CN104164440A - Plant stress reactive MYC (myelocytomatosis protein) transcription factors as well as coding genes and application thereof - Google Patents
Plant stress reactive MYC (myelocytomatosis protein) transcription factors as well as coding genes and application thereof Download PDFInfo
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- CN104164440A CN104164440A CN201410390070.2A CN201410390070A CN104164440A CN 104164440 A CN104164440 A CN 104164440A CN 201410390070 A CN201410390070 A CN 201410390070A CN 104164440 A CN104164440 A CN 104164440A
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Abstract
The invention discloses plant stress reactive MYC (myelocytomatosis protein) transcription factors as well as coding genes and application thereof. The invention firstly discloses coding genes of MYC transcription factors separated from gossypium hirsutum L., wherein the polynucleotide sequences of the coding genes are as shown in SEQ ID No.4, and the coded amino acid sequences of the coding genes are as shown in SEQ ID No.5. The invention further discloses a recombinant expression vector and a recombinant host cell which contain the coding genes of the transcription factors. By virtue of an analysis experiment on a transgenic function of arabidopsis, the coding genes, which are excessively expressed in the plant, of the gossypium hirsutum L. MYC transcription factors separated from the gossypium hirsutum L. can be used for effectively improving the resistance of the plant on abiological adversity stress such as high salinity or drought. The plant tress reactive MYC transcription factors as well as the coding genes thereof have important application prospects in improving plant adversity stress resistance, as well as culturing a novel adversity stress-resistant transgenic plant variety, and the like.
Description
Technical field
The present invention relates to transcription factor, relate in particular to MYC class transcription factor encoding gene separated from cotton, the invention still further relates to the recombinant expression vector and the recombinant host cell that contain described cotton MYC class transcription factor encoding gene, the invention further relates to cotton MYC class transcription factor and encoding gene thereof at regulating plant to environment stress resistance and cultivate the application in the transgenic plant new variety of resistance to environment stress, belong to cotton MYC class transcription factor and applied technical field thereof.
Background technology
Myelocytomatosis albumen in plant (Myelocytomatosis proteins, MYCs) is MYC class transcription factor, has multiple regulatory function, and is extensively present in animals and plants.In the MYC class transcription factor having been found that, MYC2 studies the most deep one, in model plant Arabidopis thaliana, find that at present MYC2 transcription factor, by forming CO I 1/JAZs/MYC2 mixture performance regulating and controlling effect, participates in the hormone signal transduction processes such as JA, ABA.As the AtMYC2 transcription factor of Arabidopis thaliana, under adverse circumstance, express and strengthen.Under drought stress, AtMYC2 albumen is also brought into play function as the transcription activator of ABA induction.
Drought and salt stain is, in many abiotic stresses, crop is endangered to the most serious natural disaster, has a strong impact on output and the cultivated area of crop.According to incompletely statistics, global arid area accounts for 1/3rd of the land total area, and has the trend increasing year by year.Salting of soil and Secondary Saline problem be extensively existence, particularly arid, semiarid zone worldwide, and problem is even more serious.
Cotton is one of farm crop of salt tolerant, and its salt tolerance differs greatly because of differences such as kind, growing stage, organ and soil salt kinds.Therefore, the transgenic plant new variety of cloning MYC class transcription factor encoding gene and being applied to the anti-environment stress of farm crop and cultivating resistance to environment stress from cotton, will have great importance to the raising of plant stress-resistance performance.
Summary of the invention
One of object of the present invention is to provide class plant stress response MYC class transcription factor and an encoding gene thereof;
Two of object of the present invention is to provide the recombinant expression vector that contains described MYC class transcription factor encoding gene and the recombinant host cell that contains this recombinant expression vector;
Three of object of the present invention is that described cotton MYC class transcription factor and encoding gene thereof are applied to regulating plant to the resistance of environment stress and the transgenic plant new variety of cultivating resistance to environment stress.
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
The present invention from cotton (Gossypium hirsutum L.) separated GhMYC4 transcription factor encoding gene, its polynucleotide be (a), (b), (c), (d) or (e) shown in:
(a), the polynucleotide shown in SEQ ID No.4; Or
(b), amino acid whose polynucleotide shown in coding SEQ ID No.5; Or
(c), with the polynucleotide that the complementary sequence of SEQ ID No.4 can be hybridized in rigorous hybridization conditions, the coded protein remains of these polynucleotide has GhMYC4 functional transcription factor; Or
(d), have 90% or the polynucleotide of above homology at least with the polynucleotide shown in SEQ ID No.4, preferably, have 95% or the polynucleotide of above homology at least with the polynucleotide shown in SEQ ID No.4, most preferred, have 98% or the polynucleotide of above homology at least with the polynucleotide shown in SEQ ID No.4; Or
(e), the polynucleotide variant that carries out disappearance, replacement or the insertion of one or more bases on the basis of the polynucleotide shown in SEQ ID No.4, and the coded albumen of this polynucleotide variant still has function or the activity of GhMYC4 transcription factor.
The invention also discloses the GhMYC4 transcription factor of being encoded by described transcription factor gene, its amino acid be (a) or (b) shown in:
(a), the amino acid shown in SEQ ID No.5;
(b), the replacement by one or more amino-acid residues by the amino acid shown in SEQ ID No.5, disappearance are or/and insert and derive the protein variant still with GhMYC4 functional transcription factor or activity obtaining.
Protein variant of the present invention can be produced by genetic polymorphism or manual operation, and these working method are generally this area and understand.For example, can prepare by the sudden change of DNA aminoacid sequence variant or the fragment of GhMYC4 transcription factor, wherein known by this area for the method for mutagenesis or change polynucleotide.Wherein, conservative replacement is that a kind of amino-acid residue is replaced to the another kind of amino acid with similar quality.GhMYC4 transcription factor of the present invention and encoding gene thereof comprise naturally occurring sequence and two kinds of forms of variant." variant " means substantially similar sequence, and for polynucleotide, the disappearance that variant comprises the one or more Nucleotide of one or more site in natural polynucleotide, insertion are or/and replace.For polynucleotide, conservative variant comprises due to the degeneracy of genetic code those variants of the aminoacid sequence that does not change coding.Naturally occurring variant like that can be identified by existing Protocols in Molecular Biology.Variant polynucleotide also comprise the polynucleotide in synthetic source, for example, adopt the resulting amino acid whose polynucleotide variant shown in SEQ ID No.5 of still encoding of site-directed mutagenesis, or for example, by the method (DNA reorganization) of restructuring.Those skilled in the art can be screened or be evaluated by following molecular biotechnology means function or the activity of the coded albumen of variant polynucleotide: the interaction between DNA binding activity, albumen, the effect of expressing in the activation situation of genetic expression in instantaneous research or transgenic plant etc.
The invention also discloses the recombinant expression vector that contains described GhMYC4 transcription factor encoding gene; Described recombinant expression vector is recombinant plant expression vector.
