockinger etc. (1997) take the lead in identifying the conjugated protein CBF1 of a DRE/CRT (CRT binding factor1 by yeast one-hybrid; Jaglo-Ottosen et al., 1998).In yeast, CBF1 can raise the transcription that depends on DRE/CRT as transcription factor, CBF1 comprises a conservative DNA binding domains (AP2 domain), this structural domain also is everlasting and is occurred in the albumen of EREBP (ethene-response element binding protein, ethylene-responsive element binding protein) family.Find also that simultaneously CBF1 has three homologous gene: CBF2, CBF3, CBF4.Wherein CBF1, CBF2 and CBF3 gene are arranged in series in the section (Medinaet al., 1999) of No. four chromosomal 8.7kb of Arabidopis thaliana.And CBF4 separately exists on karyomit(e) (Haake et al., 2002).

In the middle of another was independently tested, Liu etc. (1998) utilized the yeast one-hybrid technology to be separated to 5 independently cDNA clones, and encoding D RE/CRT is conjugated protein, with these albumen called afters DREBs (DRE-binding proteins).These 5 albumen are divided into two classes again, i.e. DREB1 and DREB2.Wherein, DREB1 comprises 3 protein D REB1A, DREB1B, DREB1C.And DREB1A and CBF3, DREB1B and CBF1, DREB1C and CBF2 are respectively the product of same gene.In addition, DREB2 comprises DREB2A and two albumen of DREB2B (Shinozaki et al., 2003).

(2002) such as Gao etc. (2002) and Xue have carried out the amino acid sequence homology analysis to CBF analogue and the Arabidopis thaliana CBF of barley, wheat, paddy rice, corn, rape, tobacco and Chinese sorghum (Sorghumvulgare Pers.) etc., find them except all containing conservative AP2 structural domain, also contain other conservative aminoacid sequences.Jaglo etc. (2001) find that also CBF resemblance and the Arabidopis thaliana CBF aminoacid sequence of paddy rice, wheat, rape and potato (Solanum tuberosum L.) etc. have higher homology, and action pathway is also very similar at low temperatures.Now clear, the DREB transcription factor belongs to the AP2/EREBP transcription factor family.The AP2/EREBP transcription factor is special to be present in the plant, is the general name with the closely-related class transcription factor of development of plants.There is the gene product of APETALA2, RAV, DREB, ERF (EthyleneResponsive Element Binding Factors) and 4 Unknown Function in this family, and its family member's protein sequence all contains conservative AP2 structural domain.

Though be DREB1 or DREB2 can both specificity in conjunction with the DRE/CRT element, and their start the function of transcribing all obtained confirmation in plant protoplast and Yeast systems.DREB1A/CBF3 gene and two other homologous gene can both by low temperature induction but can not be by drought-induced.DREB2A and DREB2B then can be induced by drought stress.What is interesting is that CBF4 can not but can be induced (Haake et al. by osmotic stress by cold stress-inducing, 2002), and on the homology of sequence, CBF4 belongs to the CBF/DREB1 gene family, it is to the characteristic that osmotic stress is replied, and has shown to exist on CBF/DREB1 and the DREB2 path to intersect.CBF4 gene this behavioral illustrations on function, although the gene of CBF family is homologous on sequence, they are existing a lot of overlapping but be not quite similar on function.And find also that recently in the stress response reaction, CBF2/DREB1C is as negative regulatory factor of CBF1/DREB1B and CBF3/DREB1A and work (Novillo et al., 2004).

The abduction delivering of CBF/DREB1 can activate some target gene expression, as COR (cold-regulated) genoid, the formation of the anti-adversity ability that latter's involved in plant is acquired.The transgenic plant of overexpression CBF/DREB1 have been improved the patience to cold, arid and high salt.But Liu etc. (1998) report overexpression DREB2 can not improve the resistance of Arabidopis thaliana, and the modification DREB2 after the function of this prompting CBF/DREB1 does not need to transcribe then needs post transcriptional modificaiton could exercise its function.

The clone of CBF/DREB gene

The DREB transcription factor is the transcription factor of the more resisting abiotic stress of research at present, clones dreb gene and utilizes dreb gene to carry out existing many work of transgenic research.

The Arabidopis thaliana dreb gene is a little multigene family, except that DREB1 and DREB2 gene, Sakuma etc. (2002) are according to the homology design primer of sequence, from Arabidopis thaliana DNA, amplify 3 and DREB1 homologous DREB1D, DREB1E and DREB1F with PCR method, and 6 and DREB2 homologous DREB2C------DREB2H transcription factor, wherein DREB1D, DREB1F, DREB2C, DREB2D and DREB2F are induced by high salt, DREB2E is induced by ABA, possible they participated in plant replying respectively to different adverse circumstances.The clone of the dreb gene of other plant also has a large amount of reports.

Isolate the analogue of CBF/DREB transcription factor gene from rape (Brassica campestris L.), be called BNCBF5, BNCBF7, BNCBF16 and BNCBF17 respectively, they accumulate at low temperatures, and induced gene BN28, BN115 express.The functional similarity of the function of the latter two and Arabidopis thaliana cor6.6/KIN and cor28 (Gao etc., 2002).

From paddy rice, isolated the resemblance of DREB, called after OsDREB1As, OsDREB1B, OsDREB1C, OsDREB1D and OsDREB2A.OsDREB1As and OsDREB1B are subjected to low temperature induction, and OsDREB2A is induced by arid and high salt.OsDREB1A crosses to express in transgenic arabidopsis and can cause Arabidopis thaliana DREB1A downstream target gene expression, causes plant to strengthen for the patience of arid, low temperature and high salt, and OsDREB1A and Arabidopis thaliana DREB1A functional similarity are described.But OsDREB1A more is inclined to the sequence in conjunction with GCCGAC, and the binding sequence of Arabidopis thaliana DREB1A is more prone to ACCGAC, and this may be the difference (Dubouzet etc., 2003) between monocotyledons and the dicotyledons.

Cloned coding with Arabidopis thaliana CBF/DREB1 family similar gene difference called after Hvcbf1, Hvcbf2 and Hvcbf3 from barley, they are subjected to cryogenic inducing (Xue, 2002).From the drought-induced cDNA of wheat (Triticum aestivum L.cvNorstar), clone TaDREB1, its expressed proteins comprises an AP2/EREBP structural domain, with the DNA land of the DREB of Arabidopis thaliana be primary structure (aminoacid sequence) or secondary structure all has very high similarity, gene promoter sequence has DRE elements T ACCGACAT, TaDREB1 can be by high salt, low temperature and the arid (Shen etc. that induce, 2003a, Jaglo etc., 2001).

Qin etc. (2003) utilize the method for yeast one-hybrid to separate the protein gene that obtains a coding and DRE combination of elements from the cDNA library of corn, called after maDREB1, the functional similarity of the DREB of it and Arabidopis thaliana.Found that in corn two DRE's is conjugated protein, called after DBF1 and DBF2, these two transcription factors belong to the AP2/EREBP transcription factor family, different with this family other transcription factor is, they participate in the ABA dependence approach that arid is replied, and be more prone to and ACCGAC sequence combination (Dimosthenis and Montserrat, 2002).

