CN101906155A - Protein ErNAC7 related to drought and salt resistance of plants and coding gene and application thereof - Google Patents
Protein ErNAC7 related to drought and salt resistance of plants and coding gene and application thereof Download PDFInfo
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8213—Targeted insertion of genes into the plant genome by homologous recombination
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
- C12N15/8266—Abscission; Dehiscence; Senescence
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
- Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants
Abstract
The invention relates to the field of genetic engineering, in particular to protein ErNAC7 related to drought and salt resistance of plants and a coding gene and application thereof. The protein has an amino acid sequence shown as SEQ ID NO.1 and a gene sequence shown as SEQ ID NO.2. The protein related to the drought and salt resistance and the coding gene thereof have important theoretical and actual meanings for improving and enhancing the stress resistance of tobacco, improving yield, accelerating the breeding process of stress-resisting molecules and effectively saving water resources.
Description
Technical field
The present invention relates to the genetically engineered field, particularly, the present invention relates to a kind of plant drought, protein related to salt tolerance ErNAC7 and encoding gene and application.
Background technology
Wheat occupies important status as one of China's important crops in national economy.Yet, cultivated area in China arid area is about 5.7 hundred million mu, and the saltings is about 5.54 hundred million mu, and the annual underproduction that wheat is caused because of adverse circumstance China such as arid, saline and alkaline reaches hundred million kilograms of 700-800, have a strong impact on the yield and quality of wheat, restricting the fast development of China's wheat grain-production.
Development along with modern molecular biology, utilize genetic engineering technique from the relation between molecular level further investigation plant and the abiotic stress, disclose plant to conduction of environment stress signal and gene expression regulation molecule mechanism, provide theoretical basis for cultivating the degeneration-resistant new germ plasm of crop.At present, many drought resistings, salt tolerant, low temperature resistant relevant gene are imported in the plant, improve the existing correlative study report of resistance of plant by genetic engineering technique.
In recent years, the mechanism of identifying, illustrate gene expression regulation under the various conditions by the structure and the functional analysis of transcription factor has obtained extensive concern.The NAC transcription factor is the newfound in recent years plant specific transcription factor with multiple biological function.Aida in 1996 etc. at first find the gene at petunia NAM, the albumen n end of Arabidopis thaliana ATAF1/2 and CUC2 genes encoding comprises a conservative aminoacid sequence, comprise 150 amino-acid residues approximately, the transcription regulatory region of C end for highly making a variation, the function class of CUC2 gene is similar to the NAM gene, and is relevant with the growth of plant; It is relevant that ATAF1/2 gene and plant adverse circumstance are replied, and gets three gene initials NAC (Aida et al., 1997) by name.The NAC transcription factor can with MYC-like combination of elements, the core sequence of this element (CATGTG) in the drought-induced reaction answering of Arabidopis thaliana ERD1, play an important role (Olsen et al., 2005).As a kind of important regulatory factor, the NAC transcription factor participates in regulating various important physical activities such as growth and development of plant, multiple defensive raction, hormone signal transduction.In addition, NAC class transcription factor also is subjected to multiple biology to coerce abduction delivering with abiotic stress, the environment stress responsing reaction of involved in plant.At present, from plants such as Arabidopis thaliana, paddy rice, rape, tomato, corn, peanut isolation identification many and degeneration-resistant relevant NAC class transcription factor gene.