The invention also discloses the recombinant host cell that contains described recombinant expression vector.
Be connected with expression regulation element described cotton GhMYC4 transcription factor encoding gene is exercisable, obtain in plant, expressing the recombinant plant expression vector of this encoding gene; This recombinant plant expression vector can be by 5 ' end non-coding region, and the Nucleotide shown in SEQ ID No.4 and 3 ' non-coding region form; Wherein, described 5 ' end non-coding region can comprise that promoter sequence, enhancer sequence are or/and translation enhancement sequences; Described promotor can be composition promotor, inducible promoter, tissue or organ specific promoters; 3 described ' non-coding region can comprise terminator sequence, mRNA cutting sequence etc.Suitable terminator sequence can be taken from the Ti-plasmid of agrobacterium tumefaciens, for example octopine synthetic enzyme and rouge alkali synthetase terminator.
In addition, those skilled in the art can be optimized to strengthen the expression efficiency in plant by the Nucleotide shown in SEQ ID No.4.For example, can adopt the preference codon of target plant to be optimized synthetic polyribonucleotides to strengthen the expression efficiency in target plant.
Described recombinant plant expression vector also can be containing being useful on the selected marker who selects transformant.Selected marker is for selecting the cell or tissue through transforming.Marker gene comprises: the gene of coding antibiotics resistance and the gene etc. of giving herbicidal compound resistance.In addition, described marker gene also comprises phenotypic markers, such as beta-galactosidase enzymes and fluorescin etc.
The invention still further relates to described cotton GhMYC4 transcription factor encoding gene is incorporated in plant to improve the resistance of plant to environment stress.
The present invention further discloses described cotton GhMYC4 transcription factor encoding gene and improving plant to the application in environment stress resistance, comprise the following steps: (1) builds the recombinant plant expression vector that contains described cotton GhMYC4 transcription factor encoding gene; (2) constructed recombinant plant expression vector is transformed in plant or vegetable cell; (3) cultivate screening and obtain the transgenic plant new variety that environment stress resistance is improved.
The present invention further discloses a kind of method of cultivating the transgenic plant new variety of resistance to environment stress, comprise the following steps: (1) builds the recombinant plant expression vector that contains described cotton GhMYC4 transcription factor encoding gene; (2) constructed recombinant plant expression vector is transformed in plant or vegetable cell; (3) cultivate screening and obtain the transgenic plant new variety that environment stress resistance is improved.
Wherein, described environment stress comprises the various abiotic stress such as high salt or arid.
Conversion scheme and by the visual type for the plant (monocotyledons or dicotyledons) that transforms or vegetable cell of the scheme of described polynucleotide or polypeptide introduced plant and change.The appropriate method of described polynucleotide or polypeptide being introduced to vegetable cell comprises: microinjection, electroporation, agriculture bacillus mediated conversion, direct gene transfer and bombardment of high speed trajectory etc.In specific embodiment, can utilize multiple instantaneous conversion method that GhMYC4 transcription factor gene of the present invention is offered to plant.In other embodiments, GhMYC4 transcription factor gene of the present invention can be by contacting to be incorporated in plant by plant with virus or viral nucleic acid, conventionally, such method relates in GhMYC4 transcription factor gene construct introducing viral DNA of the present invention or RNA molecule.
Utilize ordinary method can make the cell regeneration stable conversion plant (McCormick et al.Plant Cell Reports.1986.5:81-84) having transformed.The present invention can be used for transforming any floristics, includes but not limited to: monocotyledons or dicotyledons.Preferred, described target plant comprises farm crop, vegetables or ornamental plant, fruit tree etc., for example, can be corn, paddy rice, Chinese sorghum, wheat, soybean, potato, barley, tomato, Kidney bean, peanut or sugarcane etc.
In order to analyze the present invention from the function of the separated GhMYC4 transcription factor of cotton (Gossypium hirsutum L.), the present invention builds the plant expression vector that contains GhMYC4 gene (Nucleotide is shown in SEQ ID No.4) by Gateway method, inflorescence dip method arabidopsis thaliana transformation, obtains T1 for seed; By Arabidopis thaliana seed resistance, screen and PCR identifies, obtain transgenic arabidopsis.Build respectively the plant expression vector that contains GhMYC1 (Nucleotide is shown in SEQ ID No.1), GhMYC2 (Nucleotide is shown in SEQ ID No.2) and GhMYC3 (Nucleotide is shown in SEQ ID No.3) gene simultaneously, arabidopsis thaliana transformation, obtains transgenic arabidopsis.
Transgenic arabidopsis is carried out to drought resistance and salt tolerance analysis.Phenotype analytical result shows, the Arabidopis thaliana that turns GhMYC4 gene waters and drenches after 10 days at NaCl, on-bladed yellowing phenomenon, and Florescence and seed set situation is all better than NK control group, shows that the Arabidopis thaliana that turns GhMYC4 gene has good resistance to salt stress; After arid is processed, the Arabidopis thaliana plant strain growth that turns GhMYC4 gene is normal but blade is little, and plant strain growth is higher, has certain drought resisting effect; Physical signs analysis shows, after NaCl processes, the arabidopsis ' chlorophyll content and the NK that turn GhMYC4 gene remain basically stable, and between 1mg/g-1.4mg/g, fluctuates, and genus normal level, illustrates that the photosynthesis of the Arabidopis thaliana that turns GhMYC4 gene is normal; The content of propylene glycol that turns GhMYC4 gene Arabidopis thaliana is starkly lower than contrast, and the saline-alkaline tolerance of Arabidopis thaliana that shows to turn GhMYC4 gene is stronger; After arid is processed, the arabidopsis ' chlorophyll level that turns GhMYC4 gene remains on 1.21mg/g left and right substantially, and chlorophyll level is normal, illustrates that to turn the photosynthesis of GhMYC4 gene plant normal; The Arabidopis thaliana content of propylene glycol that turns GhMYC4 gene is lower, near 0.013 μ mol/g, proves drought resisting successful; Turn the soluble sugar average content of GhMYC4 gene Arabidopis thaliana higher than control group, illustrate that turning GhMYC4 gene plant improves its drought resisting level by rising soluble sugar content.
By transformation of Arabidopsis thaliana functional experiment, prove, in plant, excessively express cotton GhMYC4 transcription factor encoding gene and can effectively improve the resistance of plant to abiotic stress such as high salt or arids.Therefore, the present invention clone's cotton GhMYC4 transcription factor and encoding gene thereof have wide practical use to environment stress resistance and the aspects such as transgenic plant new variety of cultivating resistance to environment stress at regulating plant.
the term definition arriving involved in the present invention
Unless otherwise defined, otherwise all technology used herein and scientific terminology all have with those skilled in the art and conventionally understand identical implication.
Term " transcription factor " mean can with cis-acting elements specific binding in eukaryotic gene promoter region, thereby activate or suppress downstream gene and transcribe and a class DBP of expressing at specified time and space.
