CN109576289A - The application of corn glycerol-3-phosphate ZmGPDH5 and its encoding gene in regulation plant stress tolerance - Google Patents
The application of corn glycerol-3-phosphate ZmGPDH5 and its encoding gene in regulation plant stress tolerance Download PDFInfo
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Abstract
The invention discloses the application of corn glycerol-3-phosphate ZmGPDH5 and its encoding gene in regulation plant stress tolerance.The present invention is transferred to wildtype Arabidopsis thaliana and obtains T3 generation homozygous transformant using corn GPDH gene family member ZmGPDH5 as research object.It chooses two transformants of wherein OE-1 and OE-2 and carries out salt resistant function identification.It is control with wildtype Arabidopsis thaliana, studies germination rate, root long and fresh weight variation of the ZmGPDH5 transgenic arabidopsis under salt stress.The result shows that: under salt stress treatment conditions, the seed germination rate of ZmGPDH5 transgenic arabidopsis is significantly higher than wildtype Arabidopsis thaliana, and the root long of ZmGPDH5 transgenic arabidopsis, fresh weight and growing way are significantly better than control.Show that ZmGPDH5 can significantly improve the salt resistance ability of genetically modified plants, ZmGPDH5 can be applied to cultivate the resistance to inverse kind of corn as resistant gene of salt.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to corn glycerol-3-phosphate ZmGPDH5 and its coding base
Because of the application in regulation plant stress tolerance.
Background technique
Improve hot and difficult issue and mesh that the resistance to inverse ability of crop has become current agricultural and the technical research of animal husbandry field
The significant problem of preceding urgent need to resolve.In recent years, it with the development of functional genomics and molecular biology, excavates resistance to inverse crucial
Gene, cultivated using technique for gene engineering there are the New Crop Varieties of good stress tolerance to have become to effectively improve crop resistance to inverse
One of means of ability.
Glycerol 3-phosphate (Glycerol-3-phosphate/G-3-P) is an important centre during fat metabolic
Product participates in a variety of physiological and biochemical procedures in plant.Glycerol 3-phosphate can be gone by glycerol 3-phosphate phosphorylase (GPP)
Phosphoric acid turns to glycerol, is alternatively arranged as the synthesis precursor of glycerolipid (including triacylglycerol, glycerophosphatide and glyceroglycolipid).3- phosphorus
Acid glycerol dehydrogenase (Glycerol-3-phosphate Dehydrogenase, GPDH) is catalyzed glycerol 3-phosphate and phosphoric acid dihydroxy
Reversible redox reaction between acetone, this fermentoid are widely present in various organisms, and with tissue specificity and carefully
Born of the same parents' location specificity.At present in addition to model plant arabidopsis and some algae, report is studied in other higher plants by GPDH family
Road it is seldom, especially there is not been reported in corn.
Summary of the invention
The technical problem to be solved by the present invention is to how regulate and control plant stress tolerance.
In order to solve the above-mentioned technical problem, present invention firstly provides the new applications of ZmGPDH5 protein.
The present invention provides application of the ZmGPDH5 protein in regulation plant stress tolerance.
The present invention also provides ZmGPDH5 protein answering in regulation plant root long and/or fresh weight and/or germination rate
With.
In above-mentioned application, the ZmGPDH5 protein be it is following a) or b) or c) or d) shown in protein:
A) amino acid sequence is protein shown in sequence 2；
B) fused protein that the N-terminal of the protein shown in sequence 2 and/or C-terminal connection label obtain；
C) by amino acid sequence shown in sequence 2 by one or several amino acid residues substitution and/or missing and/or
Add obtained protein with the same function；
D) homology with amino acid sequence shown in sequence 2 with 75% or 75% or more and egg with the same function
White matter.
In order to make protein in a) convenient for purifying, can in sequence table the amino terminal of protein shown in sequence 2 or
Carboxyl terminal connects upper label as shown in Table 1.
The sequence of table 1, label
| Label | Residue | Sequence |
| Poly-Arg | 5-6 (usually 5) | RRRRR |
| Poly-His | 2-10 (usually 6) | HHHHHH |
| FLAG | 8 | DYKDDDDK |
| Strep-tag II | 8 | WSHPQFEK |
| c-myc | 10 | EQKLISEEDL |
It is above-mentioned c) in protein, the substitutions of one or several amino acid residues and/or deletion and/or addition is not
More than the substitution and/or deletion and/or addition of 10 amino acid residues.
It is above-mentioned c) in protein can be artificial synthesized, can also first synthesize its encoding gene, then carry out biological expression and obtain.
It is above-mentioned c) in the encoding gene of protein can be one or several by will be lacked in DNA sequence dna shown in sequence 1
The codon of amino acid residue, and/or the missense mutation of one or several base-pairs is carried out, and/or at its 5 ' end and/or 3 ' ends
The coded sequence for connecting label shown in table 1 obtains.
It is above-mentioned d) in, " homology " include with amino acid sequence shown in sequence of the invention 2 have 75% or higher, or
80% or higher or 85% or higher or 90% or higher or 95% or more high homology amino acid sequence.
