CN103173463A - Cucumis sativus L. ethylene signal transduction pathway CTR1 gene and protein coded by same - Google Patents
Cucumis sativus L. ethylene signal transduction pathway CTR1 gene and protein coded by same Download PDFInfo
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Abstract
The invention provides a cucumis sativus L. ethylene signal transduction pathway CTR1 gene (CsCTR1). The DNA (deoxyribonucleic acid) sequence of the CsCTR1 is shown as SEQID NO. 1, and the amino acid residue sequence of a protein coded by the CsCTR1 is shown as SEQID NO. 2. The CsCTR1 comprises an open reading frame of 2559bp, is capable of coding 852 amino acids and can be inserted into an expression vector to obtain an overexpression vector. The expression vector can be used for transforming a CTR1 mutation arabidopsis for functional complementation to obtain a CsCTR1 genetically-modified pure arabidopsis. The invention lays a foundation for the transcription and the expression of the cucumis sativus L. ethylene signal transduction pathway CTR1 gene and also lays a foundation for the functional study on the application of the cucumis sativus L. ethylene signal transduction pathway CTR1gene to the growth and the development of the ethylene-regulated cucumis sativus L.
Description
Technical field
The present invention relates to genetically engineered, particularly the albumen of a kind of cucumber ethylene signaling approach CTR1 gene and coding thereof.
Background technology
Cucumber (Cucumis sativus L.) is the herbaceous plant that Curcurbitaceae (Cucurbitaceae) Cucumis (cucumis) was overgrow in a year.Particularly cucumber sexual type differentiation is various, common cucumber variety be male and female flowering independent the different same plant type (monoecious) of spending of giving birth to; But hermaphrodite flower (hermaphrodite) also can appear in some kind, and on a large amount of physiology and genetic basis, cucumber becomes the type material of research plant sexual type differentiation gradually.
Ethene as a Plant Hormone to the normal development of plant and coerce the reaction play a part very important.In development process, the physiological processs such as the coming off of ethene regulation and control seed germination seedling early growth, blade and flower, fruit maturation, organ senescence, flower sex determination.In addition, comprise injury, cause of disease intrusion, arid etc. when plant is subject to external stimulus, Ethylene biosynthesis increases, and promotes the generation of defence compound, shows that ethene plays an important role in tackling biological and abiotic stress.
On the Synthesis pathway approach, done a large amount of research.And cucumber ethylene signaling Study of way is comparatively slow, and in approach, some important components are also separated and identify.
The CTR1 gene is a negative regulator gene in the ethylene signaling approach, also first ethylene signaling pathway gene that is cloned [Kieber JJ et al.CTR1, a negative regulator of the ethylene response pathway in Arabidopsis, encodes a member of the Raf family of protein kinases.Cell, 1993,72 (3): 427-441.].heredity and Biochemical Research are found, without the aminoterminal of the CTR1 of cross-film functional domain or membrane attachment structure can with endoplasmic reticulum on the ethylene receptor carboxyl terminal interact, thereby indirectly be attached to and form ETR1/CTR1 complex body and then negative regulation ethylene reaction [Gao Z et al.Localization of the Raf-like kinase CTR1to the endoplasmic reticulum of Arabidopsis through participation in ethylene receptor signaling complexes.The Journal of Biological Chemistry on endoplasmic reticulum, 2003, 278:34725-34732.] at present, the CTR1 gene is as the key ingredient in the ethylene signaling approach, from Arabidopis thaliana, summer squash, muskmelon, tomato, wheat, Root of Rocket Consolida, Chinese rose, separate in the species such as Kiwifruit.Therefore, separate and identify that cucumber CTR1 gene provides possibility for the transcript and expression of research CTR1, also laying a good foundation for the effect of functional study CTR1 in ethene regulation and control Plant Growth of Cucumber process simultaneously.
Summary of the invention
Purpose of the present invention, in order further to study the function and application of cucumber ethylene signaling approach genes involved, and the dependency between ethylene signaling pathway gene and cucumber sexual type, the albumen of a kind of cucumber ethylene signaling approach CTR1 gene and coding thereof is provided.