From Vinca (Catharanthus roseus), clone two AP2/EREBP member's gene: ORCA1 (octadecanoid responsive-catharanthus-APETALA2-domain protein) and ORCA2 gene, they mainly are subjected to induce (Menke et al., 1999) of jasmonic equal excitation.Park etc. (2001) utilize the mRNA differential display to clone from tobacco and have obtained Salt Stress-induced gene Tsi1.Shen etc. (2003b) separate the gene A hDREB1 that has obtained a coding EREBP/AP2 proteinoid from salt-tolerant plant prunella asiatica (Atriplex hortensis) the cDNA library that high salt is handled, the overexpression of the latter in tobacco significantly improved the salt resistance ability of transfer-gen plant.Zhang etc. (2004) clone cold stress-inducing gene LeDREB from tomato (Lycopersicon esculentum).Methods such as peak etc. (2005) use RACE have obtained the complete sequence of little salt mustard (Thellungiella salsuginea) CBF1 gene, with its called after TsCBF1.Huang etc. (2007) are cloned into GhDREB1L from cotton, this gene can not only be expressed by low temperature induction, also can be by arid and high salt abduction delivering.Chen etc. (2007) clone a newcomer GmDREB2 of AP2/EREBP family from soybean, its expression is subjected to arid, high salt, and low temperature and ABA induce, and cross in Arabidopis thaliana and express the drought-enduring and salt tolerance that GmDREB2 has significantly improved transfer-gen plant.

In addition, obtain Ca-DREBLP1 from capsicum (Hong etc., 2005) respectively; (D í az-Mart í n etc., 2005) obtain HaDREB2 in the Sunflower Receptacle; Isolate CmDREB1 from watermelon (Mizuno et al., 2006); From Festuca Arundinacea (Tang etc., 2005), clone FaDREB1; From pearl broomcorn millet (Agarwal etc., 2007), obtain PgDREB2A.From white poplar (Populus balsamifera) (Benedictl etc., 2006), white birch (Betula pendula) (Wellingl etc., 2006), add Ning An (Eucalyptus gunni) (Kayall etc., 2006), from blue gum (E.Maria) seeds such as (Gamboa etc., 2007), clone the CBF family gene.

The application of CBF/DREB gene in plant genetic engineering and the problem of existence

Transform with tolerance arid, low temperature and high salt inductive RD29A gene, can improve plant to arid and cryogenic patience, but can not improve the resistance (Yamaguchi-Shinozaki etc., 1994) of plant comprehensively.Compare with the proterties such as pest-resistant and disease-resistant of plant, plant wants complicated many to the resistance of abiotic stress, and the ability of plant tolerance arid, high salt and low temperature patience does not depend on the single factor, but restricted by many factors.RD29A coding and the closely similar hydrophilic albumen of lea protein, RD17 and the ERDIO second group of lea protein of encoding, KtNI is structurally very similar to the fish antifreeze protein that is rich in L-Ala with the COR6.6IKIM encoded protein, the COR15A encoded protein is distributed in chloroplast stroma and the protoplasma, can strengthen the cold tolerance of chloroplast(id) and protoplastis.From the similarity analysis of gene expression characteristics and gene product, the product of these arabidopsis gene codings is all relevant with arid, low temperature and high-salt stress patience.In the promotor of RD29A, RD17, ERDIO, KI2V1, COR6.6/KIN2 and COR15A etc., all contain the CRT/DRE element, hinting that these expression of gene are subjected to the regulation and control of transcription factor CBF/DREB.

The CBF/DREB transcription factor can be discerned the CRT/DRE element, regulates and control the relevant genetic expression of a plurality of and degeneration-resistant proterties.Therefore, can improve the resistance of plant by importing the CBF/DREB transcription factor gene.

Jaglo etc. (2001) import Arabidopis thaliana to CBF1, and the overexpression of this gene activates many anti contravariance related genes and expresses.DREB1A (CBF3) arabidopsis thaliana transformation that Liu etc. (1998) start with the CaMV 35S promoter, transfer-gen plant DREB1A composing type overexpression is comprehensively improved the resistance of plant.Gilmour etc. (2000) report CBF3 can induce cold-resistant expression of gene, increases the content of soluble substance such as sucrose, raffinose, fructose, glucose and proline(Pro), thereby improves winter resistance.Find that simultaneously its cross expressing in the transgenic arabidopsis plant not only makes the low temperature resistant ability of plant improve, and drought resisting and salt resistance also obtain improvement.Hsieh etc. (2002) change the Arabidopis thaliana CBF1 that the CaMV35S promotor starts over to tomato, find that transfer-gen plant has the signal transduction process that occurs in the Arabidopis thaliana that is analogous to, the cell free radical reduces under stress conditions, proline content raises, stomatal movement is adjusted, and drought resistance of plant and lower temperature resistance all obviously strengthen.Kang etc. (2002) discovery CaMV35S-CBF3 transfer-gen plant pore under drought stress is closed rapidly, and rising the minimizing tide over adverse circumstance smoothly, and wild-type plant has only 16% can survive under identical adverse environmental factor.The AhDREB1 that Shen etc. (2003b) will clone from prunella asiatica imports tobacco, and the salt resistance of transfer-gen plant obviously improves.Expressing DREB class homologous gene can improve the resistance (Dubouzet etc., 2003, Qin etc., 2004) of transfer-gen plant to adverse circumstance excessively in Arabidopis thaliana and corn.Chen etc. (2007) cross soybean GmDREB2 gene and express in Arabidopis thaliana, significantly improved the drought-enduring and salt tolerance of transfer-gen plant.

The CBF/DREB transcription factor gene is imported plant, can not improve the resistance of transgenic plant.Arabidopis thaliana DREB2A is induced by arid and high salt, and regulation and control downstream CRT/DRE genetic expression.Liu etc. (1998) DREB2A will be connected with composing type strong promoter CaMV35S and import Arabidopis thaliana, filter out the transgenic line that normal condition descended to express DREB2A.Northern hybridization shows, although DREB2A crosses expression under the normal condition in transgenic line, can not regulate and control downstream CRT/DRE genetic expression, infers that the product of transgenosis DREB2A and the DREB2A transcription factor that possesses physiologically active are distinguishing.Be converted into activated DREB2A transcription factor from mRNA, may also need play certain modification (as phosphorylation).The transgenic line phenotype of overexpression DREB2A changes little, and resistance improves not obvious.Dubouzet etc. (2003) utilize CaMV35S to drive the OsDREB24 arabidopsis thaliana transformation, have also obtained same conclusion.

The mechanism of action of DREB transcription factor is comparatively complicated, the function of OsDREB1A and Arabidopis thaliana DREB1A is identical in the transgenic arabidopsis plant, DREB transcription factor energy specific combination DRE (A/GC2CGAC) sequence, the expression of activation target gene, but Arabidopis thaliana DREB1A can be in conjunction with the core sequence of DRE promptly: ACCGAC and GCCGAC sequence, and OsDREB1A can only specific combination GCCGAC sequence, and can not be in conjunction with ACCGAC sequence (Dubouzet etc., 2003).The DREB transcription factor that has has very high sequence similarity as DREB1A and DREB1D, and DREB1A can be induced by the low temperature adverse circumstance, and DREB1D can not be expressed (Sakuma etc., 2002) by low temperature induction.

The DREB transcription factor gene is crossed to express in transgenic plant and is often caused the plant dwarfing, as the Arabidopis thaliana plant that changes 35S:DREB1A and commentaries on classics 35S:OsDREB1A shows the form variation under the normal growth condition, change the 35S:DREB1Aa plant and show serious dwarfing, growth retardation, supposition is to cross expression because the DREB transcription factor causes target gene, cause the excessive synthetic of associated protein, though make transgenic plant under the normal growth condition, have higher adverse circumstance endurance, but cause and make plant strain growth slow (Liu et al., 2003).Though the transfer-gen plant dwarfism can overcome (Kasuga etc., 1999) by using stress inducibility promoter, not expected result often occurs.For example, Hsieh (2002) promptly uses ABRC1 of barley and Arabidopis thaliana COR15A adverse circumstance inductive promotor to replace 35S promoter, and transgenic Fructus Lycopersici esculenti cessation of growth cessation phenomenon does not change yet, just can restore normal growth after only spraying Plant hormones regulators,gibberellins.

