CN100587070C

CN100587070C - Barbadosnut salt induced gene and application

Info

Publication number: CN100587070C
Application number: CN200610000100A
Authority: CN
Inventors: 唐明娟; 沈世华; 刘杰
Original assignee: Institute of Botany of CAS
Current assignee: Institute of Botany of CAS
Priority date: 2006-01-10
Filing date: 2006-01-10
Publication date: 2010-02-03
Anticipated expiration: 2026-01-10
Also published as: CN1807627A

Abstract

This invention discloses a Jatropha curcas slat inducing transcription factor and its coding gene and application. The related enzyme is one the following amino acid remnant radical sequences: 1) SEQID No: 1 in the sequence list; 2) the protein that can control the plant resistant reverse and replace, minus or add the acid remnant radical sequences of the SEQ I D No: 1 in the sequence list through one to ten amino acid remnant radicals. Using this transferring gene technique and transplanting the gene in the plant host can obtain the transfer gene plant that has the improved aridity resist and high salt resist ability.

Description

Cortex jatrophae salt induced 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 salt 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

Natural plant grows it and is suppressed, even cause plant death through the harm of coercing that regular meeting suffers severe environment such as arid, saline and alkaline and low temperature.The soil salinization is a main environment-stress factor that influences the plant-growth amount.According to statistics, 7.5% of ball land area is taken up an area of in the saltings, but nearly in the world 1/3rd irrigation soils no longer are fit to plant growth because saltiness is higher at present.China has more than 1,500,000,000 mu of salinification soil approximately, mainly is distributed in coastal and arid and semi-arid lands such as Shandong, Hebei, northeast, Xinjiang, Gansu.Along with the high speed development of the sharp increase of China's population and industry, the arable land sharply descends, and unreasonable irrigation, farming etc. have caused the secondary salinization in a large amount of good farmlands.Cause China to be ploughed and sharply descend year by year, grain and forest that China in serious threat produce.Therefore, how to utilize and develop more than one hundred million mu of salinization soils of China, cultivation can be on salinization soil well-grown salt tolerant new variety, 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 high salt and drought-induced expression.

Salt induced transcription factor provided by the present invention, name is called JcERF, 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 253 amino-acid residues, for conservative EREBP/AP2 structural domain, is acidic activated zone from the 81st the-the 253rd amino acids residue of aminoterminal from the 25th the-the 80th amino acids residue of aminoterminal (N end).

The gene (JcERF) of coding Cortex jatrophae salt induced transcription factor JcERF 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 759 based compositions, its encoding sequence is from the 1st the-the 759th 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 76th the-the 243rd bit base coding, from the 244th the-the 759th 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.

Arbitrary segmental primer is to also within protection scope of the present invention among the amplification JcERF.

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 salt induced transcription factor JcERF of coding, obtains the plant that resistance improves.

Described Cortex jatrophae salt induced transcription factor gene JcERF can import explant by the plant expression vector that contains described Cortex jatrophae salt induced transcription factor gene JcERF; 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 JcERF 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 salt induced transcription factor JcERF gene of structure is p3301-BI121-JcERF.

Carry gene JcERF 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 both 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 salt induced transcription factor JcERF and an encoding gene thereof that derives from EREBP/AP2 family.Experiment showed, that its encoding gene JcERF is subjected to high salt and drought-induced expression, the ability of adjustable plant opposing arid, low temperature and environment stress such as saline and alkaline, thus significantly improve the resistance of plant.JcERF 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

The agarose gel electrophoresis detected result of Fig. 1 for coercing the total RNA of young seedling of Jatropha of processing through NaCl

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 JcERF full-length cDNA of PCP amplification

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

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

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

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

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

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

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

Figure 12 is the expression pattern analytical results of JcERF under salt stress

Figure 13 is the expression pattern analytical results of JcERF under drought stress

Figure 14 is the expression pattern analytical results of JcERF under low temperature stress

Figure 15 is the expression pattern analytical results of JcERF under ABA coerces

