CN104178498B - Cotton Gossypii GhMYC1 transcription factor and encoding gene thereof and application - Google Patents
Cotton Gossypii GhMYC1 transcription factor and encoding gene thereof and application Download PDFInfo
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Abstract
The invention discloses Cotton Gossypii GhMYC1 transcription factor and encoding gene thereof and application, belong to Cotton Gossypii MYC class transcription factor and application thereof.First the present invention discloses the GhMYC1 transcription factor encoding gene separated from Cotton Gossypii, and its polynucleotide sequence is shown in SEQ ID No.1, and the aminoacid sequence of its coding is shown in SEQ ID No.2.The invention also discloses the recombinant expression carrier containing described transcription factor encoding gene and recombinant host cell.Arabidopsis transgenic functional analysis it is demonstrated experimentally that in plant process LAN Cotton Gossypii GhMYC1 transcription factor encoding gene can be effectively improved the plant resistance to the abiotic stress such as high salt or arid.The Cotton Gossypii GhMYC1 transcription factor encoding gene that the present invention is separated improve plant to environment stress resistance and cultivate resistance to environment stress new variety of plant in have important application prospect.
Description
Technical field
The present invention relates to the transcription factor relevant to stress resistance of plant, particularly relate to the GhMYC1 separated from Cotton Gossypii
Transcription factor encoding gene and the aminoacid sequence of coding thereof, the invention still further relates to containing Cotton Gossypii GhMYC1 transcribe because of
The recombinant expression carrier of sub-encoding gene and recombinant host cell, the invention further relates to Cotton Gossypii GhMYC1 and transcribe
The factor and encoding gene thereof improve plant to environment stress resistance and cultivate resistance to environment stress new variety of plant in
Application, belong to Cotton Gossypii MYC class transcription factor and applied technical field thereof.
Background technology
In plant, myelocytomatosis albumen (Myelocytomatosis proteins, MYCs) i.e. MYC class turns
The record factor, has multiple regulatory function, and is widely present in animals and plants.The MYC class having been found that transcribe because of
In son, MYC2 is to study the most deep one, at present in model plant arabidopsis find MYC2 transcribe because of
Son plays regulating and controlling effect by forming CO I 1/JAZs/MYC2 complex, participates in the hormone signals such as JA, ABA and turns
Lead process.Such as the AtMYC2 transcription factor of arabidopsis, express under adverse circumstance and strengthen.Under drought stress, AtMYC2
Albumen also serves as the transcription activator function of ABA induction.
Arid and salting are natural disasters the most serious to crop harm in many abiotic stresses, have a strong impact on crop
Yield and cultivated area.According to incompletely statistics, global arid area accounts for 1/3rd of the land gross area, and
There is the trend increased year by year.The salinization of soil and Secondary Saline problem are worldwide widely present, the most dry
Drought, semiarid zone, problem is even more serious.
Cotton Gossypii is one of crops of salt tolerant, and its salt tolerance is because of kind, growing stage, organ and soil salt kind
Classes etc. are different and differ greatly.Therefore, from Cotton Gossypii, clone MYC class transcription factor encoding gene and be applied to
Regulation and control plant is to environment stress resistance and the new variety of plant of cultivating resistance to environment stress, the raising to plant stress-resistance performance
To have great importance.
Summary of the invention
An object of the present invention is to provide the MYC class transcription factor encoding gene of a class regulation and control plant stress resistance；
The two of the purpose of the present invention be to provide recombinant expression carrier containing described MYC class transcription factor encoding gene with
And the recombinant host cell containing this recombinant expression carrier；
The three of the purpose of the present invention are that described Cotton Gossypii MYC class transcription factor and encoding gene thereof are applied to regulation and control and are planted
Thing is to the resistance of environment stress and the transgenic plant new varieties of cultivating resistance to environment stress.
The above-mentioned purpose of the present invention is achieved through the following technical solutions:
The present invention has separated GhMYC1 transcription factor encoding gene from Cotton Gossypii (Gossypium hirsutum L.),
Its polynucleotide are shown in (a), (b), (c), (d) or (e):
Polynucleotide shown in (a), SEQ ID No.1；Or
Amino acid whose polynucleotide shown in (b), coding SEQ ID No.2；Or
C the complementary series of () and SEQ ID No.1 can carry out the polynucleotide hybridized at stringent hybridisation conditions,
Coded by these polynucleotide, protein still has GhMYC1 functional transcription factor；Or
The polynucleotide of at least 90% or above homology of the polynucleotide shown in (d) and SEQ ID No.1,
Preferably, with the polynucleotide of at least 95% or above homology of the polynucleotide shown in SEQ ID No.1, optimum
Choosing, with the polynucleotide of at least 98% or above homology of the polynucleotide shown in SEQ ID No.1；Or
(e), carry out on the basis of the polynucleotide shown in SEQ ID No.1 one or more base disappearance,
Replace or the polynucleotide variant of insertion, and this albumen coded by polynucleotide variant still has GhMYC1 and transcribes
The function of the factor or activity.
The invention also discloses the GhMYC1 transcription factor encoded by described transcription factor gene, its aminoacid is (a)
Or shown in (b):
Aminoacid shown in (a), SEQ ID No.2；
(b), by the aminoacid shown in SEQ ID No.2 by the replacement of one or more amino acid residues, disappearance
Or/and insert and the derivative protein variant still with GhMYC1 functional transcription factor or activity obtained.
Protein variant of the present invention can be produced by genetic polymorphism or manual operation, and these operational approach are the most originally
Field is understood.Such as, the amino acid sequence variation of GhMYC1 transcription factor can be prepared by the sudden change of DNA
Or fragment, wherein the method for mutation or change polynucleotide is known by this area.Wherein, conservative replacement is
A kind of amino acid residue is replaced to the another kind of aminoacid with similar quality.GhMYC1 of the present invention turns
The record factor and encoding gene thereof include naturally occurring sequence and two kinds of forms of variant." variant " means the sequence of basic simlarity
Row, for polynucleotide, variant comprises the one or more nucleotide of one or more site in native polynucleotide
Disappearance, insertion are or/and replace.For polynucleotide, conservative variant include the degeneracy due to genetic code and not
Change those variants of the aminoacid sequence of coding.Naturally occurring variant like this can pass through existing molecular biosciences
Learn a skill and identify.Variant polynucleotides also includes the polynucleotide in synthesis source, obtained by direct mutagenesis
The amino acid whose polynucleotide variant still encoded shown in SEQ ID No.2, or by restructuring method (such as
DNA reorganizes).