In order to solve the above-mentioned technical problem, invention further provides the new of biomaterial relevant to ZmGPDH5 protein
Purposes.
The present invention provides application of the biomaterial relevant to ZmGPDH5 protein in regulation plant stress tolerance.
The present invention also provides biomaterial relevant to ZmGPDH5 protein regulation plant root long and/or fresh weight and/
Or the application in germination rate.
The present invention also provides biomaterials relevant to ZmGPDH5 protein in the transgenosis plant for cultivating resistance of reverse raising
Application in object.
The biomaterial is following A 1) any one of to A12):
A1 the nucleic acid molecules of ZmGPDH5 protein) are encoded；
A2) contain A1) expression cassettes of the nucleic acid molecules；
A3) contain A1) recombinant vectors of the nucleic acid molecules；
A4) contain A2) recombinant vector of the expression cassette；
A5) contain A1) recombinant microorganisms of the nucleic acid molecules；
A6) contain A2) recombinant microorganism of the expression cassette；
A7) contain A3) recombinant microorganism of the recombinant vector；
A8) contain A4) recombinant microorganism of the recombinant vector；
A9) contain A1) the transgenic plant cells systems of the nucleic acid molecules；
A10) contain A2) the transgenic plant cells system of the expression cassette；
A11) contain A3) the transgenic plant cells system of the recombinant vector；
A12) contain A4) the transgenic plant cells system of the recombinant vector.
In above-mentioned application, A1) nucleic acid molecules be it is following 1) or 2) or 3) shown in gene:
1) its coded sequence is cDNA molecule or genomic DNA molecule shown in sequence 1；
2) there is 75% or 75% or more identity with the nucleotide sequence 1) limited, and encodes ZmGPDH5 protein
CDNA molecule or genomic DNA molecule；
1) or 2) 3) and the cDNA of ZmGPDH5 protein is encoded with the nucleotide sequence hybridization that limits under strict conditions
Molecule or genomic DNA molecule.
Wherein, the nucleic acid molecules can be DNA, such as cDNA, genomic DNA or recombinant DNA；The nucleic acid molecules can also
To be RNA, such as mRNA or hnRNA.
Those of ordinary skill in the art can easily adopt by known method, such as the side of directed evolution and point mutation
Method is mutated the nucleotide sequence of coding ZmGPDH5 protein of the invention.Those are by manually modified, with volume
The code nucleotide sequence 75% of the ZmGPDH5 protein or nucleotide of higher identity, if coding ZmGPDH5 protein and
It is derived from nucleotide sequence of the invention and to be equal to sequence of the invention with identical function.
Term " identity " used herein refers to the sequence similarity with native sequence nucleic acid." identity " includes and this hair
Amino acid sequence shown in bright coded sequence 2 composition protein nucleotide sequence have 75% or higher or 85% or
Higher or 90% or higher or 95% or higher identity nucleotide sequence.Identity can with the naked eye or computer software
It is evaluated.Using computer software, identity between two or more sequences can be indicated with percentage (%), can be with
For evaluating the identity between correlated series.
Above-mentioned 75% or 75% or more identity can be 80%, 85%, 90% or 95% or more identity.
In above-mentioned application, the stringent condition is to hybridize at 68 DEG C in 2 × SSC, the solution of 0.1%SDS and wash film
2 times, each 5min, but in 0.5 × SSC, the solution of 0.1%SDS, hybridize at 68 DEG C and washes film 2 times, each 15min；
Or, hybridizing under the conditions of 65 DEG C in the solution of 0.1 × SSPE (or 0.1 × SSC), 0.1%SDS and washing film.
In above-mentioned application, A2) described in the expression cassettes of the nucleic acid molecules containing coding ZmGPDH5 protein be to refer to
The DNA of ZmGPDH5 protein is expressed in host cell, which may include not only the promoter for starting ZmGPDH5 transcription, may be used also
Terminator including terminating ZmGPDH5 transcription.Further, the expression cassette may also include enhancer sequence.It can be used for this hair
Bright promoter includes but is not limited to: constitutive promoter；It organizes, the promoter and induction type that organ and development are special start
Son.
The recombinant vector of the ZmGPDH5 expression casette can be contained with existing expression vector establishment.The plant table
It include double base agrobacterium vector and the carrier etc. that can be used for plant micropellet bombardment up to carrier.As pAHC25, pBin438,
PCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or
PCAMBIA1391-Xb (CAMBIA company) etc..The plant expression vector also may include 3 ' end non-translational regions of foreign gene
Domain, i.e., comprising polyadenylation signals and any other DNA fragmentation for participating in mRNA processing or gene expression.The polyadenylic acid letter
Number bootable polyadenylic acid is added to 3 ' ends of mRNA precursor, as Agrobacterium crown gall nodule induces (Ti) plasmid gene (such as nopaline
Synthase gene Nos), plant gene (such as soybean storage protein genes) 3 ' end transcription non-translational region all have similar functions.