Figure 16 is the structure synoptic diagram of carrier p3301-BI121-JcERF

Figure 17 cuts qualification result for the enzyme of carrier p3301-BI121-JcERF

Figure 18 is the PCR qualification result of carrier p3301-BI121-JcERF

Figure 19 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 salt induced transcription factor gene JcERF cDNA complete sequence

The acquisition of Cortex jatrophae salt induced transcription factor gene JcERF full length cDNA sequence may further comprise the steps:

One, the clone of Cortex jatrophae salt induced transcription factor gene JcERF 3 ' terminal sequence

1, the extraction of vegetable material processing and total RNA

Be material with the young seedling of Jatropha earlier, NaCl solution-treated with 300mM is 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 salt induced transcription factor gene JcERF 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:

JS1：5’-AGG(A/T)T(A/T)TGGCT(C/T)GG(A/T/G)AC(A/T)TT-3’

JS2：5’-CG(G/T)(A/G)T(T/C)TGG(T/C)T(A/T)GG(G/T)AC(C/T)TT-3’

The young seedling of Jatropha RNA through 300mM NaCl solution-treated that extracts with step 1 is a template, with PlantRNAtrip Reagent Kit2 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 treating 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, increase with dT-Adaptor primer pairing carrying out sleeve type PCR respectively with above-mentioned two degenerated primers, 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 ℃ earlier, and 5min; 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 '-JcERF, difference called after pMD-3 '-JcERF, carry out the BLAST analysis to its order-checking and to sequencing result, this fragment length is 792bp 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 salt induced transcription factor gene JcERF 5 ' terminal sequence

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

JcERF-5-gsp1：5’-GATGAATCGGAATCACTGTGG-3’

JcERF-5-gsp2：5’-GTCCGATCCAATCTGCACA-3’

The young seedling of Jatropha RNA through 300mM NaCl solution-treated 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: RNA 10ul, primer JcERF-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 3 nested primers, the PCR reaction system is: primer JcERF-5-gsp2 1ul, water 4.5. μ l, 10 * PCR damping fluid, 2.5 μ l, 25mMMgCl 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 ' JcERF, carry out the BLAST analysis to its order-checking and to sequencing result, this fragment length is 294bp as a result, has SEQ ID № in the sequence table: 4 nucleotide sequence, 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 salt induced transcription factor gene JcERF full length cDNA sequence and PCR detect

The length of utilizing step 1 and step 1 to obtain is the overlap between 792bp and the 294bp fragment, and splicing obtains the full length cDNA sequence of JcERF by DNA software DNAMAM.This sequence has SEQ ID № in the sequence table: 2 polynucleotide sequence.SEQ ID № in the sequence table: 2 by 759 based compositions, its encoding sequence is from the 1st the-the 759th 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 253 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):

JcERFW-1：5’-AAACCCGACCTTCTTTCGCT-3’

JcERFW-2：5’-GAAGTAGCCTGATTTTGA-3’

Is template through the young seedling of Jatropha RNA of 300mM NaCl solution-treated through the reverse transcription synthetic first chain cDNA with step 1, under the guiding of primer JcERFW-1 and JcERFW-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 MarkerIII 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 759bp as a result, it is checked order, sequencing result and above-mentioned splicing result are identical in the amplification part, its total length is 759bp, show that 3 ' RACE fragment and 5 ' RACE fragment of being cloned belong to same gene, with this unnamed gene is JcERF, with its proteins encoded called after JcERF.

