CN105017393A - Protein BhDNAJC2 relevant to plant adverse resistance and encoding gene and application thereof - Google Patents
Protein BhDNAJC2 relevant to plant adverse resistance and encoding gene and application thereof Download PDFInfo
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- CN105017393A CN105017393A CN201410176146.1A CN201410176146A CN105017393A CN 105017393 A CN105017393 A CN 105017393A CN 201410176146 A CN201410176146 A CN 201410176146A CN 105017393 A CN105017393 A CN 105017393A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
- C12N15/8271—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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
- C12N15/8271—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
- C12N15/8273—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 drought, cold, salt resistance
Abstract
The invention discloses protein BhDNAJC2 relevant to the plant adverse resistance and an encoding gene and application thereof. The protein is a or b, wherein a is the protein composed of an amino acid sequence shown in the sequence 1 in a sequence table, and b is the protein which is obtained through the way that the amino acid sequence of the protein a is replaced with and/or is lack of and/or is added with one or a few amino acid residues and is relevant to the plant adverse resistance. Experiments prove that transgenic arabidopsis thaliana can be obtained by introducing the encoding gene of the protein into wild-type arabidopsis thaliana, compared with the wild-type arabidopsis thaliana, the draught resistance, salt resistance and alkali resistance of the transgenic arabidopsis thaliana are obviously improved. The protein BhDNAJC2 has an important theoretical and practical significance for cultivating new varieties such as crops and forest grass with the draught resistance, salt resistance and alkali resistance improved, and can be applied to cultivating and identifying of resistance plant varieties needed in agriculture and animal husbandry and ecological environment management.
Description
Technical field
The invention belongs to genetically engineered field, relate to a kind of protein B hDNAJC2 relevant to stress resistance of plant and encoding gene thereof and application.
Background technology
Along with the shortage of global water resources and the growth of extreme weather events, adverse circumstance affects increasingly significant to the yield and quality of crop.Therefore, by molecule manipulation technology improve crop resistance day show important.
After organism is subject to drought stress or other poor environment factor, a series of stress reaction can be produced, wherein heat shock protein (heat shock protein, HSP) nascent protein can be impelled to fold on the one hand again correctly to fold with the albumen of false folding, sex change collectin can also be made to degrade on the other hand, maintain the stability of a series of enzyme and cytolemma simultaneously.Further research finds, the content of HSPs and the resistance to coercive of organism are proportionate, and can improve the stress ability of organism and the survival rate in adverse circumstance.
Resurrection plant can stand the condition of Extreme drought, when moisture content is sufficient, can recover normal activities very soon again.Resurrection plant in known angiosperm is little, and is mainly distributed in South Africa, South America and Australia.Revolving capsule lettuce tongue (Boea hygrometrica) is a kind of Gesneriaceae resurrection plant distributed in China, this plant is vigorous in the area growth of the heavy alkali of the karst height salt based on limestone, its blade has very strong drought-enduring recovery ability, grow 72 hours under room temperature, relative air humidity are the condition of 0 after, leaf r elative water content reduces to about 3%, blade area shrinkage is to less than 1/3 of former blade area, and photosynthesis stops substantially.As long as again feed water, blade just can absorb water stretching, extension, and revert to untreated before the apparent state of blade and physiological status (comprising photosynthetic recovery).
Summary of the invention
The object of this invention is to provide a kind of protein B hDNAJC2 relevant to stress resistance of plant and encoding gene thereof and application.
Protein provided by the present invention, name is called BhDNAJC2, and what derive from Gesneriaceae revolves capsule lettuce tongue (Boeahygrometrica), is following (a) or (b):
A protein that () is made up of the aminoacid sequence shown in sequence in sequence table 1;
The aminoacid sequence of b protein that (a) limits by () is through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation, and the protein relevant to stress resistance of plant.
For the ease of the purifying of BhDNAJC2 albumen, the N-terminal of the protein that the amino acid residue sequence of sequence 1 forms or C-terminal label as shown in the table can be connected in by sequence table.
Table: the sequence of label
Tag residues 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 |
Protein in above-mentioned (b) can synthetic, also can first synthesize its encoding gene, then carries out biological expression and obtain.The encoding gene of the protein in above-mentioned (b) by the codon by lacking one or several amino-acid residue in the DNA sequence dna shown in sequence in sequence table 2, and/or carries out the missense mutation of one or several base pair.
The nucleic acid molecule of described BhDNAJC2 albumen of encoding also belongs to protection scope of the present invention.
Described nucleic acid molecule can be DNA, as cDNA, genomic dna or recombinant DNA; Described nucleic acid molecule can be also RNA, as mRNA, hnRNA or tRNA etc.
In one embodiment of the invention, described nucleic acid molecule is specially the gene (called after BhDNAJC2) of described BhDNAJC2 albumen of encoding; Described BhDNAJC2 gene can be following 1) to 5) in arbitrary described DNA molecular:
1) encoding sequence is the DNA molecular shown in the 193-696 position of sequence in sequence table 2;
2) DNA molecular shown in 181-721 position of sequence 2 in sequence table;
3) DNA molecular shown in sequence 2 in sequence table;
4) under strict conditions with 1)-3) in the DNA molecule hybridize of arbitrary restriction and the DNA molecular of code for said proteins;
5) with 1)-4) in the DNA molecular of arbitrary restriction there is the DNA molecular of more than 90% homology and code for said proteins.
