CN110484542A - Arabidopsis disease-resistant related gene EIJ1 and its application - Google Patents

Arabidopsis disease-resistant related gene EIJ1 and its application Download PDF

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CN110484542A
CN110484542A CN201910697361.9A CN201910697361A CN110484542A CN 110484542 A CN110484542 A CN 110484542A CN 201910697361 A CN201910697361 A CN 201910697361A CN 110484542 A CN110484542 A CN 110484542A
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侯兴亮
刘海伦
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South China Botanical Garden of CAS
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    • C12N15/8281—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for bacterial resistance

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Abstract

The invention discloses a kind of arabidopsis EIJ1 gene and its albumen, the DNA sequence dna of the EIJ1 gene is as shown in SEQ ID NO.1, or for containing replacement, missing or the increase for there are one or several nucleotide in sequence shown in SEQ ID No.1 and sequence, and there is the nucleotide sequence with identical function shown in SEQ ID NO.1；Or the nucleotide sequence for amino acid sequence shown in coding SEQ ID NO.2.The present invention also provides recombination over-express vectors and bioengineered strain inserted with above-mentioned EIJ1 gene.Application of the arabidopsis EIJ1 albumen described in arabidopsis EIJ1 gene or claim 2 in regulation plant disease-resistant is immune.The present invention also provides the application of above-mentioned arabidopsis EIJ1 gene and albumen in the immune breeding of regulation plant disease-resistant.

Description

Arabidopsis disease-resistant related gene EIJ1 and its application
Technical field
The invention belongs to plant genetic engineering field, it is related to a kind of arabidopsis gene correlation EIJ1 gene and its in plant Application in disease-resistant.
Background technique
Disease is to influence the important limiting factor of the growth and development of plant.In agricultural production, serious disease can cause Crops significantly the underproduction or make its quality decline, therefore study disease resistance of plant and have great importance to agricultural production.
Although plant disease-resistant signal path related gene has been widely studied, the specific molecular mechanism of specific gene is still Not clear enough, some unknown resistant genes or resistance related regulatory genes are not still exploited.So excavating newly disease-resistant Gene, can both enrich plant disease-resistant important regulating and controlling mechanism, more can improve the potential economic characters of plant, it is later grain economy The crossbreeding of crop provides Research foundation and scientific basis.
Summary of the invention
One of the objects of the present invention is to provide the genes and egg of a kind of arabidopsis EIJ1 of response phytopathogen stress It is white.
Above-mentioned is to be achieved through the following technical solutions:
A kind of arabidopsis EIJ1 gene, DNA sequence dna is as shown in SEQ ID NO.1, or is containing shown in SEQ ID No.1 Sequence and sequence in have replacement, missing or the increase of one or several nucleotide, and have identical as shown in SEQ ID NO.1 The nucleotide sequence of function；Or the nucleotide sequence to be matched with SEQ ID NO.1 complete complementary；It or is coding SEQ ID The nucleotide sequence of amino acid sequence shown in NO.2.
Arabidopsis EIJ1 albumen, amino acid sequence be as shown in SEQ ID NO.2, or be SEQ ID NO.2 shown in sequence Through replacement, missing or the one or more amino acid of increase on the basis of column, and there is the amino acid with SEQ ID NO.2 identical function Sequence.
It is a further object of the present invention to provide a kind of recombination over-express vectors.
A kind of recombination over-express vector, inserted with above-mentioned arabidopsis EIJ1 gene.
The expression vector is HY105 in one of the embodiments,.
In order to achieve the above-mentioned object of the invention, the present invention also provides a kind of recombination over-express vector, the recombinant expression carriers The acceptor carrier used is HY105, and it comprises arabidopsis EIJ1 genes.The carrier is will to intend shown in SEQ ID NO.1 The plant table that genomic dna sequence possessed by southern mustard EIJ1 is obtained with recombination switching technology insertion plant expression vector HY105 Up to carrier pEIJ1:EIJ1-HA.