When using gene constructed plant expression vector of the invention, enhancer, including translational enhancer or transcriptional enhancer also can be used,
These enhancer regions can be ATG initiation codon or neighboring region initiation codon etc., but must read with coded sequence
Frame is identical, to guarantee the correct translation of entire sequence.The source of the translation control signal and initiation codon be it is extensive,
Can be it is natural, be also possible to synthesis.Translation initiation region can come from transcription initiation region or structural gene.In order to just
In transgenic plant cells or plant are identified and screened, plant expression vector used can be processed, it can as being added
The coding expressed in plant can produce the enzyme of color change or gene (gus gene, luciferase genes of luminophor
Deng), the marker gene of antibiotic (if assigned the nptII gene to kanamycins and associated antibiotic resistance, assigns to herbicide
The bar gene of phosphinothricin resistance assigns the hph gene to antibiotic hygromycin resistance, and assigns to methotrexate resistance
Dhfr gene is assigned to the EPSPS gene of glyphosate) or (such as anti-herbicide base such as anti-chemical reagent marker gene
Cause), provide metabolism mannose ability mannose-6-phosphate isomerase gene.It, can not from the security consideration of genetically modified plants
Add any selected marker, transformed plant is directly screened with adverse circumstance.
In above-mentioned application, the carrier can be plasmid, sticking grain, bacteriophage or viral vectors.
In above-mentioned application, the microorganism can be yeast, bacterium, algae or fungi, such as Agrobacterium.
In above-mentioned application, the transgenic plant cells system, Transgenic plant tissue and genetically modified plants organ are not wrapped
Include propagation material.
In above-mentioned application, the resistance of reverse is salt tolerance.
In order to solve the above-mentioned technical problem, the present invention finally provides a kind of genetically modified plants of cultivation resistance of reverse raising
Method.
The method provided by the invention for cultivating the genetically modified plants that resistance of reverse improves includes improving ZmGPDH5 in recipient plant
The expression quantity and/or activity of protein, the step of obtaining genetically modified plants；The resistance of reverse of the genetically modified plants be higher than it is described by
Body plant.
Further, the resistance of reverse is salt tolerance.
The resistance of reverse of the genetically modified plants is embodied in any in following (1)-(3) higher than the recipient plant:
(1) seed germination rate of genetically modified plants is higher than recipient plant；
(2) root long of genetically modified plants is longer than recipient plant；
(3) fresh weight of genetically modified plants is greater than recipient plant.
Further, it is described improve recipient plant in ZmGPDH5 protein expression quantity and/or active method be
ZmGPDH5 protein is overexpressed in recipient plant；The method of the overexpression is to import the encoding gene of ZmGPDH5 protein
Recipient plant；The nucleotide sequence of the encoding gene of the ZmGPDH5 protein is DNA molecular shown in sequence 1.In the present invention
Specific embodiment in, the encoding gene of ZmGPDH5 protein imports recipient plant by recombinant vector 35S:ZmGPDH5-GFP
In, the recombinant vector 35S:ZmGPDH5-GFP is by the DNA between XbaI the and SalI restriction enzyme site of pBI121-GFP carrier
Segment replaces with ZmGPDH5 gene shown in sequence 1 in sequence table, and after keeping the other sequences of pBI121-GFP carrier constant
Obtained carrier.
In the above method, the genetically modified plants are interpreted as not only including by the ZmGPDH5 genetic transformation recipient plant
Obtained first generation genetically modified plants also include its filial generation.For genetically modified plants, the gene can be bred in the species,
The gene transfer can also be entered to other kinds of same species with traditional breeding techniques, particularly including in commercial variety.It is described
Genetically modified plants include seed, callus, intact plant and cell.
In the above method or application, the plant is monocotyledon or dicotyledon.The dicotyledon is specific
It can be arabidopsis；The arabidopsis concretely arabidopsis (Columbia ecotype col-0).
The present invention with corn GPDH gene family member ZmGPDH5 (GRMZM2G063258_T05) be research object, by it
It is transferred to wildtype Arabidopsis thaliana and obtains T3 generation homozygous transformant.It chooses two transformants of wherein OE-1 and OE-2 and carries out salt resistant function mirror
It is fixed.It is control with wildtype Arabidopsis thaliana, studies germination rate, root long and fresh weight of the ZmGPDH5 transgenic arabidopsis under salt stress
Variation.As a result, it has been found that the seed germination rate of ZmGPDH5 transgenic arabidopsis is significantly higher than wild type under salt stress treatment conditions
Arabidopsis, and the root long of transgenic line, fresh weight and growing way are significantly better than control.Show that ZmGPDH5 can significantly improve transgenosis
The salt resistance ability of plant, ZmGPDH5 can be applied to cultivate the resistance to inverse kind of corn as resistant gene of salt.
Detailed description of the invention
Fig. 1 is the expression pattern analysis of ZmGPDH5 gene.
Fig. 2 is that salt stress handles the 5th day wildtype Arabidopsis thaliana (CK) and transgenic arabidopsis (OE-1 and OE-2) germination rate
Statistics.