3 different NAC genes (ANAC019, ANAC055 and ANAC072) of Arabidopis thaliana are expressed and are subjected to inducing of arid, high salt and ABA, and overexpression can significantly strengthen the drought-resistance ability of transfer-gen plant.Tadashi etc. have identified a drought resisting, resistant gene of salt SNAC1 in paddy rice, but drought stress inducing paddy rice Stomacal guard cell SNAC1 gene specific is expressed, promote stomatal closure, but do not influence photosynthetic rate, thereby the plant drought resistance greatly improves, and the rice plant salt tolerance of overexpression SNAC1 gene obviously strengthens (Tadashi et al, 2008).After this, in I in Rice RAT109, be separated to a NAC gene SNAC2, the analysis of Northern-blot promoter activity confirms that SNAC2 is subjected to arid, high salt, low temperature, physical abuse, ABA abduction delivering, wild-type and SNAC2 cross the express transgenic plant and coexist 4-8 ℃ of following subzero treatment after 5 days, wild-type is all dead, transfer-gen plant has 50% survival rate, and transfer-gen plant is to the also significantly enhancing of susceptibility of ABA.(Zhong et al such as Zhong, 2007) from paddy rice, be separated to an OsNAC6, this gene is subjected to low temperature, arid, high salt, rice blast abduction delivering, utilizes biochip method to analyze overexpression OsNAC6 gene and can raise a plurality of Expression of Related Genes such as peroxidase genes of coercing.Overexpression OsNAC6 transfer-gen plant medium drought resistant and Nai Gao salt ability are improved, the blast resisting ability strengthens, but plant is downgraded and low yield, drive down by inducible promoter, can effectively reduce the plant development negative effect, OsNAC6 can be used as the anti-effective instrument of the multiple ability of coercing of a kind of raising plant.The relevant report that does not at present also have the NAC gene in wheat, to study.
In sum, NAC class transcription factor plays crucial effects regulating the adverse circumstance reaction of plant in the resistance of raising plant.Overexpression NAC transcription factor gene has improved the anti-adversity ability of plant, and transfer-gen plant is grown and do not produced any negative effect, and this produces huge pushing effect and economic benefit to degeneration-resistant breeding and agriculture production meeting.Therefore, utilize degeneration-resistant relevant NAC class transcription factor gene improvement and the resistance of raising crop to have extremely important theory and practice meaning.
It is that the good perennial rhizome of Gramineae wheat family (Triticeae) is dredged clump type herbaceous plant that couchgrass belongs to (Elytrigia), has crucial utility value.Wherein, Xinjiang couchgrass (E.repens) has strong drought resistant, salt tolerant alkali and winter resistance, the long and flourishing rhizome of tool, and adaptive faculty is extremely strong, is the desirable plants (Gu Anlin, 2004) in improvement arid, saltings.Simultaneously, Xinjiang couchgrass platymiscium also is the sibling species of wheat (Triticum aestivum), has similar pathways metabolism, has become important wild gene storehouse (Wang Honggang, 2000 of improvement wheat at present; Lv Weidong, 2007).At present, by distant hybirdization technology, pollen tube passage method Xinjiang couchgrass medium drought resistant, resistant gene of salt resource are changed in the crop such as wheat, the resistance of improvement farm crop has obtained certain effect.Yet by genetic engineering means, the research of cloning drought resisting, resistant gene of salt and making up food crop such as plant expression vector transformed wheat from the couchgrass of Xinjiang has not yet to see relevant report.Therefore, utilize the couchgrass platymiscium germ plasm resource of characteristics such as strong drought resistant, salt tolerant alkali, excavate, filter out the resistance of good drought resisting, salt-resistant related gene improvement wheat, provide important adversity gene resource for cultivating food crop drought resisting, salt tolerant new variety such as wheat, aspects such as the improvement in salinification soil and improvement are all had important practical usage.
Summary of the invention
The purpose of this invention is to provide a kind of plant drought, protein related to salt tolerance ErNAC7.
A further object of the present invention provides the encoding gene of the above-mentioned plant drought of coding, the relevant egg ErNAC7 of salt tolerant.
Another object of the present invention provides the recombinant vectors that comprises said gene.
Another object of the present invention provides the application of above-mentioned plant drought, protein related to salt tolerance ErNAC7 and gene thereof.
Drought resisting provided by the present invention, protein related to salt tolerance ErNAC7, (its direct sources is: Beijing Grass Industry ﹠. Environment Research Development Center to derive from the Xinjiang couchgrass, its primary source: self-sow ground of plant or collection ground---Chinese Xinjiang), its aminoacid sequence is shown in SEQ ID NO.1.Transcription factor of the present invention is made up of 298 amino-acid residues, is NAC class transcription factor.From the N-terminal 10-133 of SEQ ID NO.1 amino acids residue is the NAC structure of guarding, and is nuclear localization sequence from the N-terminal 142-145 of SEQ ID NO.1 amino acids residue.