Term " polynucleotide " or " Nucleotide " mean deoxyribonucleotide, dezyribonucleoside, ribonucleoside or ribonucleotide and the polymkeric substance thereof of sub-thread or bifilar form.Unless specific limited, otherwise the nucleic acid of the known analogue that contains natural nucleotide contained in described term, described analogue has and is similar to the binding characteristic of reference nucleic acid and carries out metabolism to be similar to the mode of the Nucleotide of natural generation.Unless other specific limited, otherwise described term also means oligonucleotide analogs, it comprises PNA (peptide nucleic acid(PNA)), DNA analogue (thiophosphatephosphorothioate, phosphamide acid esters etc.) used in antisense technology.Unless otherwise, otherwise specific nucleic acid sequence is also impliedly contained its conservative varient of modifying (including, but is not limited to degenerate codon replaces) and complementary sequence and the clear and definite sequence of appointment.Specific, can through mixing sequence that base and/or Hypoxanthine deoxyriboside residue replace, realize degenerate codon and replace (people such as Batzer, Nucleic Acid Res.19:5081 (1991) by producing one of them or above selected (or all) codon the 3rd; The people such as Ohtsuka, J.Biol.Chem.260:2605-2608 (1985); With the people such as Cassol, (1992); The people such as Rossolini, Mol Cell.Probes 8:91-98 (1994)).
Term " polypeptide ", " peptide " and " albumen " exchange in this article and use to mean the polymkeric substance of amino-acid residue.That is, for the description of polypeptide, be equally applicable to describe peptide and describe albumen, and vice versa.Described term is applicable to natural generation aminoacid polymers and one of them or an aminoacid polymers that above amino-acid residue is non-naturally encoded amino acids.As used herein, the amino acid chain of any length contained in described term, and it comprises full-length proteins (being antigen), and wherein amino-acid residue connects via covalency peptide bond.
Predicate " rigorous hybridization conditions " means low ionic strength known in affiliated field and the condition of high temperature.Conventionally, under rigorous condition, probe and the hybridization of its target sequence can detection level than with other sequence hybridization can detection level higher (for example surpassing at least 2 times of backgrounds).Rigorous hybridization conditions is sequence dependent, will be different under different envrionment conditionss, and longer sequence is specific hybrid under comparatively high temps.The preciseness of hybridizing by control or wash conditions can be identified the target sequence with probe 100% complementation.Detailed guidance for nucleic acid hybridization can be with reference to related documents (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 specifically, described rigorous condition is selected as conventionally lower than distinguished sequence the about 5-10 ℃ of heat fusion joint (Tm) under regulation ionic strength pH.Tm be under equilibrium state 50% with the probe hybridization of target complementation residing temperature (under specify ion intensity, pH and nucleic acid concentration) during to target sequence (because of the excessive existence of target sequence, thus under Tm under equilibrium state 50% probe be occupied).Rigorous condition can be following condition: wherein in 7.0 to 8.3 times salt concn of pH lower than about 1.0M Na ion concentration, be generally approximately 0.01 to 1.0M Na ion concentration (or other salt), and temperature is at least about 30 ℃ for short probe (including, but is not limited to 10 to 50 Nucleotide), and is at least about 60 ℃ for long probe (including, but is not limited to be greater than 50 Nucleotide).Rigorous condition also can realize by adding such as the destabilizing agent of methane amide.For selectivity or specific hybrid, positive signal can be at least background hybridization of twice, is optionally 10 times of background hybridizations.Exemplary rigorous hybridization conditions can be as follows: 50% methane amide, and 5 * SSC and 1%SDS cultivate at 42 ℃; Or 5 * SSC, 1%SDS cultivates at 65 ℃, washs and in 0.1%SDS, wash at 65 ℃ in 0.2 * SSC.Described washing can be carried out 5,15,30,60,120 minutes or the longer time.
Term " recombinant host cell strain " or " host cell " mean the cell that comprises polynucleotide of the present invention, and no matter use which kind of method to insert to produce recombinant host cell, for example, directly absorb, other known method in transduction, f pairing or affiliated field.Exogenous polynucleotide can remain the nonconformity carrier of plasmid for example or can be integrated in host genome.Host cell can be prokaryotic cell prokaryocyte or eukaryotic cell, and host cell also can be unifacial leaf or dicotyledons cell.
Term " conversion " means heterology DNA sequence dna to be incorporated into host cell or organic method.
Term " expression " means endogenous gene or transgenosis transcribing and/or translating in vegetable cell.
Term " encoding sequence " means to be transcribed into the nucleotide sequence of RNA.
Term " recombinant plant expression vector " means one or more for realizing the DNA vector of Plant Transformation; In this area, these carriers are often called as binary vector.Binary vector is to be mostly usually used in agrobacterium-mediated conversion together with the carrier with helper plasmid.Binary vector generally includes: T-DNA shifts needed cis acting sequence, through through engineering approaches, processes so that the selectable marker that can express heterology DNA sequence dna to be transcribed etc. in vegetable cell.
