CN100355894C - Method for cultivating perennial ryegrass with improved drought resistance - Google Patents

Abstract

The present invention discloses a method for cultivating the perennial ryegrass with improved drought resistance, which comprises the following steps: coding genes of DREB transcription factors are inserted into a plant expression vector, and a recombinant expression vector of the coding genes containing the DREB transcription factors is obtained; the recombinant expression vector is introduced into the perennial ryegrass, and the perennial ryegrass with improved drought resistance is obtained. The present invention uses adversity inducible promoters (RD29B) for increasing the expression of exogenous DREB1B, CBF1 genes, and can obviously improve the drought resistance of the perennial ryegrass.

Description

A kind of method of cultivating drought resistance enhanced rye grass

Technical field

The purpose of this invention is to provide a kind of method of cultivating drought resistance enhanced rye grass.

Background technology

Northern China belongs to the arid and semi-arid area, because precipitation deficiency, eluviation are weak, phreatic evaporation and rising strong or rainfall are brought salinity into to soil, the arid of soil, salinification problem are very serious.The concrete manifestation of this problem on turfgrass causes the phenomenons such as evening, early ageing in autumn, poor growth of germinateing in general spot, spring to take place exactly, influences the attractive in appearance of lawn, reduces the value on lawn.Although the lawn grass variety itself that present northern urban afforestation is used has certain defence arid, cryogenic ability, but since planting lawn to soil and moisture require too high, caused plantation, management costs too big, these factors have seriously limited the use of turfgrass, conserving water and soil, preventing aspect sand and dust, the urban afforestation irreplaceable effect is arranged because of it again, so research is cultivated novel anti arid lawn grass variety and is seemed by for urgent.Because the drought stress resistance belongs to quantitative character, adopt traditional breeding method to cultivate drought-resistant variety, time and effort consuming, difficulty is big.Compare with traditional breeding way, utilize molecular breeding, improve or the transcription factor that increases a key can improve the many-sided resistance of plant.

Plant is being subjected to adverse circumstance (arid, high salt, low temperature) when coercing, and can produce a series of albumen and protect and oneself escape injury, to keep vigor.At present; utilize model plant mouseearcress research adverse circumstance factor to induce the serial albumen of generation rather deep; discovery mouseearcresses such as Mie Kasuga are being subjected to arid; high salt; when adverse circumstance factors such as low temperature are coerced; can activate associated transcription factor such as DREB (DRE; dehydration responsiveelement; binding protein)/CBF (C-repeat/DRE-binding factor); start the corresponding resistant gene transcription; as DREB1A; rd29A; kin1; cor6.6; cor15a; rd17; P5CS; erd1; rd22; rd29B etc.; thereby improved viability (the Foolad MR of plant under adverse circumstance greatly; LP Zhang; Lin GY.Identification and validat ion of QTLs for salt tolerance duringvegetative growth in tomato by selective genotyping.Genome; 2001; 44:444-454); Liu Qiang etc. forward the DREB1A gene in the mouseearcress to; can give expression to albumen under the home; and in arid; its expression amount shows that than the remarkable enhancing of wild-type same transcription factor can be regulated and control a plurality of genetic expressions simultaneously by corresponding cis-acting elements in the plant under the condition of subzero treatment.Under drought condition, rd29B expresses one of tangible albumen (Foolad MR, LP Zhang, Lin GY.Identification andvalidation of QTLs for salt tolerance during vegetative growth in tomatoby selective genotyping.Genome, 2001,44:444-454), the functional element of taking advantage of a situation that in its promoter sequence, has an arid reaction that depends on ABA at least, and in the expression that can induce rd29B under high salt and the cryogenic condition (.DREB transcription factor such as Zhao Liu Qiang Nanming is improving the effect in the plant stress-resistance.Science Bulletin, 2000,45 (1): 11-16).

DREB, CBF transcription factor belong to the EREBP type transcription factor in the AP2/EREBP family that has found at present, comprise many members such as DREB1A～C (CBF3,1,2) and DREB2A～B.They all contain the AP2/EREBP structural domain, both can both with core sequence DRE combination of elements.The CBF transcription factor is CTR/DRE element and the combination with it that can discern among the anti-freeze gene COR (cold-regulated gene), thereby opens serial degeneration-resistant protein expression.At present, a series of CBF family members have been isolated, CBF1, CBF2, CBF3 and CBF4.Wherein CBF1, CBF2, CBF3 gene are named as DREB1B, DREB1C and DREB1A (Eric J.Stockinger again respectively, etal.Transcriptional adaptor and histone acetyltransferase proteins inArabidopsis and their interactions with CBF1, a transcriptional activatorinvolved in cold-regulated gene expression.Nucleic Acids Res., 2001,29:1524-1533; Volker Haake, Daniel Cook, JoseLuis Riechmann, Transcriptionfactor CBF4 is a regulator of drought adaptation in Arabidopsis.PlantPhysiology, 2002,130:639-648).Had a large amount of evidences to prove, with DREB, CBF transcription factor gene by changing the ability of environment stresses such as the drought resisting that can obviously improve plant in the plant, anti-low temperature, anti-salt over to.

Select promotor to express the problem of growing that transcription factor protein need be considered transfer-gen plant, the strong promoter 35S overexpression transcription factor DREB 1A albumen of use composing types such as Liu Qiang has caused the mouseearcress plant growth obstruction to occur, lodging, solid few phenomenon, thereby this may be because (the Mie Kasug that the stress-inducing genetic expression of DREB1A albumen control too much causes under non-stress conditions, Qiang Liu, Setsuko Miura, etal.Improving plant drought, salt, and freezing tolerance by gene transferof a single stress-inducible transcription factor.Nature Biotechnology1999,17:287-292).Therefore, can anti-drought gene successfully change over to, and can goal gene express, and can the acceptor plant show the proterties that foreign gene is controlled, and also needs to identify by a series of experiment.