Above-mentioned stringent condition can be the solution with 6 × SSC, 0.5%SDS, and hybridize at 65 DEG C, then use 2 × SSC, 0.1%SDS and 1 × SSC, 0.1%SDS respectively wash film once.
Wherein, sequence 2 is made up of 913 Nucleotide, and 193-696 position is ORF, the BhDNAJC2 albumen in polynucleotide shown in sequence 1.
Recombinant vectors containing above-mentioned nucleic acid molecule, expression cassette, transgenic cell line or recombinant bacterium also belong to protection scope of the present invention.
Described recombinant vectors can be recombinant expression vector, also can be recombinant cloning vector.
Described recombinant expression vector can use existing plant expression vector construction.Described plant expression vector comprises double base agrobacterium vector and can be used for the carrier etc. of plant micropellet bombardment, as pGreen0029, pCAMBIA3301, pCAMBIA1300, pBI121, pBin19, pCAMBIA2301, pCAMBIA1301-UbiN or other derivative plant expression vector.Described plant expression vector also can comprise 3 ' end untranslated region of foreign gene, namely comprises the DNA fragmentation of polyadenylation signals and any other participation mRNA processing or genetic expression.The bootable polyadenylic acid of described polyadenylation signals joins 3 ' end of mRNA precursor.When using described gene constructed recombinant expression vector, any one enhancement type, composing type, organizing specific type or inducible promoter can be added before its transcription initiation Nucleotide, such as cauliflower mosaic virus (CAMV) 35S promoter, ubiquitin gene Ubiquitin promotor (pUbi), stress induced promoter rd29A etc., they can be used alone or are combined with other plant promoter; In addition, when using gene constructed recombinant expression vector of the present invention, also enhanser can be used, comprise translational enhancer or transcriptional enhancer, these enhanser regions can be ATG initiator codon or neighboring region initiator codon etc., but must be identical with the reading frame of encoding sequence, to ensure the correct translation of whole sequence.The source of described translation control signal and initiator codon is widely, can be natural, also can be synthesis.Translation initiation region can from transcription initiation region or structure gene.For the ease of identifying transgenic plant cells or plant and screening, can process recombinant expression vector used, the coding can expressed in plant as added can produce enzyme or the gene of luminophor, the antibiotic marker thing with resistance or the chemical resistance reagent marker gene etc. of colour-change.Also any selected marker can not be added, directly with adverse circumstance screening transformed plant.
In an embodiment of the present invention, the promotor starting described BhDNAJC2 genetic transcription in described recombinant expression vector is specially pA35S promotor; The terminator stopping described BhDNAJC2 genetic transcription is specially pA35S terminator.
More specifically, described recombinant expression vector is between recombination site attR1 and attR2 of pLeela carrier, insert the recombinant plasmid that described BhDNAJC2 gene obtains.
Described expression cassette by the promotor that can start described BhDNAJC2 genetic expression, described BhDNAJC2 gene, and transcription termination sequence composition.
Described BhDNAJC2 albumen, or described nucleic acid molecule, or the application in arbitrary as follows of described recombinant expression vector, expression cassette or recombinant bacterium also belongs to protection scope of the present invention:
(a1) regulating plant resistance;
(a2) plant variety of seed selection resistance raising.
In the present invention, described regulating plant resistance is embodied in: in described plant materials, if the expression amount of described BhDNAJC2 gene is higher, then the resistance of described plant is stronger.
In the present invention, the method for the plant variety that described seed selection resistance improves, specifically can comprise and plant higher for described BhDNAJC2 gene expression amount is carried out the step of hybridizing as parent.
Another object of the present invention is to provide a kind of method of cultivating the transgenic plant that resistance improves.
The method of the transgenic plant that cultivation resistance provided by the present invention improves, specifically can comprise the steps:
A) in object plant, import the encoding gene of described BhDNAJC2 albumen, obtain the transgenic plant of expressing described encoding gene;
B) obtain from step a) gained transgenic plant compared with described object plant, the transgenic plant that resistance improves.
The expression amount of described BhDNAJC2 albumen in described transgenic plant is higher than described object plant.The gene (i.e. described BhDNAJC2 gene) of described BhDNAJC2 albumen of encoding specifically can be following 1) to 5) in arbitrary described DNA molecular:
1) encoding sequence is the DNA molecular shown in the 193-696 position of sequence in sequence table 2;
2) DNA molecular shown in 181-721 position of sequence 2 in sequence table;
3) DNA molecular shown in sequence 2 in sequence table;
4) under strict conditions with 1)-3) in the DNA molecule hybridize of arbitrary restriction and the DNA molecular of code for said proteins;
5) with 1)-4) in the DNA molecular of arbitrary restriction there is the DNA molecular of more than 90% homology and code for said proteins.
Above-mentioned stringent condition can be the solution with 6 × SSC, 0.5%SDS, and hybridize at 65 DEG C, then use 2 × SSC, 0.1%SDS and 1 × SSC, 0.1%SDS respectively wash film once.