The technical solution used in the present invention is as follows:
A kind of cucumber ethylene signaling approach CTR1 gene, called after CsCTR1, its DNA sequence dna is as shown in SEQID NO.1.
The albumen of cucumber ethylene signaling approach CTR1 genes encoding, its amino acid residue sequence is as shown in SEQID NO.2.
Above-mentioned cucumber ethylene signaling approach CTR1 gene, wherein, described CsCTR1 contains the open reading frame of a 2559bp, and this open reading frame is the 1st to the 2559th Nucleotide.
Above-mentioned cucumber ethylene signaling approach CTR1 gene, wherein, the initiator codon of described open reading frame is ATG, terminator codon is TGA.
The albumen of above-mentioned cucumber ethylene signaling approach CTR1 genes encoding, wherein, with replacement and/or disappearance or the interpolation of the amino acid residue sequence shown in SEQID NO.2 through an above amino-acid residue, obtain having the derived protein with the identical activity of described albumen.
Above-mentioned cucumber ethylene signaling approach CTR1 gene, wherein, the total length of this gene is obtained by following primer pair amplification:
Article one, the nucleotide sequence of primer is as shown in SEQID NO.3, and the nucleotide sequence of another primer is as shown in SEQID NO.4.
The recombinant vectors that contains said gene is described gene to be inserted between expression vector pBI121CaMV35S promotor and terminator no obtain.
The transformation of Arabidopsis thaliana pure strain that contains said gene.
The acquisition of above-mentioned cucumber ethylene signaling pathway gene CsCTR1 and proteins encoded thereof, that the aminoacid sequence (GenBank accession No.NP_850760) that utilizes Arabidopis thaliana CTR1 gene is information probes, by the BlastP program in Cucumber germplasm website (http://cucumber.genomics.org.cn), acquisition and information probes consistence are 67% csa011978, and it is positioned at karyomit(e) No. 6.Utilize the primer of csa011978 sequences Design amplification ORF sequence, extract total RNA from the mature leaf of cucumber S52 strain, through reverse transcription, ORF with conventional PCR method amplification CsCTR1, order-checking draws the ORF2559bp of CsCTR1 gene, 852 amino acid of derivation coding, initiator codon position ATG, terminator codon is TGA.Albumen prediction size is 94.521KDa, and iso-electric point is 5.77.Find by the BlastP program in Cucumber germplasm website (http://cucumber.genomics.org.cn), CsCTR1 is single copy in Cucumber germplasm.The Cucumber germplasm sequence shows that this gene comprises 15 exons, 14 introns.
With Genscan (http://genes.mit.edu/GENSCAN.html), the ORF of CsCTR1 is analyzed, obtain the similar sequences of other species by BLASTP (http://www.ncbi.nlm.nih.gov/BLAST/), carry out sequence alignment and systematic evolution tree analysis and show CsCTR1 and other plant CTR1 gene height homology, originate from during evolution same ancestors, and with pumpkin, muskmelon cluster in cluster.The protein homology modeling result shows that CTR1 contains the structure similar to Arabidopis thaliana CTR1 protein kinase structural domain height.
The invention discloses the central CTR1 gene order of cucumber ethylene signaling approach, not only the transcript and expression for CTR1 in research cucumber ethylene signaling approach lays the foundation, and also provides the basis for the dependency between research ethylene signaling pathway gene and cucumber sexual type simultaneously.In addition, also provide condition for further studying the effect of cucumber CTR1 in other relative growth growth courses except the differentiation of flower sexual type.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.These embodiment only are not used in for explanation the present invention and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, for example the Sambrook equimolecular is cloned: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
The acquisition of embodiment 1 cucumber CTR1 open reading frame (ORF)
1.RNA extraction (RNAisolation)
The south China kind that cucumber S52 collects for us, synoecy, getting its mature leaf grinds in liquid nitrogen, add in 1.5ml EP pipe, extract to specifications total RNA (Aidlab with TRIpure Reagent reagent, China), get afterwards 1ul RNA and utilize its content of spectrophotometric determination.