Summary of the invention

The purpose of this invention is to provide a kind of method of cultivating drought resistance enhanced rye grass.

The method of cultivation drought resistance enhanced rye grass provided by the present invention, it is encoding gene insertion plant expression vector with the DREB transcription factor, obtain containing the recombinant expression vector of the encoding gene of DREB transcription factor, this recombinant expression vector is imported in the rye grass, obtain drought resistance enhanced rye grass.

Described rye grass is meant Gramineae lolium plant, comprises the kind more than 20 of English ryegrass and annual ryegrass.

Described plant expression vector can be Ti class plasmid vector or virus vector or the conventional carrier of particle gun conversion.

The encoding gene of described DREB transcription factor can derive from mouseearcress or wheat or paddy rice or corn or soybean or rape etc.

The encoding gene of described DREB transcription factor is preferably and derives from mouseearcress or wheat.

The encoding gene of described DREB transcription factor is the DREB1B gene that derives from mouseearcress, as has the nucleotide sequence of Genbank Access No.AB013816; Or for having the dna fragmentation of Genbank AccessNo.AB013816 from 5 ' end the 612nd to 1320 bit base.For the ease of inserting expression vector, can add the BamHI site in 5 ' design, add the SacI site in the design of 3 ' end, sequence total length 727bp, as the sequence in the sequence table 1, from the 1st to the 6th deoxynucleotide of 5 ' end is the BamHI recognition site, is the SacI recognition site from the 722nd to the 727th deoxynucleotide of 5 ' end.

The encoding gene of described DREB transcription factor is the CBF1 gene that derives from wheat, has the sequence of GenbankAccess No.AF376136, or has the dna fragmentation from 5 ' end the 8th to 657 bit base of Genbank Access No.AF376136.For the ease of inserting expression vector, can add the BamHI site in 5 ' design, add the SacI site in the design of 3 ' end, sequence total length 669bp is as the sequence in the sequence table 2.

In described recombinant expression vector, starting the promotor that the encoding gene of described DREB transcription factor transcribes is the promotor of the hydrophilic albumen RD29B of mouseearcress gene, or the promotor of the hydrophilic albumen RD29A of mouseearcress gene, or stress induced promoter such as mouseearcress Induced by Salicylic Acid gene SAG12; Or corn polyubiqutin gene promoter, or constitutive promoter such as cauliflower mosaic virus CaMV 35S promoter.

The promotor of the hydrophilic albumen RD29B of described mouseearcress gene, contain Genbank Access No.D013044 from 5 ' dna fragmentation of end the 85th to 1784 bit base.For the ease of inserting expression vector, can add the HindIII site in 5 ' design, add the BamHI site in the design of 3 ' end, sequence total length 1706bp is as the sequence in the sequence table 3.

The recombinant expression vector pBAC117 that obtains between the recognition site of the recombinant expression vector of the encoding gene of the described CBF1 of containing transcription factor for the restriction endonuclease sites BamHI that the encoding gene of described CBF1 transcription factor inserted pBPC18 and SacI; Or the recombinant expression vector pBAC122 that obtains between recognition site for the restriction endonuclease sites HindI that described RD29B promotor inserted pBAC117 and BamHI; Or for plasmid pBAC122 cuts with the HindIII enzyme, the CaMV 35S promoter that inserts two ends and be the HindIII sticky end is the fragment of terminator with the bar gene of corn Adhlintron driving and with NOS 3 ', the plasmid pBAC127 that obtains.

The recombinant expression vector pBAC118 that obtains between the recognition site of the recombinant expression vector of the encoding gene of the described DREB of containing transcription factor for the restriction endonuclease sites BamHI that the encoding gene of described DREB transcription factor inserted pBPC18 and SacI; Or the recombinant expression vector pBAC123 that obtains between recognition site for the restriction endonuclease sites HindI that described RD29B promotor inserted pBAC118 and BamHI; Or for pBAC123 is cut with the HindIII enzyme, the CaMV 35S promoter that inserts two ends and be the HindIII sticky end is the fragment of terminator with the bar gene of corn Adhl intron driving and with NOS 3 ', the plasmid pBAC128 that obtains.

In the described method, be to be explant with rye grass mature embryo or rataria, the recombinant expression vector that will contain the encoding gene of DREB transcription factor imports rye grass.

The recombinant expression vector that carries the encoding gene of DREB transcription factor can be by particle bombardment, use that Ti-plasmids, Ri plasmid, plant viral vector, microinjection, electricity be led, conventional biological method such as agriculture bacillus mediated transforms the rye grass cell or tissue.

The described recombinant expression vector that carries the encoding gene of DREB transcription factor preferably imports rye grass by particle bombardment.

Method of the present invention is particularly suitable for English ryegrass, especially sharpshooter's kind.

Kind-lawn tailored version English ryegrass kind sharpshooter (Topgun) is a material with the rye grass representativeness in the present invention, will be by the plasmid pBAC122 (containing the CBF1 gene) of the DREB transcription factor of the promoters driven of the hydrophilic albumen RD29B of mouseearcress gene, pBAC123 (containing the DREB1B gene), pBAC127 (containing the CBF1 gene) is in the rataria of pBAC128 (containing the DREB1B gene) importing English ryegrass kind Topgun, the mature embryo callus.Through weedicide Bialaphos resistance screening and plant regeneration, 98 transfer-gen plants have finally been obtained.Through the Molecular Detection of PCR, Dot-blotting, CBF1 gene and DREB1B gene have been incorporated in the genome of English ryegrass part transgenic line respectively.Smear the rye grass blade with the weedicide of five kinds of different concns, the non-transgenic plant cannot not show as anti-ly, can resist 135-200mg/L and transfer-gen plant is the highest.The blade proline content is measured and is shown, there are 31 strain proline contents to be higher than the non-transgenic plant in 62 commentaries on classics DREB1B gene strain systems, have the proline content of 13 strains to be higher than the non-transgenic plant in 36 commentaries on classics CBF1 gene strain systems, part plant increase rate can reach 2-4 doubly.Handle through 25 days artificial greenhouse arids, have 9 plant to show the survival sign, after the rehydration, have 4 plant to recover growth.Experimental result shows, utilizes adverse circumstance inducible promoter (RD29B) to strengthen external source DREB1B, CBF1 expression of gene, can significantly improve the drought-resistant ability of rye grass.