Using HY105 construct plant expression vector when, for the ease of to transgenic plant cells or plant carry out identification and Screening, can process plant expression vector used, as the selected marker (Basta that can be expressed in plant is added Gene, luciferin kinase gene etc.) or antibiotic marker (kanamycins marker, Hygromycin marker object etc.) resistance base Cause.From the security consideration of genetically modified plants, any selected marker can be not added, transformed plant is directly screened with adverse circumstance.
The primer for expanding the arabidopsis EIJ1 gene in one of the embodiments, includes SEQ ID NO.3 and SEQ Sequence shown in ID NO.4.
It is a further object of the present invention to provide a kind of bioengineered strains.
A kind of bioengineered strain is that above-mentioned recombination over-express vector is transferred to Agrobacterium tumefaciems GV3101 and is obtained.
It is a further object of the present invention to provide the application of above-mentioned arabidopsis EIJ1 gene or albumen in regulation plant disease-resistant.
It is a further object of the present invention to provide arabidopsis EIJ1 genes or albumen in the immune breeding of regulation plant disease-resistant Application.
Beneficial effect
The invention belongs to plant genetic engineering field, it is related to a kind of arabidopsis gene EIJ1 and its in plant disease-resistant Using.This invention is cloned into a kind of arabidopsis EIJ1 gene from arabidopsis for the first time, and demonstrates its function.Utilize plant table Up to carrier, its corresponding mutant eij1 (SALK_142975) by EIJ1 channel genes of the invention.Disease resistance experiments have shown that, Relative to wild type Col (Columbia is environmental), mutant eij1 disease resistance is remarkably reinforced；And stablizes and imports pEIJ1: After EIJ1-HA to eij1 mutant, the disease-resistant phenotype of eij1 mutant can be replied.This shows that EIJ1 gene is adjusting Genes For Plant Tolerance Sick immunology plays an important role.The present invention regulates and controls plant disease resistance genes EIJ1, can both enrich plant disease-resistant important regulating and controlling Mechanism can more improve the potential economic characters of plant, and the crossbreeding for later grain economy crop provides Research foundation and section Learn foundation.
Detailed description of the invention
The identification of Fig. 1 arabidopsis eij1 mutant；
A. the structural schematic diagram of mutant eij1；B. mutant detects, the primer P1 sequence such as SEQ ID NO.5；P2 Sequence such as SEQ ID NO.6；P3 sequence such as SEQ ID NO.7；C. EIJ1 gene expression amount in mutant eij1 and wild type Col Detection (P < 0.05 *；**P<0.01).
The screening of Fig. 2 transgenic positive seedling；
Sowed material is (eij1 pEIJ1:EIJ1-HA) and its negative control (eij and Col).Black arrow institute It is identified as the positive young plant of normal growth.
The Protein Detection of Fig. 3 transgenic line；
Extract eij1 pEIJ1:EIJ1-HA#1, #2, #4 and #5Each strain and negative control eij1 material total protein carry out Western Blot detection.
Fig. 4 Arabidopsis Mutants eij1 and its reply disease-resistant phenotypic evaluation of material；
A. Arabidopsis Mutants eij1 and its material inoculation Pst DC3000 pathogen Phenotypic Observation after 5 days is replied；B. intend After 5 days clump count statistics (P < 0.05 * for southern mustard mutant eij1 and its reply material inoculation Pst DC3000 pathogen；**P< 0.01)。
Fig. 5 Arabidopsis Mutants eij1 and its disease-resistant important Marker gene expression amount measurement of reply material.
Specific embodiment
Unless otherwise specified, practice of the invention will use molecular biology, microbiology, cell biology, immunology With the traditional technology of recombinant DNA, belong to art technology range.See, for example, Sambrook, Fritsch and Maniatis, Molecular Cloning:A Laboratory guide, the 3rd edition (2002) in the following examples, the experimental methods for specific conditions are not specified, usually according to routine Condition, or according to the normal condition proposed by manufacturer.Used various common chemical reagent, are commercially available production in embodiment Product.
Unless otherwise defined, all technical and scientific terms used in the present invention and belong to technical field of the invention The normally understood meaning of technical staff it is identical.Term used in the description of the invention is intended merely to describe specific reality The purpose for applying example is not used in the limitation present invention.Term "and/or" used in the present invention includes one or more relevant listed Any and all combinations of project.