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

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

Utilize DNAMAN software that the full length cDNA sequence of the JcERF 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 the acid activatable zone), this sequence total length 759bp is ORF from the 1st the-the 759th bit base of 5 ' end, the protein that coding is made up of 253 amino-acid residues, infer that its molecular weight is 27.474kDa, iso-electric point pI value 9.09.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 25th the-the 80th amino acids residue of aminoterminal (N end), and this structural domain is made up of 56 amino-acid residues.In addition, there is a typical acidic activated zone (acidic activationregion in this proteic C-end; AAR), SEQ ID № in the sequence table: 1 from the 81st the-the 253rd amino acids residue of aminoterminal, and this zone has and is beneficial to this genetic transcription.Above-mentioned analytical results shows that JcERF 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 JcERF and the plant

(the GenBank accession number is: the little salt mustard of AAM19703 to the aminoacid sequence of the ERF proteinoid that other has been cloned in JcERF and the plant to utilize DNAMAN software; The AAQ96342 pigeon grape; The NP_188139 Arabidopis thaliana; AAR84424 dish green pepper; The AA034705 tomato; The BAD99476 tobacco; AAD09248 flower beans; AAD00708 flower beans; AAV51938 upland cotton; The ABB51576 Caulis et Folium Brassicae capitatae; The AAV66332 cucumber; The AAV98701 paddy rice; The AAY82590 Cortex jatrophae) carries out homology analysis and systematic evolution tree analysis, the homology analysis result as shown in Figure 6, the homology of JcERF and monocotyledons ERF proteinoid OsERF3 is: 39%, the homology of itself and dicotyledons Arabidopis thaliana, cotton, grape proteinoid is respectively: 55%, 51%, 57%, the homology that shows JcERF 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 39%, show that bigger difference has appearred in ERF proteinoid during evolution, but this albumen is relatively conservative in each class plant.

Three, the structural domain of JcERF and structure prediction

Analyze the structural domain of JcERF 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 254 amino-acid residues, from the 25th the-the 80th 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 JcERF, the result is shown in the figure B among Fig. 8, and JcERF contains a typical α spiral and three βZhe Die structures as a result.

The structural analysis of embodiment 3, JcERF genomic dna

Extract among the embodiment 1 through the genomic dna of the young seedling of Jatropha of 300mM NaCl solution-treated and as template, primer JcERFW-1 and and the guiding of JcERFW-2 under, carry out pcr amplification and analyze the structure of JcERF 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 JcERF 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 JcERF and corresponding cDNA sequence are identical, shows in this gene inside and does not contain intron.

Embodiment 4, JcERF homologous gene are analyzed

Extract among the embodiment 1 genomic dna through the young seedling of Jatropha of 300mM NaCl solution-treated, use XbaI, BamH I, Hind III and four kinds of restriction enzyme enzymolysis of EcoR I respectively, JcERFcDNA with the Digoxigenin mark is that probe carries out Southern hybridization detection, detected result shows that JcERF is a single copy gene as shown in figure 10.

The tissue specific expression analysis of embodiment 5, JcERF gene

Analyze the tissue specific expression pattern of JcERF, concrete experimental technique is: extract root, the stem of the young seedling of Jatropha of handling through salt among the embodiment 1, total RNA of three kinds of tissues of leaf respectively, utilize total length primer JcERFW-1 and JcERFW-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 JcERF 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 JcERF expression pattern analysis under different abiotic stress factor conditions

Analyze the relation of JcERF in the different abiotic stress factors with some of transcriptional level, concrete experimental technique is: the low temperature (4 ℃), high salt (250mM 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 in batches, extract total RNA, earlier with the expression pattern of Nortern Blot methods analyst JcERF under various stress conditions, to express abundance under above-mentioned different stress conditions all lower for this gene as a result, Nortern Blot results of hybridization no signal or signal a little less than, other handles equal amixia signal.For analyzing the expression pattern of JcERF under other adverse environmental factor, use the expression pattern of JcERF under the above-mentioned several stress conditions of RT-PCR methods analyst again, with the Actin gene as interior mark, the cDNA template is carried out sxemiquantitative, RT-PCR result such as Figure 12-shown in Figure 15, show that JcERF is induced by salt obviously at transcriptional level, 300mM NaCl hybridization signal just occurs after handling 0.5h, lengthening along with the treatment time, expression amount increases sharply, reach maximum value to 6h, expression level reduces gradually afterwards, returns to originally expression level (seeing Figure 12) again to 24h.Under the inducing of PEG, this gene changes fainter at the beginning, and expression amount rises rapidly then, and 2-6h reaches the climax, and (seeing Figure 13) then descends rapidly again.Under low temperature and ABA processing, the transcriptional expression of JcERF does not change (seeing Figure 14 and Figure 15) basically.Above-mentioned experimental result shows that the transcriptional expression of JcERF is subjected to salt and drought-induced, and low temperature and ABA are inoperative to it.

