CN1982333A - Populus diversifolia PeCBL1 gene and its use - Google Patents

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

Populus diversifolia PeCBL 1 gene and application

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.