The drought-enduring genetically engineered progress of corn salt tolerant

The genetically engineered purpose of corn is to obtain transgenic plant by importing useful foreign gene, is used for its genetic improvement.The corn gene engineering has mainly obtained significant achievement at aspects such as pest-resistant (anti insect gene that changes over to mainly is the Bt toxoprotein gene), antiweed, disease-resistant (viral diseases or resistance to fungal disease) and drought resistings in corn, transgenic insect-resistant corn and antiweed corn in America big area promote also popularizing planting in Europe.The corn gene engineering of resisting abiotic stress and quality-improving also obtains remarkable progress.Summarize the progress of the drought-enduring resistant gene of salt engineering of corn below.

Liu Yan etc. (1998) change intestinal bacteria 6-phosphoric acid sorbitol dehydrogenase gene gutD over to corn, detect the synthetic and accumulation of sorbyl alcohol in transgenic corns, and salt tolerance is apparently higher than contrast.What clean grade of strontium (1999) changes betaine aldehyde dehydrogenase gene over to corn, and transfer-gen plant has obtained salt tolerance.Jeanneau etc. (2002) cross expression C4-PEPC gene in corn, improved the drought-resistant maize ability.For example significantly improved the salt tolerance of corn.Quan etc. (2004) change the trimethyl-glycine synthase gene betA of bacterium over to corn, and drought tolerance experiment and some Physiology and biochemistries detect and show that the drought tolerance of transgenic corns obviously improves.The NPK1 protein kinase gene that Shou etc. (2004) will have the MAPKKK characteristic changes corn over to, and the milpa drought tolerance is improved.Yin etc. (2004) will be from the vacuole skin Na of Arabidopis thaliana ⁺/ H ⁺Antiport protein gene NXH1 changes corn over to, has improved the salt tolerance of plant.Recently, Qin etc. (2007) report is crossed expression with the ZmDREB2A gene that clone in the corn obtains in corn, improved drought tolerance and the thermotolerance of plant.

The drought-enduring genetically engineered progress of wheat salt tolerance

The Drought-resistance in Wheat salt tolerant breeding is mainly taked following several approach: conventional breeding, distant hybirdization, molecular mark and genetic engineering breeding etc.For want of high germplasm anti-and that utilize easily, and very long breeding cycle, Drought-resistance in Wheat (salt) conventional breeding is made slow progress, and far can not satisfy the demand of production.Be accompanied by the clone of the development of plant genetic engineering and many drought resistings, salt-resistant related gene, it is more and more to utilize genetic engineering means to improve the research of crop drought resistance, salt tolerance.1991, Vasil etc. obtained the callus that transforms with particle gun bombardment wheat cells, and the first routine wheat transforms successful report and is born at this point.Vasil in 1993 etc. directly bombard rataria with particle bombardment again, have obtained the transgenic wheat strain system that can educate.In the same year, Weeks etc. have also obtained the commentaries on classics bar dna triticum plant that can educate by particle gun bombardment WHEAT CALLUS.Obtained in the report of wheat transgenic plant before 2002, particle bombardment accounts for about 90%, and additive method only accounts for 10% (Wang et al., 2002).The foundation of particle gun transformation system, promoted the development that wheat genetic transforms, but this conversion process also is subjected to all multifactor impacts, as poor repeatability, cost height, conversion back mosaic is many, the polygene copy is integrated, the gene silencing probability is sweet, foreign gene is lost in the offspring easily etc. its application is restricted.1997, Cheng etc. utilize the success of agrobacterium-mediated transformation transformed wheat, in recent years, agrobacterium-mediated transformation is higher because of its repeatability, transformation frequency, the low copy of transgenosis inserts and transfer-gen plant waits advantage to become the mainstream technology that wheat genetic transforms stably and rapidly.Sivamani etc. (2000a) import wheat with barley HVA1 gene (member of lea protein gene family 3), under the situation of soil lack of water, improved plant strain growth, transfer-gen plant offspring's field experiment draws, some biomass and contrasts of rate ratio non-transgenic of expressing the transgenic line of HVA1 gene significantly improve (Bahieldin et al., 2005).Sawahel and Hasson (2002) import wheat with the proline(Pro) synthase gene and have obtained the transfer-gen plant that salt tolerance significantly improves.Abebe etc. (2003) import wheat with the N.F,USP MANNITOL synthase gene and have obtained the salt tolerant of synthetic N.F,USP MANNITOL and the wheat that water-fast branch is coerced.These transfer-gen plants have accumulated the osmotic protection material of different levels, and its salt tolerance shows raising in various degree.Xue etc. (2004) are by the agrobacterium-mediated transformation transformed calli, change Arabidopis thaliana vacuole skin Na+/H+Antiport gene over to wheat, discovery is under condition of salt stress, accumulated higher K+ and lower Na+ in the blade of transfer-gen plant than non-transgenic plant, 100-grain weight and the contrast of rate ratio non-transgenic that the saltings test also draws transgenic wheat strain system significantly improve.

Summary of the invention

At present present Research, the purpose of this invention is to provide the application of a kind of TsCBF1 of commentaries on classics gene alteration corn, wheat resistance.

The application method of commentaries on classics TsCBF1 gene alteration corn of the present invention, wheat resistance is, the TsCBF1 gene that to clone from the salt mustard is recombinated with just form in the plant expression vector, adopt transgenic technology that recombination is imported vegetable cell, obtain transfer-gen plant; By detecting transgene expression and plant being carried out resistance identify, therefrom filter out transfer-gen plant and offspring thereof that resistance obviously improves or reduces, create the new germ plasm and the new variety that in corn, wheat breeding, have application prospect.

Wherein, described TsCBF1 gene has cDNA form or genomic gene form from halophytes salt mustard, and its encoding sequence is built into fusion gene with just form, and changes in the plant materials.

Wherein, the described TsCBF1 gene that is used for gene fusion construct can be the full length sequence of gene, also can be partial sequence, also can be the deoxy polynucleotide according to the partial sequence synthetic.

Wherein, described application method is the transfer-gen plant that improves by the resistance of changeing the TsCBF1 gene or obtain according to the polynucleotide sequence that the TsCBF1 gene order designs.

Wherein, described application method can also be by changeing transfer-gen plant and the offspring that the acquisition of TsCBF1 gene improves stress resistance.

The said TsCBF1 gene clone of the present invention is in the halophytes salt mustard of different ecological type, and sequence has high similarity each other.This gene is generally with cDNA form or genomic gene form clone, recombinates in the plant expression vector with forward.In plant expression vector, this genetic transcription can be started by former promotor, or by starting from the special promotor of the plant of other gene, is 3 ' tail district of plant gene behind the coding region.The fusion gene that produces should be able to be in vegetable cell effective expression.

The said gene forward of the present invention form is inserted the complete encoder block that is meant TsCBF1 gene in the correct connection in the promoter sequence back of plant, and promptly first codon is near promotor, and transcription product is just mRNA.Require transcription product basicly stable in cell.Be not limited by is constitutive promoter or inducible promoter.

The said stress resistance of plant of the present invention is meant anti-(resist) drought, anti-(resisting) salt, resistance to cold, winter resistance that plant shows, resists characteristic and combinations thereof such as (anti-) radiativity, disease resistance, insect-resistance, antiweed on whole strain, organ and/or cell levels.This anti-(anti-) property can show some etap or the time of infertility on the plant level, can weigh or estimate by multiple index.