Described BhDNAJC2 gene specifically imports in described object plant by above-mentioned arbitrary described recombinant expression vector, obtains described transgenic plant.Specifically by using the conventional biology methods such as Ti-plasmids, Ri plasmid, plant viral vector, directly delivered DNA, microinjection, conductance, agriculture bacillus mediated, particle gun by described recombinant expression vector transformed plant cells or tissue, and the plant tissue of conversion is cultivated into plant.The agriculture bacillus mediated biological method that waits is transformed in vegetable cell or tissue.
In above-mentioned application or method, described resistance all specifically can be at least one as follows: drought resistance, salt resistance, alkali-resistivity.
In above-mentioned application or method, described plant can be both dicotyledons, also can be monocotyledons.
In one embodiment of the invention, described plant is dicotyledons, is specially Arabidopis thaliana, as wildtype Arabidopsis thaliana Col-0.
The primer pair of described BhDNAJC2 full length gene or its arbitrary fragment of increasing also belongs to protection scope of the present invention.Experiment proves, the present invention is revolved capsule lettuce tongue (Boea hygrometrica) from resurrection plant and is screened a BhDNAJC2 gene by drought-induced expression, by this channel genes wildtype Arabidopsis thaliana, obtain BhDNAJC2 transgenic arabidopsis, compared with wildtype Arabidopsis thaliana, drought resisting, anti-salt and alkali-resistivity significantly improve, and illustrate that BhDNAJC2 is and plant drought, albumen that anti-salt is relevant with alkali resistant.Therefore the new variety such as crop, woods grass that BhDNAJC2 albumen and encoding gene thereof improve for cultivation drought resisting, anti-salt and alkali-resistivity have important theory and practical significance, can be used for cultivation and the qualification of the resistance plant kind needed for husbandry and ecological environment treatment.
Accompanying drawing explanation
Fig. 1 is that BhDNAJC2 gene is revolving the expression in capsule lettuce tongue (Boea hygrometrica) arid resuscitation process.In figure, between different lowercases, represent significant difference (p<0.05).
Fig. 2 is the RT-PCR detected result of T3 for BhDNAJC2 genetic expression in BhDNAJC2 transgenic arabidopsis homozygous lines OE4-5, OE7-7, OE8-7.
Fig. 3 is that T3 identifies for the drought-resistant ability of BhDNAJC2 transgenic arabidopsis homozygous lines OE4-5, OE7-7, OE8-7.Wherein, A is each plant phenotype in 1/2MS (pH5.6) control medium; B is that on 1/2MS+20%PEG substratum, each plant phenotype C is the long statistics of root; D is Ion leakage result; E is Fv/Fm result.In figure, between different lowercases, represent significant difference (p<0.05).
Fig. 4 is that T3 identifies for the saline-alkaline tolerance of BhDNAJC2 transgenic arabidopsis homozygous lines OE4-5, OE7-7, OE8-7.Wherein, A is each plant phenotype in 1/2MS (pH5.6) control medium; B is 1/2MS+150mmolL
-1each plant phenotype in NaCl culture medium; C is the long statistics of root; D is Ion leakage result; E is Fv/Fm result.In figure, between different lowercases, represent significant difference (p<0.05).
Fig. 5 is that T3 identifies for the alkali resistant ability of BhDNAJC2 transgenic arabidopsis homozygous lines OE4-5, OE7-7, OE8-7.Wherein, A is each plant phenotype in 1/2MS (pH5.6) control medium; B is each plant phenotype on 1/2MS substratum (pH9.0) substratum; C is the long statistics of root; D is Ion leakage result; E is Fv/Fm result.In figure, between different lowercases, represent significant difference (p<0.05).
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
RNase inhibitor (purchased from Takara company).ThermoScript II SuperScript TM III (purchased from Invitrogen company).PENTR/D-TOPO carrier (purchased from Invitrogen company).Phusion High-Fidelity DNAPolymerase (purchased from NEB company).
Plant expression vector pLeela: be recorded in " A novel role for histone methyltransferaseKYP/SUVH4in the control of Arabidopsis primary seed dormancy; New Phytologist (2012) 193:605-616 " civilian, the public can obtain from Institute of Botany, Chinese Academy of Sciences.
Agrobacterium Gv3101 (Agrobacterium tumefaciens strain GV3101): be recorded in " BinaryAgrobacterium vectors for plant transformation; M Bevanin; Nucleic Acids Research (1984) 12 (22): 8711-8721 " civilian, the public can obtain from Institute of Botany, Chinese Academy of Sciences.
Wildtype Arabidopsis thaliana Col-0 (ecotype Columbia-0, Arabidopsis thaliana): be recorded in " Arabidopsis; a useful weed.Meyerowitz EM; Cell (1989) 56:263-270. " civilian, the public can obtain from Institute of Botany, Chinese Academy of Sciences.
