CN116987704A - AtDPBF4 and bZIP recombinant gene fragment and application thereof - Google Patents

AtDPBF4 and bZIP recombinant gene fragment and application thereof Download PDF

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CN116987704A
CN116987704A CN202310523614.7A CN202310523614A CN116987704A CN 116987704 A CN116987704 A CN 116987704A CN 202310523614 A CN202310523614 A CN 202310523614A CN 116987704 A CN116987704 A CN 116987704A
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arabidopsis
seq
4iid4bzip
thiana
4iid5bzip
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马建忠
陈欣
薛才
冯翀
王永刚
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Lanzhou University of Technology
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Lanzhou University of Technology
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
    • C12N15/8205—Agrobacterium mediated transformation
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
    • C12N15/8267—Seed dormancy, germination or sprouting
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance

Abstract

The application provides AtDPBF4 and bZIP recombinant gene fragments 4IID3bZIP, 4IID4bZIP, 4IID5bZIP, 4II910bZIP and application thereof. The above 4 simple transcription factors with transcriptional activation activity were obtained by ligating CRII fragments of AtDPBF4 in the ABI5 subfamily of arabidopsis with bZIP fragments of AtDPBF3, atDPBF4, atDPBF5, at5G42910, respectively. The recombinant fragments are connected with a vector and respectively introduced into agrobacterium to carry out genetic transformation on the arabidopsis, and finally the arabidopsis plant with the over-expressed transgene is realized. Through detection, the transgenic plant has the application of advancing seed germination, increasing the number of bolting plants, increasing the number of rosette leaves, enhancing drought resistance, enhancing salt tolerance and the like.

Description

AtDPBF4 and bZIP recombinant gene fragment and application thereof
Technical Field
The application relates to the field of plant genetic engineering, in particular to an arabidopsis AtDPBF4 and bZIP recombinant gene fragment, which comprises four recombinant gene fragments, namely 4IID3bZIP, 4IID4bZIP, 4IID5bZIP, 4II910bZIP and application thereof.
Background
Identification from the 5 th, ABA-unresponsive arabidopsis mutant (ABA-insensitive mutant) gene ABI5 has been done for twenty years so far. As leucine zipper-type (bZIP) transcription factors, there are also 8 genes in the Arabidopsis genome that are highly homologous to ABI5, which are collectively classified as the ABI5 subfamily (ABI 5 subfamilies). The Thomas group reported the first 5 homologous genes of the ABI5 subfamily: atDPBF1, atDPBF2, atDPBF3, atDPBF4 and AtDPBF5. Wherein AtDPBF1 is ABI5, and AtDPBF5 is a different splice of ABF 3. An alignment of the whole genome of Arabidopsis shows that there are 9 members in the Arabidopsis ABI5 subfamily: ABI5/AtDPBF1, atDPBF2, atDPBF3/AREB3, atDPBF4/EEL, ABF1, ABF2/AREB1, ABF3/AtDPBF5, ABF4/AREB2 and At5G42910. Wherein the 9 th member, at5G42910, was found by sequence homology alignment.
The ABI5 subfamily genes are involved in multiple stages of plant growth such as seed maturation, seed germination, root growth, flowering time, and stress response, among others. Although there are reports of drought and salt resistance obtained from plants which overexpress the ABI5 subfamily member genes, the individual mutants or overexpressed plants of the 9 members of the ABI5 subfamily have no or very weak phenotypic changes. Among them, the biological functions and functional molecular mechanisms of AtDPBF3, atDPBF4, atDPBF5, and At5G42910 are not yet fully understood. In the case of gene redundancy, to see the effect of single gene overexpression on the phenotype of plant growth and development, on the basis of earlier work (CRII fragment of AtDPBF4 in ABI5 subfamily has strongest transcriptional activation activity), recombinant transcription factor genes 4IID3bZIP, 4IID4bZIP, 4IID5bZIP, 4II910bZIP with stronger transcriptional activation activity were obtained by recombining CRII fragment of transcriptional activation region of AtDPBF4 with DNA binding region fragment of AtDPBF3, atDPBF4, atDPBF5, at5G42910 (alkaline leucine zipper region, bZIP). The effect of AtDPBF3, atDPBF4, atDPBF5 and At5G42910 on plant growth and development and stress adaptability is deeply known by constructing an Arabidopsis plant over-expressing the recombinant transcription factor genes 4IID3bZIP, 4IID4bZIP, 4IID5bZIP and 4II910bZIP. The Arabidopsis plants (A.thaliana D3-1-A.thaliana D3-8, A.thaliana D4-1-A.thaliana D4-6, A.thaliana D5-1-A.thaliana D5-6) over-express recombinant gene segments 4IID3bZIP, 4IID4bZIP, 4IID5bZIP, and show genetic phenotypes of seed pre-germination, increased number of bolting, increased number of rosette leaves, salt tolerance, and drought resistance compared with wild Arabidopsis plants (Columbia ecotype). Arabidopsis plants (A.thaliana 910-1 to A.thaliana 910-8) overexpressing the recombinant gene fragment 4II910bZIP exhibit a salt-tolerant genetic phenotype compared to wild type Arabidopsis plants (Columbia ecology).
Problems of the prior art: the biological functions of 4IID3bZIP, 4IID4bZIP, 4IID5bZIP, 4II910bZIP have not been reported in the prior studies.
Disclosure of Invention
The key technical problem to be solved by the application is to provide the sequences of 4 recombinant gene fragments 4IID3bZIP, 4IID4bZIP, 4IID5bZIP and 4II910bZIP and the application thereof, and the application of the transgenic arabidopsis plant is finally realized by respectively connecting the CRI fragments of AtDPBF4 in the arabidopsis ABI5 subfamily with the bZIP fragments of AtDPBF3, atDPBF4, atDPBF5 and At5G42910, wherein the seed germination in advance, the number of bolting, the number of rosette leaves, the salt tolerance, drought resistance and the like of the transgenic arabidopsis plant are finally realized.