Fig. 3 is the resistance of reverse inspection of wildtype Arabidopsis thaliana and transgenic arabidopsis.
Fig. 4 is the root long statistics of salt stress Wildtype Arabidopsis thaliana and transgenic arabidopsis.
Fig. 5 compares for the root long of salt stress Wildtype Arabidopsis thaliana and transgenic arabidopsis.
Fig. 6 is the fresh weight statistics of salt stress Wildtype Arabidopsis thaliana and transgenic arabidopsis.
Fig. 7 compares for the fresh weight of salt stress Wildtype Arabidopsis thaliana and transgenic arabidopsis.
Specific embodiment
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified certainly
What routine biochemistry reagent shop was commercially available.Quantitative test in following embodiment is respectively provided with three repeated experiments, as a result makes even
Mean value.
Data statistical analysis method in following embodiments: it utilizes SPSS 16.0 (SPSS Inc, Chicago, IL, USA)
Software is for statistical analysis.The more different strains of one-way analysis of variance (CK, OE-1 and OE-2) are poor under same treatment conditions
It is different whether to there is conspicuousness (P < 0.05, one-way ANOVA).
PBI121-GFP carrier in following embodiments is the product of abundant Biotechnology Co., Ltd, upper Hisense, catalogue
Number or article No. be XY2117.
EHA105 Agrobacterium in following embodiments is the product of only the biological Co., Ltd in Shanghai, catalog number or goods
Number be AC1010S.
Expression pattern of the ZmGPDH5 gene in corn root and leaf under embodiment 1, salt stress
1, the processing of plant sample
Three leaves wholeheartedly period corn (corn closes 344 self-mating systems) seedling is handled using 200mM NaCl, when handling different
Between point (0,1,3,6,12 and for 24 hours) leaf and root system are sampled, liquid nitrogen flash freezer, -80 DEG C save for extracting RNA.
2, the extraction and reverse transcription of RNA
Total RNA is extracted using TRIzol reagent (Invitrogen).Use ReverTra Ace qPCR RT Master
Mix with gDNA Remover (TOYOBO) reverse transcription, obtains cDNA.
3, real-time quantitative PCR
Real-time quantitative PCR is carried out using Bio-Rad Chromo4real-time PCR system.Corn ZmGAPDH (XM_
020551757) and ZmACTIN (XM_008656735.2) is used as internal reference standardized data.Primer sequence is as shown in table 1.Relatively
Expression utilizes 2-△△CTMethod calculates.Expression of the ZmGPDH5 in 0h root is set as 1, and others processing is accordingly counted
It calculates.Each sample carries out independent biology three times and repeats, and carries out technology three times every time and repeats.
Table 1, real-time quantitative PCR primer sequence
| Primer | Primer sequence (5 ' -3 ') |
| ZmGPDH5-RT-F | 5'-CAACCTTTCGCGGAATAGA-3' |
| ZmGPDH5-RT-R | 5'-TTGACCTTGTACTTCTGC-3' |
| ZmGAPDH-F | 5'-AAGGGAGAGTTGAGTCCTG-3' |
| ZmGAPDH-R | 5'-AGTATTCTGTAAAGCCTC-3' |
| ZmACTIN-F | 5'-ATCCAGGCTGTTCTTTCGTT-3' |
| ZmACTIN-R | 5'-CATTAGGTGGTCGGTGAGGT-3' |
Reaction system is as follows: 2 × SYBR Green Realtime PCR Master Mix (Toyobo) 12.5 μ l, upstream
Primer (0.5 μM), downstream primer (0.5 μM), 2 μ l of cDNA template (RNA for being equivalent to 100ng), total volume are 25 μ l.
Reaction condition is as follows: 94 DEG C, 30s；45 circulations: 94 DEG C, 12s；58 DEG C, 30s；72 DEG C, 30s.Last 80 DEG C, 1s.
As a result as shown in Figure 1.The result shows that: after NaCl Stress treatment 1h, ZmGPDH5 gene is in significant in leaf and root
Up-regulated expression, when NaCl Stress treatment 3h, the expression intensity of ZmGPDH5 gene reaches maximum value in leaf；NaCl Stress treatment
When 1h, the expression intensity of ZmGPDH5 gene reaches maximum value in root.The above result shows that ZmGPDH5 can be to some extent
It is expressed by Salt treatment, illustrates that ZmGPDH5 is that corn adapts to an important regulatory factor during abiotic stress.
Embodiment 2, the acquisition of ZmGPDH5 transgenic arabidopsis and its resistance of reverse analysis
One, the acquisition and identification of ZmGPDH5 transgenic arabidopsis
1, using corn root cDNA as template, PCR expansion is carried out using ZmGPDH5-GFP-F and ZmGPDH5-GFP-R primer
Increase, obtains pcr amplification product.Primer sequence is following (sequence shown in underscore is digestion recognition site):
ZmGPDH5-GFP-F:5'-GCTCTAGAATGGCCGCCGCCGCCGC-3'；
ZmGPDH5-GFP-R:5'-ACGCGTCGACGACTTCCTCAACCTGGGGAAG-3'。
2, the pcr amplification product that pBI121-GFP carrier and step 1 are obtained with restriction enzyme XbaI and SalI into
Row double digestion, connection, obtains recombinant plasmid 35S:ZmGPDH5-GFP.And sequence verification is carried out to it.
