CN100357319C - Barbadosnut cold-induced transcription factor, its encoding gene and uses - Google Patents

Abstract

The present invention discloses a jatropha curcas cold induction transcription factor and a coding gene and the application thereof. The present invention has the purpose of providing the jatropha curcas cold induction transcription factor, the coding gene of the jatropha curcas cold induction transcription factor and the application of jatropha curcas cold induction transcription factor for cultivating plants with enhanced stress resistance. The transcription factor is a protein with one of the following amino acid residue sequences: firstly, SEQ ID No.: 1 is in a sequence table; secondly, a plant stress resistance regulating and controlling protein which has a transcriptional activation function and is formed by replacing, removing or adding one to ten amino acid residues to the amino acid sequence of SEQ ID No.: 1 in the sequence table. Genes of the present invention are transferred into the hosts of plants through transgenic technology; transgenic plants (crops) with enhanced capability of resisting the adverse stress of drought, high salt, etc. are obtained; the jatropha curcas cold induction transcription factor has important theory significance and actual significance for cultivating new species of crops with enhanced stress resistance, and the jatropha curcas cold induction transcription factor can be applied to the cultivation and the identification of stress-resistance plant species required by agriculture, animal husbandry and ecological environment governing; the present invention has high practical application value.

Description

Cortex jatrophae cold-induced transcription factor and encoding gene and application

Technical field

The present invention relates in the plant a kind of with coerce relevant transcription factor and encoding gene thereof and application, particularly relate to and Cortex jatrophae cold-induced transcription factor that one derives from EREBP/AP2 family and encoding gene thereof and its application in cultivating the plant that resistance improves.

Background technology

Bad physical environment such as low temperature, high temperature, arid, salt marsh etc. act on plant, will cause a series of physiological metabolism reaction takes place in the plant materials, the reversibility that shows as metabolism and growth suppresses, and when serious even cause irreversible injury, causes whole plant death.In various the coercing, arid, saline and alkaline, cold (freezing) evil is particularly outstanding to the influence of plant, is the topmost environmental factor that influences plant-growth and crop yield.As early as possible and effectively utilize genetically engineered and improve crop arid, high salt and low temperature patience, avoid as far as possible or alleviate the harm of poor environment factor, being the research focus in current biology and modern agricultural technology field, also is the key subjects that current China and world agriculture are badly in need of solution.Plant is called resistance to opposing of coercing or the ability of restraining oneself, and is the adaptability to poor environment that plant forms in the long-term evolution process.For many years, people study the relation between plant and the high-salt stress from all angles.To ecological, hereditary research, arrive Physiology and biochemistry, metabolic research from the research of initial physiological phenomenon again, accumulated the data of enriching.Particularly along with development of molecular biology, make people on genomic constitution, expression regulation and signal conduction equimolecular level, be familiar with the patience mechanism of plant, and opened up new approach for utilizing genetic engineering means improvement plant anti-salt to coerce performance to coercing.Because the complicacy of plant stress-resistance proterties, adopt the resistance of traditional breeding method raising plant very difficult, along with development of molecular biology, opened up the new way of plant stress-resistance breeding by the resistance of gene process means improvement plant, but efficient adversity gene be separated into the engineered main factor of restriction plant stress-resistance.In the past, what clone and use mainly is single functional gene, and as trimethyl-glycine synthase gene and proline(Pro) synthase gene etc., though obtained certain effect, the resistance of plant is not comprehensively improved.

Stress resistance of plant is subjected to controlled by multiple genes, numerous functional genes that the plant stress-resistance proterties is exerted an influence are given full expression to and plays a role, and could improve stress resistance of plant effectively.Along with the continuous development of biotechnology, research emphasis has turned to the regulatory factor (as promotor and transcription factor) of each species specificity or high efficiency from general functional gene.Because a transcription factor can be regulated and control a plurality of and resistance Expression of Related Genes in the plant, strengthens the effect of a transcription factor, just can make the degeneration-resistant proterties of plant obtain comprehensive improvement.

Summary of the invention

The purpose of this invention is to provide a transcription factor that is subjected to the Cortex jatrophae EREBP/AP2 family of low temperature induction expression.

Cold-induced transcription factor provided by the present invention, name is called JcDREB, derives from Cortex jatrophae (Jatrophacurcas), is the protein with one of following amino acid residue sequences:

1) the SEQ ID № in the sequence table: 1;

2) with SEQ ID № in the sequence table: 1 amino acid residue sequence is through replacement, disappearance or the interpolation of one to ten amino-acid residue and have the protein of the regulation and control stress resistance of plant of transcriptional activation function.

SEQ ID № in the sequence table: 1 is made up of 256 amino-acid residues, for conservative EREBP/AP2 structural domain, is acidic activated zone from the 116th the-the 256th amino acids residue of aminoterminal from the 58th the-the 115th amino acids residue of aminoterminal (N end).

The gene (JcDREB) of coding Cortex jatrophae cold-induced transcription factor JcDREB is one of following nucleotide sequence:

1) SEQ ID № in the sequence table: 2 dna sequence dna;

2) SEQ ID № in the code sequence tabulation: 1 dna sequence dna;

3) under the rigorous condition of height can with SEQ ID № in the sequence table: the nucleotide sequence of the 2 dna sequence dnas hybridization that limit.

The rigorous condition of described height is at 6 * SSC or (6 * SSPE), 0.1%SDS in 2 * Denhardt solution, is hybridized under 65 ℃ of conditions; At 0.1 * SSC, in the 0.1%SDS solution, wash film under 65 ℃ of conditions.