Description of drawings

Fig. 1 is the expression vector synoptic diagram

Fig. 2 is a callus differentiation and seedling emergence photo

Fig. 3 obtains the transfer-gen plant photo for the Herbicid resistant screening

Fig. 4 is part T ₀The PCR that generation is changeed the DREB1B gene plant detects

Fig. 5 is part T ₁In generation, changeed the dot blot analysis of DREB1B gene plant

The PCR that Fig. 6 changes CBF1 gene strain system for part detects

Fig. 7 changes the dot blot analysis of CBF1 gene strain system for part

Fig. 8 changes the proline content analysis of pBAC128 gene strain system for part

Fig. 9 changes the proline content analysis of CBF1 gene strain system for part

Figure 10 is the Herbicid resistant analysis of transgenosis rye grass strain system

Figure 11 a is the transgenic line 123-21 after the rehydration

Figure 11 b is the transgenic line 122-7 after the rehydration

Figure 12 is the normal growth plant photo of 128-14

Embodiment

Experimental technique among the following embodiment if no special instructions, is ordinary method.

T ₀The transfer-gen plant that expression is obtained by the callus of rye grass, T ₁Expression T ₀The seed that produces for selfing and by plant that it grew up to.

Embodiment 1, cultivation drought resistance enhanced rye grass

The Taq archaeal dna polymerase is purchased the company in TaKaLa, T ₄Dna ligase, T-easy vector, restriction enzyme, DNA reclaim purification kit etc. and purchase the company in Promega, and conventional reagent, organic reagent be Wanyuan chemical reagents corporation too all, and particle gun and reagent consumptive material thereof are purchased the company in BIORAD.

One, the structure of plant expression vector

1, the structure of DREB1B expression vector pBAC123 and pBAC128

(1) clone of mouseearcress (Arabidopsis thaliana) DREB1B gene

DREB1B gene order (GenbankAccess No.AB013816) according to the mouseearcress of having delivered (Arabidopsis thaliana), design and synthesize the special primer of this gene: P1: upstream primer 5 '-GGATCCTGATCAATGAACTCATTTTC, downstream primer P2:5 '-GAGCTCCCATTCTAAAAAAGGAAC, genomic dna (still cDNA) with mouseearcress is a template, utilizes PCR to clone this gene.Wherein, the temperature condition of PCR is: 94 ℃ of pre-sex change 5min of elder generation; 94 ℃ of sex change 45s then, 45 ℃ of annealing 45s, 72 ℃ are extended 1min, 35 circulations; Last 72 ℃ are extended 10min.About 730bp dna fragmentation that amplification is obtained reclaims this fragment with DNA recovery purification kit, with (Promega company of pGEM-T easy vector system, the U.S.) scheme that provides by supplier is cloned this fragment, order-checking (Shanghai Shen You biotech company), sequencing result shows that this fragment has the nucleotide sequence of sequence 1 in the sequence table.By with dreb gene sequence (Genbank Access No.AB013816) relatively, confirm to be cloned into have Genbank AccessNo.AB013816 from the 612nd to 1320 gene order fragment of 5 ' end.

(2) structure of DREB1B expression vector pBAC123 and pBAC128

The construction process of plasmid pBAC123 and pBAC128, process and gene transformation are with reference to " molecular cloning " second edition (Sambrook J, Fritsch E F, Maniatis T.Molecular Cloning-A LaboratoryManual.2nd ed.New York:Cold Spring Harbor laboratory Press, 1989).

Concrete grammar is as follows:

1) with SacI and EcoRI double digestion plasmid pSP72 (available from Promega company), agrobacterium tumefaciens rouge alkali synthetase gene NOS 3 ' the terminator sequence of inserting about 260bp is (from plasmid vector pBARGUS, with SacI and EcoRI double digestion, Vasil, et al, Bio/technology, 10:667-674,1992).The plasmid that is built into is named and is pBPC18.

2) the about 730bp dreb gene fragment that is obtained by the PCR clone adopts the TA cloning process to insert pGEM-T (Promega), after the dna sequencing checking is correct, and with BamHI and SacI double digestion, the BamHI and the SacI site of inserting pBPC18.The plasmid that is built into is named and is pBAC118.

3) genomic dna by mouseearcress is a masterplate, upstream primer P1:5 '-GCGGAAGCTTCATTTTCTGCTACAG, downstream primer P2:5 '-GGATCCTTTCCAAAGCTGTGTTTTCTCTTTTTC carries out the 1.7kb RD29B promoter fragment (dna fragmentation from 5 ' end the 85th to 1784 bit base of Genbank Access No.D013044) that the PCR clone obtains, adopt the TA cloning process to insert pGEM-T (Promega), after the dna sequencing checking is correct, with HindIII and BamHI double digestion, insert between the HindIII and BamHI site of pBAC118.The plasmid that is built into is named and is pBAC123.

4) plasmid pBAC123 cuts with the HindIII enzyme, the CaMV 35S promoter that inserts two ends and be the HindIII sticky end is fragment (Vasil, et al, the Bio/technology of terminator with the bar gene of corn Adhl intron driving and with NOS 3 ', 10:667-674,1992).The plasmid forward that makes up inserts and names into pBAC128 (Fig. 1), oppositely inserts to name to be pBAC128R.The goal gene DREB1B of pBAC128 is driving with the promotor (1.7kb) of the hydrophilic albumen RD29B of mouseearcress gene, is adverse circumstance abduction delivering type.PBAC128 contains bar gene that the Intronl of CaMV 35S promoter and corn Adh1 gene drives as selective marker.