SEQ?ID?NO.1：
1 MSGGQELNLP?PGFRFHPTDE
21 ELVMHYLCRR?CAGAPIAVPI
41 ITEIDLYKFD?PWQLPKMALY
61 GEKEWYFFSP?RDRKYPNGSR
81 PNRAAGSGYW?KATGADKPVG
101 TPKPLAIKKA?LVFYAGKAPK
121 GEKTNWIMHE?YRLADVDRSA
141 RKKNSLRLDD?WVLCRIYNKK
161 GGMEKPASVD?RKPATVGGYG
181 GPPGAMVSSP?QEQKPVMGMN
201 ANGGGGVQPF?PDFAAYYDRP
221 SDSMPRLHAD?SSCSEQVLSP
241 DFPAGEVQSQ?PKISEWERSF
261 ASGGDPVNPA?AGSMLEPNGG
281 FGGDPLLQDI?LMYWGKPF
*
In order to make albumen ErNAC7 be convenient to purifying, can connect label as shown in table 1 at proteinic N-terminal of forming by the aminoacid sequence shown in the SEQ ID NO.1 or C-terminal.
The sequence of table 1 label
| Label | Residue | Sequence |
| Poly-Arg | 5-6 (being generally 5) | RRRRR |
| Poly-His | 2-10 (being generally 6) | HHHHHH |
| FLAG | 8 | DYKDDDDK |
| Strep-tag?II | 8 | WSHPQFEK |
| c-myc | 10 | EQKLISEEDL |
SEQ ID NO.1 sequence disclosed according to the present invention, but transcription factor ErNAC7 synthetic of the present invention also can be synthesized its encoding gene earlier, carry out the biology expression again and obtain.
ErNAC7 encoding gene according to the present invention has nucleotide sequence shown in SEQ ID NO.2.The expression of ErNAC7 is subjected to inducing of arid, salt stress.
SEQ?ID?NO.2
1 ATGAGCGGCG?GACAGGAGCT?GAATCTGCCG?CCGGGCTTCCGGTTCCACCC?GACGGACGAG
61 GAGCTGGTGA?TGCACTACCT?CTGCCGCCGC?TGCGCCGGCGCGCCCATCGC?CGTCCCCATC
121?ATCACCGAGA?TCGACCTCTA?CAAGTTCGAC?CCCTGGCAGCTCCCAAAGAT?GGCGCTGTAC
181?GGCGAGAAGG?AGTGGTACTT?CTTCTCCCCG?CGGGACCGCAAGTACCCCAA?CGGGTCCAGG
241?CCCAACCGGG?CCGCCGGGTC?CGGGTACTGG?AAGGCCACCGGGGCCGACAA?GCCCGTGGGC
301?ACCCCCAAGC?CGCTGGCCAT?CAAGAAGGCG?CTCGTCTTCTACGCCGGCAA?GGCCCCCAAG
361?GGCGAGAAGA?CCAACTGGAT?CATGCACGAG?TACCGCCTCGCCGACGTCGA?CCGCTCCGCC
421?CGCAAGAAGA?ACAGCCTCAG?GTTGGATGAT?TGGGTGCTGTGCCGCATCTA?CAACAAGAAG
481?GGCGGCATGG?AGAAGCCGGC?GTCCGTGGAC?CGGAAGCCGGCGACAGTGGG?CGGCTACGGG
541?GGTCCTCCTG?GGGCCATGGT?GAGCTCCCCG?CAGGAGCAGAAGCCCGTCAT?GGGGATGAAC
601?GCCAACGGCG?GCGGCGGCGT?GCAGCCGTTC?CCGGACTTCGCGGCGTACTA?CGACCGGCCG
661?TCCGACTCGA?TGCCGCGGCT?GCACGCCGAC?TCGAGCTGCTCGGAGCAGGT?GCTGTCGCCG
721?GACTTCCCGG?CCGGGGAGGT?GCAGAGCCAG?CCCAAGATCAGCGAGTGGGA?GCGCTCATTC
781?GCCTCCGGCG?GCGACCCCGT?GAACCCGGCG?GCTGGCTCCATGCTCGAGCC?CAACGGCGGG
841?TTCGGCGGCG?ACCCGCTCCT?CCAGGACATC?CTCATGTACTGGGGCAAGCC?GTTCTGA
Contain ErNAC7 expression of gene box, recombinant expression vector, transgenic cell line and reorganization bacterium and all belong to protection scope of the present invention.