SEQ ID № in the sequence table: 2 by 768 based compositions, its encoding sequence is from the 1st the-the 768th bit base of 5 ' end, coding has SEQ ID № in the sequence table: the protein of 1 amino acid residue sequence, from the conservative EREBP/AP2 structural domain of 5 ' end the 174th the-the 345th bit base coding, from the 346th the-the 768th bit base of 5 ' end acidic activated zone of encoding.

Contain expression carrier of the present invention, transgenic cell line and host bacterium and all belong to protection scope of the present invention.

Appoint among the amplification JcDREB-segmental primer is to also within protection scope of the present invention.

Another object of the present invention provides a kind of method that improves plant stress tolerance.

The method of raising plant stress tolerance provided by the present invention is gene transfered plant tissue or the cell with the described Cortex jatrophae cold-induced transcription factor JcDREB of coding, obtains the plant that resistance improves.

Described Cortex jatrophae cold-induced transcription factor gene JcDREB can import explant by the plant expression vector that contains described Cortex jatrophae cold-induced transcription factor gene JcDREB; The carrier that sets out that is used to make up described plant expression vector can be any one double base agrobacterium vector or can be used for carrier of plant micropellet bombardment etc., as p3301-BI121, pBI121, pBin19, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300 or other plant expression vector of deriving.

When using JcDREB to make up plant expression vector, before its transcription initiation Nucleotide, can add any enhancement type, composing type, organizing specific type or inducible promoter, as cauliflower mosaic virus (CAMV) 35S promoter, general living plain gene Ubiquitin promotor (pUbi) etc., they can use separately or be used in combination with other plant promoter; In addition, when using gene constructed plant expression vector of the present invention, also can use enhanser, comprise translational enhancer or transcriptional enhancer, these enhanser zones can be ATG initiator codon or neighboring region initiator codon etc., but must be identical with the reading frame of encoding sequence, to guarantee the correct translation of whole sequence.The source of described translation control signal and initiator codon is widely, can be natural, also can be synthetic.Translation initiation region can be from transcription initiation zone or structure gene.

For the ease of transgenic plant cells or plant being identified and screening, can process used plant expression vector, can produce the enzyme of colour-change or the gene of luminophor (gus gene, GFP gene, luciferase genes etc.) as adding the coding that in plant, to express, have the antibiotic marker thing (gentamicin marker, kantlex marker etc.) of resistance or anti-chemical reagent marker gene (as anti-weedkiller gene) etc.From the security consideration of transgenic plant, can not add any selected marker, directly with adverse circumstance screening transformed plant.

Be the carrier that sets out with p3301-BI121, the plant expression vector that contains described Cortex jatrophae cold-induced transcription factor JcDREB gene of structure is p3301-BI121-JcDREB.

Carry gene JcDREB of the present invention plant expression vector can Ti-plasmids, Ri plasmid, plant viral vector, directly DNA conversion, microinjection, electricity be led, conventional biological method transformed plant cells or tissue such as agriculture bacillus mediated by using, and the plant transformed cell or tissue is cultivated into plant.By the plant transformed host can be plants such as paddy rice, wheat, soybean, tobacco, corn, rape, Chinese sorghum, cotton, clover, Cortex jatrophae or Arabidopis thaliana.

The invention provides a Cortex jatrophae cold-induced transcription factor JcDREB and an encoding gene thereof that derives from EREBP/AP2 family.Experiment showed, that its encoding gene JcDREB is expressed by low temperature induction, the ability of adjustable plant opposing arid, low temperature and environment stress such as saline and alkaline, thus significantly improve the resistance of plant.JcDREB and encoding gene thereof have important theory and practical significance for Cortex jatrophae and other crop new variety of cultivating the resistance raising, can be applicable to the cultivation and the evaluation of the required resistance plant kind of husbandry and ecological environment treatment, have higher actual application value.The present invention has broad application prospects at agriculture field.

The present invention will be further described below in conjunction with specific embodiment.

Description of drawings

Fig. 1 is the agarose gel electrophoresis detected result through the total RNA of young seedling of Jatropha of low temperature stress processing

Fig. 2 is the agarose gel electrophoresis detected result of 3 ' RACE product

Fig. 3 is the agarose gel electrophoresis detected result of 5 ' RACE product

Fig. 4 is the agarose gel electrophoresis detected result of the JcDREB full-length cDNA of PCP amplification

The structural representation of the JcDREB full-length cDNA that Fig. 5 is

Fig. 6 is the homology analysis result of other ERF proteinoid aminoacid sequence of having cloned in JcDREB and the plant

Fig. 7 is the systematic evolution tree analytical results of other ERF proteinoid aminoacid sequence of having cloned in JcDREB and the plant

Fig. 8 is structural domain and the structure prediction result of JcDREB

Fig. 9 is the results of structural analysis of JcDREB genomic dna

Figure 10 is the homogenic result of JcDREB in the Southern hybridization analysis Cortex jatrophae genome

Figure 11 is the tissue specific expression analytical results of JcRREB gene

Figure 12 is the expression pattern analytical results of JcDREB under different abiotic stress factor conditions

Figure 13 is the structure synoptic diagram of carrier p3301-BI121-JcDREB

Figure 14 cuts qualification result for the enzyme of carrier p3301-BI121-JcDREB

Figure 15 is the PCR qualification result of carrier p3301-BI121-JcDREB

Figure 16 is the process of setting up of rice plant regeneration system

Embodiment

Method therefor is ordinary method if no special instructions among the following embodiment, and it is synthetic that the primer and probe are given birth to the worker by Shanghai.