The Cloning and Expression of embodiment 1, BhDNAJC2 gene
Extract and revolve capsule lettuce tongue (Boea hygrometrica) blade total serum IgE through drought stress process 8 hours, utilize
library construction Kit (Stratagene, La Jolla, CA) construction cDNA library.Therefrom random choose 4800 genes check order, and find wherein 1 coding DNA J type heat shock protein.Analyze their dynamic changes in normal condition and drought-induced rear genetic expression.Found that, this gene is by drought-induced rise, and the method obtaining this gene is specific as follows:
Extract the total serum IgE revolving capsule lettuce tongue (Boea hygrometrica) blade through drought stress process, and with it for template carries out reverse transcription.Refer to Zhennan Zhang, Bo Wang, Dongmei Sun* & Xin Deng*.2013, Molecular cloning and differential expression of sHSP gene family members from theresurrection plant Boea hygrometrica in response to abiotic stresses.Biologia68 (4): 651-661 mono-literary composition.
(1) DNA digestion (10 μ l): total serum IgE 2 μ g; 10 × DNase buffer1 μ l; DNaseI (50U/ μ l) 0.4 μ l; RNase inhibitor 0.1 μ l; DEPC-H
2o complements to 10 μ l.37℃30min。
(2) reverse transcription (25 μ l): through the total serum IgE 10 μ l of DnaseI digestion process; OligodT1 μ l; DEPC-H
2o2.5 μ l; 70 DEG C of incubation 5min, place 5min on ice; Then 5 × buffer5 μ l is added; 2mM dNTP5 μ l; RNase inhibitor 0.5 μ l; ThermoScript II SuperScript TM III1 μ l.42 DEG C of incubation 1h.
The cDNA that reverse transcription obtains dilutes 10 times as template, with BhDNAJC2-F and BhDNAJC2-R for primer, adopts Phusion High-Fidelity DNA Polymerase to carry out the amplification of full length gene.Reaction conditions is: first 98 DEG C of denaturation 45sec; Then 98 DEG C of sex change 10sec, 55 DEG C of annealing 20sec, then 72 DEG C extend 1.5min, totally 40 circulations; Last 72 DEG C extend 10min.
BhDNAJC2-F:5 '-
cACCtTTGATTTCTGAATGGAGTTC-3 ' (underscore place is the sequence of mating with pENTR/D-TOPO carrier indentation, there, and sequence is thereafter the 181-201 position of sequence 2);
BhDNAJC2-R:5 '-TTGTGAA GCTGGTACACTC GAATTT-3 ' (reverse complementary sequence of the 697-721 position of sequence 2).
After reaction terminates, PCR primer is carried out agarose gel electrophoresis detection, and result obtains the PCR primer that size is about 541bp, reclaims this PCR primer band.
This PCR primer is connected with pENTR/D-TOPO carrier, the correct plasmid of order-checking is designated as intermediate carrier BhDNAJC2-pENTR.The structrual description of intermediate carrier BhDNAJC2-pENTR is the recombinant plasmid obtained after being connected with pENTR/D-TOPO carrier by DNA sequence dna shown in the 181-721 position of sequence in sequence table 2.
Be BhDNAJC2 gene by the unnamed gene shown in sequence 2, the coding region of this gene is from 5 ' end 193-696 position Nucleotide in sequence 2, be BhDNAJC2 albumen by the protein designations of this genes encoding, the aminoacid sequence of this albumen is the sequence 1 in sequence table.
Embodiment 2, BhDNAJC2 gene are revolving the expression study in capsule lettuce tongue arid resuscitation process
Carrying out 2 kinds of Osmotic treatment in various degree to revolving capsule lettuce tongue (Boea hygrometrica), being respectively:
Fresh Plants (F): every day normally waters;
In Fresh Plants soil, arid 5 days (S5D): continuous 5 days is not watered at a slow speed;
In Fresh Plants soil, arid 14 days (S14D): continuous 14 days is not watered at a slow speed.
In Fresh Plants soil at a slow speed arid 14 days after rehydration 3 days (A): the recovery of normally watering carrying out 3 days after not watering for continuous 14 days.
The plant number of each process is at least 5 strains.
Extract total serum IgE, reverse transcription acquisition cDNA that capsule lettuce tongue blade is revolved in 3 kinds of process respectively.Q-PCR amplification is carried out with gene-specific primer BhDNAJC2-f and BhDNAJC2-r.And with 18S rRNA gene for internal reference, amplimer is 18S-f and 18S-r.
BhDNAJC2-f:5 '-GTTCTTGGTGTTCGCTCGTAC-3 ' (the 229-249 position of sequence 2);
BhDNAJC2-r:5 '-AGCAGAGCCGTACAATCCAG-3 ' (reverse complementary sequence of the 419-438 position of sequence 2).
18S-f：5’-CTTAGTTGGTGGAGCGATTTG-3’；
18S-r：5’-CCTGTTATTGCCTCAAACTTCC-3’。
Reaction system is: cDNA1 μ l, 10 μ l2 × SYBR Green Master Mix, upstream and downstream primer each 0.5 μ l, ddH
2o8.5 μ l.Response procedures is: 95 DEG C of denaturations 30 seconds; 95 DEG C of sex change 5 seconds, 55 DEG C of annealing 30 seconds, 72 DEG C extend 30 minutes, totally 40 circulations.
Result as shown in Figure 1, can find out that the expression amount of BhDNAJC2 gene in S14D group is significantly higher than F group.Visible, BhDNAJC2 gene is obviously by drought-induced.