In order to solve the technical problems, the application adopts the following technical scheme:
1. an arabidopsis AtDPBF4 and bZIP recombinant gene fragment comprising: 4IID3bZIP, 4IID4bZIP, 4IID5bZIP, 4II910bZIP, the sequence of the coding region of which comprises: atDPBF4 conserved region II (CRII) contains 22 amino acid residues, and the nucleotide residue sequence for encoding the peptide fragment is shown in a sequence table SEQ No. 1; the AtDPBF3 alkaline leucine region (bZIP) contains 87 amino acid residues, and the nucleotide residue sequence for encoding the peptide is shown in a sequence table SEQ No. 2; the total length of the recombinant gene 4IID3bZIP is 113 amino acid residues (comprising a connecting peptide RSGG between CRII and bZIP and 2 amino acid residues MG before CRII), and the nucleotide residue sequence of the coded peptide is shown as a sequence table SEQNo. 3; the AtDPBF4 alkaline leucine region (bZIP) contains 86 amino acid residues, and the nucleotide residue sequence for encoding the peptide is shown in a sequence table SEQ No. 4; the total length of the recombinant gene 4IID4bZIP is 112 amino acid residues (comprising a connecting peptide RSGG between CRII and bZIP and 2 amino acid residues MG before CRII), and the nucleotide residue sequence of the coded peptide is shown as a sequence table SEQ No. 5; the AtDPBF5 alkaline leucine region (bZIP) contains 112 amino acid residues, and the nucleotide residue sequence for encoding the peptide is shown in a sequence table SEQ No. 6; the total length of the recombinant gene 4IID5bZIP is 138 amino acid residues (comprising a connecting peptide RSGG between CRII and bZIP and 2 amino acid residues MG before CRII), and the nucleotide residue sequence of the coded peptide is shown in a sequence table SEQ No. 7; the At5G42910 alkaline leucine region (bZIP) contains 98 amino acid residues, and the nucleotide residue sequence of the coding peptide is shown in a sequence table SEQ No. 8; the total length of the recombinant gene 4II910bZIP is 124 amino acid residues (comprising a connecting peptide RSGG between CRII and bZIP and 2 amino acid residues MG before CRII), and the nucleotide residue sequence of the coded peptide is shown in a sequence table SEQ No. 9; .
2.A recombinant method of an Arabidopsis recombinant gene fragment 4IID3bZIP, 4IID4bZIP, 4IID5bZIP, 4II910bZIP, comprising: (1) total RNA extraction reverse transcription; (2) The CRII region of AtDPBF4 is amplified with bZIP regions of AtDPBF3, atDPBF4, atDPBF5 and At5G42910 respectively; (3) Construction of plant genetic transformation vectors (p 1304-4IID3bZIP, p1304-4IID4bZIP, p1304-4IID5bZIP, p1304-4II910 bZIP) and sequencing.
3.A method for genetic transformation of transgenic arabidopsis with over-expressed recombinant gene fragments 4IID3bZIP, 4IID4bZIP, 4IID5bZIP, 4II910bZIP, comprising:
(1) Construction of plant genetic transformation recombinant vectors p1304-4IID3bZIP, p1304-4IID4bZIP, p1304-4IID5bZIP and p1304-4II910 bZIP; (2) Plant genetic transformation recombinant vectors p1304-4IID3bZIP, p1304-4IID4bZIP, p1304-4IID5bZIP, p1304-4II910bZIP transformed with Agrobacterium (GV 3101); (3) Agrobacterium-dip-flower method for transforming Arabidopsis thaliana. (4) resistance screening of transgenic Arabidopsis thaliana; (5) PCR and RT-PCR identification of transgenic Arabidopsis genome and transcripts: (6) Arabidopsis plants overexpressing the transgenic fragments 4IID3bZIP, 4IID4bZIP, 4IID5bZIP, 4II910bZIP were designated as: the A.thiana D3, A.thiana D4, A.thiana D5, A.thiana 910.
4. Application of recombinant gene fragments 4IID4bZIP and 4IID5bZIP in seed germination in advance.
5. Use of recombinant gene fragments 4IID3bZIP, 4IID4bZIP, 4IID5bZIP in increasing rosette leaf numbers.
6. The application of the recombinant gene segments 4IID4bZIP and 4IID5bZIP in the increase of bolting number.
7. Use of recombinant gene fragments 4IID3bZIP, 4IID4bZIP, 4IID5bZIP in drought.
8. Use of recombinant gene fragments 4IID3bZIP, 4IID4bZIP, 4IID5bZIP, 4II910bZIP in salt tolerance.
The application has the following beneficial effects:
1. although AtDPBF4 and AtDPBF3, atDPBF4, atDPBF5 and At5G42910 respectively have different specific expression modes in the growth and development and stress response processes of arabidopsis, the over-expression transgenic plants of the over-expression transgenic plants have no reports of early seed germination, increased bolting number of plants, increased rosette number, salt tolerance, drought resistance and other phenotypic changes At present.
2. The application is based on the research work of the transcriptional activation function of ABI5 family conservation region II (CRII) and the strongest CRI activity of AtDPBF4, and the CRI fragment of AtDPBF4 is recombined with DNA binding regions of AtDPBF3, atDPBF4, atDPBF5 and At5G42910 for the first time so as to obtain genetic phenotype changes which cannot be expressed independently by AtDPBF3, atDPBF4, atDPBF5 or At5G42910.
3. The transgenic plants over-expressing the recombinant gene fragments 4IID3bZIP, 4IID4bZIP, 4IID5bZIP and 4II910bZIP show unexpected biological functions, and the variation of the genetic phenotypes such as drought resistance, salt resistance and the like has important application value in crop/vegetable cultivation. For example, drought is severe in northwest areas, which results in serious impact on agricultural production in some areas, and insufficient moisture is a major factor limiting plant growth. Meanwhile, the salinization problem is also a world problem, the soil salinization problem restricts the sustainable development of agriculture, and the improvement and utilization of the saline land are significant for guaranteeing the world grain safety.
4. The number of the transgenic plants over-expressing the recombinant gene segments 4IID4bZIP and 4IID5bZIP is obviously increased, and the number of the transgenic plants over-expressing the recombinant gene segments is increased from the wild type common 1 bolt to 3-5 bolts of the transgenic plants. The bolting is a mark for changing the plant such as arabidopsis from vegetative growth to reproductive growth, and the increased number of the bolts means more flowers and fruits, which has important application prospect for improving the yield of a plurality of crops such as rape (belonging to the same family as the arabidopsis) taking the fruits as harvest parts.
5. The genetic phenotype changes such as increased rosette leaves of transgenic plants over-expressing recombinant gene segments 4IID3bZIP, 4IID4bZIP and 4IID5bZIP can be applied to the cultivation of vegetable crops such as cabbages with stems and leaves as edible parts, and the yield of the vegetable crops can be improved.
Drawings
FIG. 1 is a sequence alignment analysis of ABI5 subfamily members. Among them, amino acid residue sequence alignment analysis of 9 members of the ABI5 subfamily using DNAMan software showed that there were 6 conserved regions (CRI-VI) in the family, and that nine members of the ABI5 subfamily were 37.79% identical, with different colors representing different degrees of homology.
FIG. 2 is an ABI5 family tree analysis. Among them, the similarity of amino acid residue sequences of 9 members of the ABI5 subfamily using MEGA-X software can be further divided into 3 subgroups, atDPBF1/ABI5, atDPBF2, atDPBF3 and AtDPBF4 as a subgroup, ABF1, ABF2, atDPBF5/ABF3 and ABF4 as a subgroup, and At5G42910 as a separate group.