2, the structure of CBF1 expression vector pBAC122 and pBAC127

(1) clone of wheat (Triticum aestivum) CBF1 gene

DREB1B gene order (Genbank AccessNo.AF376136) according to the wheat of having delivered (Triticum aestivum), design and synthesize the special primer of this gene: P3: upstream primer 5 '-GGATCCCAACTGATGGACACCGCCGCTGCCGGC, downstream primer P4:5 '-GAGCTCTTAGTCGGATAATTAGTTCCAAAGCGG, spending No. 1 genomic dna with the wheat breed capital is template, utilizes PCR to clone this gene.Wherein, the temperature condition of PCR is: 94 ℃ of pre-sex change 5min of elder generation; 94 ℃ of sex change 45s then, 45 ℃ of annealing 45s, 72 ℃ are extended 1min, 35 circulations; Last 72 ℃ are extended 10min.About 670bp dna fragmentation that amplification is obtained reclaims this fragment with DNA recovery purification kit, with (Promega company of pGEM-T easy vector system, the U.S.) scheme that provides by supplier is cloned this fragment, order-checking (Shanghai Shen You biotech company), sequencing result shows that this fragment has the nucleotide sequence of the sequence 2 in the sequence table.By with dreb gene sequence (GenbankAccess No.AF376136) relatively, confirm to be cloned into have Genbank Access No.AF376136 from the 8th to 657 deoxynucleotide gene order fragment of 5 ' end.

(2) structure of CBF1 expression vector pBAC122 and pBAC127

The construction process of plasmid pBAC122 and pBAC127, process and gene transformation are with reference to " molecular cloning " second edition (Sambrook J, Fritsch E F, Maniatis T.Molecular Cloning-A LaboratoryManual.2nd ed.New York:Cold Spring Harbor laboratory Press, 1989).

Concrete grammar is as follows:

1) with SacI and EcoRI double digestion plasmid pSP72 (available from Promega company), agrobacterium tumefaciens rouge alkali synthetase gene NOS 3 ' the terminator sequence of inserting about 260bp is (from plasmid vector pBARGUS, with SacI and EcoRI double digestion, Vasil, et al, Bio/technology, 10:667-674,1992).The plasmid that is built into is named and is pBPC18.

2) the about 670bp CBF1 gene fragment that is obtained by the PCR clone adopts the TA cloning process to insert pGEM-T (Promega), after the dna sequencing checking is correct, and with BamHI and SacI double digestion, the BamHI and the SacI site of inserting pBPC18.The plasmid that is built into is named and is pBAC117.

3) genomic dna by mouseearcress is a masterplate, upstream primer P1:5 '-GCGGAAGCTTCATTTTCTGCTACAG, downstream primer P2:5 '-GGATCCTTTCCAAAGCTGTGTTTTCTCTTTTTC carries out the 1.7kb RD29B promoter fragment (dna fragmentation from 5 ' end the 85th to 1784 bit base of Genbank Access No.D013044) that the PCR clone obtains, adopt the TA cloning process to insert pGEM-T (Promega), after the dna sequencing checking is correct, with HindI and BamHI double digestion, insert between the HindIII and BamHI site of pBAC117.The plasmid that is built into is named and is pBAC122.

4) plasmid pBAC122 cuts with the HindIII enzyme, the CaMV 35S promoter that inserts two ends and be the HindIII sticky end is fragment (Vasil, et al, the Bio/technology of terminator with the bar gene of corn Adhl intron driving and with NOS 3 ', 10:667-674,1992).The plasmid forward that makes up inserts and names into pBAC127 (Fig. 1), oppositely inserts to name to be pBAC127R.The goal gene CBF1 of pBAC127 is driving with the promotor (1.7kb) of the hydrophilic albumen RD29B of mouseearcress gene, is adverse circumstance abduction delivering type.PBAC127 contains bar gene that the Intronl of CaMV 35S promoter and corn Adhl gene drives as selective marker.

Two, plant expression vector pBAC122, pBAC123, pBAC127, pBAC128 transform rye grass

Lawn tailored version English ryegrass kind " sharpshooter " is the acceptor kind.Its outstanding feature be growth rapidly, the level ground is with property is good, humidity resistance is strong, high disease and insect resistance (the endophyte of plant infection rate is more than 90%).The north and transitional zone weather condition are had adaptability widely, splendid compatible compatibleness is arranged with other English ryegrass kind or English grass.

Selecting the mature embryo of ryegrass seed is that explant carries out callus induction, needs with 2 of 8mg/L, and 4-D carries out pre-treatment, otherwise mature embryo is difficult to induce callus.Concrete grammar is as follows: its seed is that the sterilization of 2.8% aqueous sodium hypochlorite solution is after 10 minutes through mass percent concentration, with containing 2mg/L 2, the sterilized water of 4-D soaked 1-2 days, after seed is sprouted, its mature embryo is seeded in W14+2, callus induction in the callus inducing medium of 4-D 8mg/L+NAA 4mg/L+KT 0.1mg/L.The result is in 617 callus that induce, there are 36 callus on screening culture medium (MS+NAA 0.5mg/L+KT 0.5mg/L+Bialaphos2mg/L), to grow green bud point, green bud point differentiation rate is 5.8%, 34 clumps of final one-tenth seedlings, seedling rate is 5.8%, and Fig. 2 has shown the situation of callus differentiation and seedling emergence.