Accompanying drawing explanation
Fig. 1 for cloning GhMYC1 and GhMYC2 electrophorogram from cotton gene group and cDNA; Wherein, M is DNA Marker; 1,2 is the PCR result of cotton gene group to MYC1 total length primer; 3 is the PCR result of cotton cDNA to MYC1 total length primer; 4,5 is the PCR result of cotton gene group to MYC2 total length primer; 6 is the PCR result of cotton cDNA to MYC2 total length primer;
Fig. 2 for cloning GhMYC3 and GhMYC4 electrophorogram from cotton gene group and cDNA; Wherein, 1,2 is the PCR result of cotton cDNA to MYC3 total length primer; 3 is the PCR result of cotton gene group to MYC3 total length primer; 4,5,6 is the PCR result of cotton gene group to MYC4 total length primer; 7,8,9 is the PCR result of cotton cDNA 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; + positive contrast;-be 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 the schema that Gateway method builds the plant expression vector that contains GhMYC1, GhMYC2, GhMYC3 or GhMYC4 gene;
Fig. 8 is the bacterium liquid PCR detection figure that the entry clones of GhMYC1, GhMYC2, GhMYC3 and GhMYC4 gene transforms coli strain DH5 α; Wherein, M is DNA Marker; 1-3 is the PCR detected result that the entry clones of GhMYC1 transforms No. 3 bacterium colonies of bacillus coli DH 5 alpha; 2-3 and 2-4 are the PCR detected result that the entry clones of GhMYC2 transforms No. 3 and No. 4 bacterium colonies of bacillus coli DH 5 alpha; 3-2,3-3 and 3-4 are the PCR detected result that the entry clones of GhMYC3 transforms 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 are the PCR detected result that the entry clones of GhMYC4 transforms No. 1, No. 2, No. 3, No. 4 and No. 5 bacterium colony of bacillus coli DH 5 alpha; + positive contrast;
Fig. 9 is the bacterium liquid PCR detection figure that the plant expression vector of GhMYC1, GhMYC2, GhMYC3 and GhMYC4 gene transforms coli strain DH5 α; Wherein, M is DNA Marker; 1-1,1-2 and 1-3 are the PCR detected result that the plant expression vector of GhMYC1 transforms No. 1, No. 2 and No. 3 bacterium colony of bacillus coli DH 5 alpha; 2-1,2-1,2-3 and 2-4 are the PCR detected result that the plant expression vector of GhMYC2 transforms No. 1, No. 2, No. 3 and No. 4 bacterium colony of bacillus coli DH 5 alpha; 3-1,3-2 and 3-3 are the PCR detected result that the plant expression vector of GhMYC3 transforms No. 1, No. 2 and No. 3 bacterium colony of bacillus coli DH 5 alpha; 4-1,4-2,4-3 and 4-4 are the PCR detected result that the plant expression vector of GhMYC4 transforms No. 1, No. 2, No. 3 and No. 4 bacterium colony of bacillus coli DH 5 alpha; + positive contrast;-be blank;
Figure 10 is the bacterium liquid PCR detection figure that the plant expression vector of GhMYC1, GhMYC2, GhMYC3 and GhMYC4 gene transforms Agrobacterium GV3101; Wherein, M is DNA Marker; 1-1,1-2,1-4 and 1-5 are the PCR detected result that the plant expression vector of GhMYC1 transforms No. 1, No. 2, No. 4 and No. 5 bacterium colony of Agrobacterium GV3101; 2-1,2-3,2-4 and 2-5 are the PCR detected result that the plant expression vector of GhMYC2 transforms No. 1, No. 3, No. 4 and No. 5 bacterium colony of Agrobacterium GV3101; 3-1,3-2,3-3 and 3-4 are the PCR detected result that the plant expression vector of GhMYC3 transforms No. 1, No. 2, No. 3 and No. 4 bacterium colony of Agrobacterium GV3101; 4-2 and 4-5 are the PCR detected result that the plant expression vector of GhMYC4 transforms No. 2 and No. 5 bacterium colonies of Agrobacterium GV3101; + positive contrast;
The PCR of Figure 11 transgenic arabidopsis identifies figure; Wherein, M is DNA Marker; 1-1-1-6 is the detected result that turns 6 strain Arabidopis thalianas of GhMYC1 gene; 3-1-3-2 is the detected result that turns 2 strain Arabidopis thalianas of GhMYC3 gene; 4-1-4-12 is the detected result that turns 12 strain Arabidopis thalianas of GhMYC4 gene;
Figure 12 is the histogram of the transgenic arabidopsis Chlorophyll content after 200mmol/L NaCl processes; Wherein NK is for contrasting not transfer-gen plant; GhMYC1-1 and GhMYC1-2 are that the Arabidopis thaliana that turns GhMYC1 gene is planted strain No.1 and No. 2; GhMYC2-1 and GhMYC2-2 are that the Arabidopis thaliana that turns GhMYC2 gene is planted strain No.1 and No. 2; GhMYC3-1 and GhMYC3-2 are that the Arabidopis thaliana that turns GhMYC3 gene is planted strain No.1 and No. 2; GhMYC4-1 and GhMYC4-2 are that the Arabidopis thaliana that turns GhMYC4 gene is planted strain No.1 and No. 2;
Figure 13 is the histogram of the transgenic arabidopsis Chlorophyll content after arid is processed; Wherein NK is for contrasting not transfer-gen plant; GhMYC1-1 and GhMYC1-2 are that the Arabidopis thaliana that turns GhMYC1 gene is planted strain No.1 and No. 2; GhMYC3-1 and GhMYC3-2 are that the Arabidopis thaliana that turns GhMYC3 gene is planted strain No.1 and No. 2; GhMYC4-1 and GhMYC4-2 are that the Arabidopis thaliana that turns GhMYC4 gene is planted strain No.1 and No. 2;
Figure 14 is the content of propylene glycol histogram of the transgenic arabidopsis after 200mmol/L NaCl processes; Wherein NK is for contrasting not transfer-gen plant; GhMYC1-1 and GhMYC1-2 are that the Arabidopis thaliana that turns GhMYC1 gene is planted strain No.1 and No. 2; GhMYC2-1 and GhMYC2-2 are that the Arabidopis thaliana that turns GhMYC2 gene is planted strain No.1 and No. 2; GhMYC3-1 and GhMYC3-2 are that the Arabidopis thaliana that turns GhMYC3 gene is planted strain No.1 and No. 2; GhMYC4-1 and GhMYC4-2 are that the Arabidopis thaliana that turns GhMYC4 gene is planted strain No.1 and No. 2;
Figure 15 is the content of propylene glycol histogram of the transgenic arabidopsis after arid is processed; Wherein NK is for contrasting not transfer-gen plant; GhMYC1-1 and GhMYC1-2 are that the Arabidopis thaliana that turns GhMYC1 gene is planted strain No.1 and No. 2; GhMYC2-1 and GhMYC2-2 are that the Arabidopis thaliana that turns GhMYC2 gene is planted strain No.1 and No. 2; GhMYC3-1 and GhMYC3-2 are that the Arabidopis thaliana that turns GhMYC3 gene is planted strain No.1 and No. 2; GhMYC4-1 and GhMYC4-2 are that the Arabidopis thaliana that turns GhMYC4 gene is planted strain No.1 and No. 2;
Figure 16 is the histogram of the soluble sugar content of the transgenic arabidopsis after 200mmol/L NaCl processes; Wherein NK is for contrasting not transfer-gen plant; GhMYC1-1 and GhMYC1-2 are that the Arabidopis thaliana that turns GhMYC1 gene is planted strain No.1 and No. 2; GhMYC2-1 and GhMYC2-2 are that the Arabidopis thaliana that turns GhMYC2 gene is planted strain No.1 and No. 2; GhMYC3-1 and GhMYC3-2 are that the Arabidopis thaliana that turns GhMYC3 gene is planted strain No.1 and No. 2; GhMYC4-1 and GhMYC4-2 are that the Arabidopis thaliana that turns GhMYC4 gene is planted strain No.1 and No. 2;
Figure 17 is the soluble sugar content histogram of the transgenic arabidopsis after arid is processed; Wherein NK is for contrasting not transfer-gen plant; GhMYC1-1 and GhMYC1-2 are that the Arabidopis thaliana that turns GhMYC1 gene is planted strain No.1 and No. 2; GhMYC2-1 and GhMYC2-2 are that the Arabidopis thaliana that turns GhMYC2 gene is planted strain No.1 and No. 2; GhMYC3-1 and GhMYC3-2 are that the Arabidopis thaliana that turns GhMYC3 gene is planted strain No.1 and No. 2; GhMYC4-1 and GhMYC4-2 are that the Arabidopis thaliana that turns GhMYC4 gene is planted strain No.1 and No. 2.