FIG. 3 is an electropherogram of the A.thaliana D3 genome identification of an overexpressed transgenic Arabidopsis plant. Genome identification results were carried out on 8 strains after 14D of seedlings of the A.thaliana D3 over-expression transgenic Arabidopsis plants of the present application. Wherein M is a marker, DNA standard molecular weight mark; negative control (template wild-type genome); + is a positive control (recombinant plasmid p1304-4IID3bZIP as template). 1-8 are each numbered 8 different transgenic Arabidopsis plants. .
FIG. 4 is an electropherogram of the A.thaliana D4 genome identification of an overexpressed transgenic Arabidopsis plant. Genomic identification results were performed on 15 lines after 14D of seedlings of the A.thaliana D4 overexpressing transgenic Arabidopsis of the present application. Wherein M is a marker, DNA standard molecular weight mark; negative control (template wild-type genome); + is a positive control (recombinant plasmid p1304-4IID4bZIP as template). 1-15 are each 15 different transgenic Arabidopsis plants line numbered.
FIG. 5 is an electropherogram of the A.thaliana D5 genome identification of an overexpressed transgenic Arabidopsis plant. Genome identification results were carried out on 4 strains after 14D of seedlings of the A.thaliana D5 over-expression transgenic Arabidopsis plants of the present application. Wherein M is a marker, DNA standard molecular weight mark; negative control (template wild-type genome); + is a positive control (recombinant plasmid p1304-4IID5bZIP as template). 1-4 are each numbered for 4 different transgenic Arabidopsis plants. .
FIG. 6 is an electrophoretogram of genomic A.thaliana910 over-expressed transgenic Arabidopsis plants. Genome identification results were carried out on 5 lines after 14d of seedlings of the A.thaliana910 over-expression transgenic Arabidopsis thaliana plants of the present application. Wherein M is a marker, DNA standard molecular weight mark; negative control (template wild-type genome); + is a positive control (recombinant plasmid p1304-4II910bZIP as template). 1-5 are each numbered for 5 different transgenic Arabidopsis plants.
FIG. 7 is an electrophoretogram of RT-PCR identification of overexpressing transgenic Arabidopsis plant A.thaliana D3. After 14D of seedlings of the A.thaliana D3 over-expression transgenic Arabidopsis thaliana of the application grow, 4 strains are randomly selected for RT-PCR identification. Wherein M is a marker, DNA standard molecular weight mark; taking housekeeping gene Actin2 as a reference, 1-4 are the expression levels of Actin2 of wild type and transgenic Arabidopsis plants A.thaliana D3-1, A.thaliana D3-2 and A.thaliana D3-4 respectively, and lane 5 has no band and is the expression level of the wild type target gene. Lanes 6-8 are the target gene expression levels of transgenic Arabidopsis plants A.thaliana D3-1, A.thaliana D3-2, and A.thaliana D3-4, respectively, further illustrating that the target gene has been integrated into the plant genome and can be transcribed. The intensity of the bands of the different strains constructed in the same species may cause different gene expression effects due to different chromosome sites into which the recombinant plasmid is inserted when the Columbia wild type is transfected.
FIG. 8 is a diagram of the RT-PCR identification electrophoresis of the overexpressing transgenic Arabidopsis plant A.thaliana D4. After 14D of seedlings of the A.thaliana D4 over-expression transgenic Arabidopsis thaliana of the application grow, 4 strains are randomly selected for RT-PCR identification. Wherein M is a marker, DNA standard molecular weight mark; taking housekeeping gene Actin2 as a reference, 1-4 are the expression levels of Actin2 of wild type and transgenic Arabidopsis plants A.thaliana D4-1, A.thaliana D4-2 and A.thaliana D4-5 respectively, and lane 7 has no band and is the expression level of the wild type target gene. Lanes 5, 8 and 9 are the target gene expression levels of transgenic Arabidopsis plants A.thaliana D4-1, A.thaliana D4-2, and A.thaliana D4-5, respectively, further illustrating that the target gene has been integrated into the plant genome and can be transcriptionally expressed. The intensity of the bands of the different strains constructed in the same species may cause different gene expression effects due to different chromosome sites into which the recombinant plasmid is inserted when the Columbia wild type is transfected.
FIG. 9 is an electrophoretogram of RT-PCR identification of overexpressing transgenic Arabidopsis plant A.thaliana D5. After 14D of seedlings of the A.thaliana D5 over-expression transgenic Arabidopsis thaliana of the application grow, 4 strains are randomly selected for RT-PCR identification. Wherein M is a marker, DNA standard molecular weight mark; taking housekeeping gene Actin2 as a reference, 1-4 are the expression levels of Actin2 of wild type and transgenic Arabidopsis plants A.thaliana D5-1, A.thaliana D5-4 and A.thaliana D5-5 respectively, and lane 7 has no band and is the expression level of the wild type target gene. Lanes 5, 8, 9 are the target gene expression levels of transgenic Arabidopsis plants A.thaliana D5-1, A.thaliana D5-4, and A.thaliana D5-5, respectively, further illustrating that the target gene has been integrated into the plant genome and can be transcriptionally expressed. The intensity of the bands of the different strains constructed in the same species may cause different gene expression effects due to different chromosome sites into which the recombinant plasmid is inserted when the Columbia wild type is transfected.
FIG. 10 is an electrophoresis chart of RT-PCR identification of an over-expressed transgenic Arabidopsis plant A.thaliana 910. After 14d of seedlings of the A.thaliana910 overexpression transgenic Arabidopsis thaliana plants of the application are grown, 5 lines are randomly selected for RT-PCR identification results. Wherein M is Maker; negative control (template wild-type genome); + is a positive control (using the correctly sequenced recombinant plasmid p1304-4II910bZIP as template). Lanes 1-5 are the target gene expression levels of transgenic Arabidopsis plants A.thaliana910-1, A.thaliana 910-2, A.thaliana 910-3, A.thaliana910-4, and A.thaliana910-5, respectively, further illustrating that the target gene has been integrated into the plant genome and can be transcriptionally expressed. The intensity of the bands of the different strains constructed in the same species may cause different gene expression effects due to different chromosome sites into which the recombinant plasmid is inserted when the Columbia wild type is transfected.
FIG. 11 is a plot of germination rate for A.thaliana D4 and A.thaliana D5. Wherein, the germination condition is observed in a 1/2MS culture medium by the seed point of the wild type and over-expressed transgenic Arabidopsis A.thaliana D4 and A.thaliana D5. Seed germination was observed by growing on 1/2MS medium (16 hours light, 8 hours dark) for 44h-54h, and the germination rate of the seeds on 1/2MS medium was statistically counted for different periods of time (n=30, error bars ± SE,16 hours light and 8 hours dark).
FIG. 12 is a chart of the number and size of A.thaliana D4 rosette leaves. Photographs were taken of 4 week old wild type and A.thaliana D4-2 transgenic Arabidopsis growth (16 hours light, 8 hours dark).