Available existing plant expression vector construction contains the recombinant expression vector of ErNAC7 gene.
Described plant expression vector comprises the double base agrobacterium vector and can be used for the carrier etc. of plant micropellet bombardment.Described plant expression vector also can comprise 3 ' end untranslated zone of foreign gene, promptly comprises the dna fragmentation of polyadenylic acid signal and any other participation mRNA processing or genetic expression.The bootable polyadenylic acid of described polyadenylic acid signal joins 3 ' end of mRNA precursor, and the non-translational region of inducing (Ti) plasmid gene (as kermes synthetic enzyme Nos gene), plant gene 3 ' end to transcribe as the Agrobacterium crown-gall nodule all has similar functions.
When using ErNAC7 to make up the recombinant plant expression vector, before its transcription initiation Nucleotide, can add any enhancement type promotor or constitutive promoter, as the ubiquitin promoter (Ubiquitin) of cauliflower mosaic virus (CaMV) 35S promoter, corn, 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, 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.
Another object of the present invention provides a kind of method of cultivating plant with adverse resistance.
The method of cultivation plant with adverse resistance provided by the present invention is that the above-mentioned recombinant expression vector that any contains the ErNAC7 gene is imported in the vegetable cell, obtains plant with adverse resistance.
Utilize any carrier that can guide foreign gene in plant, to express, the encoding gene of NAC transcription factor ErNAC7 provided by the present invention is imported vegetable cell, can obtain abiotic stress stress-tolerance power enhanced transgenic cell line and transfer-gen plants such as arid and salt.Carry encoding gene 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 tissue cultivating is become plant.By the plant transformed host both can be monocotyledons, also can be dicotyledons, as: tobacco, wheat, Xinjiang couchgrass, Arabidopis thaliana, paddy rice, corn, cucumber, tomato, willow, turfgrass, lucerne place etc.
Described plant stress tolerance specifically can be the resistance of reverse to abiotic stress, as to or the resistance of reverse of salt stress.
The present invention is an experiment material with drought resisting, Xinjiang couchgrass (Elytrigia repens L.) that salt tolerance is stronger, has obtained degeneration-resistant relevant NAC transcription factor ErNAC7 (Elytrigia repens
NAM-
ATAF1/2-
CUC27) albumen and encoding gene thereof, and, significantly improved drought resisting, the salt tolerance of plant with ErNAC7 gene importing tobacco.Drought resisting of the present invention, protein related to salt tolerance and encoding gene thereof to improvement, strengthen tobacco resistance, improve output, quicken degeneration-resistant molecular breeding process, and effectively save water resources and have important theoretical and practical significance.
Description of drawings
Fig. 1 is the cDNA clone of drought resisting, protein related to salt tolerance ErNAC7 encoding gene, is template with Xinjiang couchgrass cDNA, and pcr amplification contains the cDNA fragment in the conservative territory of NAC, 1,2: Xinjiang couchgrass fragment; M:DL2000marker (100,250,500,750,1000,2000bp).
Fig. 2 analyzes the expression characteristic of ErNAC7 under arid, salt stress are handled, the expression characteristic under the A. drought stress is handled for quantitative fluorescent PCR; B. the expression characteristic under salt stress is handled.
Fig. 3 infects, breaks up screening and culturing, plant regeneration and PCR for the tobacco Agrobacterium and detects.A. screening, the differentiation culture of tobacco after Agrobacterium is infected; B. through the regeneration plant root culture of screening, differentiation back acquisition; The PCR of C.pBI35S-ErNAC7 transgenic tobacco plant detects.