The invention also discloses the recombinant expression carrier containing described GhMYC1 transcription factor encoding gene；Described
Recombinant expression carrier is recombinant plant expression vector.
The invention also discloses the recombinant host cell containing described recombinant expression carrier.
It is connected exercisable for described Cotton Gossypii GhMYC1 transcription factor encoding gene with expression regulation element, obtains
The recombinant plant expression vector of this encoding gene can be expressed in plant；This recombinant plant expression vector can be by 5 '
End noncoding region, the nucleotide shown in SEQ ID No.1 and 3 ' noncoding regions composition；Wherein, described 5 ' non-volume is held
Code district can include promoter sequence, enhancer sequence or/and translate enhancement sequences；Described promoter can be group
Constitutive promoter, inducible promoter, tissue or organ specific promoters；3 ' described noncoding regions can comprise
Terminator sequence, mRNA cutting sequence etc..
It addition, the nucleotide shown in SEQ ID No.1 can be optimized to strengthen plant by those skilled in the art
In expression efficiency.Such as, the preference codon of target plant can be used to be optimized synthetic polyribonucleotides to strengthen
Expression efficiency in target plant.
Described recombinant plant expression vector also can be containing for selecting to convert the selected marker of cell.Marker gene
Including: the gene of coding antibiotic resistance and the gene etc. of imparting herbicides compounds resistance.
The invention still further relates to be incorporated in plant described Cotton Gossypii GhMYC1 transcription factor encoding gene and plant to improve
The thing resistance to environment stress.
The present invention further discloses described Cotton Gossypii GhMYC1 transcription factor encoding gene and improve plant to adverse circumstance
Application in stress resistance, comprises the following steps: (1) builds and encodes containing described Cotton Gossypii GhMYC1 transcription factor
The recombinant plant expression vector of gene；(2) constructed recombinant plant expression vector is transformed into plant or plant is thin
In born of the same parents；(3) the transgenic plant new varieties that screening obtains improving environment stress resistance are cultivated.
A kind of method that the present invention further discloses transgenic plant new varieties cultivating resistance to environment stress, including following
Step: (1) builds the recombinant plant expression vector containing described Cotton Gossypii GhMYC1 transcription factor encoding gene；(2)
Constructed recombinant plant expression vector is transformed in plant or plant cell；(3) cultivate screening to obtain adverse circumstance
The transgenic plant new varieties that stress resistance improves.
Wherein, described environment stress includes high salt or arid.
Conversion scheme and by the scheme of described polynucleotide or polypeptide introduced plant visually be used for convert plant or plant
The type of thing cell and change.The appropriate method that described polynucleotide or polypeptide introduce plant cell is included: micro-note
Penetrate, electroporation, Agrobacterium-medialed transformation, direct gene transfer and high velocity ballistic bombardment etc..
Utilize cell regeneration stable conversion plant (the McCormick et al.Plant Cell that conventional method can make to have converted
Reports.1986.5:81-84).The present invention can be used for converting any floristics, includes but not limited to: unifacial leaf
Plant or dicotyledon.It is furthermore preferred that described target plant includes crops, vegetable or ornamental plant, fruit tree
Deng, for example, it may be Semen Maydis, Oryza sativa L., Sorghum vulgare Pers., Semen Tritici aestivi, Semen sojae atricolor, Rhizoma Solani tuber osi, Fructus Hordei Vulgaris, Fructus Lycopersici esculenti, Kidney bean, flower
Life or Caulis Sacchari sinensis etc..
In order to analyze GhMYC1 transcription factor that the present invention separates from Cotton Gossypii (Gossypium hirsutum L.)
Function, present invention Gateway method builds containing GhMYC1 gene (nucleotide is shown in SEQ ID No.1)
Plant expression vector, inflorescence dip method arabidopsis thaliana transformation, it is thus achieved that transgenic arabidopsis.Build the most respectively and contain
GhMYC2 (nucleotide is shown in SEQ ID No.3), GhMYC3 (nucleotide is shown in SEQ ID No.4)
With the recombinant plant expression vector of GhMYC4 (nucleotide is shown in SEQ ID No.5) gene, arabidopsis thaliana transformation,
Obtain transgenic arabidopsis.Transgenic arabidopsis is carried out drought resistance and salt tolerance analysis.Phenotype analytical result shows, turns
The arabidopsis of GhMYC1 gene NaCl water pouring 10 days after, on-bladed yellowing phenomenon, bloom with solid situation all
It is better than NK matched group；After Osmotic treatment 10 days, the Arabidopsis plant turning GhMYC1 gene grows normally but blade
Little, plant strain growth is higher, has certain drought resisting effect.After NaCl and Osmotic treatment, turn GhMYC1 gene
Arabidopsis ' chlorophyll content, all at normal level, illustrates that the photosynthesis of transfer-gen plant is normal.NaCl turns after processing
The arabidopsis content of propylene glycol of GhMYC1 gene is minimum, illustrates to turn the saline-alkaline tolerance of GhMYC1 gene arabidopsis
By force；After Osmotic treatment, the arabidopsis content of propylene glycol turning GhMYC1 gene is relatively low, near 0.013 μm ol/g,
Prove that drought resisting effect is obvious.
In a word, proved by arabidopsis function transformation experiment, process LAN Cotton Gossypii GhMYC1 transcription factor in plant
Encoding gene can be effectively improved the plant resistance to the environment stress such as high salt or arid.Therefore, the cotton of present invention clone
Flower GhMYC1 transcription factor encoding gene to environment stress resistance and is cultivated resistance to environment stress and is turned base improving plant
Because new variety of plant has important application prospect.
The term definition that the present invention relates to
Unless otherwise defined, all technology the most used herein and scientific terminology all have with of the art
Those of ordinary skill is generally understood identical implication.
Term " transcription factor " means to tie with cis acting element specificity in eukaryotic gene promoter region
Close, thus activate or suppress the class DBP that downstream gene is transcribed at special time and space and expressed.