EIJ1 full-length genome (SEQ ID NO.1)
TTGGTTAAATTAAAAATATTTAACAATCTATGTAAGAACAAAAAAAAATGTTATATTATCATCTTAAAA CTAGTAAATTTATACATAGAAAAAAATTATTTTCTCAATCATATAAGCAAACCAAAAACAAACAAGCAATCTGGATT TTGTTTTGACTGTAGGAAGAATCCAAGGAATTGTAGAGGAGATAATGAGTTGGTTTAAATCATTGAATCAATCATTT TAGCAAACAAAAAGATCCAACTTTAATGGGCCCAGGGTCCAATAACCCAATTGGGTTATTCCTAGACTTTTCTCCTC CTCCTGTTGTCTTCCAATTTGGTATGCGAGAAAAATAAAATAAAATAAAAATAAGCCAAAGACAAATTTAAACCTCC GATTTCAACCTTCTCGGCGAAGAAGAAGAAGAAGAAGAGAAAATGACGATTTGCATGTGTTGCGATTCACATCTCGT AGCGAAATCGATTCTAAATCCATGGAATTCTCCTAGGATAACGAAGCTAGGTTTCGTACCAGTCGTTCGCCGTTTTC CAGCTACTACTACCGTCAAGGCTTCGGCGGTGGATTCACCTGAGAGTTCTTCAAATTTCGCCAAGCGTATGGATCAA GCTTGGATTATCTCACAGGTTCGTATCTCCAGAGAGTTGTTGATGTGTATAATTTTGTTGAATCTGAGTAATCACAA GAACTGATCCTTGATGTAGTCATGTACGAGTGAAGAGCTATATGCAATTTTGTATGAGATTAAGAACTTGGATCTGT GATGAAATGTTGTAAATTTCAGATTATGAGGTGAATAGTATCAGCAAGTTTCAAAAGGATTTGTATCGAAAATCTGT AATTGCAAAGTACCTTTTAGTTGGTTTCTAGCAAAAAATGTTTGTTCACAATTCATTTGATTTGTGCATATGATGAG AACATGTTAGCTACTTAATCAGGTTGTAGCTGTTCTCTATGATCCCTGAGTGATGAGGCTTAAGCAGGCTTATCTCG CAACGCAATGTAGTTTTGTAGCTTTGCATGTACAATTGTTGGATTCTCTGCTTTTTCTCTAATATTGCTCTTAGAGA ATCTATCAAAAGTTCAGACTTTGAAGCCTTTCTTCTAGCACTTGCAATTTGATATTGAGCTCGTGTGAATTTTCTAG ATGTTACTTGTTGACCTGTGTTTCGATTCAATGATTTTCTTTGTCTGTTTTTTTCTTCACTGCAGCAACCAAGTCCT GTTGGATGTTCATCGTGCAATTCAAAGGGTCATGTGGAATGCAAATGGTGTGCGGGTACAGGATTCTTCATCCTTGG TGATAACATGCTTTGCCAGGTTCCCTCAAGGAATACTTCATGTGTAATCTGCTCTGGACAGGTTTGTTATCTAATAA AAACGAAAGTGCAGACACATTATTGTGTCTTCTTGTTTATATATCTTCCCAAGTATGAACAAATTCGATTCTTTCAA TGGTTTTAGGGTTCTGCTAGCTGTAGCGATTGCAAAGGAACGGGGTTTCGTGCCAAGTGGTTGGAAAAACCTCCAGT GCCAACA
EIJ1 amino acid sequence (SEQ ID NO.2)
MTICMCCDSHLVAKSILNPWNSPRITKLGFVPVVRRFPATTTVKASAVDSPESSSNFAKRMDQAWIISQ QPSPVGCSSCNSKGHVECKWCAGTGFFILGDNMLCQVPSRNTSCVICSGQGSASCSDCKGTGFRAKWLEKPPVPT
The clone of 1 arabidopsis EIJ1 gene of embodiment and identification
Testing arabidopsis material used is Columbia ecotype (Columbia, Col).Design primer carries out arabidopsis The clone of EIJ1 genome.