The acquisition of embodiment 1, Cortex jatrophae cold-induced transcription factor gene JcDREB cDNA complete sequence

The acquisition of Cortex jatrophae cold-induced transcription factor gene JcDREB full length cDNA sequence may further comprise the steps:

One, the clone of Cortex jatrophae cold-induced transcription factor gene JcDREB 3 ' terminal sequence

1, the extraction of vegetable material processing and total RNA

Be material with the young seedling of Jatropha earlier, 4 ℃ of subzero treatment are extracted total RNA after 12 hours, it is carried out 1% agarose gel electrophoresis detects, detected result as shown in Figure 1, the total RNA that is extracted has 2 tangible electrophoretic bands, be respectively 28s RNA and 18s RNA from top to bottom, show to have obtained higher, the more complete total RNA of purity.

2, the clone of Cortex jatrophae cold-induced transcription factor gene JcDREB 3 ' terminal sequence

The amino acid residue sequence of discloseder DREB, seek conservative region, and according to conservative region sequences Design pair of degenerate primers, primer sequence is as follows:

JC1：5’-GC(A/G/T)GCTCG(T/G)GC(T/G/C)CA(C/T)GACGT(G/C)GC(G/C)GC-3’

JC2：5’-GCTGCT(T/C)(T/G)AGC(T/A)(T/C)ATGATG(A/T)TGCTGC-3’

The young seedling of Jatropha RNA through deepfreeze that extracts with step 1 is a template, with Plant RNAtrip ReagentKit2 test kit (coming gene company limited) and synthetic its first chain cDNA of reference reagent box specification sheets reverse transcription available from Beijing Puli, reaction system and condition are: oligodT (10uM) 1ul, DEPC-treated Water 10ul, RNA 2ul, 5 * AMV damping fluid 4ul, dNTP (10mM) 2ul, AMV 1ul, 42 ℃ were reacted 1 hour.With the synthetic first chain cDNA be stored in-20 ℃ standby.

Be template with the first chain cDNA that obtains again, article two, degenerated primer increases with dT-Adaptor primer pairing carrying out sleeve type PCR respectively, the PCR reaction system is: primer 1ul, dT primer 1ul, template cDNA 2ul, 10 * Taq damping fluid 2ul, dNTP (10mM) 2ul, Taq (2.5U/ul) 0.2ul, water 11.8ul, reaction conditions is: 94 ℃ of 5min of elder generation; 94 ℃ of 30s then, 57 ℃ of 30s, 72 ℃ of 50s, totally 30 circulations; Last 72 ℃ of 10min.After reaction finishes, the PCR product is carried out 1% agarose gel electrophoresis detect, detected result is (swimming lane M is molecular weight standard Marker III, and swimming lane 1 is 3 ' RACE product) as shown in Figure 2, has obtained the purpose fragment of the about 800bp of length through pcr amplification.Reclaim and purifying 3 ' RACE product, connect in the pMD-18T carrier, to connect product transformed into escherichia coli DH5 α competent cell, screening positive clone upgrading grain, obtain containing the recombinant plasmid of 3 ' UTR, difference called after pMD18-3 ' Jc-DREB, carry out the BLAST analysis to its order-checking and to sequencing result, this fragment length is 813bp as a result, has SEQ ID № in the sequence table: 3 nucleotide sequence, have higher homology with 3 ' terminal sequence of known erf gene in the plant, show that this fragment may be 3 ' terminal sequence of Cortex jatrophae AP2 class DNA binding-protein gene.

Two, the clone of Cortex jatrophae cold-induced transcription factor gene JcDREB 5 ' terminal sequence

The JcDREB 3 ' that obtains according to step 1 holds a pair of nested primer of cDNA sequences Design: JcDREB-5-gsp1 and JcDREB-5-gsp2, and sequence is as follows:

JcDREB-5-gsp1：5’-ACCGTACACGACTCTGGCTT-3’

JcDREB-5-gsp2：5’-AACCTCCGCCACCAAGGGTC-3’

The young seedling of Jatropha RNA through deepfreeze that extracts with step 1 is a template, adopt 5 ' the RACE test kit and synthetic its first chain cDNA of reference reagent box specification sheets reverse transcription of Invitrogen company, reaction system and condition are: RNA10ul, primer JcDREB-5-gsp1 1ul, water 4.5 μ l, 10 * PCR damping fluid, 2.5 μ l, 25mM MgCl ₂2.5 μ l, 10mM dNTP 1.0 μ l, DTT 2.5 μ l, 42 ℃ were reacted 1 hour.With the synthetic first chain cDNA be stored in-20 ℃ standby.

Be template with the first chain cDNA that obtains again, carry out the sleeve type PCR amplification under the guiding of above-mentioned nested primer JcDREB-5-gsp2, the PCR reaction system is: primer JcDREB-5-gsp2 1ul, water 4.5 μ l, 10 * PCR damping fluid, 2.5 μ l, 25mM MgCl ₂2.5 μ l, 10mM dNTP 1.0 μ l, DTT 2.5 μ l, the first chain cDNA 1ul, reaction conditions is: 94 ℃ of 5min of elder generation; 94 ℃ of 30s then, 57 ℃ of 30s, 72 ℃ of 50s, totally 30 circulations; Last 72 ℃ of 10min.After reaction finishes, the PCR product is carried out 1% agarose gel electrophoresis detect, detected result is (swimming lane M is molecular weight standard Marker III, and swimming lane 1 is 5 ' RACE product) as shown in Figure 3, has obtained the purpose fragment of the about 600bp of length through pcr amplification.Reclaim and purifying 5 ' RACE product, connect in the pMD-18T carrier, to connect product transformed into escherichia coli DH5 α competent cell, screening positive clone upgrading grain, obtain containing the recombinant plasmid of 5 ' UTR, difference called after pMD-5 ' JcDREB, carry out the BLAST analysis to its order-checking and to sequencing result, this fragment length is 507bp as a result, has SEQ ID № in the sequence table: 4 nucleotide sequence, with above-mentioned 3 ' RACE fragment the overlap of 323bp is arranged, The sequencing results shows that 5 ' end of known DREB2 genoid has higher homology in this fragment and the plant, shows that this fragment may be 5 ' terminal sequence of Cortex jatrophae DREB class DNA binding-protein gene.