Term " polynucleotide " or " nucleotide " mean sub-thread or the deoxyribonucleotide of bifilar form, deoxyribose core
Glycosides, ribonucleotide or ribonucleotide and polymer thereof.Unless specific restriction, the most described term is contained containing natural
The nucleic acid of the known analog of nucleotide, described analog has and is similar to the binding characteristic of reference nucleic acid and to be similar to
The mode of naturally-produced nucleotide carries out metabolism.Unless other specific restriction, the most described term also means oligonucleoside
Acid-like substance, it include PNA (peptide nucleic acid(PNA)), DNA analog used in antisense technology (thiophosphate,
Phosphamide acid esters etc.).Unless otherwise, otherwise specific nucleic acid sequence the most impliedly contains its conservative variation modified
Body (include, but is not limited to degenerate codon replace) and complementary series and the sequence clearly specified.Particularly,
Can by produce one of them or more than one selected by the 3rd the blended base of (or all) codon and/or de-
The substituted sequence of oxygen inosine residue realizes degenerate codon and replaces (Batzer et al., Nucleic Acid Res.19:5081
(1991)；Ohtsuka et al., J.Biol.Chem.260:2605-2608 (1985)；With Cassol et al., (1992)；
Rossolini et al., Mol Cell.Probes8:91-98 (1994)).
Predicate " stringent hybridisation conditions " means known low ionic strength and the condition of high temperature in the art.Generally,
Under high stringency conditions, probe and its target sequence hybridize can detection level than with other sequence hybridization can detection level more
High (such as exceeding background at least 2 times).Stringent hybridisation conditions is sequence dependent, under different environmental conditions
Will be different, longer sequence specific hybrid at relatively high temperatures.The preciseness hybridized by control or wash conditions
Can identify and the target sequence of probe 100% complementation.For the detailed guidance of nucleic acid hybridization refer to relevant document (Tijssen,
Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Probes,
"Overview of principles of hybridization and the strategy of nucleic acid assays.1993).More
Concrete, described high stringency conditions is typically selected to be less than distinguished sequence heat fusion joint under regulation ionic strength pH
(Tm) about 5-10 DEG C.Tm is that 50% probe complementary with target hybridizes to during target sequence residing in the state of the equilibrium
Temperature (under specifying ionic strength, pH and nucleic acid concentration) (because target sequence is present in excess, so at Tm
Under in the state of the equilibrium 50% probe be occupied).High stringency conditions can be following condition: wherein at pH7.0 to 8.3
Lower salinity is below about 1.0M Na ion concentration, and typically about 0.01 arrives 1.0M Na ion concentration (or other salt),
And temperature is at least about 30 DEG C for short probe (including, but is not limited to 10 to 50 nucleotide), and
It is at least about 60 DEG C for long probe (including, but is not limited to more than 50 nucleotide).High stringency conditions is also
Can realize by adding the destabilizing agent of such as Methanamide.For selectivity or specific hybrid, positive signal can
For the background hybridization of at least twice, it is optionally 10 times of background hybridizations.Exemplary stringent hybridisation conditions can be as follows: 50%
Methanamide, 5 × SSC and 1%SDS, cultivate at 42 DEG C；Or 5 × SSC, 1%SDS, cultivate at 65 DEG C,
Washing and washing in 0.1%SDS at 65 DEG C in 0.2 × SSC.Described washing can carry out 5,15,30,60,
120 minutes or longer time.
Term " recombinant host cell strain " or " host cell " mean to comprise the cell of polynucleotide of the present invention, regardless of making
By which kind of method carry out inserting to produce recombinant host cell, the most directly absorb, transduce, f pairing or art
Other method known in.Exogenous polynucleotide can remain the non-integrated vector of such as plasmid or can be integrated into place
In major gene group.Host cell can be prokaryotic cell or eukaryotic cell, and host cell can be also unifacial leaf or dicotyledonous plant
Thing cell.
Term " recombinant plant expression vector " means that one or more are for the DNA vector realizing Plant Transformation；Ability
In territory, these carriers are commonly referred to as binary vector.Binary vector is mostly to be usually used in soil together with the carrier with helper plasmid
Earth bacillus mediated transformation.Binary vector generally includes: T-DNA transfer required for cis acting sequence, through work
Journeyization process so as in plant cell express selectable marker, heterologous DNA sequence etc. to be transcribed.
Accompanying drawing explanation
Fig. 1 is clone's GhMYC1 and GhMYC2 electrophoretogram from cotton gene group and cDNA；Wherein, M is
DNA Marker；1,2 is the cotton gene group PCR result to MYC1 total length primer；3 is cDNA pair, Cotton Gossypii
The PCR result of MYC1 total length primer；4,5 is the cotton gene group PCR result to MYC2 total length primer；6
For the Cotton Gossypii cDNA PCR result to MYC2 total length primer；
Fig. 2 is clone's GhMYC3 and GhMYC4 electrophoretogram from cotton gene group and cDNA；Wherein, 1,2
For the Cotton Gossypii cDNA PCR result to MYC3 total length primer；3 is that cotton gene group is to MYC3 total length primer
PCR result；4,5,6 is the cotton gene group PCR result to MYC4 total length primer；7,8,9 is Cotton Gossypii
The cDNA PCR result to MYC4 total length primer；
Fig. 3 is the positive bacterium colony detection figure of GhMYC1；Wherein, M is DNA Marker；11,13 is 11 and
No. 13 bacterium colonies；+ for positive control；-for blank；
Fig. 4 is the positive bacterium colony detection figure of GhMYC2；
Fig. 5 is the positive bacterium colony detection figure of GhMYC3；