Fig. 4 is that transgene tobacco drought tolerance, salt tolerant are identified.A.pBI35S-ErNAC7 transgene tobacco and W38 are at the growing state that contains on the 200mM NaCl substratum; B.pBI35S-ErNAC7 transgene tobacco and W38 are at the growing state that contains the 2%PEG substratum.
Embodiment
Make the experimental methods of molecular biology specify in following examples, all carry out, perhaps carry out according to test kit and product description with reference to listed concrete grammar in " molecular cloning experiment guide " (third edition) J. Sa nurse Brooker one book.
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.
Embodiment 1: the cDNA clone of Xinjiang couchgrass drought resisting, the relevant ErNAC7 gene of salt tolerant.
The Xinjiang couchgrass seedling that grows about 45 days is carried out arid processing 5 hours, extract the total RNA of Xinjiang couchgrass with Trizol.Use 5 ' RACE test kit (5 ' RACE System for Rapid Amplification of cDNA EndsKit) (GIBCOBRL, CAT.NO.18374-058) and 3 ' RACE test kit (3 ' RACE System for RapidAmplification of cDNA Ends Kit) (GIBCOBRL CAT.NO.18373-019) obtains the full length sequence 894bp of ErNAC7 gene.
Extract total RNA of Xinjiang couchgrass seedling with Trizol, acquire cDNA with the reverse transcription of superscript II (invitrogen) ThermoScript II.According to ErNAC7 gene coding region sequences Design primer P1 and P2.The cDNA that obtains with reverse transcription is a template, carries out pcr amplification with primer P1 and P2.The sequence of primer P1 and P2 is as follows:
P1：5’-ATGAGCGGCGGACAGGAGCT-3’，
P2：5’-GAGATTGAACGGCTTGCCCCAGT-3’。
The PCR product is carried out 0.8% agarose gel electrophoresis detect, obtain molecular weight and be about band about 0.8-1kb, conform to expected results.Reclaim test kit (TIANGEN) with sepharose and reclaim this fragment.Should reclaim fragment is connected with pGEM-T Easy (Promega), method (Proc Natl Acad Sci with reference to Cohen etc., 69:2110), to connect product transformed into escherichia coli DH5 α competent cell, according to the acillin resistance marker screening positive clone on the pGEM-T Easy carrier, obtain containing the segmental recombinant plasmid of recovery.With T7 on this recombinant plasmid vector and SP6 promoter sequence is that primer carries out nucleotide sequencing to it, the open reading frame (ORF) that sequencing result shows the ErNAC7 gene that increases for SEQ ID No.2 from 5 ' terminal the 1st to 894 deoxyribonucleotide, encoding amino acid sequence is the protein of SEQ ID No.1.The recombinant vectors called after pTE-ErNAC7 that will contain ErNAC7 gene shown in the sequence SEQ ID No.2, its cDNA clone result as shown in Figure 1.
The sequence of ErNAC7 gene is compared on Genabnk, and NAC class transcription factor has higher homology in this gene and the Arabidopis thaliana, and does not find the homologous protein gene in the couchgrass of Xinjiang, proves that the ErNAC7 gene is a new gene.
Embodiment 2: environment stress is handled Xinjiang couchgrass ErNAC7 expression of gene feature down
Xinjiang couchgrass seed kind in basin, is got seedling and carried out the following processing of coercing after 3 weeks of growing: 1) arid is handled: Xinjiang couchgrass seedling is taken out the moisture that blots on the root from soil, place on the exsiccant filter paper; 2) salt is handled: Xinjiang couchgrass seedling is placed 200mM NaCl solution; In the sampling in 0,1,2,5,10,24 hour of above-mentioned various processing back, use liquid nitrogen flash freezer respectively ,-80 ℃ of preservations are standby.
Respectively arid processing, salt being handled 0,1,2,5,10,24 hour the total RNA of sample extraction in back, is probe with ErNAC7DNA, carries out quantitative fluorescence analysis.