2. reverse transcription PCR (RT-PCR)
Take the total RNA of cucumber as template, adopt ReverTra
QPCR RT Kit amplification kit (TOYOBO, China) carries out reverse transcription cDNA the first chain, and reverse transcription step is with reference to the user manual of this test kit.
3. the acquisition of open reading frame
The aminoacid sequence (GenBank accession No.NP_850760) that utilizes Arabidopis thaliana CTR1 gene is information probes, by the BlastP program in Cucumber germplasm website (http://cucumber.genomics.org.cn), acquisition and information probes consistence are 67% csa011978, and it is positioned at karyomit(e) No. 6.Utilize the primer (seeing sequence table sequence 3 and 4) of csa011978 sequences Design amplification ORF sequence.
PCR reaction system: 40ng cDNA, the 0.5 forward and reverse primer of μ M, 25 μ l2 * PrimeSTAR Max (TaKaRa, China), reaction system 50 μ l.Reaction conditions: 95 ℃ of sex change 10s, 55 ℃ of annealing 15s, 72 ℃ are extended 30s, carry out 30 circulations; Last 72 ℃ are extended 20min (adding Taq enzyme 0.5ul, dNTP3ul).The PCR product of gained after separating, is reclaimed 1% agarose gel electrophoresis.Reclaimer operation is according to the agar gel recovery test kit specification sheets of giving birth to the biological company limited of work.
Embodiment 2CsCTR1 gene-correlation albumen carries out the evaluation of eukaryotic expression and transfer-gen plant in arabidopsis mutant strain CTR1
1, contain the destination gene expression Vector construction
Utilize restriction enzyme XmaI and SacI double digestion CsCTR1DNA recovery fragment and plant binary expression vector pBI121 simultaneously, CsCTR1 is inserted between expression vector pBI121CaMV35S promotor and terminator no.The endonuclease reaction system is as shown in table 1:
Table 1
Mix and slightly centrifugal, be placed in 37 ℃ of constant-temperature metal baths and process 3h, process 20min for 65 ℃ afterwards.The DNA fragmentation electrophoresis reclaims purifying; Agarose gel electrophoresis detects it and reclaims quality and relative concentration.
Gene fragment is mixed to final volume 10 μ l with the plasmid fragment in the quality ratio of 3: 1～5: 1 respectively, then carries out ligation by the T4 ligase enzyme, reaction system is as shown in table 2:
Table 2
Mix and slightly centrifugal, after being placed in 22 ℃ of constant-temperature metal baths and processing 1h, 4 ℃ are spent the night.Reaction finishes rear 65 ℃ and processes 10min deactivation ligase enzyme.
To connect product with electric shocking method and be transformed into the bacillus coli DH 5 alpha competent cell.Concrete grammar is as follows:
1. will connect product and dilute 10 times with cold (0 ℃-4 ℃) deionized water.
2. draw the 1.5ml centrifuge tube that 1ml SOB substratum is placed in sterilization.
3. get the centrifuge tube that connection product after 1 μ l dilution adds splendid attire 50 μ l competent cell DH5 α, mixing.
4. will be added with the competent cell that connects product and add electric shock cup (specification is 1mm), the conversion of shocking by electricity of the electric shock instrument of packing into (EppendorfInc.), 1700V5ms.
5. after the electric shock, bacterium liquid is washed in 1ml SOB substratum.
6. 37 ℃, 200r/min cultivates 1h.
After bacterium liquid is cultivated 1h, bacterium liquid is spread upon equably on the flat board of LB solid medium (this substratum contains kantlex 50mg/L), be inverted in 37 ℃ of incubator overnight incubation.
Toothpick with the bacterium of going out after incubated overnight is chosen the bacterium colony on flat board in the LB liquid nutrient medium that 1ml contains kantlex (50mg/L), and 37 ℃, 150r/min carries out bacterium liquid PCR after cultivating 4h, and residue bacterium liquid continues at 37 ℃, the 150r/min overnight incubation.
The system of bacterium liquid PCR is: the forward and reverse primer of CsCTR1 gene fragment (Fw:cttcaccatt gcgatttcc, Re:tcagttgggc tgctttttt) each 0.4 μ M, 2 * GoldStar Best MasterMix (Beijing health is the reagent product), bacterium liquid 1 μ l, the total reaction system is 20 μ l.Bacterium liquid PCR program is: 95 ℃ of 10min; 95 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 30s, 30 circulations; 72 ℃ of 5min.