Fig. 6 is the positive bacterium colony detection figure of GhMYC4；
Fig. 7 is that Gateway method builds containing GhMYC1, GhMYC2, GhMYC3 or GhMYC4 gene
The flow chart of plant expression vector；
Fig. 8 is that the entry clones of GhMYC1, GhMYC2, GhMYC3 and GhMYC4 gene converts large intestine bar
The bacterium solution PCR detection figure of bacteria strain DH5 α；Wherein, M is DNA Marker；1-3 is the introduction of GhMYC1
The PCR testing result of No. 3 bacterium colonies of Cloning Transformation bacillus coli DH 5 alpha；2-3 and 2-4 is entering of GhMYC2
The PCR testing result of No. 3 and No. 4 bacterium colonies of door Cloning Transformation bacillus coli DH 5 alpha；3-2,3-3 and 3-4 are
The entry clones of GhMYC3 converts the PCR testing result of No. 2, No. 3 and No. 4 bacterium colonies of bacillus coli DH 5 alpha；
4-1,4-2,4-3,4-4 and 4-5 be GhMYC4 entry clones convert No. 1 of bacillus coli DH 5 alpha, No. 2,
The PCR testing result of No. 3, No. 4 and No. 5 bacterium colonies；+ for positive control；
Fig. 9 is that the plant expression vector of GhMYC1, GhMYC2, GhMYC3 and GhMYC4 gene converts big
The bacterium solution PCR detection figure of enterobacteria bacterial strain DH5 α；Wherein, M is DNA Marker；1-1,1-2 and 1-3 are
The plant expression vector of GhMYC1 converts the PCR detection of No. 1, No. 2 and No. 3 bacterium colony of bacillus coli DH 5 alpha
Result；2-1,2-1,2-3 and 2-4 be the plant expression vector of GhMYC2 convert No. 1 of bacillus coli DH 5 alpha,
The PCR testing result of No. 2, No. 3 and No. 4 bacterium colonies；3-1,3-2 and 3-3 are the plant expression vector of GhMYC3
Convert the PCR testing result of No. 1, No. 2 and No. 3 bacterium colony of bacillus coli DH 5 alpha；4-1,4-2,4-3 and 4-4
Plant expression vector for GhMYC4 converts No. 1, No. 2, No. 3 and No. 4 bacterium colony of bacillus coli DH 5 alpha
PCR testing result；+ for positive control；-for blank；
Figure 10 is that the plant expression vector of GhMYC1, GhMYC2, GhMYC3 and GhMYC4 gene converts
The bacterium solution PCR detection figure of Agrobacterium GV3101；Wherein, M is DNA Marker；1-1,1-2,1-4 and 1-5
Plant expression vector for GhMYC1 converts No. 1, No. 2, No. 4 and No. 5 bacterium colony of Agrobacterium GV3101
PCR testing result；2-1,2-3,2-4 and 2-5 are that the plant expression vector of GhMYC2 converts Agrobacterium GV3101
The PCR testing result of No. 1, No. 3, No. 4 and No. 5 bacterium colony；3-1,3-2,3-3 and 3-4 are GhMYC3
Plant expression vector convert the PCR testing result of No. 1, No. 2, No. 3 and No. 4 bacterium colony of Agrobacterium GV3101；
4-2 and 4-5 is the PCR of No. 2 and No. 5 bacterium colonies of the plant expression vector conversion Agrobacterium GV3101 of GhMYC4
Testing result；+ for positive control；
The PCR of Figure 11 transgenic arabidopsis identifies figure；Wherein, M is DNA Marker；1-1 1-6 is for turning
The testing result of 6 strain arabidopsiss of GhMYC1 gene；3-1 3-2 is the 2 strain arabidopsiss turning GhMYC3 gene
Testing result；4-1 4-12 is the testing result of the 12 strain arabidopsiss turning GhMYC4 gene；
Figure 12 is the block diagram of the transgenic arabidopsis Chlorophyll content after 200mmol/L NaCl processes；Wherein
NK is for compareing non-transfer-gen plant；GhMYC1-1 and GhMYC1-2 is that the arabidopsis turning GhMYC1 gene is planted
Strain No.1 and No. 2；GhMYC2-1 and GhMYC2-2 is the Arabidopsis plant 1 and 2 turning GhMYC2 gene
Number；GhMYC3-1 and GhMYC3-2 is the Arabidopsis plant No. 1 and No. 2 turning GhMYC3 gene；GhMYC4-1
It is the Arabidopsis plant No. 1 and No. 2 turning GhMYC4 gene with GhMYC4-2；
Figure 13 is the block diagram of the transgenic arabidopsis Chlorophyll content after Osmotic treatment；Wherein NK is for comparison not
Transfer-gen plant；GhMYC1-1 and GhMYC1-2 is the Arabidopsis plant No. 1 and No. 2 turning GhMYC1 gene；
GhMYC3-1 and GhMYC3-2 is the Arabidopsis plant No. 1 and No. 2 turning GhMYC3 gene；GhMYC4-1
It is the Arabidopsis plant No. 1 and No. 2 turning GhMYC4 gene with GhMYC4-2；
Figure 14 is the content of propylene glycol block diagram of the transgenic arabidopsis after 200mmol/L NaCl processes；Wherein NK
For compareing non-transfer-gen plant；GhMYC1-1 and GhMYC1-2 is the Arabidopsis plant 1 turning GhMYC1 gene
Number and No. 2；GhMYC2-1 and GhMYC2-2 is the Arabidopsis plant No. 1 and No. 2 turning GhMYC2 gene；
GhMYC3-1 and GhMYC3-2 is the Arabidopsis plant No. 1 and No. 2 turning GhMYC3 gene；GhMYC4-1
It is the Arabidopsis plant No. 1 and No. 2 turning GhMYC4 gene with GhMYC4-2；
Figure 15 is the content of propylene glycol block diagram of the transgenic arabidopsis after Osmotic treatment；Wherein NK does not turns for comparison
Gene plant；GhMYC1-1 and GhMYC1-2 is the Arabidopsis plant No. 1 and No. 2 turning GhMYC1 gene；
GhMYC2-1 and GhMYC2-2 is the Arabidopsis plant No. 1 and No. 2 turning GhMYC2 gene；GhMYC3-1
It is the Arabidopsis plant No. 1 and No. 2 turning GhMYC3 gene with GhMYC3-2；GhMYC4-1 and GhMYC4-2
For turning the Arabidopsis plant No. 1 and No. 2 of GhMYC4 gene；
Figure 16 is the block diagram of the soluble sugar content of the transgenic arabidopsis after 200mmol/L NaCl processes；Wherein
NK is for compareing non-transfer-gen plant；GhMYC1-1 and GhMYC1-2 is that the arabidopsis turning GhMYC1 gene is planted
Strain No.1 and No. 2；GhMYC2-1 and GhMYC2-2 is the Arabidopsis plant 1 and 2 turning GhMYC2 gene
Number；GhMYC3-1 and GhMYC3-2 is the Arabidopsis plant No. 1 and No. 2 turning GhMYC3 gene；GhMYC4-1
It is the Arabidopsis plant No. 1 and No. 2 turning GhMYC4 gene with GhMYC4-2；
Figure 17 is the soluble sugar content block diagram of the transgenic arabidopsis after Osmotic treatment；Wherein NK is for comparison not
Transfer-gen plant；GhMYC1-1 and GhMYC1-2 is the Arabidopsis plant No. 1 and No. 2 turning GhMYC1 gene；
GhMYC2-1 and GhMYC2-2 is the Arabidopsis plant No. 1 and No. 2 turning GhMYC2 gene；GhMYC3-1
It is the Arabidopsis plant No. 1 and No. 2 turning GhMYC3 gene with GhMYC3-2；GhMYC4-1 and GhMYC4-2
For turning the Arabidopsis plant No. 1 and No. 2 of GhMYC4 gene.
