CN1982333A - Populus diversifolia PeCBL1 gene and its use - Google Patents
Populus diversifolia PeCBL1 gene and its use Download PDFInfo
- Publication number
- CN1982333A CN1982333A CN 200510130448 CN200510130448A CN1982333A CN 1982333 A CN1982333 A CN 1982333A CN 200510130448 CN200510130448 CN 200510130448 CN 200510130448 A CN200510130448 A CN 200510130448A CN 1982333 A CN1982333 A CN 1982333A
- Authority
- CN
- China
- Prior art keywords
- sequence
- gene
- acid residue
- dna
- pecbl1
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
CBL1 gene of Populus euphratica Olive and its use are disclosed. The CBL1 gene is named PeCBL1, which has amino-acid residue sequence of sequence 2 or derived protein having same activity with amino-acid residue sequence substituted, lost or added by one or several amino-acid residue. It has anti-reversible performance, 90% homology with DNA sequence defined by sequence 1 and important regulating function in breeding drought-resisting, cold-resisting and salt-resisting plant variety.
Description
Technical field
The present invention relates to PeCBL1 encoding gene and application from diversiform-leaved poplar (Populus euphratica Oliver).
Background technology
Plant can be subjected to the influence of many adverse environment factors in process of growth, damage to plants caused by sudden drop in temperature, and arid and salt damage etc. can the serious normal growths that disturbs forest, farm crop.Cultivate the resistance of reverse new variety of plant is one of major objective of agricultural scientific and technical research always.Organism self can experience the stimulation of the adverse circumstance environment that comes from the outside, and regulates and control a series of resistance expression of gene.The CBL1 gene is as a factor relevant with the calcium signal transduction, and abduction delivering strengthens when being subjected to environment-stress, thereby combines the expression that starts downstream adverse circumstance response gene with calcium ion.
Diversiform-leaved poplar is to be grown in arid, saline and alkaline geographic a kind of arbor.Utilize drought resisting, the resistant gene of salt resource of diversiform-leaved poplar, for cultivating the forest new variety, it is significant to improve its resistance.
Summary of the invention
The purpose of this invention is to provide to have has CBL1 encoding gene than the strongly expressed characteristic to adverse circumstance, and this gene has the important regulating and controlling effect plant in to the adverse circumstance signal transduction.
Gene source provided by the present invention is in diversiform-leaved poplar, name is called: PeCBL1, protein with sequence 2 amino acid residue sequences in the sequence table, or with the amino acid residue sequence of sequence 2 through replacement, disappearance or the interpolation of one or several amino-acid residue and with the identical active sequence 2 deutero-protein that have of amino acid residue sequence of sequence 2.
The protein that sequence 2 amino acid residue sequences are made up of 213 amino-acid residues in the sequence table has 4 conservative EF-hand structural domains.This genetic expression is induced by low temperature, arid and salt mainly.
The encoding gene of PeCBL1 is one of following nucleotide sequences:
1) dna sequence dna of sequence 1 in the sequence table.
2) with sequence table in the dna sequence dna that limits of sequence 1 have 90% above homology, and the identical function protein dna sequence of encoding.
Gene with coding PeCBL1 provided by the present invention, use any expression vector (these plant expression vectors comprise double base agrobacterium vector and the carrier that is used for the unifacial leaf micropellet bombardment) transformed plant of can induction exogenous gene in plant, expressing, can obtain stronger to low temperature, arid and salt tolerant transfer-gen plant.
Gene of the present invention can add any strong promoter or inducible promoter in being building up to plant expression vector the time before its transcription initiation Nucleotide.These promotors are a lot, and as cauliflower mosaic virus (CAMV 35S) and Ubiqutin promotor etc., they can use separately or be used in combination with other plant promoter.Gene of the present invention also can use enhanser when being building up to plant expression vector, comprise translational enhancer or transcriptional enhancer.These enhanser zones can make ATG initiator codon and neighboring region initiator codon etc., but must be identical with the reading frame of encoding sequence, to guarantee the translation of whole sequence.Translation control signal and initiator codon can be multiple different sourcess such as natural or synthetic.The translation initiation district can be from transcription initiation zone or structure gene.
For the ease of transgenic plant cells or plant being identified and screening, can process employed carrier, comprise adding the alternative mark of plant.The selected marker of using can be encoded to the gene of antibiotics resistance enzyme, and microbiotic comprises gentamicin, Totomycin, kantlex etc.Also can be to produce the enzyme of colour-change or the gene of luminophor, as GUS, luciferase, can also be the gene of anti-chemical reagent (for example weedkiller).Certainly, also can any selection markers.