With high-pressure helium particle gun PDS1000 (particle gun and reagent consumptive material thereof are purchased the company in BIORAD) with recombinant plasmid pBAC122 with contain the plasmid pBARGUS of bar genescreen mark, or pBAC123 and contain the plasmid pBARGUS of bar genescreen mark, or pBAC127, or pBAC128 imports the rye grass callus respectively, concrete operations are with reference to (Zhang Xiaodongs such as Zhang Xiaodongs, Liang Rongqi, Chen Xuqing, Yang Fengping etc. the acquisition of high-quality HMW gluten subunit transgenic wheat and genetic stability thereof and quality trait analysis. Science Bulletin, 2003,48 (5): method 474～479) is carried out.Wherein, pBAC122 or pBAC123 respectively with the plasmid pBARGUS that contains bar genescreen mark (Vasil, et al, Bio/technology, 10:667-674,1992) cotransformation, the mol ratio of pBAC122 and pBARGUS is 4: 1, and the mol ratio of pBAC123 and pBARGUS is 4: 1.

The callus that particle gun is transformed changes callus of induce screening culture medium W14+2 over to, cultivate 1-2 week among the 4-D 8mg/L+NAA4mg/L+KT 0.1mg/L+Bialaphos 1mg/L, induce callus and change differentiation and seedling emergence among the differentiation screening culture medium MS+NAA 0.5mg/L+KT 0.5mg/L+Bialaphos 2mg/L again over to.At last seedling is taken root containing on the strong seedling culture base MS+NAA 0.5mg/L+Bialaphos 2mg/L of 2mg/L paclobutrazol, Cheng Miaohou transplants to the field.The result shows the screening through the weedicide Bialaphos of 2mg/L, obtains 98 strain transfer-gen plant (T altogether ₀Generation), the plant of its transfer CBF1 gene has 36 strains, comprises plant 28 strains of changeing pBAC122 and plant 8 strains of changeing pBAC127, and the plant that changes the DREB1B gene obtains 62 strains, comprises plant 8 strains of changeing pBAC123 and plant 54 strains of changeing pBAC128.Herbicid resistant effect such as Fig. 3 of transgenosis rye grass.

Three, the analysis of molecules of transfer-gen plant

1, the PCR that changes the DREB1B gene plant detects and the dot blot analysis

(1) T ₀PCR for transfer-gen plant detects

The CTAB method is extracted transgenosis rye grass plant leaf genomic dna, with the negative contrast of unconverted rye grass plant, the positive contrast of plasmid pBAC123, PCR product 1% detected through gel electrophoresis.The PCR of transfer-gen plant detects, and uses DREB1B upstream region of gene primer P1 and downstream primer P2.Reaction system is: cumulative volume is 25 μ, 1,10 * PCR damping fluid, 2.5 μ l, each 1 μ l of primer (10 μ M), dNTPs 2 μ l, ddH ₂O 13 μ l, Taq enzyme 1U, MgCl ₂2 μ l, template DNA 1 μ g.Its cyclic amplification program is: 94 ℃ of pre-sex change 5min of elder generation; 94 ℃ of sex change 45s then, 45 ℃ of annealing 45s, 72 ℃ are extended 1min, 35 circulations; Last 72 ℃ are extended 10min.The PCR detected result shows that 8 strains change the specific fragment that the plant of pBAC123 has 4 strains to increase to obtain the DREB1B gene about 727bp, and the plant that pBAC128 is changeed in 54 strains has 14 strains to increase to obtain the specific fragment of the DREB1B gene about 727bp.Part T ₀For the PCR detected result of transfer-gen plant as shown in Figure 4, show the part transfer-gen plant (swimming lane 3 that is obtained, 4,7,8,10,14 etc.) can both amplify the specific fragment of the DREB1B gene about 727bp with positive control plasmid (swimming lane 1), negative control (non-transgenic plant, swimming lane 2) does not have amplified band, shows that external source DREB1B gene has entered transfer-gen plant.Among Fig. 4, swimming lane 15 is the 1kb plus of a Gibco company dna molecular amount standard, 1 positive contrast, and 2 negative contrasts, all the other are transfer-gen plant, and wherein, swimming lane 3-8 is for changeing the plant of pBAC123, and swimming lane 9-14 is for changeing the plant of pBAC128.

(2) T ₁Dna molecule hybridize for transfer-gen plant detects

To detect the T of the specific fragment (PCR test positive) that obtains the DREB1B gene about 727bp through above-mentioned PCR ₀Plant for plant, obtain T ₁For transfer-gen plant, extract the blade genomic dna respectively, with digoxin (DIG, Roche) (the DREB1B gene of this 727bp is that the genomic dna with mouseearcress is a template to the dreb gene of mark 727bp, utilize primer P1 and P2 pcr amplification to obtain) as probe, carry out dot blot and detect.This dot blot is analyzed, use the Bio-Rad Bio-Dot Microfiltration of company dot blot instrument to carry out a film, hybridize and detect by the DIG DNA Labeling and Detection Kit of Roche company, the DREB1B gene is carried out the DIG mark with the PCR method.The dot blot result shows that it is strong signal that part point is arranged as shown in Figure 5, part signal (comprising transfer-gen plant) a little less than, this explanation also exists similar DREB1B transcription factor or the higher dna sequence dna of homology probably in the genome of rye grass.Among Fig. 5, some A1: positive control (pBAC123), B1: blank, D8: negative control CK (not transfer-gen plant), all the other are transfer-gen plant (plant that changes pBAC123 has 4 strains, and the plant that changes pBAC128 has 25 strains).