The acquisition of embodiment 3, BhDNAJC2 transfer-gen plant and resistance qualification
One, the acquisition of BhDNAJC2 transgenic arabidopsis
1, the acquisition of recombinant expression vector pLeela-BhDNAJC2
The PCR primer of embodiment 1 is imported plant expression vector pLeela by LR reaction (LR clones enzyme, purchased from Invitrogen company) by the intermediate carrier BhDNAJC2-pENTR built in embodiment 1, and homologous recombination obtains recombinant vectors.
To show to insert between attR1 and the attR2 recombination site of plant expression vector pLeela the recombinant vectors called after pLeela-BhDNAJC2 of the PCR primer (i.e. shown in the 181-721 position of sequence 2 DNA fragmentation) of embodiment 1 through order-checking.
In recombinant expression vector pLeela-BhDNAJC2, the promotor starting described BhDNAJC2 genetic transcription is pA35S promotor, and the terminator stopping described BhDNAJC2 genetic transcription is pA35S terminator.
2, the acquisition of recombinational agrobacterium
Above-mentioned recombinant expression vector pLeela-BhDNAJC2 is transformed in Agrobacterium Gv3101, with containing 50 μ gmL
-1sulphuric acid kanamycin, 50 μ gmL
-1gentamicin and 50 μ gmL
-1the YEB substratum of Rifampin screens, and obtains recombinational agrobacterium.
The plasmid extracting recombinant bacterium sends to order-checking, this plasmid of result is pLeela-BhDNAJC2 (recombinant plasmid of DNA fragmentation shown in the 181-721 position that order-checking shows to insert sequence 2 between attR1 and the attR2 recombination site of pLeela carrier), be illustrated as positive recombinant bacterium, by its called after of recombinant bacterium Gv3101/pLeela-BhDNAJC2.
Experiment arranges the contrast proceeding to pLeela empty carrier in Agrobacterium Gv3101, gained Agrobacterium called after Gv3101/pLeela simultaneously.
3, the acquisition of BhDNAJC2 transgenic arabidopsis and qualification
(1) acquisition of BhDNAJC2 transgenic arabidopsis
Recombinant bacterium Gv3101/pLeela-BhDNAJC2 and Gv3101/pLeela is adopted respectively inflorescence infusion method transformed wild type Arabidopis thaliana Col-0 (ecotype Columbia-0, Arabidopsis thaliana), obtaining T0 generation turns BhDNAJC2 transgenic arabidopsis and T0 generation proceeds to the Arabidopis thaliana of pLeela empty carrier.
Get T0 for BhDNAJC2 transgenic arabidopsis seed, be all seeded in equably containing 10mgL
-1on the 1/2MS substratum of glufosinates, the surviving seedling (T1 generation) with resistance is moved to hot-house culture (culture temperature 22 DEG C, 16/8 hour photoperiod), collect the seed of T1 for BhDNAJC2 transgenic arabidopsis.
Get 100 T1 respectively for BhDNAJC2 transgenic arabidopsis planting seed in containing 10mgL
-1in the 1/2MS substratum of glufosinates, be that the T2 of 3:1 moves to hot-house culture for the surviving seedling (5-10) of BhDNAJC2 transgenic arabidopsis by segregation ratio, collect the seed of T2 for BhDNAJC2 transgenic arabidopsis.
Get 100 T2 respectively for BhDNAJC2 transgenic arabidopsis planting seed in containing 10mgL
-1in the 1/2MS substratum of glufosinates, moving to hot-house culture by there is not the seedling (about 10) that survives of separation T3 for BhDNAJC2 transgenic arabidopsis, collecting the seed (homozygote) of T3 for BhDNAJC2 transgenic arabidopsis.
From the T3 obtained for random selecting 3 strain BhDNAJC2 transgenic arabidopsis homozygous lines, be designated as respectively: OE4-5, OE7-7, OE8-7.
(2) qualification of BhDNAJC2 transgenic arabidopsis
Extract the total serum IgE of above-mentioned T3 for BhDNAJC2 transgenic arabidopsis homozygous lines OE4-5, OE7-7, OE8-7 and wildtype Arabidopsis thaliana Col-0 of 10 days seedling ages respectively, and with it for template, with BhDNAJC2-F and BhDNAJC2-R (sequence is shown in embodiment 1) for primer carries out RT-PCR.Pcr amplification program is: 95 DEG C of denaturation 4min; 94 DEG C of sex change 30 seconds, 55 DEG C of annealing 30 seconds, 72 DEG C extend 2 minutes, totally 30 circulations; Last again 72 DEG C extend 10 minutes.
Experiment is with 18S rRNA gene as internal reference, and amplimer is 18S-f and 18S-r (sequence is shown in embodiment 2).Pcr amplification program is: 95 DEG C of denaturation 4min; 94 DEG C of sex change 30 seconds, 55 DEG C of annealing 30 seconds, 72 DEG C extend 30 seconds, totally 30 circulations; Last again 72 DEG C extend 10 minutes.
Setup Experiments is with not genetically modified wildtype Arabidopsis thaliana Col-0 and proceed to the Arabidopis thaliana of pLeela empty carrier in contrast.