Detailed description of the invention
Below in conjunction with specific embodiment further describe the present invention, advantages of the present invention and feature will be with describe and
Apparent.It should be understood that described embodiment is only exemplary, the scope of the present invention is not constituted any restriction.
It will be understood by those skilled in the art that can be to the technology of the present invention side under without departing from the spirit and scope of the present invention
The details of case and form are modified or replace, but these amendments or replacement each fall within protection scope of the present invention.
1, experiment material
The blade of Cotton Gossypii silver cotton variety, silver cotton variety buys vegetables fully stocked wood flowers market in Beijing；Arabidopsis seed
For Colombia's type, the present inventor's laboratory preserves；PMD-19T cloning vehicle, Taq enzyme are purchased from Quan Shi King Company；
Bacillus coli DH 5 alpha and high-purity Plasmid Miniprep Kit are purchased from Beijing hundred Tyke Bioisystech Co., Ltd；Agriculture
Bacillus strain GV3101 is that the present inventor's laboratory preserves；pDONRTM207 carriers are purchased from tall and handsome Jetta company；
Expression vector pH7WG2D is purchased from VIB (Belgian) company；The BP reaction kit of Gateway clone and LR
Reaction kit is purchased from tall and handsome Jetta company；DNA glue reclaims test kit and draws company purchased from Plutarch；Trizol reagent is purchased from
Tian Gen Science and Technology Ltd.；CDNA Reverse Transcription box is purchased from Fermentas company；Primer synthesis and order-checking are by north
AudioCodes prosperous Bioisystech Co., Ltd in capital completes.
The clone of embodiment 1 Cotton Gossypii MYC class transcription factor encoding gene
1, experimental technique
1.1 electronic clonings obtain MYC class transcription factor
According to homologous genes exists the characteristic guarding similar sequences, by the sequence of arabidopsis MYC class transcription factor gene
It is classified as information probes, est database Cotton Gossypii in NCBI (Gossypium hirsutum) is carried out Tblastx homology
Property retrieval, utilize DNAStar software will retrieve come from Cotton Gossypii est sequence splicing obtain connector；So
Again carry out Homology search and the splicing of Tblastx afterwards with this connector, repeat above procedure until non-overlapping EST
Sequence, the final cDNA sequence fragment obtaining 4 GhMYC genes of Gossypium hirsutum L..
1.2RT-PCR method obtains genes of interest
CDNA sequence design RT-PCR primer (table according to 4 GhMYC genes of total length that electronic cloning obtains
1)。
Table 1 GhMYC Cloning of full length primer
Utilize Trizol-isopropanol method to extract the cotton total serum IgE of silver, operate according to the explanation of test kit.Extract Cotton Gossypii
After total serum IgE, with it as template, Oligo-dT (5pmol/ μ L) is primer 1 μ L, mixes latter 65 DEG C and hatches 5min,
It is put on ice, adds 5 × Reaction Buffer4 μ L, RNasin Inhibitor1 μ L, M-MLVRT1 μ L and dNTP
After Mix2 μ L, and gentle mixing wink from, hatch 60min for 42 DEG C, 70 DEG C of 5min terminate reaction, i.e. reverse transcription and close
Becoming cDNA the first chain ,-20 DEG C save backup.
After reverse transcription product Preliminary detection, take 1 μ L and carry out PCR amplification as template by following system and condition.
RT-PCR reaction system is: total system is 20 μ L, and wherein containing 10 × Buffer is 2 μ L, 2mmol/L dNTP
It is 1 μ L, is diluted to each 1 μ L of primer of 10 μm ol/L, Taq enzyme 0.5U (reaction system of general 20 μ L adds 1 μ L),
Add ddH2O to 20 μ L.The response procedures of RT-PCR is: 94 DEG C of denaturations 5min；94 DEG C of degeneration 30s, annealing
45s, 72 DEG C extend 60s, and circulate 29 times；72 DEG C extend 10min.
The fragment of expection size, through 1% agarose gel electrophoresis, is reclaimed and is cloned into pMD-19T gram by amplified production
Grand carrier, converts coli strain DH5 α.Picking positive bacteria carries out bacterium solution PCR and checks order.
It addition, utilize CTAB method to extract cotton genomic dna, utilize the primer of table 1 from cotton genomic dna
Clone 4 GhMYC transcription factor genes.
2, experimental result
Four GhMYC DNA sequence design primers of Cotton Gossypii according to electronic cloning, by RT-PCR reaction respectively
Go out four GhMYC transcription factor genes (Fig. 1, Fig. 2) from cDNA or genomic dna cloning, it is thus achieved that 4
Individual MYC class transcription factor gene is named respectively: GhMYC1, GhMYC2, GhMYC3 and GhMYC4, its
Nucleotide sequence is respectively shown in SEQ ID No.1, SEQ ID No.3, SEQ ID No.4 and SEQ ID No.5.
Wherein, the aminoacid sequence of the derivation of GhMYC1 gene is shown in SEQ ID No.2.Find from Fig. 1, Fig. 2
Four genes of GhMYC transcription factor do not contain intron in the cotton genome of silver.