2, the PCR that changes the CBF1 gene plant detects and the dot blot analysis

(1) T ₀PCR for transfer-gen plant detects

The CTAB method is extracted transgenosis rye grass plant leaf genomic dna, with the negative contrast of unconverted rye grass plant, the positive contrast of plasmid pBAC122, PCR product 1% detected through gel electrophoresis.The PCR of transfer-gen plant detects, and uses CBF1 upstream region of gene primer P3 and downstream primer P4.Reaction system is: cumulative volume is 25 μ l, 10 * PCR damping fluid, 2.5 μ l, each 1 μ l of primer (10 μ M), dNTPs 2 μ l, ddH ₂O 13 μ l, Taq enzyme 1U, MgCl ₂2 μ l, template DNA 1 μ g.Its cyclic amplification program is: 94 ℃ of pre-sex change 5min of elder generation; 94 ℃ of sex change 45s then, 45 ℃ of annealing 45s, 72 ℃ are extended 1min, 35 circulations; Last 72 ℃ are extended 10min.The PCR detected result shows the specific fragment that 28 strains are changeed 12 strains to increase in the plant of pBAC122 to obtain the CBF1 gene about 649bp, and the specific fragment that has 4 strains to increase in the plant of pBAC127 to obtain the CBF1 gene about 649bp is changeed in 8 strains.Part T ₀For the PCR detected result of transfer-gen plant as shown in Figure 6, show the specific fragment that the part transfer-gen plant that obtained and positive control plasmid can both amplify the CBF1 gene, the part plant has non-special band, and this may be because not high the causing of specificity of CBF1 primer.Among Fig. 6, the swimming lane 11 1kb plus of Gibco company are dna molecular amount standard, 9 positive contrasts, and 10 negative contrasts (not transfer-gen plant), all the other are transfer-gen plant, wherein, swimming lane 1-8 is for changeing the plant of pBAC122.

(2) T ₁Dna molecule hybridize for transfer-gen plant detects

To detect the T of the specific fragment (PCR test positive) that obtains the CBF1 gene about 690bp through above-mentioned PCR ₀Plant for plant, obtain T ₁For transfer-gen plant, extract the blade genomic dna respectively, with digoxin (DIG, Roche) (the CBF1 gene of this 690bp is that the genomic dna with mouseearcress is a template to the CBF1 gene of mark 690bp, utilize primer P3 and P4 pcr amplification to obtain) as probe, carry out dot blot and detect.This dot blot is analyzed, use the Bio-Rad Bio-Dot Microfiltration of company dot blot instrument to carry out a film, hybridize and detect by the DIG DNA Labeling and Detection Kit of Roche company, the CBF1 gene is carried out the DIG mark with the PCR method.The dot blot result shows that it is strong signal that part point is arranged as shown in Figure 7, part signal (comprising transfer-gen plant) a little less than, this explanation also exists similar CBF1 transcription factor or the higher dna sequence dna of homology probably in the genome of rye grass.Among Fig. 7, some C-7: positive control (pBAC122), B-7: blank, A-7: negative control CK (not transfer-gen plant), all the other are transfer-gen plant.

Four, the mensuration of rye grass blade free proline content

1, T ₀In generation, changeed the mensuration of the blade free proline content of DREB1B gene plant

Proline(Pro) is in close relations as the drought resistance of a kind of Physiology of Drought Resistance index and plant.A large amount of evidences show, in arid, low temperature is high oozes etc. under the adverse circumstance factor condition, can cause increasing sharply of the interior concentration of proline of plant body, thereby strengthened the resistance (Liu Ning of plant, Gao Yubao, Jia Caixia etc. the variation of the relative property of Itanlian rye leaf endoperoxide enzymic activity under the osmotic stress with proline content and plasma membrane. Plant Physiology Communications, 2000,36 (1): 11-14; Gao Yubao, Ren Anzhi, Liu Feng etc. free proline content is for the physiological ecological response of the water stress of dissimilar and intensity in the rye blade of grass. Plant Physiology Communications, 1999,23 (3): 193-204; High beautiful Liu Bao Renanzhi honeybee. the interrelationship study in the simulation meadow between rye blade of grass free proline content and leaf water content, the soil moisture content, Nankai University's journal (natural science edition), 1999,32 (3): 169-176)

According to document (Xu Xiaofeng, Zhu are. the research of PA method in the wheat leaf, biotechnology .1997,7 (1): method 40～42), the T of DREB1B gene is changeed in 62 strains that determination step two obtains ₀For rye grass plant leaf free proline content, concrete grammar is as follows: the method that adopts the lab simulation drought condition, after plant stops to water 25 days, take by weighing blade 0.1g, put into test tube after shredding, add 5ml 3% (quality percentage composition) sulphosalicylic acid solution, lixiviate is 10 minutes in boiling water bath, is chilled to room temperature.Draw vat liquor 2ml in another test tube, add 2ml water, 2ml glacial acetic acid and 4ml 2.5% (quality percentage composition) acid ninhydrine solution (is that solvent is prepared with 3: 2 glacial acetic acids and 6mol/L phosphoric acid) developed the color 60 minutes to boiling water bath, was cooled to room temperature.Add 4ml toluene, vibration is with the extraction red material.After leaving standstill, draw toluene layer, survey its OD value in 721 type spectrophotometer 520nm wavelength place colorimetrics.The making of typical curve: compound concentration is the serial proline(Pro) standardized solution of ten of 1～10 μ g/ml.Get 2ml vat liquor and 2ml water in standardized solution 2ml and the 2ml 3% sulphosalicylic acid solution replacement sample determination, develop the color, extraction and colorimetric (wavelength 520nm), last drawing standard curve by said procedure.

Calculate the content of the free proline(Pro) of rye grass blade according to the value that records:

Proline content (μ g/g)=C*2.5/W

In the formula: C-----is with the proline content (μ g) of the sample of typical curve acquisition

W-----rye grass leaf weight (g)

2.5---vat liquor 3% sulphosalicylic acid volume (5ml) and mensuration time institute's sample thief liquid long-pending (2ml) ratio when extracting proline(Pro)

The measurement result that DREB1B gene rye grass arid processing rear blade proline content is changeed in 62 strains shows, wherein the proline content of 1 strain commentaries on classics pBAC123 and 30 strains commentaries on classics pBAC128 rye grass is higher than contrast CK (not transfer-gen plant), has shown the measurement result of partly changeing the pBAC128 plant among Fig. 8.CK-P is contrast (not transgenosis rye grass) among Fig. 8, and all the other are the pBAC128 plant of walking around.