The specific method is as follows: the forward primer such as SEQ ID NO.3 of amplification EIJ1 full-length genome (TTGGTTAAATTAAAAATATTTA) shown in；Expand the reverse primer SEQ ID NO.4 of EIJ1 full-length genome (TGTTGGCACTGGAGGTTTTTCC) shown in.Take three weeks blades of Col material, be placed in liquid nitrogen and grind, extracting genome DNA according to It is carried out according to TIANGEN plant genome DNA extracts kit (DP305).PCR amplification is carried out by template of genomic DNA.PCR Reaction system are as follows: 10 μ Lmix, each 1 μ L of upstream and downstream primer (10pmol), 1 μ L of template (greatly containing about 0.03 μ g) add ddH2O 7μ L, total volume are 20 μ L.Its program is 95 DEG C of denaturation 5min；94 DEG C of 30sec again, 58 DEG C of 30sec, 72 DEG C of 1:30min, totally 34 A circulation；Then 72 DEG C of extension 5min；4 DEG C of preservations.
PCR product recycling after through connection PMD19-T carrier (Takara), conversion Escherichia coli Top10, shake bacterium, sequencing and The sequencing results show that PCR product has sequence shown in SEQ ID NO.1, and the amino acid sequence of the protein of coding is such as Shown in SEQ ID NO.2.
The identification of 2 arabidopsis eij1 mutant of embodiment
Identify arabidopsis eij1 mutant design of primers: in left wing's design primer P1 sequence of eij1 mutant insertion point As shown in SEQ ID NO.5 (CAGCAAGTTTCAAAAGGATTTG)；In the right flank design primer of eij1 mutant insertion point Shown in P3 sequence such as SEQ ID NO.7 (TTCCGTTGAGCTACTCCAATG)；The design primer on eij1 mutant insetion sequence Shown in P2 sequence such as SEQ ID NO.6 (ATTTTGCCGATTTCGGAAC).
Mutant eij1 material extracting genome DNA: taking three weeks blades of eij1 mutant material, be placed in liquid nitrogen and grind, Extracting genome DNA is carried out according to TIANGEN plant genome DNA extracts kit (DP305).
PCR identifies the combination of arabidopsis eij1 mutant primer: using eij1 mutant material genomic DNA as template, with drawing Object P1+P2 and P1+P3 carry out PCR amplification, PCR product electrophoresis detection.
Mutant Col and eij1 material RNA is extracted, and first chain synthesis and the detection of EIJ1 gene expression amount: is taken prominent Three weeks blades of variant eij1 material, are placed in liquid nitrogen and grind, and RNA is extracted according to Promega plant RNA extraction kit (Promega Cat#LS1040 it) carries out.The synthesis of the first chain of cDNA is according to Takara Reagent Company cDNA the first chain synthetic agent box (TaKaR D6110A), is specifically detailed in operational manual.Using obtained cDNA segment as template, design EIJ1 gene RT-PCR is just To primer SEQ ID NO.8 (GCAATTCAAAGGGTCATG) and reverse primer SEQ ID NO.9 (TATGTTGGCACTGGAGGT) RT-PCR amplification is carried out.PCR reaction system are as follows: 5 μ L mix, upstream and downstream primer (10pmol) Each 0.5 μ L, 1 μ L of template (greatly containing about 0.03 μ g), adds ddH23 μ L of O, total volume are 10 μ L.Its program is 95 DEG C of denaturation 5min； 94 DEG C of 30sec again, 60 DEG C of 30sec, 72 DEG C of 1min, totally 30 recycle；Then 72 DEG C of extension 5min；4 DEG C of preservations.
Experimental result is see Fig. 1.Figure 1A is as it can be seen that its mutational site mutant homozygosis eij1 is on third exon.Through Crossing PCR and detecting visible eij1 is homozygous mutation (Figure 1B).RT-PCR detects the discovery of EIJ1 gene expression amount, relative to wild type EIJ1 gene is hardly expressed in Col, mutant eij1.These results suggest that buying and the mutant identified is homozygous eij1 Mutant, mutational site are authentic and valid.