FIG. 13 is a chart of the number and size of A.thaliana D5 rosette leaves. Photographs were taken of 4 week old wild type and A.thaliana D5-5 transgenic Arabidopsis growth (16 hours light, 8 hours dark).
FIG. 14 is a chart of the number and size of A.thaliana D5 rosette leaves. Photographs were taken of 7 week old wild type and A.thaliana D5-5 transgenic Arabidopsis growth (16 hours light, 8 hours dark).
FIG. 15 is a chart of the number and size of A.thaliana D3 rosette leaves. Photographs were taken of 6 week old wild type and A.thaliana D3-4 transgenic Arabidopsis growth (16 hours light, 8 hours dark).
FIG. 16 is a phenotype chart showing the increase of bolting number of A.thaliana D4 and A.thaliana D5. The T4 generation seeds of the A.thiana D4 and A.thiana D5 transgenic arabidopsis lines are placed in a greenhouse for 6 weeks for culture, and the result shows that the number of the arabidopsis plants bolting of the overexpression recombinant gene segments 4IID4bZIP and 4IID5bZIP all show 3-4 increased phenotypes.
FIG. 17 is a phenotype plot of drought resistance for A.thaliana D3, A.thaliana D4, and A.thaliana D5. And (3) placing the T4 generation seeds of the A.thiana D3, A.thiana D4 and A.thiana D5 transgenic arabidopsis strain in a greenhouse for culturing for 3 weeks, stopping watering, taking a photo after 9 days of drought, taking a photo after 12 days of drought, and taking a photo after 12 days of drought.
FIG. 18 is a phenotype diagram of salt tolerance of A.thaliana D3. The T4 generation seeds of the wild type and A.thiana D3 transgenic Arabidopsis lines were sown on 1/2MS medium containing NaCl (0, 100, 150, 200 mM) at different concentrations for 14 days, and their growth conditions were observed.
FIG. 19 is a phenotype diagram of salt tolerance of A.thiana D4. The T4 generation seeds of the wild type and A.thiana D4 transgenic Arabidopsis lines were inoculated on 1/2MS medium containing NaCl (0, 100, 150, 200 mM) at different concentrations for 14 days, and their growth conditions were observed.
FIG. 20 is a phenotype diagram of A.thiana D5 salt tolerance. The T4 generation seeds of the wild type and A.thiana D5 transgenic Arabidopsis lines were inoculated on 1/2MS medium containing NaCl (0, 100, 150, 200 mM) at different concentrations for 14 days, and their growth conditions were observed.
FIG. 21 is a phenotype diagram of salt tolerance of A.thiana 910. The T4 generation seeds of the wild type and A.thiana 910 transgenic Arabidopsis lines were sown on 1/2MS medium containing NaCl (0, 100, 150, 200 mM) at different concentrations for 7 days, and their growth conditions were observed.
FIG. 22 is a phenotype diagram of A.thiana D3, A.thiana D4 and A.thiana D5 salt tolerance. Seeds of the transgenic A.thaliana lines T4 of A.thaliana D3, A.thaliana D4 and A.thaliana D5 were placed in a greenhouse and cultured for 3 weeks, and then 50ml of 200mM NaCl solution was poured, and the growth conditions of the wild-type and transgenic A.thaliana lines A.thaliana D3, A.thaliana D4 and A.thaliana D5 were observed.
Detailed description of the preferred embodiments
The methods and apparatus used in the following examples of the present application are conventional methods and apparatus unless otherwise specified; the equipment and the reagent are conventional equipment and reagents purchased by reagent companies. In order to make the objects, technical solutions and advantages of the present patent more apparent, the following detailed description of the present patent refers to the field of 'electric digital data processing'. Examples of these preferred embodiments are illustrated in the specific examples. It should be noted that, in order to avoid obscuring the technical solutions of the present application due to unnecessary details, only the technical solutions and/or processing steps closely related to the solutions according to the present application are shown in the embodiments, and other details having little relation are omitted.
Example 1
The embodiment provides a recombinant and vector construction method of recombinant gene fragments 4IID3bZIP, 4IID4bZIP, 4IID5bZIP and 4II910bZIP, which comprises the following steps:
1. total RNA extraction and reverse transcription
Total RNA extraction was performed using the entire Columbia ecological Arabidopsis thaliana (Arabidopsis thaliana Columbia) grown on 1/2MS solid medium for about one week according to the total RNA extraction kit (Tiangen, DP 437). The concentration of the RNA was measured by NanoDrop2000, and reverse transcription was performed in the following order, and the following were mixed on ice:
after 5min of reaction at 65 ℃, the ice bath was rapidly removed for 10min, and the following were added to the tube and gently mixed:
after 60min of reaction at 37℃the reaction was stopped by placing at 70℃for 15 min. After completion of the reverse transcription, ddH was added 2 The cDNA concentration was diluted to 20 ng/. Mu.l with O.
Amplification of CRII fragment of atdpbf4
Specific primer pairs were designed based on the sequence of CRII gene encoding AtDPBF4, and PCR amplification was performed with the cDNA template obtained in (1). The PCR system is as follows:
the amplification parameters of PCR were: pre-denaturation at 95℃for 5min,30 cycles consisting of denaturation at 95℃for 15s, annealing at 57℃for 30s, extension at 72℃for 25s, and extension at 72℃for 10min. Agarose gel electrophoresis was performed after the completion of PCR.
Amplification of bZIP fragments of AtDPBF3, atDPBF4, atDPBF5, at5G42910
And (3) designing specific primer pairs according to sequences of the genes encoding AtDPBF3bZIP, atDPBF4bZIP, atDPBF5bZIP and At5G42910bZIP respectively, and carrying out PCR amplification by using the cDNA template obtained in the step (1). The PCR system is as follows:
the amplification parameters of PCR were: pre-denaturation at 95℃for 5min,30 cycles consisting of denaturation at 95℃for 15s, annealing at 57℃for 30s, extension at 72℃for 50s, and extension at 72℃for 10min. Agarose gel electrophoresis was performed after the completion of PCR.