Carry PeCBL1 expression carrier of the present invention and can import vegetable cell (Weissbach by using conventional biotechnological means such as Ti granulation, Ri plasmid, the direct DNA conversion of plant viral vector, microinjection, electroporation, 1998, Method for Plant Molecular Biology VIII, Academy Press, New York, pp.411-463:Geiserson and Corey, 1998, Plant Molecular Biology (2
NdEdition).
Can use the plant expression vector that comprises PeCBL1 gene of the present invention to transform plant host (both can be monocotyledons, also can be dicotyledons), cultivate the plant variety of cold-resistant, drought resisting, salt tolerant.
The invention will be further described below in conjunction with specific examples.
Description of drawings
Fig. 1 is the full length gene electrophorogram that obtains by RT-PCR technology clone
M: for the marker size is 1000bp, 750bp, 500bp; 1 is PeCBL1 gene PCR product, learns that according to figure gained PCR product size is about 750bp.
Fig. 2 is the RT-PCR detected result of PeCBL1 gene expression status under conditions such as low temperature, arid, salt damage.
Analyze by RT-PCR, the PeCBL1 gene transcription level is all obviously strengthened under salt, arid and cold inductive condition.Wherein last figure be PeCBL1 gene transcription level diversity ratio, figure below is Action genetic transcription contrast, finds that by contrast the quantity of employed each RNA sample is suitable.
Fig. 3 be with PeCBL1 gene constructed in the PBI121 expression vector by RT-PCR technology sieve bacterium electrophoresis result.
By detecting, finding has a PeCBL1 gene forward to be inserted into PBI121 carrier (A), and one is oppositely inserted (B).The used PCR primer of last figure is in the forward primer gained result who inserts the upstream design of fragment carrier.The used PCR primer of figure below is on the PeCBL1 gene.A left side is labeled as the Marker size.
Embodiment
Embodiment 1, PeCBL1 gene clone
At first extract the total RNA of diversiform-leaved poplar leaflet tablet that arid is handled, detect it by spectrophotometer and extract quality by CTAB (Sa nurse Brooker, molecular cloning experiment guide, the third edition) method.Similarity according to plant CBL1 genes such as Arabidopis thaliana, paddy rice, willows has designed a pair of primer 5 '-AGTCTGGTATTTGGCTGCC-3 ', 5 '-ACTCAGACCAACACAGTTCTC-3 '.(consider the high similarity of CBL1 and CBL9 gene during the design primer, so primer will be at the lower non-coding region of similarity) obtain the total length of this gene by RT-PCR technology clone, and product is connected in the pMD18-T conversion carrier, and carrier construction is detected and order-checking.
Embodiment 2, the Populus diversifolia PeCBL 1 gene expression characteristic in adverse circumstance
Material processing:
The simulation salt damage is handled: in the flowerpot with the disposable adding of the NaCl solution of 2% (NaCl quality/soil property amount) diversiform-leaved poplar seedling (life in 2 years) normal growth, respectively at 0 hour, 1 hour, 3 hours, 6 hours, 12 hours, blade sampling in 24 hours, back liquid nitrogen flash freezer, after be saved in-80 ℃ the Ultralow Temperature Freezer, with standby.
Simulating drought is handled: choose 2 years living diversiform-leaved poplar seedlings of normal growth, the simulating drought that dewaters naturally at ambient temperature is respectively dehydration 0 hour, 3 hours, 6 hours, 12 hours, the sampling of 24 hours blades is saved in behind the liquid nitrogen flash freezer in-80 ℃ the Ultralow Temperature Freezer, with standby.
Processing is damaged to plants caused by sudden drop in temperature in simulation: chooses 2 years living seedling of normal growth, places between-4 ℃ of dark places, and respectively at 0 hour, 3 hours, 6 hours, 12 hours, got its blade in 24 hours, be saved in behind the liquid nitrogen flash freezer in-80 ℃ the Ultralow Temperature Freezer, with standby.
The RT-PCR expression characteristic is analyzed:
The diversiform-leaved poplar blade of handling is extracted total RNA with the CTAB method, and detect its quality and concentration, detect its expression characterization by sxemiquantitative RT-PCR method then by spectrophotometer.
The structure of embodiment 3, Populus diversifolia PeCBL 1 gene expression vector
With gene (PCR product) and the PBI121 conversion carrier that the clone obtains, under 37 ℃, cut 1 hour by the Xbal enzyme.The product that cuts is spent the night with the ligase enzyme connection under 16 ℃, connector is transformed into competent escherichia coli cell.Last PCR detects the result of vector construction.