The acquisition of 3 transgenic arabidopsis of embodiment
With reference to the ClonExpress II One Step Cloning Kit kit of Vazyme company, handed over using recombination It changes after EIJ1 genome sequence is inserted into plant expression vector HY105 by method and converts bacillus coli DH 5 alpha, conversion fluid, which is coated on, to be contained 50mg/L card receive penicillin LB solid medium on screening positive clone.Plasmid order-checking verifying is extracted, correct carrier is verified Name are as follows: pEIJ1:EIJ1-HA.PEIJ1:EIJ1-HA is transferred in Agrobacterium tumefaciens strain GV3101 with freeze-thaw method.It will PEIJ1:EIJ1-HA passes through its corresponding eij1 mutant of Agrobacterium GV3101 mediated transformation.Harvest above institute's arabidopsis thaliana transformation kind Son, and saved after being dried in 37 DEG C.By transgenic arabidopsis seed obtained (eij1 pEIJ1:EIJ1-HA) and Basta resistance culture ware juxtaposition is seeded in the light incubator after the disinfection of its negative control seed (eij and Col), it is warm round the clock 22/20 DEG C is spent, round the clock time 12/12h.Each material is grown 5 days or so in Basta resistance screening culture dish, the positive out to be screened Plant.As a result see Fig. 2, as a result visible to pass through Basta resistance screening, eij1 pEIJ1:EIJ1-HA material can filter out the positive Plant is (shown in arrow.Positive seedling is being capable of normal growth in Basta culture medium), control Col and eij1 all material exists It is withered and yellow on Basta culture medium.
The Protein Detection of 4 transgenic arabidopsis of embodiment
The positive seedling (eij1 pEIJ1:EIJ1-HA) that example 3 to be performed is filtered out is grown on Basta screening and culturing medium To 10 days or so, it is transferred in 8cm small flower.Take 3 weeks or so transgenic arabidopsis eij1 pEIJ1:EIJ1-HA and its feminine gender Eij1 blade about 100mg is compareed, 100 μ L plant total protein extraction Buffer are added in liquid nitrogen after grinding, according to Solarbio plant Total protein extraction kit (BC3720) extracts.Western Blot detection is carried out after total protein extraction.Briefly are as follows: albumen Electrophoresis constant pressure 60V, about 30min, ran concentration glue to albumen, changed voltage into 120V, electrophoresis to bromophenol blue is run to PAGE glue Bottom terminates electrophoresis.Afterwards by protein delivery to pvdf membrane, condition is constant current 300mA electrophoresis 75min.After transferring film, take out Pvdf membrane is placed in 10mL TBST (10mM Tris-Hcl, 150mM NaCl, 0.05% (v/v) tween20PH=7.5)；It abandons Fall TBST, is added confining liquid (10mL TBST+5% skimmed milk power), is incubated for 1h-1.5h at room temperature；Confining liquid is discarded, TBST is used Cleaning pvdf membrane 3 times, each 5min；1 μ L HA antibody (Santa Cruz, Cat#sc-7392) is added in the pvdf membrane closed It is incubated for 1-2h；Pvdf membrane 3 times are cleaned with TBST, each 5min；10mL TBST is added, while the corresponding secondary antibody of a certain concentration is added, It is incubated for 1-2h at room temperature；Secondary antibody is discarded, cleans pvdf membrane 3 times with TBST, each 5min；Developing result is shown in Fig. 3.The results show that Target EIJ1-HA albumen is able to detect that in #1, #2, #3 and #5 strain of eij1 pEIJ1:EIJ1-HA material；Its yin Property control eij1 mutant in can not detect EIJ1-HA albumen.The above result shows that transgenic line (eij1 obtained It pEIJ1:EIJ1-HA is) correct.Transgenic line i.e. obtained has been transferred to EIJ1 gene and successful expression EIJ1-HA Albumen.