Preparation of E.coli DH5α competent cells
(1) Activating the strain. Selecting DH5 alpha single colony in 3mL LB culture medium, and shake culturing at 37 ℃ overnight;
(2) Inoculating the activated strain into 50mL of non-resistant LB culture medium at a proportion of 1%, performing shake culture at 37 ℃, and measuring OD600 every 15min after the culture starts to be turbid until OD600 = 0.6;
(3) Transferring the culture solution into a precooled 50mL centrifuge tube, ice-bathing for 10min, and centrifuging at 5000rpm for 10min at 4 ℃;
(4) Removing the supernatant, lightly suspending the thalli until uniform by using 20mL of ice-precooled 0.1mol/L CaCl2 solution, and standing on ice for 30min;
(5) Centrifuging at 5000rpm at 4deg.C for 10min;
(6) Removing supernatant, lightly suspending thalli to be uniform by using 2mL of ice precooled 0.1mol/L CaCl2 solution, and placing on ice for 1-2min to prepare competent cells;
construction of CRII plant genetic transformation vector of AtDPBF4 and sequence determination
According to the conserved region sequence of CRII for encoding AtDPBF4, a pair of specific amplification primers are designed as follows:
D4II-F1:5’-CATGCCATGGGTGGAAAACCACTAGGAAGCAT-3’；
D4II-R1:5’-GAAGATCTTTCCTCAGCTGGTGGCAAGA-3’。
the 5' end of the upstream and downstream primers was introduced with restriction enzyme sites (underlined) NcoI and BglII, respectively, and 2-3 protecting bases were added. The PCR product amplified by using cDNA as template and specific primer pair is recovered, and then is cut with vector pCAMBIA1304 by restriction enzymes EcoRI and PstI respectively, and is connected by T4 DNA ligase to transform E.coli DH5 alpha competent cells, 6 single colonies are selected for colony PCR identification and double cutting identification, and then sent to Suzhou Jin Weizhi Biotechnology Co., ltd for sequencing, and the recombinant plasmid with correct sequencing is named pCAMBIA1304-D4II.
6.4IID3bZIP, 4IID4bZIP, 4IID5bZIP, 4II910bZIP plant genetic transformation vector construction and sequence determination
According to the basic leucine zipper region (bZIP) sequences of coding AtDPBF3, atDPBF4, atDPBF5 and At5G42910, specific amplification primers are designed as follows:
D3-F1:5’-GAAGATCTggtggaGGAAGGAAGAGGGTAGCTTC-3’；
D3-R1:5’-GACTAGTGAAAGGAGCCGAGCTTGTC-3’；
D4-F1:5’-GAAGATCTGGTGGAGGGAGGAAAAGAGTAGCTGG-3’；
D4-R1:5’-GACTAGTGAGAGAAGCAGAGTTTGTTC-3’；
D5-F1:5’-GAAGATCTGGTGGACCAAAGAGCGCCCTGGATG-3’；
D5-R1:5’-GACTAGTCCAGGGACCCGTCAATGTCC-3’；
910-F1:5’-GAAGATCTGGTGGAGGTAGTACaAGTACAAGAGG-3’；
910-R1:5’-GACTAGTCTTGATGTCCGATTTCGTTC-3’；
the 5' end of the upstream and downstream primers was introduced with restriction enzyme sites (underlined) BglII and SpeI, respectively, and 1-2 guard bases were added. The PCR product amplified by using cDNA as a template and a specific primer pair is recovered and subjected to double digestion with a vector pCAMBIA1304-D4II, the PCR product is respectively connected with restriction enzymes BglII and SpeI by T4 DNA ligase, E.coli DH5 alpha competent cells are transformed, single colony is selected for carrying out colony PCR identification and double digestion identification, and then the single colony is sent to Suzhou Jin Weizhi Biotechnology Co., ltd for sequencing, and recombinant plasmids with correct sequencing are respectively named p1304-4IID3bZIP, p1304-4IID4bZIP, p1304-4IID5bZIP and p1304-4II910bZIP.
Example 2
The present example provides a method for genetic transformation of recombinant gene fragments 4IID3bZIP, 4IID4bZIP, 4IID5bZIP, 4II910bZIP Arabidopsis, comprising:
1. taking p1304-4IID3bZIP overexpression vector as an example, agrobacterium GV3101 is transformed:
mu.l of recombinant plasmid p1304-4IID3bZIP was taken and mixed with competent cells of Agrobacterium tumefaciens GV 3101. After being placed on ice for 30min, the liquid nitrogen is quickly frozen for 1min, and the ice is quickly pre-frozen for 2min after heat shock for 90s at 42 ℃. 800 μl of fresh LB liquid medium was added and shake cultured at 28deg.C for 4h. 100 μl was plated on a resistant LB plate (containing Gen 40 μg/ml, rif 20 μg/ml, kan 40 μg/ml) and cultured upside down at 28℃for 2-3d. 3 single colonies were selected and cultured overnight at 28℃and 220rpm in 3mL of fresh LB (containing 40. Mu.g/mL Gen, 20. Mu.g/mL Rif, 40. Mu.g/mL Kan) liquid medium. 1 μl of each bacterial liquid was taken for colony PCR identification. Colony PCR positive transformants further genetically transformed Arabidopsis thaliana. The methods for transforming agrobacterium GV3101 by using the p1304-4IID4bZIP, p1304-4IID5bZIP and p1304-4II910bZIP overexpression vectors are the same as above.
2. Agrobacterium-dip method for transforming arabidopsis thaliana
(1) The columbia ecological type arabidopsis plants are ready to grow for about 25 days before transformation, and after the arabidopsis plants begin bolting, the top ends of main inflorescences of the arabidopsis are cut off while avoiding damaging axillary inflorescences so as to induce the generation of lateral inflorescences. Transformation is carried out when the lateral branches extend out by 2-10 cm. And watering the Arabidopsis plants to be impregnated, wherein the Arabidopsis plants are subject to more flower buds which are not yet bloomed.
(2) Agrobacterium containing the overexpression vectors of AtDPBF3, atDPBF4, atDPBF5 and At5G42910, p1304-4IID3bZIP, p1304-4IID4bZIP, p1304-4IID5bZIP and p1304-4II910bZIP, were inoculated into 5ml of fresh LB (containing 40. Mu.g/ml Gen, 20. Mu.g/ml Rif and 40. Mu.g/ml Kan) liquid medium, and shake cultured At 30℃for about 15 hours with a shaking table of 200 r/min.
(3) Transferring the culture obtained in the last step into 200ml fresh LB liquid medium containing corresponding antibiotics at a ratio of 1:50, and shake culturing at 28deg.C for about 15 hr to OD 600 ＝0.8-1.0。
(4) The bacterial cells are collected by centrifugation at 4000r/min for 15min, and the OD of the solution is regulated by using a permeation buffer (1/2 MS culture medium+5% sucrose) 600 =0.8-1.0, and Silwett L-77 (SINOPCR S5605) was added to the final concentration of 0.03% and mixed.
(5) The pollinated flowers, pods were removed with scissors and the plants were watered one day in advance. Each flower of the prepared Arabidopsis plants was dip-stained with an Agrobacterium suspension containing the four plasmids to be transformed, respectively, by aspiration with a 5ml syringe. The impregnated plants (T0 generation plants) were covered with a fresh-keeping bag to maintain humidity.
(6) Placing the impregnated Arabidopsis plants in a dark place for culturing for 12 hours, then placing the Arabidopsis plants in normal conditions (22 ℃ C., 16h of illumination and 8h of darkness) for culturing, removing a fresh-keeping bag after 2 days, and continuously culturing until pods are mature, wherein the dip dyeing can be repeated for 2-3 times during the period to improve the conversion efficiency.