Quantitative fluorescence analysis the results are shown in Figure 2.A is the sample that arid is handled; B is the sample that NaCl handles.
The result shows: peak in drought stress ErNAC7 genetic expression in 10 hours; Coerce ErNAC7 genetic expression in 1 hour at high salt (200mMNaCl) and peak, illustrate that this gene is subjected to arid, high-salt stress abduction delivering.
Embodiment 3: the drought resisting, the salt tolerance that strengthen plant with the ErNAC7 gene
1, the structure of recombinant expression vector
1) structure of 35S-ErNAC7 recombinant expression vector
The cDNA that obtains with total RNA reverse transcription of Xinjiang couchgrass is a template, carries out pcr amplification with the special primer that contains SmaI and XbaI joint sequence; SmaI and XbaI double digestion PCR product reclaim then, enzyme are cut between the CaMV 35S promoter SmaI and XbaI enzyme cutting site afterwards of product forward insertion carrier pBI121, obtain recombinant vectors 35S-ErNAC7.
Primer sequence is as follows:
ErNAC7[SmaI]5’-GCGCCCGGGATGAGCGGCGGACAGGAGCT-3’
ErNAC7[XbaI]5’-TGCTCTAGAGAGATTGAACGGCTTGCCCCAGT-3’
2, the acquisition of transgene tobacco and evaluation
1) acquisition of transgene tobacco
The recombinant expression vector 35S-ErNAC7 of above-mentioned structure is transformed agrobacterium tumefaciens EHA105 with freeze-thaw method respectively, to be integrated with the agrobacterium tumefaciens EHA105 transformation of tobacco W38 of 35S-ErNAC7 more respectively with leaf dish method, carry out 2 with the MS substratum that contains the 100mg/L kantlex and take turns screening, the every wheel screened 10-15 days, obtained positive transfer-gen plant (Fig. 3).The positive transfer-gen plant that screening obtains is done further evaluation and screening with PCR, and the used a pair of primer of PCR is P3 and P4.
P3 (upstream primer): 5 '-GGCGCTGTACGGCGAGAAGGA-3 ',
P4 (downstream primer): 5 '-CGCCGTTGGCGTTCATCCC-3 '.
The 35S-ErNAC7 transgene tobacco is carried out PCR identify that positive transfer-gen plant can obtain 1Kb left and right sides band through pcr amplification, the result obtains to change 40 strains of 35S-ErNAC7 tobacco.
Simultaneously the pBI121 empty carrier is imported tobacco W38, method is the same, in contrast, obtains commentaries on classics empty carrier tobacco (the transgene tobacco T that screening obtains of 10 strain systems
0Representative is shown).
2) drought-enduring, the salt tolerance evaluation of commentaries on classics ErNAC7 gene plant
With T
0In generation, changeed 35S-ErNAC7 genetic tobacco plant and T
0Generation change the empty carrier adjoining tree 3 age in week seedling root system move into respectively carry out salt in the substratum that contains 200mM NaCl, 2%PEG, drought stress was handled 35 days, observe phenotype and also take pictures.
The result shows: coerce and handle after 35 days, the 35S-ErNAC7 transfer-gen plant can normal growth under arid, condition of salt stress, and well developed root system, leaf color are dark green; All empty carrier rotaring gene plant blades all wilting or chlorosis, turn white and cause death.Transgene tobacco is drought-enduring, salt tolerance qualification result proof ErNAC7 gene can improve the drought-enduring and salt tolerance of plant.
Fig. 4 is that transgene tobacco is drought-enduring, salt tolerance is identified, shows as Fig. 4 A: 35S-ErNAC7 transfer-gen plant, 35 days photo of empty carrier transfer-gen plant 200mM NaCl substratum growth; Fig. 4 B is that pBI35S-ErNAC7 transgene tobacco and W38 are in 35 days the photo situation of growth that contains on the 2%PEG substratum.