After reaction terminates, agarose gel electrophoresis detection is carried out to PCR primer, often organize sample and carry out 2 repetitions.
Result as shown in Figure 2, can find out, what do not have BhDNAJC2 gene in not genetically modified wildtype Arabidopsis thaliana Col-0 (WT) amplifies band, T3 is in BhDNAJC2 transgenic arabidopsis homozygous lines OE4-5, OE7-7, OE8-7, all have size to be about the object fragment of 541bp, visible T3 is positive for BhDNAJC2 transgenic arabidopsis homozygous lines OE4-5, OE7-7, OE8-7.And the Arabidopsis plant proceeding to pLeela empty carrier is consistent with not genetically modified wildtype Arabidopsis thaliana Col-0 (WT), do not amplify the object fragment that size is about 541bp.
Two, the resistance qualification of BhDNAJC2 transgenic arabidopsis
1, experimental technique
Step one is identified positive T3 is for BhDNAJC2 transgenic arabidopsis homozygous lines OE4-5, OE7-7, OE8-7, proceed to the Arabidopsis plant of pLeela empty carrier, and not genetically modified wildtype Arabidopsis thaliana Col-0, be sowed on 1/2MS substratum (pH5.6), 4 DEG C of vernalization are after 3 days, cultivate at 22 DEG C, 50% humidity, under the condition of illumination 16h and dark 8h, after 4 days, seedling is transferred to respectively following three kinds of substratum for Analysis of Resistance, take a picture after 10 days, statistics root is long, measure Ion leakage (relative conductivity) and PSII Efficiency of primary conversion of light energy (Fv/Fm), thus analyze the drought resisting of each strain, the ability of anti-salt and alkali resistant.Each strain 6-8 strain, experiment establishes three repetitions, results averaged altogether.
For analyzing the substratum of drought resistance: 1/2MS+20%PEG (mass percentage) substratum, pH5.6;
For analyzing the substratum of salt resistance: 1/2MS+150mmolL
-1naCl culture medium, pH5.6;
For analyzing alkali-resisting substratum: 1/2MS substratum (pH9.0).
In experiment, 1/2MS (pH5.6) is all set during often kind of Analysis of Resistance in contrast.
Cytolemma has to be selected permeability and remains stable, but when plant suffers environment-stress, cell membrane system is first destroyed, cytolemma occurs even breaking in a large amount of cavity, cell Dissolve things inside spills into extracellular in a large number, cell vat liquor specific conductivity is caused sharply to increase, relative conductivity is as index (the Wang YC whether measure of cell plasma membrane comes to harm, Qu GZ, Li HY, Wu YJ, Wang C, Liu GF, Yang CP (2010) .Enhanced salt tolerance oftransgenic poplar plants expressing a manganese superoxide dismutase from TamarixandrossowiI.Molecular Biology Reports37, 1119-1124.).Fv/Fm reflects PS II reactive center light energy conversion efficiency, the size of its parameter value and variation characteristic are often used for judging and infer that plant is to the resistance of environmental factor, also someone is used as Heat Resistance of Plant, low temperature resistant, salt tolerant, resistance to high light, drought-resistant, important indicator (the Yang CW of the aspects such as antipollution, Peng CL, Duan J, Lin GZ, Chen YZ (2002) .Responses of chlorophyllfluorescence and carotenoids biosynthesis to high light stress in rice seedling leaves atdifferent leaf position.Acta Botanica Sinica44, 1303-1308.).
Wherein, relative conductivity measuring method is as follows: utilize conductivitimeter (EC215, HANNA company of Italy) measure the relative conductivity of plant, the seedling of complete each strain is cultivated under getting different treatment, with distilled water flushing 3 times, blot surface-moisture with filter paper, respectively get 0.1g (fresh weight), be placed in the scale test tube of 6ml deionized water respectively, screw a lid on and be placed in immersion treatment 4h under room temperature.Measure vat liquor conductance A with conductivity meter, then boiling water bath heating 30min, shakes up after being cooled to room temperature, again measures vat liquor conductance B.Relative conductivity=A/B × 100%.
Fv/Fm measuring method is as follows: utilize modulated chlorophyll fluorescence imaging system (MAXI-Imaging-Pam, Walz company of Germany) measure PSII Efficiency of primary conversion of light energy (Fv/Fm), after the seedling cultivating complete each strain under different treatment processes 20min under dark, measure chlorophyll fluorescence Fv/Fm.Data ImagingWin v2.32 reads, and imports in excel and analyzes.
2, experimental result
(1) Drought Resistance Analysis
Result as shown in Figure 3.
A. the long result of root
As shown in A-C in Fig. 3.Wherein, in Fig. 3, A is that in 1/2MS (pH5.6) control medium, each plant phenotype is observed, and can find out, the root long basically identical (p>0.05) of each plant.In Fig. 3, B is that on 1/2MS+20%PEG (mass percentage) substratum, each plant phenotype is observed, can find out, compared with wildtype Arabidopsis thaliana Col-0 (WT), T3 is for the root longer (p<0.05) turning BhDNAJC2 Arabidopis thaliana strain OE4-5, OE7-7, OE8-7.In Fig. 3, B adds up the long result of root as shown in C in Fig. 3, on 1/2MS+20%PEG (mass percentage) substratum, T3 is respectively 5.00cm, 5.00cm, 5.17cm for the root length of BhDNAJC2 transgenic arabidopsis homozygous lines OE4-5, OE7-7, OE8-7, be longer than the long 4.28cm of root of wildtype Arabidopsis thaliana Col-0 (WT), and long without significant difference (p>0.05) with the root without each plant in 1/2MS (pH5.6) control medium of Osmotic treatment.