(7) After the plants grow for 2-3 weeks, the top pods turn yellow, the arabidopsis seeds (T1 generation) are collected according to the number before the pods crack, and the arabidopsis seeds are stored after being sufficiently dried to screen transgenic arabidopsis plants.
Screening and identification of 4IID3bZIP, 4IID4bZIP, 4IID5bZIP, 4II910bZIP over-expressed transgenic Arabidopsis plants
Adding a small amount of sterile water to immerse the seeds of the T1 generation, and placing the seeds in a refrigerator at 4 ℃ for vernalization for 3-4 days. After disinfection in an ultra-clean workbench, the mixture is spread on a 1/2MS solid culture medium containing 25 mug/ml hygromycin and placed in a plant illumination incubator for cultivation (22 ℃,16h illumination, 8h darkness, humidity of about 60 percent, illumination intensity of 3000-5000 Lx). After two weeks of growth, the suspected positive transgenic arabidopsis plants can grow normally, while the non-transgenic plants show yellow cotyledons and short roots and cannot grow into the culture medium. Transplanting the positive transgenic arabidopsis plants into nutrient soil (Danish's substrate No.5, pH 5.5) for culture (22 ℃,16h illumination, 8h darkness, humidity of about 60%, illumination intensity of 3000-5000 Lx), and harvesting mature seeds (T2 generation seeds) singly. The T2 generation seeds were screened on MS medium containing 30 μg/ml hygromycin resistance for further T3 generation followed by T4 generation for phenotyping.
Specific primers were designed based on the sequences of the recombinant plasmids p1304-4IID3bZIP, p1304-4IID4bZIP, p1304-4IID5bZIP and p1304-4II910bZIP, respectively, and genomes were extracted from 14-day-old wild-type and 4IID3bZIP, 4IID4bZIP, 4IID5bZIP and 4II910bZIP overexpressing transgenic Arabidopsis plants (cultured on 1/2MS plates) and were identified by the specific primers to determine whether the gene of interest was transferred, and the specific primers were as follows:
D3-F:5’-GCCACCAGCTGAGGAAAGAT-3’；
D3-R:5’-AGCTCATGAGTGTAAGCCTGT-3’
D4-F:5’-AGACTTTTCAACAAAGGGTAATATCCG-3’
D4-R:5’-GAGAGAAGCAGAGTTTGTTCGC-3’
D5-F:5’-GAGACTTTTCAACAAAGGGTAATATCCG-3’
D5-R:5’-TGCCTTTTGCATCCCATTCC-3’
910-F:5’-TGAGACTTTTCAACAAAGGGTAATATCCG-3’
910-R:5’-CTTGATGTCCGATTTCGTTCTCC-3’
RT-PCR is used for respectively identifying the expression status of 4IID3bZIP, 4IID4bZIP, 4IID5bZIP and 4II910bZIP genes in the antibiotic positive Arabidopsis plant. Total RNA was extracted from 14-day-old wild-type and 4IID3bZIP, 4IID4bZIP, 4IID5bZIP, 4II910bZIP overexpressing transgenic Arabidopsis plants (cultured on 1/2MS plates), reverse transcribed into cDNA, and amplified with specific primers, respectively. Specific primers were as follows:
D3-F:5’-GCCACCAGCTGAGGAAAGAT-3’
D3-R:5’-AGCTCATGAGTGTAAGCCTGT-3’
D4-F:5’-TGCCACCAGCTGAGGAAAGA-3’
D4-R:5’-TGAACGTGCTGCAGATTCTC-3’
D5-F:5’-AACCACTAGGAAGCATGAACCT-3’
D5-R:5’-AGGAACAGGGGACAAAGATGC-3’
910-F:5’-GGAAAACCACTAGGAAGCATGAAC-3’
910-R:5’-CTTGATGTCCGATTTCGTTCTCC-3’
Actin2-F:5’-TGTGCCAATCTACGAGGGTTT-3’
Actin2-R:5’-TTTCCCGCTCTGCTGTTGT-3’
expression of the overexpressed gene in transgenic Arabidopsis was analyzed by genome. The genome PCR electrophoresis result shows that the specific band with the corresponding molecular weight is displayed, the band size of the over-expression strain A.thiana D3-1-A.thiana D3-8 is 705bp,A.thaliana D4-1-A.thiana D4-15, the band size of 702bp,A.thaliana D5-1-A.thiana D5-4 is 750bp,A.thaliana 910-1-A.thiana 910-5 and 740bp. The expression of the overexpressed gene in transgenic Arabidopsis was analyzed by RT-PCR. The RT-PCR electrophoresis result shows that the specificity band with the corresponding molecular weight is displayed, the band size of the overexpression line A.thiana D3 band is 133bp,A.thaliana D4 band size 122bp,A.thaliana D5 band size 104bp,A.thaliana 910 band size 372bp, and the band size of the housekeeping gene Actin2 band is 137bp, as shown in the attached figures 3-10. The results show that: the recombinant genes 4IID3bZIP, 4IID4bZIP, 4IID5bZIP and 4II910bZIP are successfully embedded into the Arabidopsis genome and can be overexpressed.
Example 3
The embodiment provides an application of recombinant genes 4IID4bZIP and 4IID5bZIP in improving germination rate of Arabidopsis seeds, which comprises the following steps: the harvested mature T3 generation seeds and wild type seeds were added with ddH2O and placed in a refrigerator at 4℃for vernalization for 3-4 days. After disinfection in an ultra-clean workbench, the mixture is spread on the same 1/2MS solid culture medium containing 25 mug/ml hygromycin, and the mixture is placed in a plant illumination incubator for cultivation (22 ℃,16h illumination, 8h darkness, humidity of about 60 percent and illumination intensity of 3000-5000 Lx). And counting the germination rate of the seeds for 44-54 hours. The germination rate of the transgenic Arabidopsis lines A.thiana D4-1, A.thiana D4-2 and A.thiana D4-5) is obviously higher than that of the wild Arabidopsis plants, and the germination rate of the transgenic Arabidopsis lines A.thiana D5 (A.thiana D5-1, A.thiana D5-4 and A.thiana D5-5) is obviously higher than that of the wild Arabidopsis plants, as shown in figure 11. The results show that: the germination rate of the arabidopsis seeds can be obviously improved by over-expressing the recombinant genes 4IID4bZIP and 4IID5 bZIP.
Example 4
The present embodiment provides the use of recombinant genes 4IID3bZIP, 4IID4bZIP, 4IID5bZIP in rosette leaf augmentation comprising:
(1) All plants are selected from flowerpots with square calibers of the same specification, a certain amount of nutrient soil (Denmark's substrate No.5, pH 5.5) is placed in each flowerpot, the flowerpots are placed in the same flat-bottom tray, and water is added into the tray so that the soil in the flowerpots in the tray can absorb water automatically.