Compare with changing single degeneration-resistant relevant functional gene over to, change the expression that an adverse circumstance inductive transcription factor can be regulated and control a plurality of and degeneration-resistant related gene over to.This invention clones the TsCBF1 gene from halophytes salt mustard, adopt agrobacterium-mediated transformation or particle gun blast technique to change corn over to, wheat, its offspring plant is carried out Molecular Detection identifies and relevant physiological index determining with salt tolerance of drought, whether checking TsCBF1 has DREB transcription factor family member's function, can improve drought tolerance and the salt tolerance of plant with the expression in transfer-gen plant, and cross the transgenic corn plant express the TsCBF1 gene and whether when drought-enduring salt tolerance comparison obviously improves according to plant, grow normally, dwarfism does not appear.

This invention has filtered out transgenic corns, the wheat strain system of good stress resistance, for certain basis has been established in the work of transgenic culturing corn, the degeneration-resistant new variety of wheat.The transfer-gen plant that utilize to obtain, we also can study plant to the signal transduction network of coercing reaction and the variation of genetic expression, for the drought resistance and salt tolerance mechanism that discloses plant can provide suitable material.

The essential characteristic of TsCBF1 sequence

CBF1 gene number of registration is gi:41351816.Find that by ORF finder on-line analysis among the NCBI 1 to 201 is 5 ' non-translational region of this gene in 1040 Nucleotide that we checked order; 202 to 852 is protein coding region, 216 amino acid of encoding altogether; 853 to 1040 is 3 ' non-translational region and part poly A+ tail.This genes encoding one contains 216 amino acid whose protein sequences, by the Blast compare of analysis, and the CBF1 in itself and the Arabidopis thaliana, CBF2 and CBF3 have 84%, 81% and 82% similarity respectively.From 60 amino acid of the 49th to 109 is an AP structural domain.And carry out cluster analysis by Clustal W, and showing clone's the TsCBF1 gene and the CBF1 of Arabidopis thaliana, CBF2 and CBF3 gene have very near evolutionary relationship.

With the speculating acid sequence of TsCBF1 gene with from the AtCBF1 of Arabidopis thaliana, AtCBF2, AtCBF3, AtDREB2A, AtDREB2B and carry out cluster analysis (analytical results shows through software Treeview) by Clustal W from the CBF1 of paddy rice and DREB1B shows that the evolutionary relationship of AtCBF1 of TsCBF1 gene and Arabidopis thaliana is nearest.

The reorganization of TsCBF1 gene

Adopt conventional molecule clone technology that the full length sequence of TsCBF1 is recombined into the recombinant plasmid that obtains the forward insertion in the plant expression vectors such as plant binary expression vector pROK2 or pCam series.Can be according to recipient plant and required TsCBF1 expression of gene intensity and adjustable degree, fusion gene can be selected the composition promotor that is suitable for single, double cotyledon plant and different unicellular lower eukaryotes respectively for use, can select specific promotor of plant organ or etap, or the inducible promoter that special signal (comprising adverse circumstance signal, chemical signal, physical signalling etc.) is reacted etc.Fusion gene can be selected different 3 ' tails district, also can insert enhancer sequence, also can insert different introns in the coding region.Recombinant plasmid can import propagation in intestinal bacteria or the Agrobacterium respectively or/and preserve.

Transform plant with the plant expression vector that contains the TsCBF1 gene

Select for use different transgenic methods to obtain transgenic plant according to transgenic acceptor.

Method for transformation commonly used has 3 classes: 1) direct conversion method, promptly extract recombinant plasmid dna, and adopt particle gun blast technique, polyoxyethylene glycol revulsion, electro fusion method, silicon-carbon fibre method etc. directly with the plasmid DNA transfered cell.2) agriculture bacillus mediated heredity method.3) planting is conversion method, comprises pollen tube passage method, ovary injection etc.In addition, also has the Transformation Program that the inhomogeneity method is used in combination, as particle gun blast technique and Agrobacterium are used in combination to improve transformation efficiency.Below be that material adopts the particle gun blast technique and is that material adopts agrobacterium-mediated transformation to carry out genetic transformation with wheat aseptic seedling stem apex with the corn inbred line embryo callus.

A. corn inbred line embryo callus genetic transformation

Corn (Zea mays L.) self-mating system plant behind pollination self 9-12 days, get and in 70% alcohol, soak 5min after fruit ear is peelled off bract, the rataria of the about 1.0-1.2mm size of picking is inoculated on the inducing culture under the aseptic condition, cultivate and obtain crisp, flaxen II type callus 4-6 week, the every 10-15 of subculture medium days succeeding transfer culture once then.Resulting II type callus is as the acceptor material of genetic transformation.

Adopt ordinary method to prepare the particle gun bullet.Promptly take by weighing the bronze of 1.0 μ m sizes, after 70% washing with alcohol, left standstill 15 minutes, centrifugal removal supernatant liquor; Thoroughly clean 3 times with sterilized water, 50% sterile glycerol (final concentration is the little bullet of 60mg/ml) is stored standby then again.Vortex was broken the bronze aggegation in 5 minutes during use, added 5 μ l plasmid DNA (1 μ g/ μ l), 50 μ l 12.5M CaCl2,20 μ l 0.1M spermidines successively, application of sample limit, limit vortex.Continued vortex then 2～3 minutes, left standstill 1 minute.Centrifugal abandon supernatant liquor after, add 70% ethanol and leave standstill.The centrifugal then supernatant liquor of abandoning uses dehydrated alcohol resuspended again, and sampling is added on little missile-borne body.Little bullet consumption is every bullet 0.5mg.

In the culture dish of diameter 9cm, pour the thick substratum of 0.4cm into, then the callus high-density is put into the every ware bombardment of culture dish once.The bombardment parameter is got: the distance that can split disk and carrier is 2.5cm, carrier and stop that the distance of net is 0.8cm, little bullet flying distance 6--9cm.Other parameter is pressed the working instructions value.Bombardment back material recovered to cultivate 3 days darkling, then material was changed in the new substratum of components unchanged and cultivated for 3 weeks, and the target gene that changes over to is given full expression to.Material is changed over to the enterprising row filter of substratum that is added with selective agent (as 1-5ppm weedicide chlorsulfuron).The step sizing three generations, 15 days per generations.Eliminate the tissue block of browning death during subculture.Change on the substratum that does not add selective agent after 1 generation of cultivation is recovered in illumination 16 hours/world through the resistant calli of screening, change over to and break up seedling on the division culture medium.The seedling that callus produces is taken root in root media, moves into flowerpot after strong sprout, grows to about 10cm and plants big Tanaka, self-fertility when high.Transfer-gen plant adopts the screening of PCR detection method, Southern blotting checking.Produce pure lines by number for Molecular Detection and self-fertility, selecting to obtain the transgenosis elite clone by resistance evaluation and field.

B. wheat aseptic seedling stem apex genetic transformation

Wheat seed soaks 7-10min with 0.1% mercuric chloride solution again with 70% alcohol immersion 1-3min, then with sterilized water washing 5 times.Constantly rock seed during the sterilization, thorough to guarantee surface sterilization.The sterilization seed is placed on to fill with sterilized water and soaks in the Cans of filter paper, is placed under the dark condition (24 ± 2 ℃) 1-2 days after sealing.Treat seed sprout grow radicle and plumule after, place it on the M1 substratum in dark condition (24 ± 2 ℃) and continue down to cultivate.When treating that plumule is stretched to 2-5cm, peel off coleoptile and 1-2 sheet spire, expose the point that sprouts with scalper, again with scalper from the middle part vertical cut wound shoot tip meristem.The aseptic seedling of cut wound shoot tip meristem (germinating seed) is used for Agrobacterium-mediated Transformation.