By clone GhMYC1, GhMYC2, GhMYC3 and GhMYC4 gene PCR primer respectively with
PMD-19T carrier connects, and converts bacillus coli DH 5 alpha.The DH5 α resistance bacterium turning GhMYC1 gene is carried out
PCR bacterium colony detects, and finds that 11 and No. 13 bacterium colonies are positive (Fig. 3).To the DH5 α turning GhMYC2 gene
Resistance bacterium carries out PCR bacterium colony detection, finds that No. 3 bacterium colonies are positive (Fig. 4).To the DH5 α turning GhMYC3 gene
Resistance bacterium carries out PCR bacterium colony detection, find No. 2,18 bacterium colonies such as No. 3 be positive (Fig. 5).To turning GhMYC4
The DH5 α resistance bacterium of gene carries out the detection of PCR positive bacterium colony, finds No. 9, No. 14 bacterium colonies be the positive (Fig. 6).
Embodiment 2 contains plant expression vector construction and the arabidopsis thaliana transformation of MYC class transcription factor encoding gene
1, experimental technique
1.1Gateway method builds plant expression vector
According to Gateway clone technology primer sequence design require and embodiment 1 clone GhMYC1,
GhMYC2, GhMYC3 and GhMYC4 gene order design Gateway reaction primer (table 2).
Table 2 Gateway design of primers
Using archaeal dna polymerase, carry out PCR reaction with the upstream and downstream primer of table 2, total system is 20 μ L, wherein
Contain connection GhMYC1, GhMYC2, GhMYC3 and GhMYC4 gene of embodiment 1 preparation respectively
PMD-19T cloning vector plasmids 1 μ L, 10 × Buffer are 2 μ L, and 2mmol/L dNTP is 1 μ L, is diluted to
The each 1 μ L of primer of 10 μm ol/L, Taq enzyme 0.5U (reaction system of general 20 μ L adds 1 μ L), add ddH2O is extremely
20μL.Upstream and downstream at GhMYC1, GhMYC2, GhMYC3 and GhMYC4 full-length gene adds attB
Sequence.Reaction condition is as follows: 94 DEG C of denaturations 5min, 94 DEG C of degeneration 30s, and anneal 45s, and 75 DEG C extend 1min,
Totally 30 circulations；75 DEG C extend 10min, and 4 DEG C terminate reaction.Product is detected with agarose gel electrophoresis, and
Purification reclaims.
BP react: BP reaction purpose be the PCR primer containing attB joint that amplification is obtained be cloned into containing
The donor vehicle pDONR of attPTMOn 207, to produce entry clones.Gateway according to invitrogen company
BP reaction system in BP Clonase II enzyme mix test kit, including PCR primer 15-150ng, pDONRTM
207 carrier 1 μ L (150ng/ μ L), supplement TE buffer (pH8.0) and mix to 8 μ L, quick whirlpool concussion enzyme system
Close liquid twice, add 1 × BP ClonaseTM enzymatic mixture 2 μ L.Reaction system adds 2 μ L after putting 25 DEG C of incubation 60min
E.C. 3.4.21.64 liquid, 37 DEG C of incubation 10min, to terminate BP reaction.Obtain entry clones pDONRTM207-GhMYC1、
pDONRTM207-GhMYC2、pDONRTM207-GhMYC3 and pDONRTM207-GhMYC4.To pass through
The entry clones that BP reaction obtains converts coli strain DH5 α, picking resistant clones, carries out PCR detection,
The bacterium colony of test positive extracts plasmid, i.e. obtains entry clones carrier.
LR reacts: the purpose of LR reaction is will to be recombined into entry vector pDONRTM207-GhMYC1、
pDONRTM207-GhMYC2、pDONRTM207-GhMYC3 and pDONRTMThe target base of 207-GhMYC4
Because GhMYC1, GhMYC2, GhMYC3 and GhMYC4 are cloned in expression vector pH7WG2D again.LR
Reaction system includes Entry clone plasmids 15-150ng, pH7WG2D carrier 1 μ L (150ng/ μ L), supplements TE
Buffer (pH8.0) to 8 μ L, quick whirlpool concussion enzyme system mixed liquor twice, add 1 × LR ClonaseTM enzyme mixes
Compound 2 μ L.Reaction system adds 2 μ L Proteinase K Solution after putting 25 DEG C of incubation 60min, 37 DEG C of incubation 10min with
Terminate LR reaction, obtain final expression vector pH7WG2D-GhMYC1, pH7WG2D-GhMYC2,
PH7WG2D-GhMYC3 and pH7WG2D-GhMYC4 (builds flow chart and sees Fig. 7).
By the expression vector pH7WG2D-GhMYC1 of LR product, pH7WG2D-GhMYC2,
PH7WG2D-GhMYC3 and pH7WG2D-GhMYC4 converts coli strain DH5 α, picking positive bacterium colony,
Carry out PCR detection, to determine positive colony.
1.2 inflorescence dip method arabidopsis thaliana transformations
1.2.1 the cultivation of arabidopsis and process
It is placed on the arabidopsis seed of the couple of days of vernalization in 4 DEG C of refrigerators in growth pot, waters and epiphragma is to keep
Moisture.When arabidopsis is bloomed for the first time, for promoting the hypertrophy of the more spray of side shoot, the alabastrum on stem top is cut.
The flowers being suitable for converting are not have maturation, the most do not produce angle fruit (about growth time is about 30 days) being fertilized.
Before turning arabidopsis with inflorescence infestation method, cut off the angle fruit grown up to, in order to avoid reducing positive rate.
1.2.2 inflorescence dip method arabidopsis thaliana transformation
By Gateway method build plant expression vector pH7WG2D-GhMYC1, pH7WG2D-GhMYC2,
PH7WG2D-GhMYC3 and pH7WG2D-GhMYC4 converts Agrobacterium GV3101, picking positive bacterium colony respectively
Carry out PCR detection.Positive strain is inoculated into respectively in YEB fluid medium, adds kanamycin and rifampicin,
Shake bacterium 1 day in a large number until OD value is 1.2.5000rpm centrifugal enrichment Agrobacterium, then with 5 sucrose+1/2MS
A large amount of suspend (surfactant SilwetL-77 of addition 0.1% in sucrose solution), until OD value is about 0.8.
The inflorescence of arabidopsis is immersed in bacterium solution and infects 1 minute；Arabidopsis black plastic bag or thin film after infecting cover
Lid, light culture 24 hours.After infecting for the first time, within 7 days, carry out secondary infection, infect method with for the first time.Treat
Arabidopsis plant cuts one's eye-teeth, and gathers in the crops seed, be T1 for seed after the fruit natural cracking of angle.