5 arabidopsis eij1 of embodiment and its reply material Disease Resistance Identification
By T2 transgenic arabidopsis seed (eij1 pEIJ1:EIJ1-HA) obtained and its negative control seed (eij and Col) is seeded in 8cm small flower, long to 3 weeks or so to each material, select grow fine, material of the same size, It is inoculated with Pseudomonas syringae pv.tomato strain DC3000 (Pst DC3000) (suspension of OD600=0.05) and control Mock (10mM MgCl is set2), after inoculation Pst DC3000 5 days, observes disease-resistant phenotype and takes pictures, Each material clump count is counted simultaneously.Count bacterium colony method particularly includes: each 10 blades (about 5 young plant) acquired for materials are rear each 1ml 10mM MgCl is added2It is ground in grinding sufficiently, lapping liquid is coated with after repeatedly diluting and (the addition of King ' s B culture medium 25mg ml-1Rifampin).The plate of coating counts clump count after cultivating three days at 28 DEG C.6 biology weights are arranged in each material It is multiple.Experimental result is see Fig. 4.Compared with Col, less downright bad symptom is presented in eij1 mutant, is shown to Pst DC3000 Apparent resistance (Fig. 4 A).In addition, under the treatment conditions of identical Pst DC3000, two complementary materials eij1 pEIJ1: EIJ1-HA#1 and eij1 pEIJ1:EIJ1-HA#4 has replied the disease resistance of eij1 mutant, shows and Col no significant difference (Fig. 4 A).In order to further confirm that eij1 mutant enhances the resistance to Pst DC3000, we are measured 0 and 5 after infection The quantity (dpi) of bacterium in its blade.As shown in Figure 4 B, Col and eij1 mutant does not have on initial inoculum (0dpi) Significant difference, but in 5dpi, germ number ratio col contained by eij1 mutant is substantially reduced.These statistics indicate that, eij1 be mutated The growth of Pst DC3000 is obviously inhibited in body.It is transferred to the disease-resistant phenotype for capableing of revertant eij1 after EIJ1.
6 arabidopsis eij1 mutant of embodiment and its detection for replying the disease-resistant important Marker gene of material
RNA is extracted, first chain synthesis is with detailed in Example 2.Material therefor be three weeks seedling Col, eij1 mutant and The reply material eij1 pEIJ1:EIJ1-HA of eij1.It is inoculated with pathogen Pst DC3000 (OD600=0.1) and Mock (10mM MgCl2), after processing 24 hours, sampling, which is placed in liquid nitrogen, immediately is saved.ICS1 and PR1 is the weight in plant disease-resistant immune defense Want gene.It is analysed using bioinformatics, design primer, carries out RT-PCR.The RT-PCR forward primer sequence such as SEQ of ICS1 gene ID NO.10(TCCCGCAAGAAGTATGAG)；Its reverse primer sequences such as SEQ ID NO.11 (GAGACGGCGGAGATTAGC)；The RT-PCR forward primer sequence such as SEQ ID NO.12 of PR1 gene (CACTCTGGTGGGCCTTAC), reverse primer sequences such as SEQ ID NO.13 (CCTCACTTTGGCACATCC).RT-PCR Used internal reference forward primer sequence such as SEQ ID NO.14 (AGTGTCTGGATCGGTGGTTC), reverse primer sequences are such as SEQ ID NO.15(CCCCAGCTTTTTAAGCCTTT).RT-PCR reaction system are as follows: 5 μ L mix, upstream and downstream primer (10pmol) each 0.5 μ L, 0.5 μ L of template (greatly containing about 0.03 μ g), adds ddH23.5 μ L of O, total volume are 10 μ L.Its program is 95 DEG C denaturation 2min；95 DEG C of 10sec again, 60 DEG C of 30sec are recycled for 40 totally.RT-PCR result is shown in Fig. 5.The result shows that In After PstDC3000 handles plant, compared with Col or replying material (eij1 pEIJ1:EIJ1-HA), SA is raw in eij1 mutant The expression of object synthesis gene ICS1 and SA approach Marker gene PR1 is significantly raised.And Col replys storeroom without significant with two Sex differernce.This shows that eij1 has apparent resistance to phytopathogen, further demonstrates that EIJ1 gene is exempted from adjusting plant disease-resistant It plays an important role in terms of epidemic disease.
Can any element not specifically disclosed herein, limit suitably be not present in the invention illustrated in the present invention Implement in the case where system.Thus, for example term " comprising/include " etc. be interpreted as it is open and there is no limit.In addition, this hair The terms and expressions of the bright middle use term for being described rather than limiting, and be not intended to shown and description using excluding Such terms and expressions of any equivalent feature of feature or part thereof, it should be recognized that claimed in the present invention Range can carry out a variety of modifications.It will be understood, therefore, that although specifically disclosing this hair by preferred embodiment and optional feature It is bright, but the modification and change programme that invention is wherein embodied disclosed in the present invention can be used in those skilled in the art, and And such modification and change programme are deemed to be within the scope of the present invention.