To have agrobacterium tumefaciens lba4404 28 ℃ of concussion cultivations down in additional antibiotic YEP substratum of purpose plasmid, concussion speed is 180rpm, makes bacterium be in logarithmic phase.Under 3000rpm centrifugal 5 minutes then, abandon supernatant.(acetosyringone, MMS liquid nutrient medium AS) is resuspended with adding the 100mg/l Syringylethanone with thalline again.Then bacterium liquid is poured in the culture dish of diameter 15cm, the inclination culture dish makes to be immersed in the bacterium liquid by the aseptic seedling stem end of cut wound, vacuumizes (0.05MPa) and infects 7～10min.

Stem apex after the dip-dye blots with aseptic filter paper, the seedling primary root is prescinded back insertion wheat germination substratum (M1 substratum) went up dark culturing 3 days.Culture temperature is 22-24 ℃.Aseptic seedling after will cultivating altogether after 3 days is transplanted in the flowerpot of upper strata vermiculite lower soil, and covers the plant top with loose moistening vermiculite.Allow plant under natural lighting, grow then, warm 22-28 ℃ of day, temperature 15-21 ℃ of night.Water the inorganic salt solution of 1/2MS substratum every other day.When being transplanted to transformed plant length in the vermiculite to tri-leaf period, the weedicide aqueous solution that evenly sprays suitable concn screens.Sprinkling is fallen drop with plant leaf and is advisable.Spray the back plant and be placed on the greenhouse growth, day temperature 15-20 ℃, about 10 ℃ of nocturnal temperatures about day illumination 12h, watered 1 time in per 3 days.Statistics survival rate of plant when moving into the field.Field plant is according to Routine Management measure management, the vernalization of surviving the winter under field conditions (factors).Period of seedling establishment is got leaf DNA and is carried out the PCR detection.

The conversion seedling that with bar is the selection markers gene is selective agent with weedicide Finale (trade(brand)name), and its activeconstituents is broad spectrum weeding agent glufosinate ammonium (ammonium salt of careless fourth phosphine), and concentration is 5.78% (w/v).Working concentration is to add 3ml Finale, i.e. the 0.3%Finale aqueous solution in every liter of tap water.After the sprinkling not the transgenosis adjoining tree begin death after 7 days, mortality ratio is more than 90% after 14 days; Transfer-gen plant shows resistance, is transplanted to the field after 15 days.Transformed plant (T0) selfing of antiweed screening produces T1 for seed, and the latter sprouts in flowerpot, and seedling is long to 3-4 leaf spraying herbicide aqueous solution during the phase, and weedicide concentration and plant strain growth condition are with T0 generation.The survival plant grows to draw materials behind 3 young leaves and carries out Molecular Detection.PCR detects positive plant and partly is transplanted into flowerpot, every strain one basin; Part is transplanted to the big Tanaka with isolation facility.Manage according to the Routine Management measure, plant survives the winter naturally, gets leaf DNA after turning green and carries out Molecular Detection.Individual plant is isolated pollination self and is got T2 for seed.At field seeding, survive the winter naturally by seedling for seed for part T2, gets young leaves after turning green and carry out PCR detection, Southern hybridization and sxemiquantitative RT-PCR checking.The seed of transgenosis homozygous lines is used for salt tolerant, drought-enduring physiological index determining and saltings planting experiment.

The detection of salt tolerance drought and the utilization of transgenic corn plant

Adopt PCR method to detect the corn positive plant that contains justice and antisense TsCBF1 gene respectively, bagging selfing or sisters hand over solid.The planting seed of results in the sand basin, the pouring 0.8%NaCl aqueous solution, and be contrast with transgenosis self-mating system seed not.The not homophyletic of transfer-gen plant ties up to salt solution pouring and shows different salt tolerances down, and the become a full member strain system of adopted TsCBF1 gene of majority shows the comparison illumination and shows the salt tolerance that improves.In the strain of the adopted TsCBF1 gene of becoming a full member system, 5 leaf phase surviving rates of strains more than half system are more than 70%, and contrast self-mating system plant emerge after very fast death, the surviving rate of 5 leaf phases is less than 20%, and plant seriously is injured.The Northern hybridization analysis shows that the TsCBF1 gene cross to express in these transfer-gen plants, at transfer-gen plant not with do not contain in the empty carrier transformed plant of goal gene, TsCBF1 expression of gene level does not have significant difference.The transfer-gen plant bagging selfing of selecting the salt tolerance excellence is isozygotied, and carries out combining ability test, selects combining ability identical with the donor self-mating system or the transgenosis self-mating system of raising is arranged, and is used for the haloduric corn culturing cenospecies.

With the seed that changes the corn seed of TsCBF1 justice gene and not genetically modified contrast self-mating system broadcast flowerpot in, carry out drought stress in seedling stage (3～7 leaf phase), female tassel growth period (9～13 leaf phase), the pollination phase of blooming respectively and handle, detect arid and handle the difference of influence, damaged membrane degree, plant single plant yield and other economic characters and the physical signs of corn growth etc.Simultaneously, the seed of transgenic line and contrast is sprouted in being added with 18%PEG solution, selected the high transgenic line of germination rate.Comprehensive many-sided test result, the corn comparison of changeing TsCBF1 justice gene shows the drought tolerance of obvious raising according to plant.The transfer-gen plant bagging selfing of selecting the drought tolerance excellence is isozygotied, and carries out combining ability test, and selection combining ability is identical with the donor self-mating system or have the transgenosis self-mating system of raising to be used for the seed selection of the corn seed of single cross.

Detection of salt tolerance drought and the utilization of transgenic wheat plant

With the wild-type plant is contrast, selects 10-25 strain system to carry out physiology and measures.In seed germination test, be sowed at (diameter 10cm, high 15cm) in the sand basin of the same size after seed dried, respectively pouring contain different concns NaCl (0,100mM, 200mM, aqueous solution 300mM), each strain is 300 seeds of every concentration kind, 25 in every basin.Be consistent between each basin of irrigation amount, observe the reguarity of seed sprouting situation and seedling every day, and avoid drenching with rain.The final seedling rate of statistics seed after 20 days.The standard of sprouting is the sand bed that coleoptile exposes covering.

According to the mensuration of seed bud ratio, select the salt concn during 200mM NaCl salts solution is handled as salt stress.Choose transgenic line and wild-type plant respectively and carry out salt tolerance physiology mensuration.With each strain is planting seed (diameter 10cm, high 15cm) in sand basin of the same size, 20 of every basin kinds, and each strain system kind of 8 basin waters the Hoagland nutritive medium every day, and plant is long to the final singling during phase of 3 leaves, stays seedling 15 strains of big or small growing way unanimity in each basin.Plant is long during the phase, to be divided into two groups with husky basin by each strain system to 5 leaves at random.Wherein use the Hoagland nutritive medium pouring that contains 200mM NaCl for one group, continuous pouring 10 days, another group continues to water with the Hoagland nutritive medium, as the untreated control group.In the salt stress treating processes, measure GB content and other physical signs of handling the plant and the plant of being untreated, comprise photosynthesis index, ion content, RWC, chlorophyll content, cytosol gesture, soluble sugar and proline content, cytolemma ion seepage, mda content and biomass.The Seedling Salt-tolerance determination test carries out in the controlled environment chamber, day temperature and night temperature be respectively 22-25 ℃ and 15-20 ℃, illumination every day 12 hours, light intensity 500 μ mol m ^-2s ^-1Select the strong transgenic line of salt tolerance according to test-results.