The acquisition of embodiment 7, JcERF transgenic paddy rice

One, the structure of JcERF plant expression vector

The JcERF 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 JcERF, called after pMD18-JcERF.PMD18-JcERF is carried out single endonuclease digestion with restriction enzyme Xba I, mend and put down, oneself connects then, thereby removes the Xba I restriction enzyme site on the pMD18-JcERF, use Sal I and this carrier of Sac I double digestion again, obtain the small pieces that contains JcERF 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-JcERF is about 800bp, obtain a recombinant plasmid, called after pGEX-KG-JcERF.With Xba I and Sac I double digestion plasmid pGEX-KG-JcERF and plant expression vector p3301-BI121 (available from CAMBIA), the large stretch of phase failure company of the small segment that will contain JcERF and the 11.4kb that downcuts from plasmid p3301-BI121 from the 800bp that plasmid pGEX-KG-JcERF downcuts, promptly be built into the high-efficiency plant binary expression vector that contains the complete single open reading frame of JcERF, called after p3301-BI121-JcERF, its building process as shown in figure 16.The plant expression vector p3301-BI121-JcERF that builds is carried out double digestion with Xba I and Sac I 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 17, 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-JcERF plasmid again, utilizing JcERF Auele Specific Primer JcERFW-1 and JcERFW-2 to carry out PCR detects, the PCR product is carried out 1% agarose gel electrophoresis to be detected, (swimming lane M is MarkerIII to detected result as shown in figure 18, swimming lane 1 is the PCR product), pcr amplification goes out the band of a 800bp, proves that further the JcERF gene fragment correctly is connected in the carrier, has obtained the JcERF 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 cultureof rice through 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 19) 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 19) 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 19).

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 JcERF transgenic paddy rice

The plant expression vector p3301-BI121-JcERF that step 1 is made up imports among the Agrobacterium EHA105 by freeze-thaw method, the screening positive recombinant.Rice paddy seed is in dark culturing on the N6D substratum after 2 weeks, 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-JcERF 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 salt resistance experiment of embodiment 8, JcERF transgenic paddy rice

The JcERF transgenic paddy rice seed of wild-type rice paddy seed (contrast) and embodiment 7 acquisitions is seeded in 1/4MS 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, cultivate at room temperature and carry out NaCl after two weeks and coerce processing, with the NaCl pouring plant of 300mM, pouring in two days once.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 at the NaCl of 300mM and two weeks yellow leaf phenomenon all do not occurred as a result, 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 JcERF of the present invention can strengthen the salt tolerance of plant, is applied thereby can be used as the improvement of plant salt-tolerant engineering candidate gene, is used for improveing the salt-tolerance character of plant.

Sequence table

<160>4

<210>1

<211>253

<212>PRT

＜213〉Cortex jatrophae (Jatropha curcas)