Reaction product is delivered to Shanghai Sani biotech company with the bacterium liquid that amplifies the big or small fragment of expection and is checked order with 1.5% agarose gel electrophoresis analysis, and sequencing primer is pBI121 plasmid 35S the preceding paragraph sequence (SEQ ID NO.5).After being shaken bacterium, the correct positive strain of order-checking extracts plasmid.
2, the conversion of Agrobacterium
Adopt electric shocking method to be transformed into agrobacterium strains GV3101 competent cell through the expression vector CTR1-pBI121 that order-checking is identified.Concrete grammar is as follows:
1. plasmid is diluted 10 times with cold (0 ℃-4 ℃) deionized water.
2. draw the 1.5ml centrifuge tube that 1ml SOB substratum is placed in sterilization.
3. the plasmid of getting after 1 μ l dilutes adds in the centrifuge tube of splendid attire 40 μ l competent cell GV3101, mixing.
4. will be added with the competent cell that connects product and add electric shock cup (specification is 1mm), the conversion of shocking by electricity of the electric shock instrument of packing into (EppendorfInc.), 1700V5ms.
5. after the electric shock, bacterium liquid is washed in 1ml SOB substratum, 28 ℃, 150r/min cultivates 2h.Afterwards bacterium liquid is spread upon equably on the flat board of LB solid medium and (contain 50mgL
-1Rifampin, 50mgL
-1Kantlex, 15mgL
-1Gentamicin), be inverted in 28.℃ incubator is cultivated 2d, carries out PCR with the toothpick picking part mono-clonal of the bacterium of going out and detects, positive clone strain-70.C preserves, and is used for the Arabidopis thaliana genetic transformation.
3, mouseearcress sterile culture:
The Arabidopis thaliana seed was sterilized in thimerosal (drift ice 10ml+ distilled water 40ml+Tween20 2-3 drips) approximately 15 minutes, afterwards in super clean bench with aseptic water washing 5-6 time to remove remaining thimerosal, after be layered on equably on MS solid medium (MS4.43g+ sucrose 20g+ agar 8g, PH5.8).In 4 ℃ of refrigerators after vernalization 3d, flat board is changed over to 22 ℃, 16h illumination 8h are dark, the white light intensity of illumination is about 140 μ molm
-2S
-1Phytotron in cultivate 5-6d, connect afterwards root with tweezers and take out seedling from culture dish, transplant to the matrix that is full of nutritive medium (vermiculite, peat soil and perlite 7: 2: 1, nutritive medium: 3g/10L spends intact), with removing lid after a transparent cover lid 5d.
4, the genetic transformation of Arabidopis thaliana
Picking 5-6 Agrobacterium clone inserts in antibiotic LB liquid nutrient medium of containing of 20ml (LB+ Rifampin 50 μ g/ml, gentamicin 15 μ g/ml, kantlex 50 μ g/ml), and 28 ℃, shaking culture 24h; Add 200ml to contain (LB+ kantlex 25 μ g/ml) in antibiotic LB liquid nutrient medium, 28 ℃, shaking culture 24h culture; Room temperature 4000r/min, centrifugal 15min collects thalline; Thalline is mixed with transforming Buffer (MS2.15g/L+ sucrose 50g/L+Silwet L-77400 μ l/L+6-BA20 μ l (mother liquor 1mg/ml) PH5.8), suspends standby.
Transformation of Arabidopsis thaliana adopts the colored method of dipping in.Concrete steps are as follows:
1. get growth about month, the upgrowth situation good stand transforms and waters sufficient water the day before yesterday.
When 2. transforming, the Arabidopis thaliana over-ground part is soaked in advance with transforming 15-30s in the bacterium liquid that Buffer suspends, guarantees that whole petals all are submerged.Lie against in the dish of cave plant and cover lid, to keep humidity, lucifuge is spent the night.
3. second day uncovers, and plant is taken out, and props up, and transfers under normal condition and grows.