Sterile petri dish is spread double-deck filter paper, filter paper is used the aseptic MS inorganic salt solution (osmotic potential :-0.55MPa) infiltration that contains 20% (W/V) PEG-6000 respectively again.Get each 300 surface sterilization of seed of transgenic line and contrast, place then on the double-deck filter paper and sprout in 23 ℃ of dark.Added up the rudiment number of seed from second day every day, it is long and maximum root is long to measure bud when germination rate no longer changes by the time.Every strain is that 30 of every processing picked at random are sprouted seedling and measured and compare, and calculating root, bud length ratio value.With the planting seed of transgenic line and wild-type plant in the flowerpot that same loam is housed, carrying out drought stress handles, measure the physical signs of handling the plant and the plant of being untreated, comprise photosynthesis index, blade relative water content (RWC), chlorophyll content, cytosol gesture, soluble sugar and proline content, cytolemma ion seepage, mda content, superoxide dismutase (SOD) and peroxidase (POD) activity etc.In addition, the seed of all strain systems is broadcast respectively in onesize husky basin, the Hoagland nutritive medium is watered in 3 leaf phase final singlings every day.Seedling 5 leaves are disposable during the phase to water no longer pouring after the sufficient nutritive medium, until the blades of all plant permanent wilting takes place, and recovers then to water, and the phenotype of observing each strain and be plant changes.Composite score is selected the strong transgenic line of salt tolerance and is used for wheat breeding.

Embodiment

Embodiment 1: change the TsCBF1 gene and create corn salt tolerant self-mating system and application

1) foundation of receptor system is material with key selfing used in China's agriculture production, as Zheng 58, tuck in 478, tuck in 515 etc. selfed seed.Isolated culture induces stem apex to produce the sprout tuber that grows thickly, and is that acceptor carries out genetic transformation with the sprout tuber that grows thickly.Used substratum has:

Seed germination substratum: KNO ₃1900mg/l, NH4NO ₃1650mg/l, CaCl ₂2H ₂O 440mg/l, MgSO ₄7H ₂O 370mg/l, KH ₂PO ₄H ₂O 170mg/l, FeSO ₄7H ₂O 27.8mg/l, ZnSO ₄7H ₂O10mg/l, MnSO ₄4H ₂O 22.3mg/l, H ₃BO ₃10mg/l, KI 0.83mg/l, Na ₂MoO ₄2H ₂O 0.5mg/l, CuSO ₄5H ₂O 0.025mg/l, CoCl ₂6H ₂O 0.025mg/l, vitamin 10.0mg/l, pyridoxine hydrochloride 1.0mg/l, nicotinic acid 1.0mg/l, glycine 2.0mg/l, inositol 100.0mg/l, vitamin H 0.05mg/l, casein hydrolysate 500mg/l, sucrose 30g/l, agar powder 7g/l, pH 5.8～6.0.Be used for seed germination.Liquid nutrient medium then removes agar powder.

The A substratum: the seed germination substratum adds 6-BA 4.5～9.0 μ mol/l and 2, and 4-D 1.0～3.0 μ mol/l are used to induce isolated culture bud point to produce grow thickly the bud tissue block and the bud tissue block succeeding transfer culture of growing thickly.

B substratum: additional 6-BA 4.5 μ mol/l of seed germination substratum and IBA (indolebutyric acid) 1.8 μ mol/l, the bud tissue block that is used to grow thickly differentiation seedling.

Become the seedling substratum: additional 6-BA 2.25 μ mol/l of seed germination substratum and IBA 3.6 μ mol/l, the budlet that is used to grow thickly develops into seedling.

Root media: the seed germination substratum adds IBA 2.8～3.6 μ mol/l, is used for the little seedling rooting of unrooted.

Minimum medium and plant growth regulating substance pressure sterilizing; Microbiotic and weedicide isoreactivity composition filtration sterilization add behind medium sterilization.

Seed sterilization and sprouting: corn seed with 0.1% mercury chloride immersion 10--15 minute, washs 3--5 time with sterilized water more then with 70% alcohol immersion 10 minutes.Sterilization back seed is placed in the aseptic triangular flask to be sprouted, and puts into a small amount of (30---40 milliliter/250 milliliter triangular flask) sterilized water in the bottle, is placed under the dark condition (23---30 ℃) 1--2 days after sealing.(showing money or valuables one carries unintentionally) back seed of sprouting is placed on the minimum medium and continues to sprout down in dark condition.

Stem tip culture and grow thickly that the bud tissue block is induced, subculture, differentiation: when the plumule elongation of germinating seed ends 3---5 centimetre, peel off coleoptile and spire, cut the epicotyl and the stem apex that are about 5 millimeter, be inoculated into (24--27 ℃) cultivation under dark on the A substratum, and in time cut the plumular axis of elongation and peel off spire.Cultivate after 6--10 days, stem apex begins the irregular growth of expanding, and occurs several wartys or digitation on the meristematic tissue that expands.After 20 days, begin to form indefinite bud and embryoid on the surface of warty or digitation.General per 4 all succeeding transfer culture once.In succeeding transfer culture, budlet is on the high side if the bud tissue block of growing thickly is grown thickly, and 2,4-D concentration is got 3.0 μ mol/l; Bud tissue block callusization is heavier if grow thickly, though a large amount of meristematic cell groups is arranged, indefinite bud seldom appears in the surface, can be with 2, and 4-D concentration is reduced to 1.0 μ moll, continues to cultivate then to produce a large amount of wartys or digitation again.The bud tissue block of growing thickly on the A substratum, minority has the generation of adventive root.Exist equally with spire, adventive root also influences the generation of expanding growth and embryoid and the budlet that grows thickly of tissue block, need remove early.The bud tissue block of growing thickly was transferred on the B substratum after 2--3 days, and color and luster is flavescence gradually, and quality is more pliable and tougher, and microspike appearred in the rear surface in 5--6 days.Observe visible each phase embryoid and indefinite bud under the scanning electron microscope.Embryoid and indefinite bud are grown rapidly, form the budlet that grows thickly on the tissue block surface.

2) with the bud tissue block of growing thickly be the conversion and the plant regeneration of acceptor

(every liter of substratum contains: tryptone 10g at additional antibiotic LB substratum will to have the agrobacterium tumefaciens (as AGL0 and LBA4404) of binary vector (the Mini--Ti plasmid has selective agent resistant gene and TsCBF1 justice gene), yeast extract 5g, NaCl 10g, pH 7.0, pressure sterilizing) 28 ℃ of concussion cultivations down in, concussion speed be 110rpm (rev/min), make bacterium be in logarithmic phase.Under 3000rpm centrifugal 10 minutes then, abandon supernatant liquor.Thalline washs centrifugal again collection with the liquid seeds germination medium (be that the seed germination medium component reduces by half, remove agar powder) of 1/2 concentration.(acetosyringone, As) the liquid inducing clumping bud substratum of 100 μ mol/l suspends, and dilutes 5--20 and doubly is used for transforming with adding Syringylethanone with thalline again.