Sequence table
＜110〉Beijing City Agriculture and Forestry Institute
＜120〉a kind of plant drought, protein related to salt tolerance ErNAC7 and encoding gene and application
<160>2
<210>1
<211>298
<212>PRT
＜213〉Xinjiang couchgrass (Elytrigia repens L.)
<400>1
MSGGQELNLP?PGFRFHPTDE?ELVMHYLCRR?CAGAPIAVPI?ITEIDLYKFD
PWQLPKMALY 60
GEKEWYFFSP?RDRKYPNGSR?PNRAAGSGYW?KATGADKPVG?TPKPLAIKKA
LVFYAGKAPK 120
GEKTNWIMHE?YRLADVDRSA?RKKNSLRLDD?WVLCRIYNKK?GGMEKPASVD
RKPATVGGYG 180
GPPGAMVSSP?QEQKPVMGMN?ANGGGGVQPF?PDFAAYYDRP?SDSMPRLHAD
SSCSEQVLSP 240
DFPAGEVQSQ?PKISEWERSF?ASGGDPVNPA?AGSMLEPNGG?FGGDPLLQDI
LMYWGKPF 298
<210>2
<211>897
<212>DNA
＜213〉Xinjiang couchgrass (Elytrigia repens L.)
<400>2
atgagcggcg?gacaggagct?gaatctgccg?ccgggcttcc?ggttccaccc?gacggacgag 60
gagctggtga?tgcactacct?ctgccgccgc?tgcgccggcg?cgcccatcgc?cgtccccatc 120
atcaccgaga?tcgacctcta?caagttcgac?ccctggcagc?tcccaaagat?ggcgctgtac 180
ggcgagaagg?agtggtactt?cttctccccg?cgggaccgca?agtaccccaa?cgggtccagg 240
cccaaccggg?ccgccgggtc?cgggtactgg?aaggccaccg?gggccgacaa?gcccgtgggc 300
acccccaagc?cgctggccat?caagaaggcg?ctcgtcttct?acgccggcaa?ggcccccaag 360
ggcgagaaga?ccaactggat?catgcacgag?taccgcctcg?ccgacgtcga?ccgctccgcc 420
cgcaagaaga?acagcctcag?gttggatgat?tgggtgctgt?gccgcatcta?caacaagaag 480
ggcggcatgg?agaagccggc?gtccgtggac?cggaagccgg?cgacagtggg?cggctacggg 540
ggtcctcctg?gggccatggt?gagctccccg?caggagcaga?agcccgtcat?ggggatgaac 600
gccaacggcg?gcggcggcgt?gcagccgttc?ccggacttcg?cggcgtacta?cgaccggccg 660
tccgactcga?tgccgcggct?gcacgccgac?tcgagctgct?cggagcaggt?gctgtcgccg 720
gacttcccgg?ccggggaggt?gcagagccag?cccaagatca?gcgagtggga?gcgctcattc 780
gcctccggcg?gcgaccccgt?gaacccggcg?gctggctcca?tgctcgagcc?caacggcggg 840
ttcggcggcg?acccgctcct?ccaggacatc?ctcatgtact?ggggcaagcc?gttctga 897
Claims (7)
1. a plant drought, protein related to salt tolerance ErNAC7 is characterized in that its aminoacid sequence is shown in SEQID NO.1.
2. a plant drought, salt-resistant related gene ErNAC7 is characterized in that, the described plant drought of coding claim 1, protein related to salt tolerance ErNAC7.
3. plant drought as claimed in claim 2, salt-resistant related gene ErNAC7 is characterized in that, its base sequence such as SEQ ID NO.2.
4. the recombinant vectors that comprises claim 2 or 3 described plant droughts, salt-resistant related gene ErNAC7.
5. the application of the described plant drought of claim 1, protein related to salt tolerance ErNAC7.
6. the application of the described plant drought of claim 2, salt-resistant related gene ErNAC7.
7. method of cultivating plant with adverse resistance, described method comprise that the recombinant expression vector that will contain the ErNAC7 gene imports in the vegetable cell, obtains the step of plant with adverse resistance.
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Application publication date: 20101208 |