Embodiment
Below in conjunction with specific embodiment, further describe the present invention, advantage and disadvantage of the present invention will be more clear along with description.It should be understood that described embodiment is only exemplary, does not form any restriction to scope of the present invention.It will be understood by those skilled in the art that lower without departing from the spirit and scope of the present invention and can the details of technical solution of the present invention and form be modified or be replaced, but these modifications or replacement all fall into protection scope of the present invention.
1, experiment material
The blade of cotton silver cotton variety, silver-colored cotton variety is bought vegetables fully stocked wood flowers market in Beijing; Arabidopis thaliana seed is Colombia's type, and preserve in inventor laboratory; PMD-19T cloning vector, Taq enzyme are purchased from Quan Shi King Company; Bacillus coli DH 5 alpha and high pure plasmid are prepared test kit in a small amount all purchased from Beijing hundred Tyke Bioisystech Co., Ltd; Agrobacterium strains GV3101 is for preserving in inventor laboratory;
PDONR
tM207 carriers are purchased from tall and handsome Jetta company; Expression vector pH7WG2D is purchased from VIB (Belgium) company; Gateway clone's BP reaction kit and LR reaction kit are 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 test kit is purchased from Fermentas company; Primer synthesizes and order-checking is completed by the prosperous Bioisystech Co., Ltd of Beijing AudioCodes.
The clone of embodiment 1 cotton MYC class transcription factor encoding gene
1, experimental technique
1.1 electronic clonings obtain MYC class transcription factor
According to there being the characteristic of conservative similar sequences in homologous gene, by the sequence of Arabidopis thaliana MYC class transcription factor gene, it is information probes, est database cotton in NCBI (Gossypium hirsutum) is carried out to Tblastx homology search, utilize DNAStar software that the est sequence splicing that comes from cotton retrieving is obtained to linker; Then with this linker, again carry out the homology retrieval and splicing of Tblastx, repeat above process until non-overlapping est sequence, finally obtain the cDNA sequence fragment of 4 GhMYC genes of upland cotton.
1.2RT-PCR method obtains goal gene
The cDNA sequences Design RT-PCR primer (table 1) of 4 the GhMYC genes of total length that obtain according to electronic cloning.
Table 1 GhMYC clone total length primer
Utilize Trizol-isopropanol method to extract the cotton total RNA of silver, according to the explanation of test kit, operate.Extract after the total RNA of cotton, take it as template, Oligo-dT (5pmol/ μ L) is primer 1 μ L, mixes latter 65 ℃ and hatches 5min, is put on ice, add 5 * Reaction Buffer, 4 μ L, RNasin Inhibitor 1 μ L, M-MLVRT 1 μ L and dNTP Mix 2 μ L, and gentleness mix rear wink from, hatch 60min for 42 ℃, 70 ℃ of 5min termination reactions, be synthetic cDNA the first chain of reverse transcription ,-20 ℃ save backup.
By after reverse transcription product Preliminary detection, get 1 μ L and by following system and condition, carry out pcr amplification as template.
RT-PCR reaction system is: be totally 20 μ L, wherein containing 10 * Buffer is 2 μ L, and 2mmol/L dNTP is 1 μ L, and dilution is each 1 μ L of primer of 10 μ mol/L, and Taq enzyme 0.5U (reaction system of general 20 μ L adds 1 μ L), adds ddH
2o to 20 μ L.
The response procedures of RT-PCR is: 94 ℃ of denaturation 5min; 94 ℃ of sex change 30s, annealing 45s, 72 ℃ are extended 60s, and circulate 29 times; 72 ℃ are extended 10min.
Amplified production, through 1% agarose gel electrophoresis, reclaims and is cloned into pMD-19T cloning vector by the big or small fragment of expection, then transforms coli strain DH5 α.Picking positive bacteria carries out bacterium liquid PCR order-checking.
In addition, utilize CTAB method to extract cotton genomic dna, utilize the primer of table 1 to clone 4 GhMYC transcription factor genes from cotton genomic dna.
2, experimental result
According to four GhMYC DNA sequence dna design primers of cotton of electronic cloning, by RT-PCR, react respectively and go out four GhMYC transcription factor genes (Fig. 1, Fig. 2) from cDNA or genomic dna cloning, 4 MYC class transcription factor genes that obtain are named respectively: GhMYC1, GhMYC2, GhMYC3 and GhMYC4, its nucleotide sequence is respectively shown in SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4.Wherein, the aminoacid sequence of the derivation of GhMYC4 gene is shown in SEQ ID No.5.Four genes finding GhMYC transcription factor from Fig. 1, Fig. 2 do not contain intron the cotton genome of silver.
The PCR product of clone's GhMYC1, GhMYC2, GhMYC3 and GhMYC4 gene is connected with pMD-19T carrier respectively, transforms bacillus coli DH 5 alpha, carry out positive bacteria liquid PCR check.
To turning the DH5 α resistance bacterium of GhMYC1 gene, carry out the detection of PCR bacterium colony, find 11 and No. 13 bacterium colonies positive (Fig. 3).
To turning the DH5 α resistance bacterium of GhMYC2 gene, carry out the detection of PCR bacterium colony, find No. 3 bacterium colonies positive (Fig. 4).
To turning the DH5 α resistance bacterium of GhMYC3 gene, carry out the detection of PCR bacterium colony, 18 bacterium colonies positive (Fig. 5) such as find No. 2, No. 3.
To turning the DH5 α resistance bacterium of GhMYC4 gene, carry out the positive bacterium colony detection of PCR, find No. 9, No. 14 bacterium colonies positive (Fig. 6).
Plant expression vector construction and arabidopsis thaliana transformation that embodiment 2 contains MYC class transcription factor encoding gene
1, experimental technique
1.1Gateway method builds plant expression vector
According to the primer sequence design requirements of Gateway clone technology and embodiment 1 clone's GhMYC1, GhMYC2, GhMYC3 and GhMYC4 gene order design Gateway reaction primer (table 2).
Table 2 Gateway design of primers
Adopt archaeal dna polymerase, with the upstream and downstream primer of table 2, carry out PCR reaction, the pMD-19T cloning vector plasmids that connects respectively GhMYC1, GhMYC2, GhMYC3 and the GhMYC4 gene 1 μ L that wherein contains embodiment 1 preparation, 10 * Buffer is 2 μ L, 2mmol/L dNTP is 1 μ L, dilution is each 1 μ L of primer of 10 μ mol/L, and Taq enzyme 0.5U (reaction system of general 20 μ L adds 1 μ L), adds ddH
2o to 20 μ L.Upstream and downstream at GhMYC1, GhMYC2, GhMYC3 and GhMYC4 full-length gene adds attB sequence.Reaction conditions is as follows: 94 ℃ of denaturation 5min, and 94 ℃ of sex change 30s, annealing 45s, 75 ℃ are extended 1min, totally 30 circulations; 75 ℃ are extended 10min, 4 ℃ of termination reactions.With agarose gel electrophoresis detection reaction product, and purifying reclaims.