4. with the arabidopsis thaliana transformation seed of results, be inoculated in after sterilization on the 1/2MS solid medium, move to Arabidopis thaliana culturing room in 4 ℃ of refrigerators after vernalization 3d and cultivate, approximately after the week, green resistance seedling (T1 generation) is moved to continued growth in matrix.The plant to be planted rear extraction DNA that grows up carries out the PCR detection validation.
5. the T1 of test positive extracted RNA for the Arabidopis thaliana plant, by the expression of sxemiquantitative and its gene of Real time PCR, choose the high plant of expression amount and proceed the pure lines screening.
6. the T1 that gathers in the crops test positive continues the sowing screening for Arabidopis thaliana plant seed (T2 generation), chooses segregation ratio and be the strain of 3: 1, and its resistant plant is continued to cultivate results seeds (T3 generation), does not have separative strain to be pure lines after screening.
5, the analysis that has complementary functions of CsCTR1 Arabidopis thaliana transgenosis strain
The strain of pure lines Arabidopis thaliana transgenosis, Colombia's wild-type (col) and mutant strain CTR1 seed are carried out disinfection, be taped against on the MS solid medium, in 4 ℃ of refrigerators after vernalization 3d, a part puts into that 22 ℃, 16h illumination 8h are dark, the white light intensity of illumination is about 140 μ molm
-2S
-1Phytotron in cultivate 5d, another part is put into 5d under dark surrounds.Observe afterwards the phenotype of mutant strain CTR1 under illumination and dark surrounds, transgenic line and wild-type col, take pictures and the root of measuring seedling under the long and dark surrounds of the root of seedling under photoenvironment is grown and hypocotyl length.
Draw three strain CsCTR1-15, CsCTR1-33 and CsCTR1-47 that expression amount is high by Molecular Detection, they are continued the sowing screening, and segregation ratio is 3: 1, and it is continued to cultivate results seed (T3 generation), do not separate after screening, prove that these three strains are single pure lines of inserting.The Phenotypic Observation discovery, the part phenotype of cucumber CTR1 gene pairs arabidopsis mutant strain CTR1 is recovered.The arabidopsis mutant strain CTR1 seedling of growth 5d under light, the long wild-type that significantly is shorter than of root, transformant pure lines CsCTR1-15, CsCTR1-33 and CsCTR1-47 recover this phenotype; The seedling that grows under dark surrounds shows triple response, and 3 transformant pure lines of dark growth 5d have been carried out recovery in various degree to this proterties, the maximum that transformant CsCTR1-15 recovers hypocotyl length.The transformant of growing under light has also carried out recovery to a certain degree to mutant strain lotus throne leaf size and gynoecium.
Sequence table
＜110〉Shanghai Communications University
＜120〉albumen of cucumber ethylene signaling approach CTR1 gene and coding thereof
<160>5
<210>1
<211>2559
<212>DNA
＜213〉cucumber (Cucumis sativus L.)