(2) T3 generation seeds and wild seeds of D3 transgenic Arabidopsis lines (A.thiana D3-1, A.thiana D3-2 and A.thiana D3-4), A.thiana D4 transgenic Arabidopsis lines (A.thiana D4-1, A.thiana D4-2 and A.thiana D4-5) and A.thiana D5 transgenic Arabidopsis lines (A.thiana D5-1, A.thiana D5-4 and A.thiana D5-5) were sown on nutrient soil (Danish substrate No.5, pH 5.5) after purification, respectively, and then placed in greenhouse culture (22 ℃,16h light, 8h darkness, humidity about 60%, light intensity 3000-5000 Lx). And removing the seedlings with poor growth conditions and redundant seedlings after one week, so that 4 seedlings with relatively consistent growth conditions are reserved in each flowerpot.
(3) Observing and counting arabidopsis plant rosettes She Zhuangkuang
At the time of 4 and 7 weeks of growth, the A.thiana D3 transgenic Arabidopsis lines (A.thiana D3-1, A.thiana D3-2 and A.thiana D3-4), the A.thiana D4 transgenic Arabidopsis lines (A.thiana D4-1, A.thiana D4-2 and A.thiana D4-5) and the A.thiana D5 transgenic Arabidopsis lines (A.thiana D5-1, A.thiana D5-4 and A.thiana D5-5) exhibited the phenotype of increased rosette leaf gain (FIGS. 12-15). The results show that: the number of rosette leaves can be obviously increased by over-expressing recombinant genes 4IID3bZIP, 4IID4bZIP and 4IID5bZIP, and the size of rosette leaves can be increased.
Example 5
The embodiment provides an application of recombinant genes 4IID4bZIP and 4IID5bZIP in increasing bolting number, which comprises the following steps:
(1) All plants are selected from flowerpots with square calibers of the same specification, a certain amount of nutrient soil (Denmark's substrate No.5, pH 5.5) is placed in each flowerpot, the flowerpots are placed in the same flat-bottom tray, and water is added into the tray so that the soil in the flowerpots in the tray can absorb water automatically.
(2) The T3 generation seeds and wild type seeds of the A.thiana D4 transgenic Arabidopsis strains (A.thiana D4-1, A.thiana D4-2 and A.thiana D4-5) and the A.thiana D5 transgenic Arabidopsis strains (A.thiana D5-1, A.thiana D5-4 and A.thiana D5-5) were placed at 4℃for vernalization for 3-4 days, on demand in nutrient soil (Danish substrate No.5, pH5.5), and placed in a greenhouse for cultivation (22 ℃,16h light, 8h darkness, humidity 60% or so, light intensity 3000-5000 Lx). And removing the seedlings with poor growth conditions and excessive seedlings after one week, so that 1 seedling with similar growth conditions is reserved in each flowerpot.
(3) And after growing for 4 weeks, observing and counting the bolting state of the arabidopsis thaliana. The number of shoots of the transgenic Arabidopsis lines A.thiana D4 (A.thiana D4-1, A.thiana D4-2 and A.thiana D4-5) and A.thiana D5 (A.thiana D5-1, A.thiana D5-4 and A.thiana D5-5) showed more phenotypes than the wild type strain (FIG. 4).
The results show that: overexpression of the recombinant genes 4IID4bZIP and 4IID5bZIP significantly increased the number of bolting in Arabidopsis plants (FIG. 16).
Example 6
The embodiment provides application of recombinant genes 4IID3bZIP, 4IID4bZIP and 4IID5bZIP in drought resistance, which comprises the following steps:
(1) All plants are selected from flowerpots with square calibers of the same specification, a certain amount of nutrient soil (Denmark's substrate No.5, pH 5.5) is placed in each flowerpot, the flowerpots are placed in the same flat-bottom tray, and water is added into the tray so that the soil in the flowerpots in the tray can absorb water automatically.
(2) T3 generation seeds and wild seeds of A.thiana D3-1, A.thiana D3-2 and A.thiana D3-4, A.thiana D4 transgenic Arabidopsis lines (A.thiana D4-1, A.thiana D4-2 and A.thiana D4-5) and A.thiana D5 transgenic Arabidopsis lines (A.thiana D5-1, A.thiana D5-4 and A.thiana D5-5) were placed in vernalization at 4℃for 3-4 days, dibble in nutrient soil (Danish substrate No.5, pH 5.5), and incubated in a greenhouse (22℃for 16h light, 8h darkness, 60% or so, and light intensity of 3000-5000 Lx), respectively. And removing the seedlings with poor growth conditions and excessive seedlings after one week, keeping 12 seedlings with similar growth conditions in each flowerpot, stopping watering the arabidopsis cultivated in the soil after three weeks of growth, and carrying out rehydration observation after 12 days of drought.
(3) The growth of wild type and transgenic Arabidopsis was observed for 9 days and 12 days of drought. The transgenic Arabidopsis lines A.thiana D3-1, A.thiana D3-2 and A.thiana D3-4, A.thiana D4-transgenic Arabidopsis lines A.thiana D4-1, A.thiana D4-2 and A.thiana D4-5 and A.thiana D5 transgenic Arabidopsis lines A.thiana D5-1, A.thiana D5-4 and A.thiana D5-5 all exhibited drought-resistant phenotypes compared to the wild type strain. The results show that: overexpression of the recombinant genes 4IID3bZIP, 4IID4bZIP, 4IID5bZIP significantly increased drought resistance in Arabidopsis plants (FIG. 17).
Example 7
The present example provides the use of recombinant genes 4IID3bZIP, 4IID4bZIP, 4IID5bZIP in salt tolerance, comprising:
(1) All plants are selected from flowerpots with square calibers of the same specification, a certain amount of nutrient soil (Denmark's substrate No.5, pH 5.5) is placed in each flowerpot, the flowerpots are placed in the same flat-bottom tray, and water is added into the tray so that the soil in the flowerpots in the tray can absorb water automatically.
(2) T3 generation seeds and wild type seeds of the A.thiana D3 transgenic Arabidopsis lines (A.thiana D3-1, A.thiana D3-2 and A.thiana D3-4), the A.thiana D4 transgenic Arabidopsis lines (A.thiana D4-1, A.thiana D4-2 and A.thiana D4-5), the A.thiana D5 transgenic Arabidopsis lines (A.thiana D5-1, A.thiana D5-4 and A.thiana D5-5) and the A.thiana transgenic plants (A.thiana 910-1, A.thiana 910-2, A.thiana 910-4) were placed in a culture medium of 4℃for vernalization for 3-4 days, a portion of the culture medium containing different concentrations (0, 150 mM, 200mM, 5 mM, and pH of the culture medium of about 5 to about 5% in a greenhouse (pH of about 5 mM, 5) were placed in a medium of about 4℃for about 6 to about 6 mM, and the culture medium of about 5% in a dark place of about 0.5 mM, pH of about 5 to about 5 h, respectively, and the culture medium of L.5000 h, respectively. After one week, the seedlings with poor growth conditions and excessive seedlings are removed, so that 12 seedlings with similar growth conditions are reserved in each flowerpot, and 50ml of 200mM NaCl solution is irrigated after three weeks of growth of the arabidopsis cultivated in the soil.