Sequence table
<110>South China Botanical Garden Chinese Academy of Sciences
<120>arabidopsis disease-resistant related gene EIJ1 and its application
<160> 15
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1539
<212> DNA
<213>arabidopsis EIJ1 gene (Arabidopsis thaliana)
<400> 1
ttggttaaat taaaaatatt taacaatcta tgtaagaaca aaaaaaaatg ttatattatc 60
atcttaaaac tagtaaattt atacatagaa aaaaattatt ttctcaatca tataagcaaa 120
ccaaaaacaa acaagcaatc tggattttgt tttgactgta ggaagaatcc aaggaattgt 180
agaggagata atgagttggt ttaaatcatt gaatcaatca ttttagcaaa caaaaagatc 240
caactttaat gggcccaggg tccaataacc caattgggtt attcctagac ttttctcctc 300
ctcctgttgt cttccaattt ggtatgcgag aaaaataaaa taaaataaaa ataagccaaa 360
gacaaattta aacctccgat ttcaaccttc tcggcgaaga agaagaagaa gaagagaaaa 420
tgacgatttg catgtgttgc gattcacatc tcgtagcgaa atcgattcta aatccatgga 480
attctcctag gataacgaag ctaggtttcg taccagtcgt tcgccgtttt ccagctacta 540
ctaccgtcaa ggcttcggcg gtggattcac ctgagagttc ttcaaatttc gccaagcgta 600
tggatcaagc ttggattatc tcacaggttc gtatctccag agagttgttg atgtgtataa 660
ttttgttgaa tctgagtaat cacaagaact gatccttgat gtagtcatgt acgagtgaag 720
agctatatgc aattttgtat gagattaaga acttggatct gtgatgaaat gttgtaaatt 780
tcagattatg aggtgaatag tatcagcaag tttcaaaagg atttgtatcg aaaatctgta 840
attgcaaagt accttttagt tggtttctag caaaaaatgt ttgttcacaa ttcatttgat 900
ttgtgcatat gatgagaaca tgttagctac ttaatcaggt tgtagctgtt ctctatgatc 960
cctgagtgat gaggcttaag caggcttatc tcgcaacgca atgtagtttt gtagctttgc 1020
atgtacaatt gttggattct ctgctttttc tctaatattg ctcttagaga atctatcaaa 1080
agttcagact ttgaagcctt tcttctagca cttgcaattt gatattgagc tcgtgtgaat 1140
tttctagatg ttacttgttg acctgtgttt cgattcaatg attttctttg tctgtttttt 1200
tcttcactgc agcaaccaag tcctgttgga tgttcatcgt gcaattcaaa gggtcatgtg 1260
gaatgcaaat ggtgtgcggg tacaggattc ttcatccttg gtgataacat gctttgccag 1320
gttccctcaa ggaatacttc atgtgtaatc tgctctggac aggtttgtta tctaataaaa 1380
acgaaagtgc agacacatta ttgtgtcttc ttgtttatat atcttcccaa gtatgaacaa 1440
attcgattct ttcaatggtt ttagggttct gctagctgta gcgattgcaa aggaacgggg 1500
tttcgtgcca agtggttgga aaaacctcca gtgccaaca 1539
<210> 2
<211> 144
<212> PRT
<213>arabidopsis EIJ1 albumen (Arabidopsis thaliana)
<400> 2
Met Thr Ile Cys Met Cys Cys Asp Ser His Leu Val Ala Lys Ser Ile
1 5 10 15
Leu Asn Pro Trp Asn Ser Pro Arg Ile Thr Lys Leu Gly Phe Val Pro
20 25 30
Val Val Arg Arg Phe Pro Ala Thr Thr Thr Val Lys Ala Ser Ala Val
35 40 45
Asp Ser Pro Glu Ser Ser Ser Asn Phe Ala Lys Arg Met Asp Gln Ala
50 55 60
Trp Ile Ile Ser Gln Gln Pro Ser Pro Val Gly Cys Ser Ser Cys Asn
65 70 75 80
Ser Lys Gly His Val Glu Cys Lys Trp Cys Ala Gly Thr Gly Phe Phe
85 90 95
Ile Leu Gly Asp Asn Met Leu Cys Gln Val Pro Ser Arg Asn Thr Ser
100 105 110
Cys Val Ile Cys Ser Gly Gln Gly Ser Ala Ser Cys Ser Asp Cys Lys
115 120 125
Gly Thr Gly Phe Arg Ala Lys Trp Leu Glu Lys Pro Pro Val Pro Thr
130 135 140
<210> 3
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
ttggttaaat taaaaatatt ta 22
<210> 4
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
tgttggcact ggaggttttt cc 22
<210> 5
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
cagcaagttt caaaaggatt tg 22
<210> 6
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
attttgccga tttcggaac 19
<210> 7
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
ttccgttgag ctactccaat g 21
<210> 8
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