<400>1
atggaaatgc ctggacggag gtcagattac tcgcttttaa gtcaaattcc ggacgaggaa 60
gttggaacgg gagcttccac ttccttttac gactctatag cagctggggg aaacgttatt 120
aaagggagaa ccgatagagt ttttgattgg gatgggattg gtgatcacag gttaaacacg 180
caggcgtatc ggacagggaa cctgtattca tggattggtt tgcagaggca ttccagtgga 240
agcagctacg atgatagctc tctctctagt gattactacg caccgacgct atcaaacgct 300
gcagcaaatg agatcaatgc attggaatat atccacgatg atgatttcag agtgatgaaa 360
gctgtgggaa gtggaggttc gtctggaaag agctgggccc agcagacgga agagagttat 420
cagttgcagc aggccttggc tcttaggctt tcttcagagg ctacttgtgc tgatgatccc 480
aactttatgg atccgatgcc agacgaggca gctttaaggt cgttatccat ttcagctgag 540
gccatctcgc atcgattctg ggtaaatgga tgtatgtcat attttgagaa agtgccagat 600
ggtttttatc taattcatgg gatggaccca tatgtatggt cattatgcac caatctgcaa 660
gaggatgggc gtataccatc atttgaatct ctgaaaactg ttgactccag catcgcttca 720
tcaattgaag tagttttgat agatcggcat agtgatgcta gcttaaaaga actgcaaaac 780
agggtgcata atattgcttc tagttgtgca actacaaaag gggttgcaga tcatttagcg 840
aagctggtat gcaatcactt ggggggttca gtttctgagg gagaagatga cttggtttct 900
tcctggaagg aatgcagcga tgacttaaag gaatgtttgg gatctgccgt tattccctta 960
tgcagcttat ctgttggcct ttgcagacat cgtgctcttt tattcaaagt cctagctgat 1020
tcaattgatt tgccctgtcg aattgctagg ggttgtaaat attgcactag agatgatgct 1080
tcatcttgcc ttgttaggtt cgggcttgat agggaatatc tcattgatct gattgggagg 1140
ccaggttgct tatgcgaacc tgattctttg ctcaatggtc catcatccat ctcaatttct 1200
tcaccattgc gatttccaag actaaaacct attgaatcta tcattgattt caggtcactg 1260
gccaaacagt atttcttgga tagccaatca cttaatgttg tatttgatga agcttcttca 1320
gggaatgttg tatctgggaa ggatgctgcg ttctctgtct atcaaaggcc attaaatagg 1380
aaggatggag atagaaaaat catagtggtt actggtgaca aggacagaaa ttctcagtta 1440
ttgaataaaa aagcagccca actgaatact caagatggaa agtctgagca atttagatca 1500
tgtgttactt ctcaatatag tgtacagtcg acccctttag tagaaaacgt agtccctttg 1560
aaccatatct cacccattgg ttccaaagat tctgagcatc tcttagcatt gtctcatcca 1620
agggtggatc atgctaacaa tttaccattt gttgacggta gtcagttgat tagaaaacca 1680
aatgatcttt cccttggctt agaagatttg gttattccat ggaaagatct tgatttgaga 1740
gagaaaattg gagcaggttc ttttgggact gtatatcatg ctgattggca tggctcagat 1800
gttgctgtga agattctgat ggaacaagac cttcatgcag aacgttttga tgaatttctt 1860
agggaggttg ccataatgaa atgtctacga catccaaaca tcgttctctt tatgggtgca 1920
gtcacagagc ctccaaactt gtccattgta acagaatact tatcaagggg tagtttgcac 1980
aggcttttgc acagacctgg ggcacgtgaa gtcttagatg agagacgacg gttaaacatg 2040
gcatatgatg ttgcaaaggg aatgaattat cttcacaaac gtaatccccc aattgttcat 2100
cgcgatttga aatcaccaaa tcttttggtt gataagaagt acactgtgaa ggtttgtgat 2160
tttgggcttt cacgcttgaa ggcacacaca tttctttcgt caaaatcagc tgctgggact 2220
cctgagtgga tggcaccaga agttctccgt gatgagccat caaatgagaa atctgatgtt 2280
tatagttttg gtgtgatact atgggagctt gctacattgc agcaaccatg gggcaacatg 2340
aacccaccgc aggttgtggc agctgttggt ttcaaaggta aaaggcttga gatcccatgt 2400
gatttagatc ctcgagttgc tactattata gaggcttgct ttgccagtga gccttggaaa 2460
cgcccttcct tttatgaaat aatggaatcg ttgaagccat tgatcaaacc tgctacacct 2520
catcaagttc gctcaaacat gtcactagtt actcagtga 2559。
<210>2
<211>852
<212>PRT
＜213〉CsCTR1 albumen
<400>2
MEMPGRRSDY SLLSQIPDEE VGTGASTSFY DSIAAGGNVI KGRTDRVFDW DGIGDHRLNT 60
QAYRTGNLYS WIGLQRHSSG SSYDDSSLSS DYYAPTLSNA AANEINALEY IHDDDFRVMK 120
AVGSGGSSGK SWAQQTEESY QLQQALALRL SSEATCADDP NFMDPMPDEA ALRSLSISAE 180