Sequencing result shows: recombinant vector 35S:ZmGPDH5-GFP is by XbaI the and SalI enzyme of pBI121-GFP carrier
DNA fragmentation between enzyme site replaces with ZmGPDH5 gene shown in sequence 1 in sequence table, and keeps pBI121-GFP carrier
Other sequences it is constant after obtained carrier.ZmGPDH5 albumen shown in recombinant vector 35S:ZmGPDH5-GFP expressed sequence 2
Matter.
3, recombinant vector 35S:ZmGPDH5-GFP is converted in Agrobacterium EHA105 using freeze-thaw method, identifies to obtain through PCR
Positive transformant, for infecting Arabidopsis plant.
4, the Agrobacterium containing recombinant plasmid 35S:ZmGPDH5-GFP is infected into wildtype Arabidopsis thaliana (brother using dipping in colored method
Rival Asia ecotype col-0).Harvest T1 is screened for seed and on the 1/2MS culture medium of the kanamycins containing 50mg/L.To screening
The next generation that obtained seedling generates carries out PCR screening again, so repeats, and the final T3 that obtains is for ZmGPDH5 transgenic arabidopsis
Homozygous lines.
5, T is extracted3For the total serum IgE of ZmGPDH5 transgenic arabidopsis homozygous lines, pass through RT-PCR method qualitative detection
The relative expression quantity of ZmGPDH5 gene, while being made with arabidopsis gene Actin2 (AT5G18780) and UBQ1 (AT4G05320)
For reference gene, primer sequence is as follows:
Actin2-F:5 '-TTACCCGATGGGCAAGTC-3 '；
Actin2-R:5 '-GCTCATACGGTCAGCGATAC-3 '；
UBQ1-F:5 '-GGCCTTGTATAATCCCTGATGAA-3 '；
UBQ1-R:5 '-AGAAGTCGACTTGTCATTAGAAAGAA-3 '.
Agarose gel electrophoresis detection is carried out to PCR product, the results showed that the RT-PCR of wild-type Arabidopsis plants is without expansion
Increase production object, and ZmGPDH5 transgenic arabidopsis homozygous lines OE-1 and OE-2 can amplify the purpose that size is 1284bp
Band shows that foreign gene ZmGPDH5 is not only smoothly integrated on the genome of arabidopsis, and can be in the quasi- south of transgenosis
Normal transcription is expressed in mustard.Choose T3For ZmGPDH5 transgenic arabidopsis homozygous lines OE-1 and OE-2 for the resistance to of next step
Inverse property is analyzed.
Two, the resistance of reverse analysis of ZmGPDH5 transgenic arabidopsis
1, germination rate is tested
1) seed disinfection: wildtype Arabidopsis thaliana (CK) and ZmGPDH5 transgenic arabidopsis strain (OE-1 and OE-2) are planted
Son is placed in 2mL centrifuge tube, and 75% dehydrated alcohol disinfection 1min is first added in centrifuge tube, during which constantly rocks oscillation, makes to disappear
It is malicious uniform.Brief centrifugation 10s precipitates arabidopsis seed, supernatant is abandoned, with sterile water wash 3-5 times.Then with 10% NaClO
10min is sterilized, sops up upper layer impurity with pipette tips, with sterile water wash 8-10 times, until colorless and odorless.
2) salt treatment: the 1/2MS that the seed after disinfection is seeded in NaCl containing 100mM or 150mM NaCl respectively is cultivated
On base, 22 ± 2 DEG C are placed in, 80-100 μm of olm of intensity of illumination-2·s-1, periodicity of illumination is the artificial gas of 8h dark, 16h illumination
It waits and is cultivated in room.Simultaneously to be cultivated in normal 1/2MS culture medium as control.Experiment uses 45-50 seeds every time, is locating
The third day of reason and germination rate of the different strains under various concentration NaCl treatment conditions was counted within the 5th day respectively, and in the 7th day
It takes pictures and observes phenotype.Experiment carries out independent biology three times and repeats.
As a result as shown in Figures 2 and 3.The result shows that: on 1/2MS (0mM NaCl) control plate, ZmGPDH5 transgenosis
The germination rate of arabidopsis seed is compared with compareing wildtype Arabidopsis thaliana strain without significant difference.In third day statistical result,
Seed germination rate of the ZmGPDH5 transgenic arabidopsis under different salinity is all remarkably higher than control wildtype Arabidopsis thaliana strain.
In 5th day statistical result, on the culture medium that NaCl concentration is 100mM and 150mM, the seed of ZmGPDH5 transgenic arabidopsis
Germination rate is significantly higher than control wildtype Arabidopsis thaliana strain (Fig. 2), and growing way is substantially better than control wildtype Arabidopsis thaliana strain (figure
3).The above results show that ZmGPDH5 participates in response of the arabidopsis sprouting to salt stress, are overexpressed ZmGPDH5 and improve transgenosis
Resistance of the arabidopsis to salt stress.