gcaattcaaa gggtcatg 18
<210> 9
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
tatgttggca ctggaggt 18
<210> 10
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
tcccgcaaga agtatgag 18
<210> 11
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
gagacggcgg agattagc 18
<210> 12
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 12
cactctggtg ggccttac 18
<210> 13
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 13
cctcactttg gcacatcc 18
<210> 14
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 14
agtgtctgga tcggtggttc 20
<210> 15
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 15
ccccagcttt ttaagccttt 20

Claims (9)

1. a kind of arabidopsis EIJ1 gene, characterized in that its DNA sequence dna is as shown in SEQ ID NO.1, or is to contain SEQ ID There are replacement, missing or the increase of one or several nucleotide in sequence and sequence shown in No.1, and has and SEQ ID NO.1 The nucleotide sequence of shown identical function；Or the nucleotide sequence to be matched with SEQ ID NO.1 complete complementary；It or is coding The nucleotide sequence of amino acid sequence shown in SEQ ID NO.2.
2. arabidopsis EIJ1 albumen, characterized in that its amino acid sequence be as shown in SEQ ID NO.2, or be SEQ ID NO.2 Through replacement, missing or increase one or more amino acid in shown sequence basis, and has and SEQ ID NO.2 identical function Amino acid sequence.
3. a kind of recombination over-express vector, characterized in that it is inserted with arabidopsis EIJ1 gene described in claim 1.
4. recombination over-express vector according to claim 3, characterized in that the expression vector is HY105.
5. recombinating the preparation method of over-express vector described in claim 3 or 4, characterized in that including by SEQ ID NO.1 institute The arabidopsis EIJ1 genomic dna sequence recombination switching technology insertion plant expression vector shown.
6. the preparation method of recombination over-express vector according to claim 5, characterized in that SEQ ID NO.1 institute The arabidopsis EIJ1 genomic dna sequence shown is expanded by primer sequence shown in SEQ ID NO.3 and SEQ ID NO.4 It arrives.
7. a kind of bioengineered strain, characterized in that it is that any one of the claim 3-4 recombination over-express vector is transferred to root Cancer Agrobacterium GV3101 and obtain.
8. arabidopsis EIJ1 albumen described in arabidopsis EIJ1 gene or claim 2 described in claim 1 is in regulation plant disease-resistant Application in immune.
9. arabidopsis EIJ1 albumen described in arabidopsis EIJ1 gene or claim 2 described in claim 1 is in regulation plant disease-resistant Application in immune breeding.
CN201910697361.9A 2019-07-30 2019-07-30 Arabidopsis thaliana disease-resistant related gene EIJ1 and application thereof Active CN110484542B (en)

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CN115806999A (en) * 2021-09-14 2023-03-17 湖南中烟工业有限责任公司 Tobacco NtEIJ1 gene and application thereof

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115806999A (en) * 2021-09-14 2023-03-17 湖南中烟工业有限责任公司 Tobacco NtEIJ1 gene and application thereof
CN115806999B (en) * 2021-09-14 2024-05-17 湖南中烟工业有限责任公司 Tobacco NtEIJ gene and application thereof

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