1.2.3 the resistance screening of arabidopsis seed
T1 is uniformly sowed for seed containing 50mg/L hygromycin or the resistant panel (1/4 of 100mg/L that penicillin of card
MS culture medium) on, after putting 4 DEG C of refrigerator cold treatment 72h, move to 25 DEG C, 16h illumination box germinates, fixed
Germination and growth of seedling situation are observed and investigated to phase.Growing about 7-10 days, the seedling growing two panels true leaf is
Resistance seedling, by resistance seedling replanting to Nutrition Soil.When long maturation, the genome extracting transfer-gen plant is carried out
PCR identifies.
2, experimental result
2.1 plant expression vector construction
Entry clones pDONRTM207-GhMYC1、pDONRTM207-GhMYC2、
pDONRTM207-GhMYC3 and pDONRTM207-GhMYC4 converts the bacterium solution of coli strain DH5 α
PCR testing result is shown in Fig. 8.
Plant expression vector pH7WG2D-GhMYC1, pH7WG2D-GhMYC2, pH7WG2D-GhMYC3
The bacterium solution PCR testing result converting coli strain DH5 α with pH7WG2D-GhMYC4 is shown in Fig. 9.Explanation
The plant expression vector construction success of GhMYC1, GhMYC2, GhMYC3 and GhMYC4 gene.
2.2 plant expression vector arabidopsis thaliana transformations
Amplification containing plant expression vector pH7WG2D-GhMYC1, pH7WG2D-GhMYC2,
The positive DH5 α bacterium colony of pH7WG2D-GhMYC3 and pH7WG2D-GhMYC4 also extracts plasmid, is transformed into
In Agrobacterium GV3101 (Figure 10).
By inflorescence dip method arabidopsis thaliana transformation, respectively obtain turn GhMYC1, GhMYC2, GhMYC3 and
The arabidopsis T1 of GhMYC4 gene is for seed.
The resistance screening of 2.3 arabidopsiss
Extracting positive seedling genome and carry out PCR qualification (Figure 11), result illustrates, is successfully obtained and turns respectively
The arabidopsis of GhMYC1, GhMYC2, GhMYC3 and GhMYC4 gene.
The drought resistance and salt tolerance analysis of 3 turns of Cotton Gossypii MYC class transcription factor encoding gene arabidopsiss of embodiment
1, experimental technique
The Stress treatment of 1.1 transgenic positive strains
1.1.1 salt stress processes
The NaCl drenching 200mmol/L is watered with the transgenic Arabidopsis plants of potted plant about 4 weeks of embodiment 2 acquisition,
Process 10d sampling detection, compare with wildtype Arabidopsis thaliana.Observe the salt stress impact on transgenic arabidopsis phenotype.
1.1.2 drought stress processes
The transgenic Arabidopsis plants of potted plant about 4 weeks obtained by embodiment 2 carries out drought stress process, 10 days
Not watering, during Osmotic treatment 10d, sampling detection, compares with wildtype Arabidopsis thaliana.Observe drought stress to turning base
Impact because of arabidopsis phenotype.
1.2 physical signs detections
1.2.1 the mensuration of chlorophyll content
Take the fresh leaf of the arabidopsis after Stress treatment to clean, shred (removing middle arteries and veins), mixing, weigh and shred fresh sample 0.05g
Put in mortar, add the quartz sand of half spoon and spoonful Paris white and the ethanol 2-3ml of 95%, grind to form homogenate,
Add 95% ethanol 4ml again to continue to be ground to tissue and turn white, the most static 3-5 minute, then enter with filter paper filtering
In 10ml volumetric flask, then drip with 95% ethanol and wash mortar, pestle and filter paper, till redgreen, finally fixed
Hold to scale, shake up, obtain chlorophyll extracting solution；646nm and 663nm wavelength is measured respectively with spectrophotometer
Under absorbance (A) value.
1.2.2 propylene glycol (MDA) and the mensuration of soluble sugar
Weigh the Arabidopsis plant 0.1g of Stress treatment, shred, powdered by liquid nitrogen grinding；Add 1.1ml's
5%TCA, whirlpool shakes 2min, 13000r/min and is centrifuged 10min, and supernatant is sample extracting solution.Draw centrifugal
Supernatant 1ml, add 1.1ml 0.6%TBA solution, shake up.Test tube is put in boiling water from test tube solution
In occur that minute bubbles start timing and boil 10min, take out test tube and also cool down, 13000r/min is centrifuged 10min, takes
Clear liquid 2ml.With 0.6%TBA solution as blank, measure the absorbance (A) at 532nm, 600nm, 450nm.
2, experimental result
The impact on transgenic arabidopsis phenotype of 2.1 salt stresses
Result shows, turns the arabidopsis of GhMYC1 gene after NaCl waters pouring 10 days, on-bladed yellowing phenomenon,
Blooming and solid situation is all better than NK matched group, matched group Arabidopsis leaf occurs in that yellow leaf, wilting and withered
Phenomenon, stem turns black, and flower and solid the most relatively transgenic group are poor；The arabidopsis turning GhMYC2 gene occurs in that
The phenomenon that partial blade is withered；Turning the arabidopsis of GhMYC3 gene, it is the slowest to grow, and sending out occurs in blade
Yellow phenomenon.
The impact on transgenic arabidopsis phenotype of 2.2 arids
Result shows, after Osmotic treatment 10 days, the plant turning GhMYC2 gene has significantly relative to matched group
The withered phenomenon of plant, blade is whole withered but has a small amount of knot of blooming；Turn GhMYC1 and GhMYC4 gene
Arabidopsis plant growth is normal but blade is little, and plant strain growth is higher, has certain drought resisting effect；Turn GhMYC3 base
The arabidopsis drought resisting effect of cause is obvious, does not has the phenomenon that obvious blade turns black, and plant strain growth is vigorous, but growth phase pair
Short and small.Relative to transfer-gen plant, NK plant leaf has blackout phenomenon, and plant strain growth is higher.
2.3 physical signs detections
2.3.1 the mensuration of Chlorophyll content
Arabidopsis to NaCl process, Osmotic treatment has carried out the mensuration of photosynthesis index chlorophyll content, result
See Figure 12 and 13.