2, root long is tested
By wildtype Arabidopsis thaliana (CK) and ZmGPDH5 transgenic arabidopsis strain (OE-1 and OE-2) seed disinfection (method
It is seeded on 1/2MS culture medium after ibid) handling, the seedling of the consistent different strains of growing way is transferred to respectively after 7 days and is contained
There are 150mM NaCl, 75mM Na2SO4And it is cultivated vertically on the 1/2MS culture medium of 15mM LiCl.Simultaneously to be trained in normal 1/2MS
It supports culture in base and is used as control.Every kind of each strain stress includes 45-50 plants of plants.Condition of culture is same as above.In Stress treatment
Root long under the conditions of each strain NaCl and treatment with mannitol was taken pictures and measured in 7th day.Experiment carries out independent life three times
Object repeats.
As a result as shown in Figure 4 and Figure 5.The result shows that: under normal operation, ZmGPDH5 transgenic arabidopsis strain and right
Growth according to wildtype Arabidopsis thaliana strain root has no difference, however, under NaCl stress conditions, ZmGPDH5 transgenic arabidopsis
The root long of strain, which is considerably longer than, compares wildtype Arabidopsis thaliana strain (Fig. 4), and growing way is substantially better than control wildtype Arabidopsis thaliana strain
It is (Fig. 5).Illustrate that ZmGPDH5 participates in the growth of regulation Plants under Salt Stress root, root long experiment further demonstrates that ZmGPDH5 can
Arabidopsis is improved to the tolerance of salt stress.
3, fresh weight is tested
By wildtype Arabidopsis thaliana (CK) and ZmGPDH5 transgenic arabidopsis strain (OE-1 and OE-2) seed disinfection (method
Be seeded on 1/2MS culture medium after ibid) handling, vernalization 2 days, after in temperature be to cultivate to transplant after a week in 22 DEG C of culturing room
In soil (vermiculite: perlite: black earth=1:1:3), after culture 3 weeks, poured respectively with water (control), 200mM NaCl
Processing, continuous pouring observes the phenotype of seedling after handling 12 days, and counts the fresh weight of different strains.Every kind of stress packet of each strain
Containing 45-50 plants of plants.Experiment carries out independent biology three times and repeats.
As a result as shown in Figure 6 and Figure 7.The result shows that: under normal operation, ZmGPDH5 transgenic arabidopsis strain and right
Difference is had no according to the fresh weight of wildtype Arabidopsis thaliana strain, however, under NaCl stress conditions, ZmGPDH5 transgenic arabidopsis strain
The fresh weight of system is noticeably greater than wildtype Arabidopsis thaliana (Fig. 6), and growing way is substantially better than control wildtype Arabidopsis thaliana strain (Fig. 7).It says
The overexpression of bright ZmGPDH5 gene improves tolerance of the arabidopsis in the growth of seedling stage to salt.
By seed germination rate to ZmGPDH5 transgenic arabidopsis, root long and fresh weight is for statistical analysis and phenotype is seen
It examines, discovery ZmGPDH5 can significantly improve the salt resistance ability of genetically modified plants.It is resistance to further to utilize transgenic approach to cultivate corn
Salt kind provides theoretical basis and gene prepares.
Sequence table
<110>Heilongjiang Bayi Agricultural Reclamation University
<120>application of corn glycerol-3-phosphate ZmGPDH5 and its encoding gene in regulation plant stress tolerance
<160>2
<170>PatentIn version 3.5
<210>1
<211>1284
<212>DNA
<213>artificial sequence (Artificial Sequence)
<400>1
atggccgccg ccgccgccgt cttctttccc tccaccccga atcctcggca ccgcctcgcc 60