Three, the acquisition of Cortex jatrophae cold-induced transcription factor gene JcDREB full length cDNA sequence and PCR detect

The length of utilizing step 1 and step 1 to obtain is the overlap between 813bp and the 507bp fragment, and splicing obtains the full length cDNA sequence of JcDREB by DNA software DNAMAM.This sequence has SEQ ID № in the sequence table: 2 polynucleotide sequence.SEQ ID № in the sequence table: 2 by 758 based compositions, its encoding sequence is from the 1st the-the 768th bit base of 5 ' end, coding has a SEQ ID № in the sequence table: the protein of 1 amino acid residue sequence, the SEQ ID № in the sequence table: 1 is made up of 256 amino-acid residues.According to the full length cDNA sequence reading frame (primer sequence is as follows for open reading frame, two ends sequences Design total length primer ORF):

JcDREBW-1：5’-GGACTCAGATAGTTGCTCCA-3’

JcDREBW-2：5’-TGATTGGGCTTCTCTTTTATCTTCTTAG-3’

Is template through the young seedling of Jatropha RNA of deepfreeze through the reverse transcription synthetic first chain cDNA with step 1, under the guiding of primer JcDREBW-1 and JcDREBW-2, carry out pcr amplification, after reaction finishes, the PCR product is carried out 1% agarose gel electrophoresis to be detected, (swimming lane M is molecular weight standard Marker III to detected result as shown in Figure 4, swimming lane 1 is a pcr amplification product), pcr amplification goes out the specific fragment that length is about 768bp as a result, it is checked order, sequencing result and above-mentioned splicing result are identical in the amplification part, its total length is 768bp, show that 3 ' RACE fragment and 5 ' RACE fragment of being cloned belong to same gene, with this unnamed gene is JcDREB, with its proteins encoded called after JcDREB.

The bioinformatic analysis of embodiment 2, JcDREB and proteins encoded thereof

One, the structure function of JcDREB Gene Sequence Analysis and proteins encoded thereof prediction

Utilize DNAMAN software that the full length cDNA sequence of the JcDREB of embodiment 1 acquisition is carried out bioinformatic analysis, (single line is represented 3 '-UTR to its structural representation as shown in Figure 5; The box indicating open reading frame, contain 2 function motifs, black box is represented the AP2 structural domain, the right shade line is represented acidic activated zone), this sequence total length 768bp is ORF from the 1st the-the 768th bit base of 5 ' end, the protein that coding is made up of 256 amino-acid residues, infer that its molecular weight is 31.731kDa, iso-electric point pI value 9.37.Utilize the SMART instrument that the amino acid residue sequence of inferring is carried out function prediction, this albumen contains a typical EREBP/AP2 structural domain as a result, be SEQ ID № in the sequence table: 1 from the 58th the-the 115th amino acids residue of aminoterminal (N end), and this structural domain is made up of 58 amino-acid residues.In addition, there is a typical acidic activated zone (acidicactivation region in this proteic C-end; AAR), SEQ ID № in the sequence table: 1 from the 116th the-the 256th amino acids residue of aminoterminal, and this zone has and is beneficial to this genetic transcription.Above-mentioned analytical results shows that JcDREB is a kind of transcription factor, belongs to the AP2 protein family.

The homology and the systematic evolution tree analysis of the DREB2 proteinoid encoding amino acid sequence that two, other has been cloned in JcDREB and the plant

(the GenBank accession number is: the AAF76898 spinach to the aminoacid sequence of the ERF proteinoid that other has been cloned in JcDREB and the plant to utilize DNAMAN software; The AA013360 tomato; The NP_177931 Arabidopis thaliana; The ABB89755 paper mulberry; AAT39542 upland cotton; The AAZ03388 soybean; The AAQ57226 soybean; The ABB89754 asparagus; The AAM80486 corn; The AAZ08560 wheat; The XP_467836 paddy rice; The AAZ14831 Cortex jatrophae) carries out homology analysis and systematic evolution tree analysis, the homology analysis result as shown in Figure 6, the homology of JcDREB and monocotyledon rice, wheat DREB proteinoid is respectively: 28.07%, 27.01%, itself and dicotyledons spinach, tomato, Arabidopis thaliana, the proteic homology of cotton are respectively: 56.13%, 45.07%, 37.14% and 35.62%, the homology that shows JcDREB and monocotyledons DREB proteinoid is very low, and higher with the albumen homology in the dicotyledons.The systematic evolution tree analytical results as shown in Figure 7, isolating DREB2 proteinoid and isolating such the proteic homology from monocotyledons are very low from monocotyledons, be about 27%, show that bigger difference has appearred in DREB proteinoid during evolution, but this albumen is relatively conservative in each class plant.