<400>1

Met?Lys?Trp?Leu?Gln?Glu?Arg?Ser?Asn?Ser?Thr?Gly?Asn?Asn?Leu?Asn

1 5 10 15

Gln?Asn?Ser?Asn?Thr?Ala?Glu?Thr?Arg?Tyr?Arg?Gly?Val?Arg?Lys?Arg

20 25 30

Pro?Trp?Gly?Arg?Tyr?Ala?Ala?Glu?Ile?Arg?Asp?Pro?Gly?Lys?Lys?Thr

35 40 45

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

50 55 60

Tyr?Asp?Ala?Ala?Ala?Arg?Glu?Phe?Arg?Gly?Ser?Lys?Ala?Lys?Thr?Asn

65 70 75 80

Phe?Pro?Thr?Val?Thr?Glu?Leu?Asn?Asn?Ala?Ala?Ala?Ala?Ala?Val?Ala

85 90 95

Ala?Gly?Ala?Val?Thr?Val?Ala?Arg?Ser?Pro?Ser?Gln?Ser?Ser?Thr?Val

100 105 110

Glu?Ser?Ser?Ser?Pro?Thr?Pro?Pro?Arg?Ala?Ala?Ser?Pro?Pro?Pro?Pro

115 120 125

Leu?Asp?Leu?Thr?Leu?Asn?Ile?Pro?Ser?His?Gln?His?His?His?Leu?Arg

130 135 140

His?Gly?His?Phe?Pro?Thr?Gly?Val?Ile?Phe?Pro?Gly?Gly?Ala?Trp?Ile

145 150 155 160

Ser?Leu?Ala?Ala?Glu?Ala?His?Pro?Val?Phe?Phe?Phe?Asp?Ala?Phe?Ser

165 170 175

Val?Gln?Gly?Glu?Ser?Asn?Asn?Asn?Asn?Lys?Asn?Asn?Ile?Ile?Asn?Asn

180 185 190

Asn?Lys?Ile?His?Ser?Lys?Asn?Ile?Asn?Leu?Cys?Arg?Leu?Asp?Arg?Thr

195 200 205

Val?Met?Val?Ser?Ser?Gly?Val?His?Ser?Asp?Ser?Asp?Ser?Ser?Ser?Val

210 215 220

Val?Val?Asp?Tyr?Asp?His?Asp?Arg?Ser?Pro?Cys?Asn?Lys?Gly?Leu?Ser

225 230 235 240

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

245 250

<210>2

<211>759

<212>DNA

＜213〉Cortex jatrophae (Jatropha curcas)

<400>2

atgaaatggc?tccaagagag?atctaacagc?actggcaata?accttaacca?gaactccaat 60

accgccgaga?cccgctacag?aggcgttagg?aaacgacctt?ggggccgata?cgccgccgag 120

atccgagacc?ctggcaagaa?aactagggtc?tggcttggta?cttttgatac?tgctgaagag 180

gcggcgcgtg?cttacgacgc?tgctgctcgt?gaatttcgtg?gctctaaggc?taagacgaat 240

tttcctacag?ttacggagct?gaacaacgcg?gctgcagccg?ccgttgccgc?gggagcagtc 300

accgtcgcac?gaagtcctag?ccaaagtagc?accgttgagt?cttcatcacc?tacacctcca 360

cgcgctgctt?ctcctccacc?gcctcttgac?cttactctca?acattcctag?tcatcaacat 420

catcatctcc?gccacggcca?tttccctacc?ggtgttattt?ttcctggtgg?cgcgtggatt 480

tcactggcgg?cggaggctca?tccagttttc?ttcttcgatg?cgttctctgt?tcaaggagag 540

agtaataaca?acaacaaaaa?taatattatc?aacaataata?aaattcactc?taaaaatatt 600

aacttgtgca?gattggatcg?gacggtgatg?gtgagcagtg?gggtccacag?tgattccgat 660

tcatcatctg?ttgtggttga?ttatgaccac?gatcgtagtc?cttgtaacaa?gggattatca 720

cttgatcttg?atcttaactt?tcctccggct?gaagtagcc 759

<210>3

<211>792

<212>DNA

＜213〉Cortex jatrophae (Jatropha curcas)