gccgccgccc gccgccctcc acccagcttc accggcgcca ccgacgccgt gccgctaccg 120
gaggacgagg actccagcga cgacgatgcc gacgacgacg gcgcgccacg caggagcggg 180
cgcagggacc ggcgccgcgc ggtgcgcgtc gcgtgggaga agctagtccg gtggtcccgc 240
tcctggcgcc gccgcaaccg cagcgacgtc ctcgagacca cgcgcaaggt ggtggttctc 300
gggggcgggt cgttcgggac ggccatggcc gctcacgtgg cggccaagaa ggccgatctc 360
gaggtggcca tgctgctcag ggacgaccac atctgccggt ccatcaacaa cgcacatgtc 420
aattgcaagt acttatcaga acacagattg ccagaaaata tcgtcgcaac aactagtgct 480
gctgatgctt tagcaggagc tgatttctgc ttccatgctg ttccggttca gttcagttca 540
tcctttcttg aaagtatttc aacacatgtt gatccaaagt tgccattcat atcacttagc 600
aaagggctgg aactcaatac ccttcggaca atgtctacaa tcatcccacg agcattggga 660
aatcgccgcc aaccgtttgt tgttctgtca ggaccatcat ttgctgtaga gttaatgaac 720
aaattgccta cagcaatggt ggtggcatcc aaagacaaca agttggcaag ttctgttcaa 780
caactgttag catccccaaa tttgaggata agcacatcaa gtgatgttac aggagtagaa 840
atcgcaggtg ccctgaagaa cgttcttgca atagctgcag gtatagtgga aggcatgagt 900
ctcgggaaca actgtatggc tgcccttgtt gcccaaggct gttcagagat acggtggttg 960
gcgacgaaga tgggagcaaa gccaaccact ctttctggct tgtctgggtc cggtgacatc 1020
atgctcacat gcttcgtcaa cctttcgcgg aatagaacag tgggactacg cctcggttct 1080
ggtgaaaagc ttgacagaat catgggttct atgaatcagg ttgctgaagg cgtatcaact 1140
gctggggctg tcattgcgtt ggcgcagaag tacaaggtca aaatgccggt attgacagcg 1200
gtggcacgga taattgataa tgagttaact ccaactaagg cggttttgga gttaatgaat 1260
cttccccagg ttgaggaagt ctga 1284
<210>2
<211>427
<212>PRT
<213>artificial sequence (Artificial Sequence)
<400>2
Met Ala Ala Ala Ala Ala Val Phe Phe Pro Ser Thr Pro Asn Pro Arg
1 5 10 15
His Arg Leu Ala Ala Ala Ala Arg Arg Pro Pro Pro Ser Phe Thr Gly
20 25 30
Ala Thr Asp Ala Val Pro Leu Pro Glu Asp Glu Asp Ser Ser Asp Asp
35 40 45
Asp Ala Asp Asp Asp Gly Ala Pro Arg Arg Ser Gly Arg Arg Asp Arg
50 55 60
Arg Arg Ala Val Arg Val Ala Trp Glu Lys Leu Val Arg Trp Ser Arg
65 70 75 80
Ser Trp Arg Arg Arg Asn Arg Ser Asp Val Leu Glu Thr Thr Arg Lys
85 90 95
Val Val Val Leu Gly Gly Gly Ser Phe Gly Thr Ala Met Ala Ala His
100 105 110
Val Ala Ala Lys Lys Ala Asp Leu Glu Val Ala Met Leu Leu Arg Asp
115 120 125
Asp His Ile Cys Arg Ser Ile Asn Asn Ala His Val Asn Cys Lys Tyr
130 135 140
Leu Ser Glu His Arg Leu Pro Glu Asn Ile Val Ala Thr Thr Ser Ala
145 150 155 160
Ala Asp Ala Leu Ala Gly Ala Asp Phe Cys Phe His Ala Val Pro Val
165 170 175
Gln Phe Ser Ser Ser Phe Leu Glu Ser Ile Ser Thr His Val Asp Pro
180 185 190
Lys Leu Pro Phe Ile Ser Leu Ser Lys Gly Leu Glu Leu Asn Thr Leu
195 200 205
Arg Thr Met Ser Thr Ile Ile Pro Arg Ala Leu Gly Asn Arg Arg Gln
210 215 220
Pro Phe Val Val Leu Ser Gly Pro Ser Phe Ala Val Glu Leu Met Asn
225 230 235 240
Lys Leu Pro Thr Ala Met Val Val Ala Ser Lys Asp Asn Lys Leu Ala
245 250 255
Ser Ser Val Gln Gln Leu Leu Ala Ser Pro Asn Leu Arg Ile Ser Thr
260 265 270
Ser Ser Asp Val Thr Gly Val Glu Ile Ala Gly Ala Leu Lys Asn Val
275 280 285
Leu Ala Ile Ala Ala Gly Ile Val Glu Gly Met Ser Leu Gly Asn Asn
290 295 300
Cys Met Ala Ala Leu Val Ala Gln Gly Cys Ser Glu Ile Arg Trp Leu
305 310 315 320
Ala Thr Lys Met Gly Ala Lys Pro Thr Thr Leu Ser Gly Leu Ser Gly
325 330 335
Ser Gly Asp Ile Met Leu Thr Cys Phe Val Asn Leu Ser Arg Asn Arg
340 345 350
Thr Val Gly Leu Arg Leu Gly Ser Gly Glu Lys Leu Asp Arg Ile Met
355 360 365
Gly Ser Met Asn Gln Val Ala Glu Gly Val Ser Thr Ala Gly Ala Val
370 375 380
Ile Ala Leu Ala Gln Lys Tyr Lys Val Lys Met Pro Val Leu Thr Ala
385 390 395 400
Val Ala Arg Ile Ile Asp Asn Glu Leu Thr Pro Thr Lys Ala Val Leu
405 410 415
Glu Leu Met Asn Leu Pro Gln Val Glu Glu Val
420 425
Claims (10)
1.ZmGPDH5 application of the protein in regulation plant stress tolerance；
Or, application of the ZmGPDH5 protein in regulation plant root long and/or fresh weight and/or germination rate.