Three, the structural domain of JcDREB and structure prediction

Analyze the structural domain of JcDREB with SMART server (http://coot.embl-heidelberg.de/SMART/), analytical results is shown in figure A among Fig. 8, in the protein sequence of forming by 256 amino-acid residues, from the 58th the-the 115th amino acids residue of aminoterminal is typical EREBP/AP2 knot leprosy territory, shows that it is a member in the EREBP/AP2 family.Protein structure with CPHmodels-2.0 server (http://genome.cbs.dtu.dk/services/CPHmodels-2_0 Server-3D.htm) prediction JcDREB, the result is shown in the figure B among Fig. 8, and JcDREB contains a typical α spiral and three βZhe Die structures as a result.

The structural analysis of embodiment 3, JcDREB genomic dna

Extract among the embodiment 1 through the genomic dna of the young seedling of Jatropha of deepfreeze and as template, primer JcDREBW-1 and and the guiding of JcDREBW-2 under, carry out pcr amplification and analyze the structure of JcDREB gene, after reaction finishes, the PCR product is carried out 1% agarose gel electrophoresis to be detected, detected result is (swimming lane M is molecular weight standard Marker III, and swimming lane 1 is a pcr amplification product) as shown in Figure 9, and pcr amplification goes out the specific band of the about 800bp of length as a result.CDNA with JcDREB is a template again, carry out pcr amplification with above-mentioned identical primer, after reaction finishes, the PCR product is carried out 1% agarose gel electrophoresis to be detected, (swimming lane M is molecular weight standard Marker III to detected result as shown in Figure 9, swimming lane 2 is a pcr amplification product), amplify the specific band of the about 800bp of length equally.Further above-mentioned two kinds of specific fragments are cloned and check order, sequencing result shows that the genome sequence of JcDREB and corresponding cDNA sequence are identical, shows in this gene inside and does not contain intron.

Embodiment 4, JcDREB homologous gene are analyzed

Extract among the embodiment 1 genomic dna through the young seedling of Jatropha of deepfreeze, use Xba I, BamH I, Hind III and four kinds of restriction enzyme enzymolysis of EcoR I respectively, JcDREB cDNA with the Digoxigenin mark is that probe carries out Southern hybridization detection, detected result shows that JcDREB is a single copy gene as shown in figure 10.

The tissue specific expression analysis of embodiment 5, JcDREB gene

Analyze the tissue specific expression pattern of JcDREB, concrete experimental technique is: extract among the embodiment 1 total RNA of root through the young seedling of Jatropha of deepfreeze, stem, three kinds of tissues of leaf respectively, utilize total length primer JcDREBW-1 and JcDREBW-2 to carry out RT-PCR and analyze.As interior mark, all cDNA templates are carried out sxemiquantitative with the Actin gene.After reaction finishes, the RT-PCR product is carried out 1% agarose gel electrophoresis to be detected, (swimming lane M is molecular weight standard Marker III to detected result as shown in figure 11, R: root, S: stem, L: leaf), show that the expression of JcDREB in three kinds of tissues there are differences, with the expression amount in the root is minimum, and the expression amount in stem and leaf is higher.

Embodiment 6, the JcDREB expression pattern analysis under different abiotic stress factor conditions

Analyze the relation of JcDREB in the different abiotic stress factors with some of transcriptional level, concrete experimental technique is: the low temperature (4 ℃), high salt (200mM NaCl), arid (20%PEG) and the dormin ABA (100 μ M) that the young seedling of Jatropha in 2 weeks of normal growth are carried out different time (0,0.5,3.0,6.0,12.0,24h) respectively coerce processing.After drawing materials, extract total RNA, with the expression pattern of Nortern Blot methods analyst JcDREB under various stress conditions, (the painted rRNA of EB represents that the applied sample amount of every swimming lane is identical to the result as shown in figure 12 earlier in batches; The Actin gene carries out sxemiquantitative as interior mark to the cDNA template), this gene is expressed abundance under different stress conditions variant, hybridization signal apparent in view (among Figure 12 A figure) under the low temperature stress condition only, other handles equal amixia signal.For analyzing the expression pattern of JcDREB under other adverse environmental factor, use the expression pattern (seeing the B-D figure among Figure 12) of JcDREB under other several stress conditions of RT-PCR methods analyst again, RT-PCR result shows that JcDREB obviously is subjected to low temperature induction at transcriptional level, hybridization signal just appears behind the subzero treatment 0.5h, along with the lengthening in treatment time, expression amount increases sharply, and reaches maximum value to 12h, expression level reduces gradually afterwards, returns to originally expression level again to 24h.Under the inducing of PEG, this gene presents faint ascendant trend at the beginning, (seeing the figure B among Figure 12) on a declining curve behind the 12h.The processing of ABA does not influence the expression (see figure C in Figure 12) of JcDREB at transcriptional level.And under high salt inductive condition, this expression of gene presents downtrending always.