Sequence table
The gene order total length of sequence 1, PeCBL1 coding and coded aminoacid sequence
1 ATGGGCTGTTTTAGTTCTAAAGTAGCAAGACAGTTCCCTGGACACGAGGACCCTGTTGCC
1 M G C F S S K V A R Q F P G H E D P V A
61 CTTGCCTCACAAACAGCTTTTAGTGTGAGTGAAGTTGAAGCACTTTTTGAACTATACAAG
21 L A S Q T A F S V S E V E A L F E L Y K
121 AGCATTAGCAGTTCTGTGGTTGATGATGGGTTAATAAGCAAGGAAGAGTTTCAGTTGGCT
41 S I S S S V V D D G L I S K E E F Q L A
181 CTTTTCAAAAACAGAAAGAAAGAGAATCTCTTTGCAAATAGGATTTTTGAGCTGTTTGAT
61 L F K N R K K E N L F A N R I F E L F D
241 GTTAAGCAAAAGGGAGTCATTGATTTCAGTGATTTTGTTAGATCACTCAATGTCTTCCAT
81 V K Q K G V I D F S D F V R S L N V F H
301 CCCAATGCCTCACAAGAAGACAAGATAGACTTCTCATTTAAACTATATGATCTGTATAAC
101 P N A S Q E D K I D F S F K L Y D L Y N
361 ACGGGATTCATTGAGCGCCAAGAGGTTAAGCAAATGTTGATTGCACTTCTCTGTGAATCG
121 T G F I E R Q E V K Q M L I A L L C E S
421 GAAATGAAGTTGGCTGATGAGACTGTTGAGATAATTCTTGATAAGACTTTCATGGACGCT
141 E M K L A D E T V E I I L D K T F M D A
481 GATGTAAATAGAGATGGGAAGATAGACAAGTCTGAGTGGGAGAACTTTGTATGTAGAAAC
161 D V N R D G K I D K S E W E N F V C R N
541 CCATCCTTATTAAAGATAATGACTCTCCCCTACTTGAGGGACATCACAACAACGTTTCCG
181 P S L L K I M T L P Y L R D I T T T F P
601 AGTTTTGTCTTTAATTCTGAGGTGGATGAGATCGCTGCATAG
201 S F V F N S E V D E I A A *
The aminoacid sequence of sequence 2, PeCBL1 genes encoding and EF-hand district (underscore part)
MGCFSSKVARQFPGHEDPVALASQTAFSVSEVEALFELYKSISS
SVVGDGLISKEEFQLA
LFKNRKENLFANRIFELF
DVKQKGVIDFSDFVRSLNVFHPNASQEDKIDFSFKLY
DLYNT
GFIERQEVKQMLIALLCESEMKLADETVEIILDKTFLDA
DVNRDGKIDKSEWENFVCRNP
SLLKIMTLPYLRDITTTFPSFVFNSEVDEIAA*
Claims (8)
1, from the PeCBL1 gene of diversiform-leaved poplar (Populus euphratica Oliver), it have with sequence table in sequence 2 amino acid residue sequence or with the amino acid residue sequence of sequence 2 through replacement, disappearance or the interpolation of one or several amino-acid residue and have identical active sequence 2 deutero-protein that have of amino acid residue sequence with sequence 2.
2, gene according to claim 1 is characterized in that: it has the amino acid residue sequence of sequence 2 in the sequence table.
3, the encoding gene of Populus diversifolia PeCBL 1, it is one of following nucleotide sequences:
1) dna sequence dna of sequence 1 in the sequence table.
2) with sequence table in the dna sequence dna that limits of sequence 1 have 90% above homology, and the identical function protein DNA sequence of encoding.
4, gene according to claim 3 is characterized in that: the gene of described PeCBL1 coding is the dna sequence dna of sequence 1 in the sequence table.
5, gene according to claim 4 is characterized in that: the reading frame of this gene is for containing 213 amino acid whose open reading frame of coding.
6, contain the described expression vector of claim 3.
7, the clone that contains the described gene of claim 3.