B. Ion leakage result
As shown in D in Fig. 3, can find out, on 1/2MS+20%PEG (mass percentage) substratum, T3 is respectively 38% for the relative conductivity of BhDNAJC2 transgenic arabidopsis homozygous lines OE4-5, OE7-7, OE8-7,31%, 28%, significantly lower than the relative conductivity 46% of (p<0.05) wildtype Arabidopsis thaliana Col-0 (WT).
C.Fv/Fm result
As shown in E in Fig. 3, can find out, on 1/2MS+20%PEG (mass percentage) substratum, T3 is respectively 0.62 for the Fv/Fm of BhDNAJC2 transgenic arabidopsis homozygous lines OE4-5, OE7-7, OE8-7,0.64,0.59, be significantly higher than the Fv/Fm value 0.5 of (P<0.05) wildtype Arabidopsis thaliana Col-0 (WT).
(2) salt resistance analysis
Result as shown in Figure 4.
A. the long result of root
As shown in A-C in Fig. 4.Wherein, in Fig. 4, A is that in 1/2MS (pH5.6) control medium, each plant phenotype is observed, and can find out, the root long basically identical (p>0.05) of each plant.In Fig. 4, B is 1/2MS+150mmolL
-1in NaCl culture medium, each plant phenotype is observed, and can find out, compared with wildtype Arabidopsis thaliana Col-0 (WT), T3 is for the root longer (p<0.05) turning BhDNAJC2 Arabidopis thaliana strain OE4-5, OE7-7, OE8-7.In Fig. 4, B adds up the long result of root as shown in C in Fig. 4, on 1/2MS+150mmolL-1NaCl substratum, T3 is respectively 1.91cm, 1.87cm, 2.69cm for the root length of BhDNAJC2 transgenic arabidopsis homozygous lines OE4-5, OE7-7, OE8-7, is obviously longer than the long 1.38cm of root of (p<0.05) wildtype Arabidopsis thaliana Col-0 (WT).
B. Ion leakage result
As shown in D in Fig. 4, can find out, on 1/2MS+150mmolL-1NaCl substratum, T3 is respectively 65% for the Ion leakage value of BhDNAJC2 transgenic arabidopsis homozygous lines OE4-5, OE7-7, OE8-7,61%, 62%, significantly lower than the Ion leakage value 77% of (p<0.05) wildtype Arabidopsis thaliana Col-0 (WT).
C.Fv/Fm result
As shown in E in Fig. 4, can find out, at 1/2MS+150mmolL
-1in NaCl culture medium, T3 is respectively 0.80 for the Fv/Fm of BhDNAJC2 transgenic arabidopsis homozygous lines OE4-5, OE7-7, OE8-7,0.81,0.78, be significantly higher than the Fv/Fm value 0.23 of (p<0.05) wildtype Arabidopsis thaliana Col-0 (WT).
(3) alkali-resistivity analysis
Result as shown in Figure 5.
A. the long result of root
As shown in A-C in Fig. 5.Wherein, in Fig. 5, A is that in 1/2MS (pH5.6) control medium, each plant phenotype is observed, and can find out, the root long basically identical (p>0.05) of each plant.In Fig. 5, B is that the upper each plant phenotype of 1/2MS substratum (pH9.0) is observed, can find out, compared with wildtype Arabidopsis thaliana Col-0 (WT), T3 is for the root longer (p<0.05) turning BhDNAJC2 Arabidopis thaliana strain OE4-5, OE7-7, OE8-7.In Fig. 5, B adds up the long result of root as shown in C in Fig. 5, on 1/2MS substratum (pH9.0), T3 is respectively 7.32cm for the root length of BhDNAJC2 transgenic arabidopsis homozygous lines OE4-5, OE7-7, OE8-7,6.04cm, 8.40cm, is obviously longer than the long 0.90cm of root of (p<0.05) wildtype Arabidopsis thaliana Col-0 (WT).
B. Ion leakage result
As shown in D in Fig. 5, can find out, on 1/2MS substratum (pH9.0), T3 is respectively 26% for the Ion leakage value of BhDNAJC2 transgenic arabidopsis homozygous lines OE4-5, OE7-7, OE8-7,25%, 20%, significantly lower than the Ion leakage value 59% of (p<0.05) wildtype Arabidopsis thaliana Col-0 (WT).
C.Fv/Fm result
As shown in E in Fig. 5, can find out, on 1/2MS substratum (pH9.0), T3 is respectively 082 for the Fv/Fm of BhDNAJC2 transgenic arabidopsis homozygous lines OE4-5, OE7-7, OE8-7,0.79,0.83, be significantly higher than the Fv/Fm value 0.17 of (p<0.05) wildtype Arabidopsis thaliana Col-0 (WT).