<400>3

gcggcgcgtg?cacacgacgt?ggctgctcgt?gaatttcgtg?gctctaaggc?taagacgaat 60

tttcctacag?ttacggagct?gaacaacgcg?gctgcagccg?ccgttgccgc?gggagcagtc 120

accgtcgcac?gaagtcctag?ccaaagtagc?accgttgagt?cttcatcacc?tacacctcca 180

cgcgctgctt?ctcctccacc?gcctcttgac?cttactctca?acattcctag?tcatcaacat 240

catcatctcc?gcctcggcca?tttccctacc?ggtgttattt?ttcctggtgg?cgcgtggatt 300

tcactggcgg?cggaggctca?tccagttttc?ttcttcgatg?cgttctctgt?tcaaggagag 360

agtaataaca?acaacaaaaa?taatattatc?aacaataata?aaattcactc?taaaaatatt 420

aacttgtgca?gattggatcg?gacggtgatg?gtgagcagtg?gggtccacag?tgattccgat 480

tcatcatctg?ttgtggttga?ttatgaccac?gatcgtagtc?cttgtaacaa?gggattatca 540

cttgatcttg?atcttaactt?tcctccggct?gaagtagcct?gattttgaag?agatctgatt 600

tctatttttg?ggaagtcgtt?tttgggtata?atgcttgtct?tttttttgtt?tttttttttt 660

ttgtctcaaa?tctcaattcg?aagacgagcc?aagaaaaatt?tagaggataa?gcaaggaagc 720

taacgtattt?tttcttttta?atgtatatat?aaagagagcc?aagataacct?ctttaaaaaa 780

aaaaaaaaaa?aa 792

<210>4

<211>294

<212>DNA

＜213〉Cortex jatrophae (Jatropha curcas)

<400>4

cgacgattgg?ccacgcgtcg?actagtacgg?ggggggggaa?acccgacctt?ctttcgcttt 60

ttctacctgt?aacaatttcc?tttccatcca?aacacgctct?cagaatccaa?aaacagaaaa 120

tggctccaag?agagatctaa?cagcactggc?aataacctta?accagaactc?caataccgcc 180

gagacccgct?acagaggcgt?taggaaacga?ccttggggcc?gatacgccgc?cgagatccga 240

gaccctggca?agaaaactag?ggtctggctt?ggtacttttg?atactgctga?agag 294

Claims

1, Cortex jatrophae salt induced gene, its base sequence is shown in SEQ ID NO:2.

2, contain the described expression carrier of claim 1.

3, the transgenic cell line that contains the described gene of claim 1.

4, the host bacterium that contains the described gene of claim 1.

5, a kind of method that improves plant stress tolerance is that the described Cortex jatrophae salt of claim 1 induced gene is imported plant tissue or cell, obtains the plant that resistance of reverse improves; Described resistance of reverse is a salt resistance.

6, method according to claim 5 is characterized in that: described Cortex jatrophae salt induced gene imports plant tissue or cell by the plant expression vector that contains described Cortex jatrophae salt induced gene.

7, method according to claim 6 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.

8, method according to claim 7, it is characterized in that: described plant expression vector is p3301-BI121-JcERF, it obtains with the following method: the described Cortex jatrophae salt of claim 1 induced gene is cloned between the EcoR V restriction enzyme site of carrier pMD18-T, obtain carrying the recombinant vectors of Cortex jatrophae salt induced gene, called after pMD18-JcERF; PMD18-JcERF is carried out single endonuclease digestion with restriction enzyme Xba I, mend and put down, from connecting, use Sal I and this carrier of Sac I double digestion more then, obtain the small segment that contains Cortex jatrophae salt induced gene that a length is about 800bp; With Sal I and Sac I double digestion plasmid pGEX-KG, obtain 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-JcERF is about 800bp, obtain a recombinant plasmid, called after pGEX-KG-JcERF; With Xba I and Sac I double digestion plasmid pGEX-KG-JcERF and plant expression vector p3301-BI121, the large stretch of phase failure company of the small segment that will contain Cortex jatrophae salt induced gene and the 11.4kb that downcuts from plasmid p3301-BI121 from the 800bp that plasmid pGEX-KG-JcERF downcuts, promptly be built into the high-efficiency plant binary expression vector that contains the complete single open reading frame of Cortex jatrophae salt induced gene, called after p3301-BI121-JcERF.

9, according to any described method among the claim 5-8, it is characterized in that: described plant tissue that is imported into Cortex jatrophae salt induced gene or cell are from paddy rice, wheat, soybean, tobacco, corn, rape, Chinese sorghum, cotton, clover, Cortex jatrophae or Arabidopis thaliana.