The acquisition of embodiment 7, JcDREB transgenic paddy rice

One, the structure of JcDREB plant expression vector

The JcDREB that embodiment 1 is obtained is cloned between the EcoR V restriction enzyme site of carrier pMD18-T (TaKaRa company), obtains carrying the recombinant vectors of JcDREB, called after pMD18-JcDREB.PMD18-JcDREB is carried out single endonuclease digestion with restriction enzyme Xba I, mend and put down, oneself connects then, thereby removes the XbaI enzyme cutting site on the pMD18-JcDREB, use Sal I and this carrier of Sac I double digestion again, obtain the small pieces that contains JcDREB that a length is about 800bp.With Sal I and Sac I double digestion plasmid pGEX-KG (available from general Jino, Beijing genome biotech firm), because pGEX-KG goes up restricted property restriction endonuclease Sal I and Sac I recognition site, and only at a distance of 6bp, so the small pieces of downcutting can manifest in agarose gel electrophoresis, so have only one and the almost equal band of original size, it is linked to each other with small pieces that the length of downcutting from above-mentioned pMD18-JcDREB is about 800bp, obtain a recombinant plasmid, called after pGEX-KG-JcDREB.With Xba I and Sac I double digestion plasmid pGEX-KG-JcDREB and plant expression vector p3301-BI121 (available from CAMBIA), the large stretch of phase failure company of the small segment that will contain JcDREB and the 11.4kb that downcuts from plasmid p3301-BI121 from the 800bp that plasmid pGEX-KG-JcDREB downcuts, promptly be built into the high-efficiency plant binary expression vector that contains the complete single open reading frame of JcDREB, called after p3301-BI121-JcDREB, its building process as shown in figure 13.The plant expression vector p3301-BI121-JcDREB that builds is carried out double digestion with Xba I and SacI to be identified, enzyme is cut product carry out the detection of 1% agarose gel electrophoresis, (swimming lane M is MarkerIII to detected result as shown in figure 14, swimming lane 1 is cut product for enzyme), cut the band that obtains a 800bp through enzyme, show that the purpose fragment correctly connects in the plant expression vector.Be template with the p3301-BI121-JcDREB plasmid again, utilizing JcDREB Auele Specific Primer JcDREBW-1 and JcDREBW-2 to carry out PCR detects, the PCR product is carried out 1% agarose gel electrophoresis to be detected, (swimming lane M is Marker III to detected result as shown in figure 15, swimming lane 1 is the PCR product), pcr amplification goes out the band of a 800bp, proves that further the JcDREB gene fragment correctly is connected in the carrier, has obtained the JcDREB plant expression vector of insertion sequence and correct position.

Two, the foundation of rice plant regeneration system and selective agent and screening concentration determines

Rice paddy seed after the sterilization is at N ₆D substratum (Chu, C.C, C.S.Wang, C.C.Sun, C.Hsu, K.C.Yin, C.Y.Chu.1975.Establishment of an efficient medium ofr anther culture of ricethrough comparative experiments on the nitrogen sources.Sci.Sinica, 18:659-668) go up dark culturing and begin germination (seeing figure A and figure A1 among Figure 16) after 2 days, begin to have callus to occur after 5 days, after 2 weeks, callus is downcut, select wherein white or flaxen densification and do not have the fritter (seeing figure B and figure B1 among Figure 16) of aquation, the small-particle that is divided into diameter and is 3-5mm places division culture medium (Hiei, Y.S.Ohta, T.Komari and T.Kumashiro.1994.Efficient transformationof rice mediated by Agrobacterium and sequence analysis of the boundaries ofthe T-DNA.Plan is J.6:271-282) on carry out differentiation culture, 15 days subcultures once, after 30-40 days, green cell group occurs on the good callus lines of individual states, and grow thus and be whole plant (seeing figure C and figure C1 among Figure 16).

Organize respectively inoculation to contain in the division culture medium of 100mg/L kantlex, (25mg/L, 50mg/L) Totomycin, (5mg/L, 10mg/L) ppt the paddy rice rice callus and carry out the antibiotics sensitivity experiment, as a result in three kinds of selective agents, kantlex to the restraining effect of rice callus tissue growth a little less than, in kantlex concentration is on the substratum of 100mg/L, the growth of obviously not observing the rice callus tissue is suppressed, and browning also do not occur.Yet, the rice callus tissue is relatively more responsive to Totomycin, cultivate containing on the substratum of Totomycin, observe after 10 days, callus lines is wherein obviously compared according to there not being the little of added with antibiotic, and be on the substratum of 50mg/L in Totomycin concentration, some callus begin brownization, after 2 weeks, are the callus that also occurs brownization on the substratum of 25mg/L in Totomycin concentration, and be on the substratum of 50mg/L in Totomycin concentration, the callus of brownization reaches 40%.The rice callus tissue is also relatively more responsive to ppt, in ppt concentration is on the substratum of 10mg/L, after 10 days, callus begins brownization, after 20 days, brownization callus reaches 50%, in ppt concentration is on the substratum of 5mg/L, and after 15 days, callus begins brownization, after 20 days, brownization callus reaches 30%.Above-mentioned experimental result shows that the screening of paddy rice resistant calli adopts the ppt of the Totomycin contain 25-50mg/L or 5-10mg/L proper.

Three, the acquisition of JcDREB transgenic paddy rice

The plant expression vector p3301-BI121-JcDREB that step 1 is made up imports among the Agrobacterium EHA105 by freeze-thaw method, the screening positive recombinant.Rice paddy seed is at N ₆Dark culturing is after 2 weeks on the D substratum, downcut callus, select wherein white or flaxen densification and do not have the fritter of aquation, infect with transforming the positive bacterium bacterium liquid of recombinating of the Agrobacterium EHA105 that plasmid p3301-BI121-JcDREB is arranged, the callus after will infecting is inoculated in N ₆25-28 ℃ of dark down cultivation on the D substratum, after 3 days, bacterium colony appears around the callus, clean callus, be transferred to then on the division culture medium that ppt concentration is 5mg/L and screen, after 15 days, a small amount of callus begins brownization, and then callus is transferred on the division culture medium that ppt concentration is 10mg/L screens, up to the death of brownization callus, take out the not callus with resistance of brownization, place on the aseptic filter paper 25-28 ℃ of dry the cultivation 2 days, callus dehydration shrinkage after drying, change it over to division culture medium again, after 2 days, the callus of shrinkage recovers, after 15 days, green bud point appears in the callus surface, treats to change over to when budlet grows to 5cm root media MS ₀On take root.When treating that seedling grows to the 8-10cm left and right sides, its root system is relatively more flourishing, opens triangular flask and seals the domestication that film carries out before transplant in the greenhouse and practice seedling, after 3 days, these transplantation of seedlings to the greenhouse, is detected transfer-gen plant, and the result obtains positive transfer-gen plant 15 strains altogether.