2, T ₀In generation, changeed the mensuration of the blade free proline content of CBF1 gene plant

The T that 36 strain step 2 is obtained according to the method for step 1 ₀In generation, changeed the mensuration that CBF1 gene rye grass plant has carried out arid processing rear blade proline content, the result shows that the proline content of wherein 8 strains commentaries on classics pBAC122 and 5 strains commentaries on classics pBAC127 rye grass is higher than contrast CK (not transfer-gen plant), wherein the comparison of the proline content of some transfer-gen plant (C127-6, C127-1) has shown the measurement result of partly changeing the CBF1 gene plant according to exceeding 2-4 doubly among Fig. 9.Among Fig. 9, C122 represents to change the pBAC122 plant, and C127 represents to change the pBAC127 plant, and CK-W and CK are contrast (not transgenosis rye grass).

Five, the transfer-gen plant Herbicid resistant detects

The Basta aqueous solution of configuration 100mg/L, 125mg/L, 135mg/L, 200mg/L, five concentration gradients of 270mg/L changes CBF1 gene T with 36 strains that cotton balls difference smearing step two obtains ₀Change the T of DREB1B gene for 62 strains of rye grass plant and step 2 acquisition ₀For rye grass plant leaf two sides, observations after 8-10 days.The result is as shown in table 1, contrast (50 strains are transfer-gen plant not) to the weedicide of five concentration all cannot not show as anti-ly (among Figure 10 picture a), blade is withered and yellow or withered; Can anti-135mg/L-200mg/L or higher (picture b among Figure 10) and transfer-gen plant is the highest, except that the blade flavescence that 270mg/L handles, the blade that other concentration is handled still be a green.Among Figure 10, the concentration of the Basta aqueous solution that numeric representation is handled blade.

The transgenosis rye grass strain number (strain) of the anti-different concns Basta of table 1.

Six, the drought resisting effect of transfer-gen plant is identified

Can rye grass kind drought resisting, and the drought resisting degree is much, though can identify in the relevant physiological index, whether it really has drought resistance, still needs the check by reality.Adopt indoor (room temperature is not higher than 26 ℃) simulation to do the method for condition early, 27 strain T ₀In generation, changeed pBAC122 plant, 7 strain T ₀In generation, changeed pBAC123 plant, 4 strain T ₀In generation, changeed pBAC127 plant, 8 strain T ₀In generation, changeed pBAC128 plant and not transgenosis contrast of 4 strains, stops to water 25 days in seedling period (disposable water sufficient 50ml water), recovers then to water.Arid through 25 days artificial greenhouses is handled, and has 9 transfer-gen plants to show survival sign (it is green that the bottom of plant center vane still keeps, and some is withered for rest blade wilting or top), and whole adjoining tree blades are chlorosis, dried-up then.After the rehydration, have 4 transfer-gen plant 123-21 (change the pBAC123 plant, Figure 11 a), 128-14 (changeing the pBAC128 plant), 122-7 (changeing the pBAC122 plant, Figure 11 b) and 128-24 (changeing the pBAC128 plant) recover growth, and whole adjoining tree death.Plant in the right side basin among Figure 11 a and Figure 11 b is not transfer-gen plant contrast, and the plant in the basin of left side is transfer-gen plant.Explanation is compared the part transgenic line with not genetically modified contrast and is had stronger drought resistance.

Finishing screen has been selected the comparatively significant transgenosis rye grass strain of 4 drought resisting effects system: 123-21 (changeing the pBAC123 plant), 128-14 (changeing the pBAC128 plant), 122-7 (changeing the pBAC122 plant) and 128-24 (changeing the pBAC128 plant).Wherein, normal growth plant photo such as Figure 12 of 128-14.