BP reaction: the object of BP reaction is that the PCR product cloning that contains attB joint that amplification is obtained is to the donor carrier pDONR that contains attP
tMon 207, to produce entry clones.According to BP reaction system in the Gateway BP Clonase II enzyme mix test kit of invitrogen company, comprise PCR product 15-150ng, pDONR
tM207 carrier 1 μ L (150ng/ μ L), supplement TE damping fluid (pH8.0) to 8 μ L, and whirlpool concussion enzyme system mixed solution twice fast, adds 1 * BP ClonaseTM enzyme mixture, 2 μ L.Reaction system adds 2 μ L Proteinase K liquid after putting 25 ℃ of incubation 60min, and 37 ℃ of incubation 10min, to stop BP reaction.Obtain entry clones pDONR
tM207-GhMYC1, pDONR
tM207-GhMYC2, pDONR
tM207-GhMYC3 and pDONR
tM207-GhMYC4.
The conversion of BP reaction product and the evaluation of positive colony: will by BP, react the entry clones pDONR obtaining
tM207-GhMYC1, pDONR
tM207-GhMYC2, pDONR
tM207-GhMYC3 and pDONR
tM207-GhMYC4 transforms coli strain DH5 α, and is applied in spectinomycin LB resistant panel, and picking resistance bacterium colony, carries out PCR detection, and the bacterium colony of test positive extracts plasmid, obtains entry clones carrier.
LR reaction: the object of LR reaction is to be recombined into entry vector pDONR
tM207-GhMYC1, pDONR
tM207-GhMYC2, pDONR
tM207-GhMYC3 and pDONR
tMtarget gene GhMYC1, GhMYC2, GhMYC3 and the GhMYC4 of 207-GhMYC4 are cloned in expression vector pH7WG2D again.LR reaction system comprises introduction cloned plasmids 15-150ng, pH7WG2D carrier 1 μ L (150ng/ μ L), supplement TE damping fluid (pH8.0) to 8 μ L, whirlpool concussion enzyme system mixed solution twice fast, adds 1 * LR ClonaseTM enzyme mixture, 2 μ L.Reaction system adds 2 μ L Proteinase K solution after putting 25 ℃ of incubation 60min, 37 ℃ of incubation 10min, to stop LR reaction, obtain final expression vector pH7WG2D-GhMYC1, pH7WG2D-GhMYC2, pH7WG2D-GhMYC3 and pH7WG2D-GhMYC4 (build schema and see Fig. 7).
The conversion of LR reaction product and the evaluation of positive colony: expression vector pH7WG2D-GhMYC1, the pH7WG2D-GhMYC2 of LR reaction product, pH7WG2D-GhMYC3 and pH7WG2D-GhMYC4 are transformed to coli strain DH5 α, and be applied in kantlex LB resistant panel, the positive bacterium colony of picking, carry out PCR detection, to determine positive colony.
1.2 inflorescence dip method arabidopsis thaliana transformations
1.2.1 the cultivation of Arabidopis thaliana and processing
By be placed in 4 ℃ of refrigerator vernalization the Arabidopis thaliana seed of couple of days in growth pot, water and epiphragma to keep moisture.When Arabidopis thaliana is bloomed for the first time, for promoting side shoot more to spend more the hyperplasia of branch, the bud on stem top is cut.The flowers that are applicable to transformed plant are unripe, also there is no to produce the angle fruit (approximately growth time is about 30 days) being fertilized.
Before turning Arabidopis thaliana with inflorescence infestation method, cut off the angle fruit having grown up to, in order to avoid reduce positive rate.Infect last evening and water, keep soil humidity, in infection processs, soil is difficult for dropping out.
1.2.2 inflorescence dip method arabidopsis thaliana transformation
Plant expression vector pH7WG2D-GhMYC1, pH7WG2D-GhMYC2, pH7WG2D-GhMYC3 and pH7WG2D-GhMYC4 that Gateway method is built transform respectively Agrobacterium GV3101, and picking positive bacteria is dropped into performing PCR and detected.Positive strain is inoculated into 250ml triangular flask (add in the ratio of 1:1000 in the YEB liquid nutrient medium that 200ml is housed, add microbiotic kantlex and Rifampin), shakes in a large number bacterium 1 day until OD value is 1.2.5000rpm centrifugal enrichment Agrobacterium, then suspends (in sucrose solution, add 0.1% tensio-active agent SilwetL-77) with sucrose+1/2MS of 5 ﹪, in a large number until OD value is about 0.8.The inflorescence of Arabidopis thaliana is immersed to bacterium liquid to be infected 1 minute; Arabidopis thaliana after infecting covers with black plastic bag or film, secretly cultivates 24 hours.Within 7 days after infecting for the first time, carry out secondary infection, infect method with for the first time.Treat that Arabidopis thaliana plant cuts one's eye-teeth, after the fruit natural cracking of angle, gather in the crops seed, be T1 for seed.
1.2.3 the resistance screening of Arabidopis thaliana seed
T1 is evenly sowed in the resistant panel (1/4MS substratum) containing 50mg/L Totomycin or that penicillin of 100mg/L card for seed, put after 4 ℃ of refrigerator deepfreeze 72h, move to 25 ℃, in 16h illumination box, germinate, routine observation is also investigated seed germination and growth of seedling situation.Grow about 7-10 days, the seedling that grows two true leaves is resistance seedling, by resistance seedling replanting in Nutrition Soil.Wait growing up to when ripe, the genome that extracts transfer-gen plant carries out PCR evaluation.
2, experimental result
2.1 plant expression vector construction
Entry clones pDONR
tM207-GhMYC1, pDONR
tM207-GhMYC2, pDONR
tM207-GhMYC3 and pDONR
tM207-GhMYC4 transforms the bacterium liquid PCR detected result of coli strain DH5 α and sees Fig. 8.
Plant expression vector pH7WG2D-GhMYC1, pH7WG2D-GhMYC2, pH7WG2D-GhMYC3 and pH7WG2D-GhMYC4 transform the bacterium liquid PCR detected result of coli strain DH5 α and see Fig. 9.The plant expression vector construction success of GhMYC1, GhMYC2, GhMYC3 and GhMYC4 gene is described.
2.2 plant expression vector arabidopsis thaliana transformations
The positive DH5 α bacterium colony that amplification contains plant expression vector pH7WG2D-GhMYC1, pH7WG2D-GhMYC2, pH7WG2D-GhMYC3 and pH7WG2D-GhMYC4 also extracts plasmid, is transformed into (Figure 10) in Agrobacterium GV3101.
By inflorescence dip method arabidopsis thaliana transformation, the Arabidopis thaliana T1 that acquisition turns GhMYC1, GhMYC2, GhMYC3 and GhMYC4 gene is respectively for seed.