The resistance experiment of embodiment 8, JcDREB transgenic paddy rice

The JcDREB transgenic paddy rice seed of wild-type rice paddy seed (contrast) and embodiment 7 acquisitions is seeded in 1/4 MS respectively and contains on the resistant panel of 10mg/L ppt, make its sprouting, after two weeks of growth on the resistant panel, seedling is written in the soil, cultivates at room temperature and carry out 4 ℃ after two weeks and coerce processing.Phenotypes are observed in two week backs, and take out from compost for surveying plant above-mentioned, get over-ground part and survey fresh weight, dry weight, simultaneously, get above-mentioned vegetable material and claim dry weight at 70 ℃ after being baked to constant weight.

The transgenic paddy rice seedling is handled through 4 ℃ did not as a result all have the freeze injury phenomenon in four days, and yellow leaf has appearred in contrast.The fresh weight of transgenic paddy rice and dry weight are all compared according to heavy after two weeks, are remarkable relation.Above-mentioned experimental result shows that JcDREB of the present invention can strengthen the low temperature tolerance ability of plant, is applied thereby can be used as the cold-resistant engineered candidate gene of plant, is used for improveing the cold resistance shape of plant.

Sequence table

<160>4

<210>1

<211>256

<212>PRT

＜213〉Cortex jatrophae (Jatropha curcas)

<400>1

Met?Ser?Tyr?Ser?Asn?Pro?Asp?Pro?Pro?Asp?Pro?Asp?Pro?Asn?Pro?Leu

1 5 10 15

Pro?Glu?Ser?Lys?Trp?Ile?Gln?Phe?Pro?Glu?Phe?Pro?His?Pro?Lys?Pro

20 25 30

Thr?Trp?Leu?Asn?Phe?Leu?Gly?Pro?Lys?Pro?Val?Pro?Met?Lys?Gln?Val

35 40 45

Gly?Ser?Pro?Pro?Lys?Pro?Thr?Lys?Leu?Tyr?Arg?Gly?Val?Arg?Gln?Arg

50 55 60

His?Trp?Gly?Lys?Trp?Val?Ala?Glu?Ile?Arg?Leu?Pro?Lys?Asn?Arg?Thr

65 70 75 80

Arg?Leu?Trp?Leu?Gly?Thr?Phe?Asp?Thr?Ala?Glu?Glu?Ala?Ala?Leu?Ala

85 90 95

Tyr?Asp?Lys?Ala?Ala?Tyr?Lys?Leu?Arg?Gly?Asp?Phe?Ala?Arg?Leu?Asn

100 105 110

Phe?Pro?Asn?Leu?Arg?His?Gln?Gly?Ser?His?Ile?Glu?Gly?Ser?Phe?Gly

115 120 125

Glu?Tyr?Lys?Pro?Leu?His?Ser?Ser?Val?Asp?Ala?Lys?Leu?Gln?Ala?Ile

130 135 140

Cys?Gln?Ser?Leu?Ala?Glu?Ser?Gln?Lys?Gln?Gly?Gly?Lys?Ala?Glu?Lys

145 150 155 160

Gln?Ser?Asn?Ser?Ser?Ala?Lys?Lys?Lys?Thr?Ser?Val?Gly?Thr?Thr?Pro

165 170 175

Ala?Thr?Ala?Glu?Lys?Val?Lys?Glu?Ala?Lys?Ala?Pro?Gln?Gln?Val?Val

180 185 190

Pro?Asp?Lys?Cys?Cys?Lys?Val?Glu?Thr?Pro?Ser?Ser?Val?Leu?Thr?Glu

195 200 205

Ser?Glu?Ala?Ser?Gly?Gly?Ser?Ser?Pro?Leu?Ser?Asp?Leu?Thr?Phe?Pro

210 215 220

Asp?Leu?Glu?Glu?Ala?Pro?Leu?Asp?Val?Asp?Ser?Gly?Asn?Phe?Asn?Leu

225 230 235 240

Glu?Lys?Tyr?Pro?Ser?Tyr?Glu?Ile?Asp?Trp?Ala?Ser?Leu?Leu?Ser?Ser

245 250 255

<210>2

<211>768

<212>DNA

＜213〉Cortex jatrophae (Jatropha curcas)

<400>2

atgtcttact?ccaacccaga?tccaccagat?ccagacccaa?atccattacc?agaatcaaaa 60

tggattcaat?ttccagaatt?tccacaccca?aaaccaacat?ggcttaactt?tttaggtccg 120

aaacccgtgc?ccatgaagca?ggtgggttca?ccaccaaaac?ccactaagct?ctacagagga 180

gtaaggcagc?gacattgggg?caaatgggtt?gccgagatcc?gactacccaa?gaaccgtaca 240

cgactctggc?ttggtacttt?tgacacagca?gaagaagccg?ctttagctta?tgacaaagcg 300

gcgtacaaac?tccgtggcga?cttcgcgaga?cttaacttcc?ctaacctccg?ccaccaaggg 360

tcccacattg?aaggcagctt?cggcgagtat?aagcctctcc?attcctcggt?cgatgcgaaa 420

ctgcaagcta?tttgtcaaag?cttagcagaa?tcgcagaaac?aaggaggaaa?agcagagaag 480

caatcaaact?cgtcagcgaa?aaagaagact?tcggtgggga?ctactccagc?gacggcggag 540

aaggttaagg?aagctaaggc?accgcaacag?gttgttccgg?acaagtgttg?caaggtcgag 600

acaccatcgt?cagtgttgac?agaaagtgaa?gcctctggcg?gatcttcacc?gttgtcggat 660

cttacgtttc?cggatctaga?agaggcacca?ttggatgttg?attctggaaa?ttttaatttg 720

gagaagtacc?catcttatga?aattgattgg?gcttctcttt?tatcttct 768

<210>3

<211>813

<212>DNA

＜213〉Cortex jatrophae (Jatropha curcas)