Getting the bud tissue block of growing thickly of cultivating 13--20 days behind the subculture is transgene receptor.Transform with agrobacterium-mediated transformation, transform the back material and recover darkling to cultivate.Grow thickly budlet or the bud tissue block of growing thickly of agroinfection were cultivated 7--12 days bacteria growing inhibiting in dark on the substratum that is added with cephamycin (Cefotaxime) 250mg/l or Pyocianil (Carb) 500mg/l.Grow thickly budlet or the bud tissue block of growing thickly of recover cultivating after back or the antibacterial cultivation are being added with step sizing 3-4 generation on the substratum of selective agent, obtain transgenic cell and budlet.The exhausted big bud tissue block of growing thickly of counting is dead gradually in screening and culturing.Remove 2 with what the tissue block of survival was transferred to no selective agent, recover to cultivate generation budlet in back on the A substratum of 4-D.

Budlet is placed on into irradiation growth down on the seedling substratum, light intensity 2000-3000lx, illumination 14-15 hour/day.Seedling length changes in the root media during to 3-4 sheet leaf takes root.Cultivated about 15 days, about 40% seedling produces new root.For the seedling of not taking root, its base portion of cut wound is transferred on the new root media and is cultivated, and most of plant produce root system after 10 days.Take root behind the seedling flush away substratum, being transplanted to the vermiculite is to grow in the flowerpot of cultivation medium.Plant grows under natural lighting, day warm 22--28 ℃, at temperature 15--21 ℃ of night, waters the inorganic salt composition of the seed germination substratum of 1/2 concentration every other day.Grow about 2 weeks, nursery transplant produces flourishing root system, is colonizated in the field then.

3) resistance of transfer-gen plant detects and selects and utilizes

The blade of getting the transplant survival plant carries out molecular Biological Detection and determines transfer-gen plant.With transfer-gen plant bagging self-fertility.Get planting seed in the sand basin, carried out continuous pouring 30 days with the 0.7%NaCl aqueous solution, the seedling of will surviving is transplanted to the land for growing field crops, treats the plant back bagging self-fertility of growing up.Its next generation is proceeded molecular biology identification and salt tolerance detect, and the bagging selfing isozygotys, obtained the salt tolerant self-mating system.This self-mating system can be used for preparing corn salt tolerant cross-fertilize seed.

Embodiment 2: change TsCBF1 justice gene and create the good breeding for stress tolerance material of wheat

1. the wheat aseptic seedling obtains

The seed of wheat improved seeds with 70% alcohol immersion 1-3 minute, soaked 8-12 minute with 0.1% mercury chloride again, then with sterilized water washing 3-5 time.Constantly rock seed during the sterilization, thorough to guarantee surface sterilization.Sterilization back seed is placed in the aseptic triangular flask to be sprouted, and puts into a small amount of sterilized water in the bottle in dark condition (20-25 ℃) 1-2 days down.Treat that seed sprouts after (showing money or valuables one carries unintentionally), place it on the modified MS medium and sprout down in dark condition.When treating that the plumule elongation ends 3-4 centimetre, peel off coleoptile and 2-3 sheet spire, emergent stem pointed tip growing tip.

2. Agrobacterium is cultivated and activation

To have agrobacterium tumefaciens (AGL1 or LBA4404) 28 ℃ of concussion cultivations down in additional antibiotic LB substratum of binary vector (the Mini-Ti plasmid has herbicide resistance gene bar and TsCBF1 gene), concussion speed is 110r/min, makes bacterium be in logarithmic phase.Under 3000r/min centrifugal 10 minutes then, abandon supernatant liquor.Thalline washs with the 1/2MS liquid nutrient medium, centrifugal collection.Thalline is suspended with the 1/2MS liquid nutrient medium that adds the 100mg/l Syringylethanone, dilution 5-20 doubly is used for transforming again.

3. the wheat aseptic seedling transforms

(1) bacterium liquid is poured in the culture dish of 4.5 cm diameters, the inclination culture dish is immersed in the bacterium liquid, 0.5 * 10 the aseptic seedling of exposing the stem apex growing tip ⁵The Pa normal atmosphere was handled 3-6 minute down.

(2) the bud point after the dip-dye blots with aseptic filter paper, and germinating seed is placed on the modified MS medium of adding the 200mg/l glutamine and cultivated 2-3 days in dark, and culture temperature is 22-24 ℃.Then aseptic seedling is placed under the scattered light and cultivated 2 days.

(3) aseptic seedling after the irradiation cultivation is transplanted in the flowerpot that is covered with upper strata vermiculite lower floor loam, and covers the plant top with vermiculite.Allow plant under natural lighting, grow then, warm 22-28 ℃ of day, at temperature 15-21 ℃ of night, water 1/2 modified MS medium inorganic salt every other day.

4. transformed plant screening and field planting

After transformed plant grows 3 leaves, spraying herbicide

(Hoechst Schering AgrEvo GmbH contains weedicide glufosinate ammonium) aqueous solution, concentration is 9.6ml-10.8ml

Falling drop with plant is advisable.Unconverted adjoining tree stops growing after back 5 days in sprinkling, and is dead about 15 days.Transformed plant is after sprinkling, and some individual variations are similar with adjoining tree, and other individual continuing grow, and change not obvious.When the plant of waiting to survive length arrives the 7-8 leaf, the field is arrived in its field planting, and promoted to tiller.

5. the antiweed plant is after vernalization treatment, standing up-jointing stage gets blade and extracts DNA, adopts round pcr to detect transformed plant.The PCR positive plant carries out Southern blotting and detects.

When adopting PCR to detect transfer-gen plant, every strain is to get that individuality detects about 30 per generation at random, the ratio of statistics PCR positive plant.By the side of card (x ²) check infers the genetic behavior of foreign gene in transfer-gen plant and whether meet Mendel's regulavity of segregation.Taq archaeal dna polymerase, various restriction enzyme and damping fluid thereof,

ExScript ^TMRT-PCR Kit (Perfect Real Time) is available from TaKaRa company.Trizol reagent is given birth to the worker available from Shanghai.Primer synthesizes and order-checking is finished by Beijing Bo Shang company.

When adopting Southern hybridization analysis method to determine transfer-gen plant, detecting the positive plant that contains the TsCBF1 gene with PCR method is that material adopts the CTAB method to extract high quality DNA, thoroughly digests with the EcoRI restriction enzyme then.The endonuclease reaction system is: 50 μ l, 10 * enzyme cutting buffering liquid, and 3U restriction endonuclease/μ g DNA, 40-45 μ g wheat cdna group DNA supplies aseptic ddH2O to 500 μ l, and 37 ℃ of enzymes are cut and are spent the night, and 65 ℃ of heating 10min stop endonuclease reaction.Cut enzyme completely genomic dna carries out the separation of 0.8% agarose gel electrophoresis after dehydrated alcohol precipitates, concentrates, every swimming lane application of sample 20-30 μ g DNA, damping fluid is 1 * TBE[0.089mol/L Tris-boric acid, 0.002mol/L EDTA (pH8.0)], electrophoretic voltage treats as required that less than 1V/cm the indicator tetrabromophenol sulfonphthalein stops electrophoresis when migrating to correct position.Electrophoresis finishes, and gel is handled 20min through sex change liquid (0.5M NaOH, 1.5M NaCl), repeats once, uses rinsed with deionized water twice again, with gel be soaked in neutralizer (0.5M TrisCl, pH 7.4; 1.5M 20min NaCl), and repeat once.With 20 * SSC (0.3M Citrate Sodium; 3M NaCl, pH 7.0) making transfering buffering liquid, vacuum trace 90min is transferred to DNA on the nylon membrane, and vacuum trace instrument (Hybaid, UK) specification sheets are seen in concrete operations.After trace finishes, 6 * SSC rinsing nylon membrane 5min, room temperature is dried 30min, places 80 ℃ of oven dry 2h that DNA is fixed on the nylon membrane, and is standby.Reclaim the pcr amplified fragment of goal gene simultaneously, as follows dna fragmentation is carried out radio-labeling: dna profiling 15-50ng, 6-random primer 1 μ L (125ng/ μ L) complements to 30 μ L with the sterilization distilled water with volume, boiling water bath 10min, ice bath 5min.In above-mentioned system, add then: 5 * Buffer10 μ L, dNTP (dATP, dTTP, dGTP each 3.3nmol/ μ L) 1.5 μ L, dCTP-32P 3 μ L (10nCi/ μ L), Klenow fragment 1 μ L (about 4U), with the sterilization distilled water volume is complemented to 50 μ L, in 37 ℃ of incubation 2h, react with end mark in 95 ℃ of sex change 10min then.The nylon membrane of engram DNA put into fill an amount of prehybridization solution (0.5M Na ₂HPO ₄, use H ₃PO ₄Transfer pH to 7.2; 1mM EDTA, pH 8.0; 7%SDS; 1%BSA; 100 μ g/mL sex change salmon sperm dnas) in the hybridizing box, 65 ℃ of prehybridization 3h add the radiolabeled probe then, and fully mixing is hybridized 16-20h for 65 ℃.After hybridization finishes, with film place film washing liquid I (2 * SSC, 0.1%SDS) in, room temperature vibration rinsing 10min, place film washing liquid II (1 * SSC then successively, 0.1%SDS), room temperature vibration rinsing 10min, film washing liquid III (0.5 * SSC, 0.1%SDS), 65 ℃ of water-bath vibration rinsing 10min blot Hybond membrane at last with filter paper, carry out conventional compressing tablet and the flushing of X-ray sheet behind the preservative film parcel.