The resistance screening of 2.3 Arabidopis thalianas
Extract positive seedling genome and carry out PCR evaluation (Figure 11).Presentation of results, successfully obtains the Arabidopis thaliana that turns respectively GhMYC1, GhMYC2, GhMYC3 and GhMYC4 gene.
Embodiment 3 turns the drought resistance and salt tolerance analysis of cotton MYC class transcription factor encoding gene Arabidopis thaliana
1, experimental technique
The Stress treatment of 1.1 transgenic positive strains
1.1.1 salt stress is processed
The transgenic arabidopsis plant of potted plant about 4 weeks obtaining with embodiment 2 is watered the NaCl that drenches 200mmol/L as research object, when salt is processed 10d, sampling detects, and with wild-type Arabidopis thaliana, compares.Observe the impact of salt stress on transgenic arabidopsis phenotype.
1.1.2 drought stress is processed
With the transgenic arabidopsis plant of potted plant about 4 weeks that embodiment 2 obtains, as research object, carry out drought stress processing, in 10 days, do not water, when arid is processed 10d, sampling detects, and with wild-type Arabidopis thaliana, compares.Observe the impact of drought stress on transgenic arabidopsis phenotype.
1.2 physical signs detect
1.2.1 the mensuration of chlorophyll content
The fresh leaf of Arabidopis thaliana of getting after Stress treatment is cleaned, shred (removing middle arteries and veins), mix, take and shred fresh sample 0.05g and put into mortar, add quartz sand and spoonful Paris white and 95% the ethanol 2-3ml of half spoon, grind to form homogenate, adding 95% ethanol 4ml continues to be ground to tissue and turns white again, in the dark static 3-5 minute, then enters in 10ml volumetric flask with filter paper filtering, then with 95% ethanol, drips and washes mortar, pestle and filter paper, until redgreen, finally be settled to scale, shake up, obtain chlorophyll extracting solution; With spectrophotometer, measure respectively absorbancy (A) value under 646nm and 663nm wavelength.
1.2.2 the mensuration of propylene glycol (MDA) and soluble sugar
The Arabidopis thaliana plant 0.1g that takes Stress treatment, shreds, powdered by liquid nitrogen grinding; The 5%TCA that adds 1.1ml, whirlpool concussion 2min, the centrifugal 10min of 13000r/min, supernatant liquor is sample extracting solution.Draw centrifugal supernatant liquor 1ml, add the 0.6%TBA solution of 1.1ml, shake up.Test tube is put into boiling water and from solution in vitro, occur that small bubbles start timing and boil 10min, take out test tube cooling, the centrifugal 10min of 13000r/min, gets supernatant liquor 2ml.Take 0.6%TBA solution as blank, measure the absorbance (A) at 532nm, 600nm, 450nm place.2, experimental result
The impact of 2.1 salt stresses on transgenic arabidopsis phenotype
Result shows, the Arabidopis thaliana that turns respectively GhMYC1, GhMYC4 gene waters and drenches after 10 days at NaCl, on-bladed yellowing phenomenon, Florescence and seed set situation is all better than NK control group, there is yellow leaf, wilting and withered phenomenon in control group Arabidopsis leaf, stem's blackout, flower and solid all poor compared with transgenosis group; There is the phenomenon that partial blade is withered in the Arabidopis thaliana that turns GhMYC2 gene, and control group leaf growth is less, also has the withered phenomenon of partial blade; Turn the Arabidopis thaliana of GhMYC3 gene, grow obviously slow, and there is the phenomenon of jaundice in blade.
To sum up, phenotype, can find out, the Arabidopis thaliana that turns respectively GhMYC1, GhMYC4 gene has good effect to the resistance of salt stress.
The impact of 2.2 arids on transgenic arabidopsis phenotype
Result shows, in arid, processes after 10 days, and the plant that turns GhMYC2 gene has the withered phenomenon of obvious plant with respect to control group, and blade is whole to dry up but has a small amount of knot of blooming; The Arabidopis thaliana plant strain growth that turns respectively GhMYC1 and GhMYC4 gene is normal but blade is little, and plant strain growth is higher, has certain drought resisting effect; Turn the Arabidopis thaliana drought resisting successful of GhMYC3 gene, there is no the phenomenon of obvious blade blackout, plant strain growth is vigorous, but growth phase is to short and small.With respect to transfer-gen plant, NK plant leaf has blackout phenomenon, and plant strain growth is higher.
2.3 physical signs detect
2.3.1 the mensuration of Chlorophyll content
The mensuration of the Arabidopis thaliana that NaCl processes, arid is processed having been carried out to photosynthesis index chlorophyll content, the results are shown in Figure 12 and 13.
As seen from Figure 12, after the NaCl of 10 days 200mmol/L processes, the arabidopsis ' chlorophyll content and the NK that turn respectively GhMYC1, GhMYC3 and GhMYC4 gene remain basically stable, between 1mg/g-1.4mg/g, fluctuate, belong to normal level, the photosynthesis of explanation plant after 200mmol/L NaCl processes 10 days is normal, and chlorophyll content is consistent substantially.And the Chlorophyll content of plant that turns GhMYC2 gene is obviously low compared with other values, illustrates that plant growth condition is undesirable.
As seen from Figure 13, arid was processed after 10 days, and the total chlorophyll content of NK control group obviously raises compared with the value of transfer-gen plant; The Arabidopsis leaf that turns GhMYC2 gene is dead, therefore without chlorophyll content data.May be that a large amount of dehydrations of control group NK Arabidopis thaliana alleviate leaf weight due to after arid processing, therefore chlorophyll content raises.The arabidopsis ' chlorophyll level that turns respectively GhMYC1 and GhMYC4 gene remains between 0.7mg/g-1.21mg/g substantially, and chlorophyll level is normal, illustrates that the photosynthesis of transfer-gen plant is normal.And the arabidopsis ' chlorophyll total content value that turns GhMYC3 gene is on the low side, show that photosynthesis has been subject to certain impact.
2.3.2 the mensuration of content of propylene glycol
The mensuration of the Arabidopis thaliana that NaCl processes, arid is processed being carried out to content of propylene glycol, the results are shown in Figure 14 and 15.
Figure 14 can find out, the content of propylene glycol of transgenic arabidopsis is starkly lower than control group, illustrates that control group Arabidopis thaliana is subject to the impact of environment larger after 10 days NaCl process, and MDA content is apparently higher than normal level.Contrast turns respectively the Arabidopis thaliana of GhMYC1, GhMYC2, GhMYC3 and GhMYC4 gene, can find out the better resistance after the Arabidopis thaliana that turns GhMYC4 gene is processed NaCl; And the Arabidopis thaliana saline-alkaline tolerance that turns GhMYC3 gene relatively a little less than, MDA value is in 0.018 μ mol/g left and right.