As seen from Figure 12, after the NaCl of 10 days 200mmol/L processes, GhMYC1, GhMYC3 are turned respectively
Remain basically stable with NK with the arabidopsis ' chlorophyll content of GhMYC4 gene, fluctuate between 1mg/g-1.4mg/g,
Belong to normal level, illustrate that the photosynthesis of plant is normal after 200mmol/L NaCl processes 10 days, chlorophyll content
Basic holding is consistent.And the plant Chlorophyll content that turns GhMYC2 gene is substantially low compared with other values, plant is described
Upgrowth situation is undesirable.
As seen from Figure 13, Osmotic treatment is after 10 days, the total chlorophyll content of NK matched group substantially relatively transgenic
The value of plant raises；The Arabidopsis leaf turning GhMYC2 gene is dead, therefore without chlorophyll content data.It is probably
After Osmotic treatment, a large amount of dehydration of matched group NK arabidopsis makes leaf weight alleviate, therefore chlorophyll content raises.
The arabidopsis ' chlorophyll level turning GhMYC1 and GhMYC4 gene respectively substantially remains in 0.7mg/g-1.21mg/g
Between, chlorophyll levels is normal, illustrates that the photosynthesis of transfer-gen plant is normal.And turn the plan of GhMYC3 gene
South mustard total chlorophyll content value is on the low side, shows that photosynthesis receives certain impact.
2.3.2 the mensuration of content of propylene glycol
Arabidopsis to NaCl process, Osmotic treatment carries out the mensuration of content of propylene glycol, and result is shown in Figure 14 and 15.
Figure 14, it can be seen that the content of propylene glycol of transgenic arabidopsis is significantly lower than matched group, illustrates at 10 days NaCl
After process, matched group arabidopsis is protected from environmental relatively big, and MDA content is apparently higher than normal level.Turn GhMYC1
The arabidopsis saline-alkaline tolerance of gene is the strongest, consistent with phenotype observed result；Turn the arabidopsis of GhMYC4 gene to NaCl
Better resistance after process；And the arabidopsis saline-alkaline tolerance that turns GhMYC3 gene is relatively weak, MDA value exists
About 0.018 μm ol/g.
Figure 15 is it can be seen that Osmotic treatment NK matched group relatively transfer-gen plant after 10 days, in content of propylene glycol
There is growth, illustrate that arid is bigger on the impact of NK plant.Turn GhMYC1, GhMYC3 and GhMYC4 respectively
The arabidopsis content of propylene glycol of gene is minimum, near 0.013 μm ol/g, it was demonstrated that drought resisting effect is obvious；Turn GhMYC2
The arabidopsis content of propylene glycol of gene is the highest, is 0.020 μm ol/g, illustrates that drought resisting effect is the most weak compared with other.
2.3.3 the mensuration of soluble sugar content
Arabidopsis to NaCl process, Osmotic treatment carries out the mensuration of soluble sugar content, and result is shown in Figure 16 and 17.
As can be seen from Figure 16,200mmol/L NaCl process matched group and transgenic group arabidopsis, soluble sugar
Changes of contents is little, and the soluble sugar content of transgenic group is substantially low compared with the value of matched group.It is possibly due at NaCl
Under process, the soluble sugar content of transgenic arabidopsis is little on the impact of regulation stress resistance of plant shape.Turn GhMYC3
The soluble sugar content of No. 2 plant of gene is the highest, has exceeded NK level；The solubility of remaining transgenic arabidopsis
Sugar content the most relatively NK value is low；No. 2 Arabidopsis plant soluble sugar contents turning GhMYC4 gene are minimum, for
0.077mmol/g。
As can be seen from Figure 17, Osmotic treatment, after 10 days, turns GhMYC1, GhMYC2, GhMYC3 respectively
It is higher than NK matched group (0.07mmol/g), explanation with the arabidopsis soluble sugar average content of GhMYC4 gene
Transfer-gen plant improves its drought resisting level by raising soluble sugar content.But except turning the 1 of GhMYC3 gene
The soluble sugar average content of number plant is outside 0.112mmol/g, remaining transfer-gen plant soluble sugar content and NK
Matched group compares the most notable.
Claims (8)
1. from Cotton Gossypii (Gossypium
hirsutum
L.) the GhMYC1 transcription factor encoding gene separated, it is characterised in that its polynucleotide are: shown in SEQ ID No.1.
2. the GhMYC1 transcription factor of encoding gene coding described in claim 1, it is characterised in that its aminoacid is: shown in SEQ ID No.2.
3. contain the recombinant expression carrier of encoding gene described in claim 1.
4. according to the recombinant expression carrier described in claim 3, it is characterised in that: described recombinant expression carrier is recombinant plant expression vector.
5. the encoding gene described in claim 1 is improving plant to the application in environment stress resistance；Wherein, described environment stress is selected from high salt or arid.
6. according to the application described in claim 5, it is characterised in that comprise the following steps: (1) builds the recombinant plant expression vector containing the encoding gene described in claim 1；(2) constructed recombinant plant expression vector is transformed in plant or plant cell；(3) the transgenic plant new varieties that screening obtains improving environment stress resistance are cultivated；Wherein, described environment stress is selected from high salt or arid.
7. the method for the transgenic plant new varieties cultivating resistance to environment stress, it is characterised in that comprise the following steps: (1) builds the recombinant plant expression vector containing the encoding gene described in claim 1；(2) constructed recombinant plant expression vector is transformed in plant or plant cell；(3) the transgenic plant new varieties that screening obtains improving environment stress resistance are cultivated；Wherein, described environment stress is selected from high salt or arid.
8. the GhMYC1 transcription factor described in claim 2 is improving plant to the application in environment stress resistance；Wherein, described environment stress is selected from high salt or arid.
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| CN101481410A (en) * | 2009-02-13 | 2009-07-15 | 中国农业科学院棉花研究所 | Drought resisting related transcription factor of cotton, and encoding gene and use thereof |
| WO2013057681A1 (en) * | 2011-10-18 | 2013-04-25 | University Of Manitoba | Drought-tolerant transgenic plants comprising decreased myc4 activity and methods for their use |
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| CN101481410A (en) * | 2009-02-13 | 2009-07-15 | 中国农业科学院棉花研究所 | Drought resisting related transcription factor of cotton, and encoding gene and use thereof |
| WO2013057681A1 (en) * | 2011-10-18 | 2013-04-25 | University Of Manitoba | Drought-tolerant transgenic plants comprising decreased myc4 activity and methods for their use |
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