8, the application of right 3 described genes in cultivating cold-resistant, drought resisting, salt-resistant plant.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN 200510130448 CN1982333A (en) | 2005-12-13 | 2005-12-13 | Populus diversifolia PeCBL1 gene and its use |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN 200510130448 CN1982333A (en) | 2005-12-13 | 2005-12-13 | Populus diversifolia PeCBL1 gene and its use |
Publications (1)
Publication Number | Publication Date |
---|---|
CN1982333A true CN1982333A (en) | 2007-06-20 |
Family
ID=38165208
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN 200510130448 Pending CN1982333A (en) | 2005-12-13 | 2005-12-13 | Populus diversifolia PeCBL1 gene and its use |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN1982333A (en) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102382826A (en) * | 2010-09-06 | 2012-03-21 | 尹伟伦 | Sequence and application of Populus deltoides ERECTA gene promoters |
CN102382842A (en) * | 2010-09-06 | 2012-03-21 | 尹伟伦 | Analysis and utilization for gene function of PeCBL10 of populus euphratica olive |
CN103031331A (en) * | 2012-12-06 | 2013-04-10 | 中国农业大学 | Application of OsCBL1 protein in culture of low-potassium-tolerance adversity stress plant |
CN109295029A (en) * | 2018-11-14 | 2019-02-01 | 西南大学 | Application of the cotton class calcineurin B subunit gene GhCBL1 in cotton and tobacco breeding |
-
2005
- 2005-12-13 CN CN 200510130448 patent/CN1982333A/en active Pending
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102382826A (en) * | 2010-09-06 | 2012-03-21 | 尹伟伦 | Sequence and application of Populus deltoides ERECTA gene promoters |
CN102382842A (en) * | 2010-09-06 | 2012-03-21 | 尹伟伦 | Analysis and utilization for gene function of PeCBL10 of populus euphratica olive |
CN103031331A (en) * | 2012-12-06 | 2013-04-10 | 中国农业大学 | Application of OsCBL1 protein in culture of low-potassium-tolerance adversity stress plant |
CN109295029A (en) * | 2018-11-14 | 2019-02-01 | 西南大学 | Application of the cotton class calcineurin B subunit gene GhCBL1 in cotton and tobacco breeding |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP6268104B2 (en) | Improvement of drought tolerance in plants: Pectinesterase | |
US9809827B2 (en) | Transgenic maize | |
EP2520656A2 (en) | Transgenic plants with increased stress tolerance and yield | |
CN104004767B (en) | Vector and method for WRKY transcription factor polypeptide gene application | |
US20160010107A1 (en) | Drought resistance in plants: upl3 | |
US10961545B2 (en) | Drought resistance in plants: UPL4 | |
CN101412751B (en) | Protein related to cold resistance of plant, coding genes and application thereof | |
EP2487246A9 (en) | Transgenic plants with increased stress tolerance and yield | |
CN1982333A (en) | Populus diversifolia PeCBL1 gene and its use | |
CN103172715A (en) | Plant epidermal hair controlling gene and application thereof | |
CN101712718B (en) | Protein relevant to plant drought resistance, coding gene and application thereof | |
CN102050870A (en) | Identification and function research on populus euphratica olive SCL7 gene | |
KR101431124B1 (en) | Transgenic plant with enhanced tolerance to cold stress and salt stress by introducing nrdH gene and preparation method thereof | |
CN103588867B (en) | Soybean transcription factor GmMYB174a, and coding gene and applications thereof | |
CN102174092B (en) | ABA (abscisic acid) and salt related protein STS1 (steroid sulfatase 1) and encoding genes and application thereof | |
CN1982334A (en) | Populus diversifolia PeCBL2 gene and its use | |
CN104610438A (en) | Cotton stress response associated protein GhGeBP and coding gene and application thereof | |
WO2018087301A1 (en) | Methods for improving drought resistance in plants - kgdr06, kgdr26, kgdr25, kgdr42 and kgdr37 | |
CN102964438B (en) | Stress-resistance-related protein PpLEA3-23 of plant as well as coding gene and application of protein | |
CN103725701B (en) | A kind of method of cultivating the drought-resistant plant of transgenosis | |
CN102382842A (en) | Analysis and utilization for gene function of PeCBL10 of populus euphratica olive | |
CN118064454A (en) | Gene separated from medicago truncatula, encoding protein thereof and application thereof in regulating and controlling collateral development | |
CN114214334A (en) | Application of gene EsH2A.3 from Thellungiella halophila in regulation and control of plant salt tolerance | |
CN102051367A (en) | Identification of populus euphratica olive PeCPK10 gene and function research thereof | |
CN104975035A (en) | Leymus chinensis triosephosphate isomerase gene as well as encoding protein and application thereof |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
WD01 | Invention patent application deemed withdrawn after publication |
Open date: 20070620 |