For the transgenic wheat plant, adopt quantitative RT-PCR to detect the testing goal expression of gene.Get the tender wheat leaf blade of about 0.1 gram children, liquid nitrogen grinding becomes powder, transfers in the 1.5ml centrifuge tube that fills 1ml Trizol reagent, fully mixing.Add 200 μ l chloroforms: primary isoamyl alcohol (24: 1), thermal agitation 30 seconds.Centrifugal, get supernatant, add the equal-volume Virahol ,-20 ℃ of precipitations 0.5-1 hour.Centrifugal 10 minutes of 12000rpm, precipitation is washed 2 times with 75% ethanol, and drying at room temperature is to transparence.Dnase digestion (12.5 μ l system) half an hour, in 37 ℃ of water-baths, carry out.Reaction system: RNA 15 μ l, DNaseI Buffer 2.5 μ l, RNase inhibitor 0.5 μ l; DEPC water 6 μ l.After having reacted, add DEPC water to 100 μ l, add the equal-volume chloroform: primary isoamyl alcohol (24: 1) extracting 10 minutes.Centrifugal 10 minutes of 12000rpm draws supernatant, and (3M, pH5.2) and the dehydrated alcohol of 2 times of volumes ,-20 ℃ of precipitations are spent the night to add the NaAc of 1/10 volume.Centrifugal recovery precipitation, 75% ethanol is washed 2 times, 10 μ l DEPC water dissolution.The detected through gel electrophoresis sample quality, qualified RNA extract is measured absorbance value and is calculated the RNA amount at 260nm and 280nm.Using at random, six primers carry out reverse transcription, reaction system and step are: RNA 500ng, Random hexaprimer 0.5 μ L, sterilization ddH2O complements to 7 μ L, 95 ℃ of 5min, ice bath 3min adds AMV reversetranscriptase Buffer 2 μ L, AMV reverse transcriptase 0.25 μ L, RNase inhibitor0.25 μ L, dNTP 0.5 μ L, cumulative volume 10 μ L then.42 ℃ of reverse transcription 2h, 95 ℃ of 3min.10 times of reverse transcription gained cDNA dilutions are carried out pcr amplification for template.Reaction system and the primer sequence of sxemiquantitative RT-PCR are as follows.In 25 μ l reaction systems, contain 10mM Tris-Cl, 50mM KCl, 1.5mM MgCl ₂, 200 μ MdNTP each, 0.8 μ M primer, 0.625U Taq DNA polymerase, 1 μ l template, aseptic deionized water is supplied 25 μ l.Response procedures is: 95 ℃ of pre-sex change 5min; 95 ℃ of sex change 1min, 58 ℃ of annealing 1min, 72 ℃ are extended 30s, circulate 40 times; 72 ℃ are extended 7min.The amplified production electrophoretic analysis.In adopting, RT-PCR is designated as wheat native gene Actin.Simultaneously with the empty carrier that does not contain goal gene transform the plant that produces and not transfer-gen plant be control material.Do not change the target gene plant and do not produce the specific amplified band.

6. transfer-gen plant progeny analysis

Plant by vernalization stage (the subzero treatment condition is different because of kind) was stood up under the long day, jointing, blossom and bear fruit.The seed that transfer-gen plant produces is put after immersion is sprouted in the refrigerator (0-2 ℃) and was carried out vernalization treatment 30-65 days.Seed 10.8ml after the part vernalization treatment

The aqueous solution is handled, and observes the individual ratio of statistics resistance and susceptibility; Planting seed after another part vernalization treatment carries out drought stress processing and high-salt stress respectively and handles at land for growing field crops and sand basin, screens drought-enduring and/or salt tolerant transfer-gen plant.Extract the leaf DNA of degeneration-resistant plant, adopt round pcr to detect foreign gene, and the segregation ratio of statistics foreign gene in progeny plant, obtain to be used for wheat breeding or directly to enter safety experiment and regional experiment after the pure lines.

Claims (6)

1. application of changeing TsCBF1 gene alteration corn, wheat resistance, it is characterized in that, the TsCBF1 gene that to clone from the salt mustard is recombinated with just form in the plant expression vector, adopts transgenic technology that recombination is imported corn, wheat cell, obtains transfer-gen plant; By detecting transgene expression and plant being carried out resistance identify, therefrom filter out transfer-gen plant and offspring thereof that resistance obviously improves, create the new germ plasm and the new variety that in corn, wheat breeding, have application prospect.

2. change the application of TsCBF1 gene alteration stress resistance of plant according to claim 1, it is characterized in that, described TsCBF1 gene has cDNA form or genomic gene form, and its encoding sequence is built into fusion gene with just form or has original promotor and change in corn, the wheat body.

3. as changeing the application of TsCBF1 gene alteration stress resistance of plant as described in the claim 2, it is characterized in that, the described form that is used for the TsCBF1 gene of gene fusion construct is the full length sequence of gene, partial sequence, or according to the deoxy polynucleotide of partial sequence synthetic.

4. change the application of TsCBF1 gene alteration corn, wheat salt tolerance drought tolerance according to claim 1, it is characterized in that described application is the transfer-gen plant that improves by the drought-and salt-tolerance that changes TsCBF1 or obtain according to the polynucleotide sequence that the TsCBF1 gene order designs.

5. change the application of TsCBF1 gene alteration corn, wheat salt tolerance drought tolerance according to claim 1, it is characterized in that, described application is by changeing corn, wheat transgenic plant and the offspring that the TsCBF1 gene obtains drought-and salt-tolerance.

6. as changeing the application of TsCBF1 gene alteration corn, wheat salt tolerance drought tolerance corn, wheat salt tolerance drought tolerance as described in the claim 1,4 or 5, it is characterized in that described corn, wheat salt tolerance drought tolerance comprise corn drought tolerance, corn salt tolerance, wheat drought tolerance and wheat salt tolerance.