Figure 15 can find out, arid is processed NK control group after 10 days compared with transfer-gen plant, has growth in content of propylene glycol, illustrate arid larger on the impact of NK plant.The Arabidopis thaliana content of propylene glycol that turns respectively GhMYC1, GhMYC3 and GhMYC4 gene is minimum, near 0.013 μ mol/g, proves drought resisting successful; The Arabidopis thaliana content of propylene glycol that turns GhMYC2 gene is the highest, is 0.020 μ mol/g, a little less than illustrating that drought resisting effect is omited compared with other.
2.3.3 the mensuration of soluble sugar content
The mensuration of the Arabidopis thaliana that NaCl processes, arid is processed being carried out to soluble sugar content, the results are shown in Figure 16 and 17.
As can be seen from Figure 16, control group and transgenosis group Arabidopis thaliana that 200mmol/L NaCl processes, soluble sugar content changes little, and the soluble sugar content of transgenosis group is substantially low compared with the value of control group.May be that the soluble sugar content of transgenic arabidopsis is on regulating the impact of stress resistance of plant shape little because under NaCl processes.The soluble sugar content of No. 2 plant that turns GhMYC3 gene is the highest, has surpassed NK level; The soluble sugar content of all the other transgenic arabidopsis is all low compared with NK value; No. 2 Arabidopis thaliana plant soluble sugar contents that turn GhMYC4 gene are minimum, are 0.077mmol/g.
As can be seen from Figure 17, arid was processed after 10 days, turn respectively the Arabidopis thaliana soluble sugar average content of GhMYC1, GhMYC2, GhMYC3 and GhMYC4 gene higher than NK control group (0.07mmol/g), illustrate that transfer-gen plant improves its drought resisting level by rising soluble sugar content.But except turning the soluble sugar average content of No. 1 plant of GhMYC3 gene, be 0.112mmol/g, all the other transfer-gen plant soluble sugar contents and NK control group are more not remarkable.
Claims (10)
1. from the separated GhMYC4 transcription factor encoding gene of cotton (Gossypium hirsutum L.), it is characterized in that, its polynucleotide be (a), (b), (c), (d) or (e) shown in:
(a), the polynucleotide shown in SEQ ID No.4; Or
(b), amino acid whose polynucleotide shown in coding SEQ ID No.5; Or
(c), with the polynucleotide that the complementary sequence of SEQ ID No.4 can be hybridized in rigorous hybridization conditions, the coded protein remains of these polynucleotide has GhMYC4 functional transcription factor; Or
(d), have 90% or the polynucleotide of above homology at least with the polynucleotide shown in SEQ ID No.4, preferably, have 95% or the polynucleotide of above homology at least with the polynucleotide shown in SEQ ID No.4, most preferred, have 98% or the polynucleotide of above homology at least with the polynucleotide shown in SEQ ID No.4; Or
(e), the polynucleotide variant that carries out disappearance, replacement or the insertion of one or more bases on the basis of the polynucleotide shown in SEQ ID No.4, and the coded albumen of this polynucleotide variant still has function or the activity of GhMYC4 transcription factor.
2. the GhMYC4 transcription factor of encoding gene coding described in claim 1, is characterized in that, its amino acid be (a) or (b) shown in:
(a), the amino acid shown in SEQ ID No.5;
(b), the replacement by one or more amino-acid residues by the amino acid shown in SEQ ID No.5, disappearance are or/and insert and derive the protein variant still with GhMYC4 functional transcription factor or activity obtaining.
3. the recombinant expression vector that contains encoding gene claimed in claim 1.
4. according to recombinant expression vector claimed in claim 3, it is characterized in that: described recombinant expression vector is recombinant plant expression vector.
5. the recombinant host cell that contains the recombinant expression vector described in claim 3 or 4.
6. encoding gene claimed in claim 1 is improving plant to the application in environment stress resistance.
7. according to application claimed in claim 6, it is characterized in that, comprise the following steps: (1) builds the recombinant plant expression vector that contains encoding gene claimed in claim 1; (2) constructed recombinant plant expression vector is transformed in plant or vegetable cell; (3) cultivate screening and obtain the transgenic plant new variety that environment stress resistance is improved.
8. cultivate a method for the transgenic plant new variety of resistance to environment stress, it is characterized in that, comprise the following steps: (1) builds the recombinant plant expression vector that contains encoding gene claimed in claim 1; (2) constructed recombinant plant expression vector is transformed in plant or vegetable cell; (3) cultivate screening and obtain the transgenic plant new variety that environment stress resistance is improved.
9. GhMYC4 transcription factor claimed in claim 2 is improving plant to the application in environment stress resistance.
10. according to the application described in claim 6,7 or 9, it is characterized in that: described environment stress comprises high salt or arid.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109837288A (en) * | 2019-03-18 | 2019-06-04 | 中国农业科学院植物保护研究所 | Wang grass MYC3 gene and its application |
| CN114410656A (en) * | 2021-06-03 | 2022-04-29 | 浙江农林大学 | Application of BrMYC3-2 gene overexpression in plant growth |
| CN114480430A (en) * | 2022-03-14 | 2022-05-13 | 浙江农林大学 | Dendrobium officinale MYC2b gene and application thereof in plant flowering delaying |
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-
2014
- 2014-08-08 CN CN201410390070.2A patent/CN104164440B/en active Active
Non-Patent Citations (3)
| Title |
|---|
| CHAO-MIN MENG, ET AL.: "Molecular Cloning and Characterization of a Novel Gossypium hirsutum L. bHLH Gene in Response to ABA and Drought Stresses", 《PLANT MOL BIOL REP》 * |
| MENG,C.M., ET AL.: "GenBank accession number: ACO53628.1", 《GENBANK》 * |
| 孙娜 等: "陆地棉GhbHLH4基因的克隆与功能分析", 《中国农业科技导报》 * |
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| CN114410656B (en) * | 2021-06-03 | 2023-12-01 | 浙江农林大学 | Application of BrMYC3-2 gene overexpression in plant growth |
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| CN114438098A (en) * | 2021-06-03 | 2022-05-06 | 浙江农林大学 | Application of BrMYC4-1 gene overexpression in plant growth |
| CN114410656A (en) * | 2021-06-03 | 2022-04-29 | 浙江农林大学 | Application of BrMYC3-2 gene overexpression in plant growth |
| CN114517200B (en) * | 2021-06-03 | 2023-06-23 | 浙江农林大学 | Use of BrMYC3-2 gene overexpression for increasing resistance of plants to fungal pathogens |
| CN114480430B (en) * | 2022-03-14 | 2023-09-22 | 浙江农林大学 | Dendrobium officinale MYC2b gene and application thereof in delaying flowering of plants |
| CN114480430A (en) * | 2022-03-14 | 2022-05-13 | 浙江农林大学 | Dendrobium officinale MYC2b gene and application thereof in plant flowering delaying |
| CN115807006A (en) * | 2022-09-22 | 2023-03-17 | 四川农业大学 | Application of gene fragment B in cultivation of new plant material |
| CN115807006B (en) * | 2022-09-22 | 2024-05-03 | 四川农业大学 | Application of gene segment B in cultivation of new plant material |
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