In addition, for above each result, turn empty carrier Arabidopis thaliana and non-transgenosis wildtype Arabidopsis thaliana Col-0 (WT) result without significant difference.
In sum, the drought resisting of BhDNAJC2 transfer-gen plant, anti-salt and alkali-resistivity are obviously better than not genetically modified plant, illustrate that BhDNAJC2 is and plant drought, albumen that anti-salt is relevant with alkali resistant.
Claims (10)
1. protein is following (a) or (b):
A protein that () is made up of the aminoacid sequence shown in sequence in sequence table 1;
The aminoacid sequence of b protein that (a) limits by () is through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation, and the protein relevant to stress resistance of plant.
2. the nucleic acid molecule of protein described in coding claim 1.
3. nucleic acid molecule according to claim 2, is characterized in that: described nucleic acid molecule is the gene of protein described in coding claim 1; Described gene is following 1) to 5) in arbitrary described DNA molecular:
1) encoding sequence is the DNA molecular shown in the 193-696 position of sequence in sequence table 2;
2) DNA molecular shown in 181-721 position of sequence 2 in sequence table;
3) DNA molecular shown in sequence 2 in sequence table;
4) under strict conditions with 1)-3) in the DNA molecule hybridize of arbitrary restriction and protein DNA molecule described in claim 1 of encoding;
5) with 1)-4) in the DNA molecular of arbitrary restriction there is more than 90% homology and protein DNA molecule described in claim 1 of encoding.
4. the recombinant vectors containing nucleic acid molecule described in Claims 2 or 3, expression cassette, transgenic cell line or recombinant bacterium.
5. recombinant vectors according to claim 4, is characterized in that: described recombinant vectors is recombinant expression vector or recombinant cloning vector.
6. recombinant vectors according to claim 5, is characterized in that: in described recombinant expression vector, and the promotor of transcribing starting described gene is pA35S promotor.
7. protein according to claim 1, or the nucleic acid molecule described in Claims 2 or 3, or arbitrary described recombinant vectors, expression cassette, transgenic cell line or the recombinant bacterium application in arbitrary as follows in claim 4-6:
(a1) regulating plant resistance;
(a2) plant variety of seed selection resistance raising.
8. cultivate the method for the transgenic plant that resistance improves, comprise the steps:
A) in object plant, import the encoding gene of protein described in claim 1, obtain the transgenic plant of expressing described encoding gene;
B) obtain from step a) gained transgenic plant compared with described object plant, the transgenic plant that resistance improves.
9. application according to claim 7, or method according to claim 8, is characterized in that: described resistance be following at least one: drought resistance, salt resistance, alkali-resistivity.
10., according to described application arbitrary in claim 7-9 or method, it is characterized in that: described plant is dicotyledons or monocotyledons.
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| CN109678940A (en) * | 2017-10-18 | 2019-04-26 | 中国科学院植物研究所 | Protein B hDnaJ6 and its encoding gene and application |
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| WO2018234191A1 (en) * | 2017-06-19 | 2018-12-27 | University Of Copenhagen | Increased drought resistance in plants |
| CN110669121B (en) * | 2019-11-14 | 2022-05-24 | 九圣禾种业股份有限公司 | Method for improving salt tolerance of cotton by using LeDNAJ gene and application |
| CN112772416B (en) * | 2021-01-28 | 2022-08-12 | 苏州金铁红林业科技有限公司 | Tissue culture seedling raising method for phyllostachys microphylla |
| CN113881685B (en) * | 2021-10-09 | 2024-03-29 | 江苏省农业科学院 | Gene PpHSP20-like1 for promoting plant organ to produce red color and application thereof |
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| WO2008043245A1 (en) * | 2006-09-11 | 2008-04-17 | The Chinese University Of Hong Kong | Abiotic stress tolerance conferred by j-domain-containing proteins |
| CN101921774A (en) * | 2010-05-24 | 2010-12-22 | 南京大学 | Application of Dnaj-like protein and encoded gene thereof |
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| CN103451193B (en) * | 2013-09-25 | 2015-01-21 | 大连民族学院 | Populus deltoidesx populus nigra PdHSP70 gene and application thereof |
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| WO2008043245A1 (en) * | 2006-09-11 | 2008-04-17 | The Chinese University Of Hong Kong | Abiotic stress tolerance conferred by j-domain-containing proteins |
| CN101921774A (en) * | 2010-05-24 | 2010-12-22 | 南京大学 | Application of Dnaj-like protein and encoded gene thereof |
| CN103361370A (en) * | 2012-03-27 | 2013-10-23 | 中国科学技术大学 | Desiccation tolerance and resurrection related gene BdSDMT from resurrection plant Boea densihispidula and application thereof |
| CN103361361A (en) * | 2012-03-27 | 2013-10-23 | 中国科学技术大学 | Desiccation tolerance and resurrection related gene BdBCP1 from resurrection plant Boea densihispidula and application thereof |
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| CN109678940A (en) * | 2017-10-18 | 2019-04-26 | 中国科学院植物研究所 | Protein B hDnaJ6 and its encoding gene and application |
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