AISHRFWVNG CMSYFEKVPD GFYLIHGMDP YVWSLCTNLQ EDGRIPSFES LKTVDSSIAS 240
SIEVVLIDRH SDASLKELQN RVHNIASSCA TTKGVADHLA KLVCNHLGGS VSEGEDDLVS 300
SWKECSDDLK ECLGSAVIPL CSLSVGLCRH RALLFKVLAD SIDLPCRIAR GCKYCTRDDA 360
SSCLVRFGLD REYLIDLIGR PGCLCEPDSL LNGPSSISIS SPLRFPRLKP IESIIDFRSL 420
AKQYFLDSQS LNVVFDEASS GNVVSGKDAA FSVYQRPLNR KDGDRKIIVV TGDKDRNSQL 480
LNKKAAQLNT QDGKSEQFRS CVTSQYSVQS TPLVENVVPL NHISPIGSKD SEHLLALSHP 540
RVDHANNLPF VDGSQLIRKP NDLSLGLEDL VIPWKDLDLR EKIGAGSFGT VYHADWHGSD 600
VAVKILMEQD LHAERFDEFL REVAIMKCLR HPNIVLFMGA VTEPPNLSIV TEYLSRGSLH 660
RLLHRPGARE VLDERRRLNM AYDVAKGMNY LHKRNPPIVH RDLKSPNLLV DKKYTVKVCD 720
FGLSRLKAHT FLSSKSAAGT PEWMAPEVLR DEPSNEKSDV YSFGVILWEL ATLQQPWGNM 780
NPPQVVAAVG FKGKRLEIPC DLDPRVATII EACFASEPWK RPSFYEIMES LKPLIKPATP 840
HQVRSNMSLV TQ 852。
<210>3
<211>28
<212>DNA
＜213〉artificial sequence
<400>3
tcccccgggc accccttcaa caatggcg 28。
<210>4
<211>26
<212>DNA
＜213〉artificial sequence
<400>4
cgagctcgtc ccttgggtcc cctcac 26。
<210>5
<211>20
<212>DNA
＜213〉artificial sequence
<400>5
gacgcacaat cccactatcc 20。
Claims (8)
1. cucumber ethylene signaling approach CTR1 gene, called after CsCTR1, its DNA sequence dna is as shown in SEQID NO.1.
2. the albumen of cucumber ethylene signaling approach CTR1 genes encoding claimed in claim 1, its amino acid residue sequence is as shown in SEQID NO.2.
3. cucumber ethylene signaling approach CTR1 gene according to claim 1, is characterized in that, described CsCTR1 contains the open reading frame of a 2559bp, and this open reading frame is the 1st to the 2559th Nucleotide.
4. cucumber ethylene signaling approach CTR1 gene according to claim 3, is characterized in that, the initiator codon of described open reading frame is ATG, and terminator codon is TGA.
5. the albumen of cucumber ethylene signaling approach CTR1 genes encoding according to claim 2, it is characterized in that, with replacement and/or disappearance or the interpolation of the amino acid residue sequence shown in SEQID NO.2 through an above amino-acid residue, obtain having the derived protein with the identical activity of described albumen.
6. cucumber ethylene signaling approach CTR1 gene according to claim 1, is characterized in that, the total length of this gene is obtained by following primer pair amplification:
Article one, the nucleotide sequence of primer is as shown in SEQID NO.3, and the nucleotide sequence of another primer is as shown in SEQID NO.4.
7. contain the recombinant vectors of the described gene of claim 1, it is characterized in that: will obtain between described gene insertion expression vector pBI121CaMV35S promotor and terminator no.
8. the transformation of Arabidopsis thaliana pure strain that contains the described gene of claim 1.
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| CN111549041A (en) * | 2020-04-22 | 2020-08-18 | 青岛农业大学 | Ethylene-induced BAHD acyltransferase ERAT2 gene and application thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111549041A (en) * | 2020-04-22 | 2020-08-18 | 青岛农业大学 | Ethylene-induced BAHD acyltransferase ERAT2 gene and application thereof |
| CN111549041B (en) * | 2020-04-22 | 2022-07-08 | 青岛农业大学 | Ethylene-induced BAHD acyltransferase ERAT2 gene and application thereof |
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