2. application according to claim 1, it is characterised in that:
The ZmGPDH5 protein be it is following a) or b) or c) or d) shown in protein:
A) amino acid sequence is protein shown in sequence 2；
B) fused protein that the N-terminal of the protein shown in sequence 2 and/or C-terminal connection label obtain；
C) amino acid sequence shown in sequence 2 is passed through to the substitution and/or deletion and/or addition of one or several amino acid residues
Obtained protein with the same function；
D) homology with amino acid sequence shown in sequence 2 with 75% or 75% or more and albumen with the same function
Matter.
3. application of the biomaterial relevant to ZmGPDH5 protein in regulation plant stress tolerance；
Or, biomaterial relevant to ZmGPDH5 protein answering in regulation plant root long and/or fresh weight and/or germination rate
With；
Or, biomaterial relevant to ZmGPDH5 protein is cultivating the application in the genetically modified plants that resistance of reverse improves.
4. application according to claim 3, it is characterised in that:
The biomaterial is following A 1) any one of to A12):
A1 the nucleic acid molecules of ZmGPDH5 protein) are encoded；
A2) contain A1) expression cassettes of the nucleic acid molecules；
A3) contain A1) recombinant vectors of the nucleic acid molecules；
A4) contain A2) recombinant vector of the expression cassette；
A5) contain A1) recombinant microorganisms of the nucleic acid molecules；
A6) contain A2) recombinant microorganism of the expression cassette；
A7) contain A3) recombinant microorganism of the recombinant vector；
A8) contain A4) recombinant microorganism of the recombinant vector；
A9) contain A1) the transgenic plant cells systems of the nucleic acid molecules；
A10) contain A2) the transgenic plant cells system of the expression cassette；
A11) contain A3) the transgenic plant cells system of the recombinant vector；
A12) contain A4) the transgenic plant cells system of the recombinant vector.
5. application according to claim 4, it is characterised in that: A1) nucleic acid molecules be it is following 1) or 2) or 3) shown in
Gene:
1) its coded sequence is cDNA molecule or genomic DNA molecule shown in sequence 1；
2) there is 75% or 75% or more identity with the nucleotide sequence 1) limited, and encodes the cDNA of ZmGPDH5 protein
Molecule or genomic DNA molecule；
1) or 2) 3) and the cDNA molecule of ZmGPDH5 protein is encoded with the nucleotide sequence hybridization that limits under strict conditions
Or genomic DNA molecule.
6. -5 any application according to claim 1, it is characterised in that: the resistance of reverse is salt tolerance.
7. a kind of method for cultivating the genetically modified plants that resistance of reverse improves, including improving ZmGPDH5 protein in recipient plant
Expression quantity and/or activity, the step of obtaining genetically modified plants；The resistance of reverse of the genetically modified plants is higher than the recipient plant.
8. according to the method described in claim 7, it is characterized by: the resistance of reverse is salt tolerance；
The resistance of reverse of the genetically modified plants is embodied in any in following (1)-(3) higher than the recipient plant:
(1) seed germination rate of genetically modified plants is higher than recipient plant；
(2) root long of genetically modified plants is longer than recipient plant；
(3) fresh weight of genetically modified plants is greater than recipient plant.
9. method according to claim 7 or 8, it is characterised in that: described to improve ZmGPDH5 protein in recipient plant
Expression quantity and/or active method are that ZmGPDH5 protein is overexpressed in recipient plant；
Or, the method for the overexpression is that the encoding gene of ZmGPDH5 protein is imported recipient plant；
Or, the nucleotide sequence of the encoding gene of the ZmGPDH5 protein is DNA molecular shown in sequence 1.
10. -6 any any method of application or claim 7-9 according to claim 1, it is characterised in that: institute
Stating plant is monocotyledon or dicotyledon.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107188940A (en) * | 2017-07-24 | 2017-09-22 | 黑龙江八农垦大学 | The application of GsHA12 albumen and its encoding gene in regulation and control plant stress tolerance |
| CN107383179A (en) * | 2017-09-13 | 2017-11-24 | 黑龙江八农垦大学 | A kind of and plant stress tolerance correlative protein GsSLAH3 and its encoding gene and application |
-
2018
- 2018-12-13 CN CN201811524262.2A patent/CN109576289B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107188940A (en) * | 2017-07-24 | 2017-09-22 | 黑龙江八农垦大学 | The application of GsHA12 albumen and its encoding gene in regulation and control plant stress tolerance |
| CN107383179A (en) * | 2017-09-13 | 2017-11-24 | 黑龙江八农垦大学 | A kind of and plant stress tolerance correlative protein GsSLAH3 and its encoding gene and application |
Non-Patent Citations (3)
| Title |
|---|
| YING ZHAO ET AL.,: ""Glycerol-3-phosphate dehydrogenase (GPDH)gene family in Zea mays L.: Identification,subcellul localization, and transcriptional responses to abiotic stresses"", 《PLOS ONE》 * |
| 匿名: ""GenBank: MH460966.1：REGION: 116..1399"", 《GENBANK》 * |
| 郎小强等: ""植物中3-磷酸甘油脱氢酶基因家族的物种特异性进化分析"", 《应用与环境生物学报》 * |
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