(3) The growth of Arabidopsis thaliana grown on 1/2MS medium containing NaCl at various concentrations for 14 days was observed, and the growth of wild-type and transgenic Arabidopsis thaliana was observed after 50ml of 200mM NaCl solution was irrigated. The transgenic Arabidopsis lines A.thiana D3-1, A.thiana D3-2 and A.thiana D3-4, A.thiana D4-transgenic Arabidopsis lines A.thiana D4-1, A.thiana D4-2 and A.thiana D4-5) and A.thiana D5 transgenic Arabidopsis lines A.thiana D5-1, A.thiana D5-4 and A.thiana D5-5 and A.thiana 910 transgenic plants A.thiana 910-1, A.thiana 910-2, A.thiana 910-4 all exhibited salt tolerant phenotypes compared to the wild type strains.
The results show that: overexpression of the recombinant genes 4IID3bZIP, 4IID4bZIP, 4IID5bZIP, 4II910bZIP significantly increased salt tolerance in arabidopsis plants (fig. 18, 19, 20, 21 and 22).
The foregoing is merely illustrative of the embodiments of this application and it will be appreciated by those skilled in the art that variations and modifications may be made without departing from the principles of the application, and it is intended to cover all modifications and variations as fall within the scope of the application.

Claims (9)

1. The arabidopsis AtDPBF4 and bZIP recombinant gene fragment is characterized in that the recombinant gene fragment respectively forms recombinant fragments 4IID3bZIP, 4IID4bZIP, 4IID5bZIP and 4II910bZIP by respectively connecting the CRI fragment of the arabidopsis AtDPBF4 with the bZIP fragments of the AtDPBF3, the AtDPBF4, the AtDPBF5 and the At5G42910.
2. The recombinant gene fragment according to claim 1, wherein the recombinant gene fragment 4IID3bZIP is shown in sequence table SEQ No.3, and comprises an AtDPBF4 conserved region II shown in sequence table SEQ No.1 and an AtDPBF3 basic leucine region shown in sequence table SEQ No. 2; the recombinant gene fragment 4IID4bZIP is shown in a sequence table SEQ No.5 and comprises an AtDPBF4 conserved region II shown in a sequence table SEQ No.1 and an alkaline leucine region of AtDPBF4 shown in a sequence table SEQ No. 4; the recombinant gene fragment 4IID5bZIP sequence is shown as a sequence table SEQ No.7 and comprises an AtDPBF4 conservation region II shown as a sequence table SEQ No.1 and an alkaline leucine region of AtDPBF5 shown as a sequence table SEQ No. 6; the recombinant gene fragment 4II910bZIP sequence is shown as a sequence table SEQ No.9, and comprises an AtDPBF4 conserved region II shown as a sequence table SEQ No.1 and an alkaline leucine region of At5G42910 shown as a sequence table SEQ No. 8.
3. The application of the arabidopsis recombinant gene fragments 4IID4bZIP and 4IID5bZIP in seed germination in advance is characterized in that the sequences of the recombinant gene fragments are shown in sequence tables SEQ No.5 and SEQ No. 7; the application is achieved by over-expressing the gene fragment.
4. The application of the arabidopsis recombinant gene fragments 4IID3bZIP, 4IID4bZIP and 4IID5bZIP in increasing bolting number is characterized in that the sequence of the recombinant gene fragments is shown as sequence tables SEQ No.3, SEQ No.5 and SEQ No. 7; the application is achieved by over-expressing the gene fragment.
5. The application of the arabidopsis recombinant gene fragments 4IID3bZIP, 4IID4bZIP and 4IID5bZIP in the increase of the number of rosette leaves is characterized in that the sequence of the recombinant gene fragments is shown in sequence tables SEQ No.3, SEQ No.5 and SEQ No. 7; the application is achieved by over-expressing the gene fragment.
6. The application of the arabidopsis recombinant gene fragments 4IID3bZIP, 4IID4bZIP and 4IID5bZIP in enhancing drought resistance is characterized in that the sequence of the recombinant gene fragments is shown in sequence tables SEQ No.3, SEQ No.5 and SEQ No. 7; the application is achieved by over-expressing the gene fragment.
7. The application of the arabidopsis recombinant gene fragments 4IID3bZIP, 4IID4bZIP, 4IID5bZIP and 4II910bZIP in enhancing salt tolerance is characterized in that the sequences of the recombinant gene fragments are shown in sequence tables SEQ No.3, SEQ No.5, SEQ No.7 and SEQ No. 9; the application is achieved by over-expressing the gene fragment.
8. The recombinant method of the arabidopsis thaliana recombinant gene fragment 4IID3bZIP, 4IID4bZIP, 4IID5bZIP, 4II910bZIP according to claim 1, characterized in that the recombinant method comprises: (1) total RNA extraction reverse transcription; (2) The CRII region of AtDPBF4 is amplified with bZIP regions of AtDPBF3, atDPBF4, atDPBF5 and At5G42910 respectively; (3) Construction of plant genetic transformation vectors p1304-4IID3bZIP, p1304-4IID4bZIP, p1304-4IID5bZIP, p1304-4II910bZIP and sequencing.
9. The method for genetic transformation of arabidopsis thaliana p1304 vector of arabidopsis thaliana recombinant gene fragment 4IID3bZIP, 4IID4bZIP, 4IID5bZIP, 4II910bZIP according to claim 1, comprising:
(1) Construction of plant genetic transformation recombinant vectors p1304-4IID3bZIP, p1304-4IID4bZIP, p1304-4IID5bZIP and p1304-4II910 bZIP; (2) Plant genetic transformation recombinant vectors p1304-4IID3bZIP, p1304-4IID4bZIP, p1304-4IID5bZIP and p1304-4II910bZIP are respectively transformed into agrobacterium (GV 3101); (3) Transforming arabidopsis by using an agrobacterium-mediated flower dipping method, (4) screening the resistance of transgenic arabidopsis; (5) PCR and RT-PCR identification of transgenic Arabidopsis genome and transcripts: (6) Arabidopsis plants overexpressing the transgenic fragments 4IID3bZIP, 4IID4bZIP, 4II910bZIP were designated as: the A.thiana D3, A.thiana D4, A.thiana D5, A.thiana 910.
CN202310523614.7A 2023-05-10 2023-05-10 AtDPBF4 and bZIP recombinant gene fragment and application thereof Pending CN116987704A (en)

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