Sequence table

<160>3

<210>1

<211>727

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>1

ggatcctgat?caatgaactc?attttcagct?ttttctgaaa?tgtttggctc?cgattacgag 60

cctcaaggcg?gagattattg?tccgacgttg?gccacgagtt?gtccgaagaa?accggcgggc 120

cgtaagaagt?ttcgtgagac?tcgtcaccca?atttacagag?gagttcgtca?aagaaactcc 180

ggtaagtggg?tttctgaagt?gagagagcca?aacaagaaaa?ccaggatttg?gctcgggact 240

ttccaaaccg?ctgagatggc?agctcgtgct?cacgacgtcg?ctgcattagc?cctccgtggc 300

cgatcagcat?gtctcaactt?cgctgactcg?gcttggcggc?tacgaatccc?ggagtcaaca 360

tgcgccaagg?atatccaaaa?agcggctgct?gaagcggcgt?tggcttttca?agatgagacg 420

tgtgatacga?cgaccacgga?tcatggcctg?gacatggagg?agacgatggt?ggaagctatt 480

tatacaccgg?aacagagcga?aggtgcgttt?tatatggatg?aggagacaat?gtttgggatg 540

ccgactttgt?tggataatat?ggctgaaggc?atgcttttac?cgccgccgtc?tgttcaatgg 600

aatcataatt?atgacggcga?aggagatggt?gacgtgtcgc?tttggagtta?ctaatattcg 660

atagtcgttt?ccatttttgt?actatagttt?gaaaatattc?tagttccttt?ttttagaatg 720

ggagctc 727

<210>2

<211>669

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>2

ggatcccaac?tgatggacac?cgccgctgcc?ggctccccgc?gtgaggggca?caggacggtg 60

tgctcggagc?cgcccaagag?gccggcaggg?cggaccaagt?tcagggagac?gcgccacccg 120

ctgtaccgcg?gcgtgcggcg?ccggggccgg?ctcgggcagt?gggtgtgcga?ggttcgcgtg 180

cgcggcgcgc?aagggtacag?gctctggctc?ggcaccttca?ccactgccga?gatggcggcg 240

cgcgcgcacg?actccgccgt?gctcgcgctc?ctcgaccgcg?ccgcctgcct?caacttcgcc 300

gactccgcct?ggcggatgct?gcccgtcctc?gcggctggct?cgtcccgctt?cagcagcgcg 360

cgggagatca?aggacgccgt?cgccatcgcc?gtcctggagt?tccagcggca?gcgccccgtc 420

gtgtcgacgt?cggagatgca?cgacggcgaa?aaggacgccc?aaggctcgcc?gacgccgagc 480

gagctgtcca?cgtccagcga?cttgttggac?gagcactggt?ttggcggcat?ggacgccggc 540

tcgtactacg?cgagcttggc?gcaggggatg?ctcatggagc?cgccgtccgc?cagaacgtgg 600

agcgaggatg?gcggcgaata?cagcgccgtc?tacacgccgc?tttggaacta?attatccgac 660

taagagctc 669

<210>3

<211>1706

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>3

aagcttcatt?ttctgctaca?gaagtgttgt?tctctgtaaa?gtaatcagaa?ggaatgtaat 60

caacagaata?aatctgctct?tgaatgtcct?ttgagttctg?caggataata?tctgacccct 120

catatttaac?ctgcaataag?aagactagct?aaaatcagat?gatgccaaga?acaggaacca 180

gctctaccac?tctacagcaa?aagcaaaaac?tactgttgtg?tgtgccaact?aaaatcaact 240

cagggattta?ccgggtcatt?tttgatctct?agaacttcag?agaaacattg?gagatgatcc 300

tcactcgtgc?ataagagctt?gttctcctgt?atatcaaaag?atgctttatc?tcgcaatgaa 360

aaagaaaaag?ctgaactagt?tcaattagta?caatgcttac?acagcttgag?caataccatc 420

tcaaaatcca?actctggagt?ctccaactca?ataattttag?gtgtagcttt?atccgcaatg 480

gatctccgtc?atccttcttc?gaatagatca?taacttcggt?tttcaactcg?gtattcgctt 540

aagcaccaaa?atttccaacc?caagtatgaa?tataagatct?aagcaacaat?cagaaatgga 600

aactgagaaa?acacaccaca?aatttcgaaa?aatctacaac?caatctcact?ataagaaaca 660

aaggaccgtt?gacagaaaca?gtcagcgaga?ctcaggaaat?tcgaaatttc?acctccagga 720

actgataata?tctagatcga?aggaacttta?cctcgtctga?gtaataaact?ccgagcgaag 780

agtcgtcgat?ttcaaaaact?cgatagtcca?cactgacgcg?gtcgggaacc?acgtcggaaa 840

ggaacttcga?caaagcagct?tcaataggca?aatttccgat?agggatacta?acattttcga 900

tcgagccaaa?tcggagacgg?tcttcttctc?cgttgtagac?gatgggtgcc?gggaaattat 960

caggagccgg?aagattgagg?aagcctaggg?tttcaaatac?gtgagaaggt?ggagtagaga 1020

agtaatcgat?gttgagacat?cgagttcgca?tcgtaatttt?ctagatccgt?cttgggagct 1080

cagactgtat?cagtgatgat?gatgatgatg?aagaagagaa?cgaattttga?aattggcggt 1140

tttgaatttt?taagaaatta?aaaaatatcc?cccgtcgatt?tcaagaggga?gatggagata 1200

ccaaagcaac?tctcgccact?tgtcgtcttt?taattttaat?tgagtacgtt?atgccgtttt 1260

aaatgttcaa?aacagcacac?agttgatagc?tgaattgatt?ttttcttttg?ccgttttgtt 1320

atatttaaac?aacacacagt?gcatttgcca?aataactaca?tgatgggcca?ataaacgtgg 1380

accgactaaa?actaaataat?agaagataca?tcgataggct?tctctaaaga?tcggataaaa 1440

gataatgtcg?catagccacg?tagagagcaa?ctggctgaga?cgtggcagga?cgaaacggac 1500

gcatcgtacg?tgtcagaatc?ctacagaagt?aaagagacag?aagccagaga?gaggtggttc 1560

ggccatatgt?catcgttctc?tctataaact?ttatggaact?ttgttctgat?tttctcagag 1620

acacgaaaag?aaagaaaaca?acactagaac?aaagagggtt?tgattgattc?acttgaaaaa 1680

gagaaaacac?agctttggaa?ggatcc 1706

Claims (7)

1, cultivates the method for drought resistance enhanced rye grass, be to insert plant expression vector by the CBF1 transcription factor encoding gene that derives from wheat of the promoters driven of the hydrophilic albumen RD29B of mouseearcress gene, obtain containing the recombinant expression vector of described transcription factor encoding gene, this recombinant expression vector is imported in the rye grass, obtain drought resistance enhanced rye grass.

2, method according to claim 1 is characterized in that: the described encoding gene that derives from the CBF1 transcription factor of wheat is the dna fragmentation from 5 ' end the 8th to 657 bit base of Genbank Access No.AF376136.

3, method according to claim 1 is characterized in that: the promotor of the hydrophilic albumen RD29B of described mouseearcress gene is the dna fragmentation from 5 ' end the 85th to 1784 bit base of Genbank Access No.D013044.

4, according to claim 1 or 2 or 3 described methods, it is characterized in that: described recombinant expression vector is pBAC122 or pBAC127.

5, according to claim 1 or 2 or 3 described methods, it is characterized in that: described rye grass is English ryegrass or annual ryegrass.

6, method according to claim 5 is characterized in that: described rye grass is an English ryegrass.

7, method according to claim 6 is characterized in that: described rye grass is an English ryegrass sharpshooter kind.

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