<400>3

tttccacacc?caaaaccaac?atggcttaac?tttttaggtc?cgaaacccgt?gcccatgaag 60

caggtgggtt?caccaccaaa?acccactaag?ctctacagag?gagtaaggca?gcgacattgg 120

ggcaaatggg?ttgccgagat?ccgactaccc?aagaaccgta?cacgactctg?gcttggtact 180

tttgacacag?cagaagaagc?cgctttagct?tatgacaaag?cggcgtacaa?actccgtggc 240

gacttcgcga?gacttaactt?ccctaacctc?cgccaccaag?ggtcccacat?tgaaggcagc 300

ttcggcgagt?ataagcctct?ccattcctcg?gtcgatgcga?aactgcaagc?tatttgtcaa 360

agcttagcag?aatcgcagaa?acaaggagga?aaagcagaga?agcaatcaaa?ctcgtcagcg 420

aaaaagaaga?cttcggtggg?gactactcca?gcgacggcgg?agaaggttaa?ggaagctaag 480

gcaccgcaac?aggttgttcc?ggacaagtgt?tgcaaggtcg?agacaccatc?gtcagtgttg 540

acagaaagtg?aagcctctgg?cggatcttca?ccgttgtcgg?atcttacgtt?tccggatcta 600

gaagaggcac?cattggatgt?tgattctgga?aattttaatt?tggagaagta?cccatcttat 660

gaaattgatt?gggcttctct?tttatcttct?tagatttggt?tatgttatgt?tatttatctt 720

ttatctttat?tttgcagtta?tgtttagttc?ttaggcgtgt?ggttgctgca?atgagttttt 780

ggcagttgca?gagccaaaaa?aaaaaaaaaa?aaa 813

<210>4

<211>507

<212>DNA

＜213〉Cortex jatrophae (Jatropha curcas)

<400>4

tctgatctag?aggtaccgga?tccttttttt?tttttttttt?atcatcttac?tccaacccag 60

atccaccaga?tccagaccca?aatccattac?cagaatcaaa?atggattcaa?tttccagaat 120

ttccacaccc?aaaaccaaca?tggccttaac?tttttaggtc?cgaaacccgt?gcccatgaag 180

caggtgggtt?caccaccaaa?acccactaag?ctctacagag?gagtaaggca?gcgacattgg 240

ggcaaatggg?ttgccgagat?ccgactaccc?aagaaccgta?cacgactctg?gcttggtact 300

tttgacacag?cagaagaagc?cgctttagct?tatgacaaag?cggcgtacaa?actccgtggc 360

gacttcgcgt?gacttaactt?ccctaacctc?cgccaccaag?ggtcccacat?tgaaggcagc 420

ttcggcgagt?ataagcctct?ccattcctcg?gtcgatgcga?aactgcaagc?tatttgtcaa 480

agcttagcag?aatcgcagaa?acaagga 507

Claims (10)

1, Cortex jatrophae cold-induced transcription factor is the protein with one of following amino acid residue sequences:

1) the SEQ ID № in the sequence table: 1;

2) with SEQ ID № in the sequence table: 1 amino acid residue sequence is through replacement, disappearance or the interpolation of one to ten amino-acid residue and have the protein of the regulation and control stress resistance of plant of transcriptional activation function.

2, Cortex jatrophae cold-induced transcription factor according to claim 1 is characterized in that: described albumen has SEQ ID № in the sequence table: 1 amino acid residue sequence.

3, the gene of the described Cortex jatrophae cold-induced transcription factor of coding claim 1 is one of following nucleotide sequence:

1) SEQ ID № in the sequence table: 2 dna sequence dna;

2) SEQ ID № in the code sequence tabulation: 1 dna sequence dna.

4, the gene of Cortex jatrophae cold-induced transcription factor according to claim 3 is characterized in that: described gene has SEQ ID № in the sequence table: 2 dna sequence dna.

5, contain the described expression carrier of claim 3, transgenic cell line and host bacterium.

6, a kind of method that improves plant stress tolerance is with the described Cortex jatrophae cold-induced transcription factor of claim 3 gene transfered plant tissue or cell, obtains the plant that resistance of reverse improves.

7, method according to claim 6 is characterized in that: described Cortex jatrophae cold-induced transcription factor gene imports plant tissue or cell by the plant expression vector that contains described Cortex jatrophae cold-induced transcription factor gene.

8, method according to claim 7 is characterized in that: the carrier that sets out that is used to make up described plant expression vector is p3301-BI121, pBI121, pBin19, pCAMBIA2301, pCAMBIA1301 or pCAMBIA1300.

9, method according to claim 8 is characterized in that: described plant expression vector is p3301-BI121-JcDREB.

10, method according to claim 6 is characterized in that: described plant is paddy rice, wheat, soybean, tobacco, corn, rape, Chinese sorghum, cotton, clover, Cortex jatrophae or Arabidopis thaliana.

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