CN117551656A - AcMT3b gene and application thereof - Google Patents
AcMT3b gene and application thereof Download PDFInfo
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- CN117551656A CN117551656A CN202311402546.5A CN202311402546A CN117551656A CN 117551656 A CN117551656 A CN 117551656A CN 202311402546 A CN202311402546 A CN 202311402546A CN 117551656 A CN117551656 A CN 117551656A
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- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 87
- 235000009436 Actinidia deliciosa Nutrition 0.000 claims abstract description 51
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- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 18
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- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims abstract description 5
- 229910052760 oxygen Inorganic materials 0.000 claims abstract description 5
- 239000001301 oxygen Substances 0.000 claims abstract description 5
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- 229910052751 metal Inorganic materials 0.000 claims description 4
- 239000002184 metal Substances 0.000 claims description 4
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- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 claims description 2
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 claims description 2
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 2
- 229910052802 copper Inorganic materials 0.000 claims description 2
- 108091008146 restriction endonucleases Proteins 0.000 claims description 2
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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Abstract
The invention provides an AcMT3b group shown as SEQ ID NO. 1Because of the coding gene and application thereof. The full-length cDNA of the AcMT3b gene is cloned from the kiwi fruit for the first time, the gene has high expression abundance in leaves, participates in the stress response process to pseudomonas syringae, can influence the development and the after-ripening softening of kiwi fruits, and can be used as an important target gene for kiwi fruit transgenic breeding. The gene is transferred into prokaryotic expression strain, and the transgenic strain is used for expressing heavy metal ions (Cu 2+ 、Zn 2+ ) The growth under NaCl stress, hydrogen peroxide stress and high and low temperature stress is obviously improved, which indicates that the protein coded by the AcMT3a gene can improve the tolerance of plants (such as kiwi fruits and the like) to adversity through scavenging active oxygen. Therefore, the gene can be used as an important gene resource and can be applied to stress resistance and heavy metal stress resistance genetic engineering of kiwi fruits and other plants or microorganisms.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to an AcMT3b gene and application thereof.
Background
The kiwi fruit belongs to the kiwi fruit family, and the kiwi fruit belongs to the kiwi fruit genus, has rich nutrition, high VC content, good fresh food and processing, high economic value and good reputation of the fruit. In recent years, the kiwi fruit industry has been rapidly developed, but is severely affected by soil heavy metal pollution and abiotic stress, and the growth and development of plants are severely affected (Huang Wei, wan Mingchang, qiao Rong. Development status and countermeasures of kiwifruit industry in Guizhou. Guizhou Agricultural Sciences,2012,40 (4): 184-186.).
The AcMT3b gene is identified from the kiwi fruits, is closely related to the development and the after-ripening softening of the kiwi fruits, participates in the stress response process of the kiwi fruits to pseudomonas syringae, can be used as an important target gene for transgenic breeding of the kiwi fruits, can improve the growth of prokaryotic strains under heavy metal stress, sodium chloride stress, hydrogen peroxide stress and high and low temperature stress, can be used as an important gene resource, and can be applied to stress resistance and heavy metal stress resistance genetic engineering of the kiwi fruits and other plants or microorganisms.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides an AcMT3b gene and application thereof.
The first aspect of the invention provides a kiwi fruit AcMT3b gene, the nucleotide sequence of which is shown as SEQ ID NO. 1.
In a second aspect, the invention provides the protein coded by the actinidia chinensis AcMT3b gene in the first aspect, and the amino acid sequence of the protein is shown as SEQ ID NO. 2.
In a third aspect, the invention provides a recombinant vector or host bacterium containing the coding region of the actinidia AcMT3b gene according to the first aspect of the invention.
Among them, the original vector may be a vector commonly used in the field of gene recombination, such as a virus, a plasmid, etc. The invention is not limited in this regard. In one embodiment of the invention, the original vector is a PET28a vector plasmid or a pMD18-T vector plasmid, but it is to be understood that the invention may be used with other plasmids, or viruses, etc.
Preferably, the original vector is a PET28a expression vector, and the kiwi fruit AcMT3b gene coding region is positioned between EcoR I and HindIII restriction enzyme sites of the PET28a expression vector.
In a fourth aspect, the invention provides the use of an AcMT3b gene according to the first aspect of the invention, or a protein according to the second aspect of the invention, or a recombinant expression vector according to the third aspect of the invention, to increase copper metal stress, and/or zinc metal stress, and/or NaCl stress, and/or hydrogen peroxide stress, and/or high temperature stress, and/or low temperature stress resistance of a prokaryotic expression strain.
The prokaryotic expression strain may be a prokaryotic expression strain commonly used in the field of gene recombination, and the invention is not limited thereto, such as E.coli BL21 (DE 3), E.coli Rosetta, E.coli Origami B (DE 3), and the like. In a specific embodiment of the invention, the prokaryotic expression strain is E.coli BL21 (DE 3).
In a fifth aspect, the invention provides the use of the AcMT3b gene according to the first aspect of the invention, or the protein according to the second aspect of the invention, or the recombinant expression vector or host bacterium according to the third aspect of the invention, in regulating and controlling the development and/or post-ripening softening of kiwi fruits.
In a sixth aspect, the invention provides the use of an AcMT3b gene according to the first aspect of the invention, or a protein according to the second aspect of the invention, or a recombinant expression vector or host bacterium according to the third aspect of the invention, for modulating the stress response of kiwi to pseudomonas syringae.
In a seventh aspect, the present invention provides the use of an AcMT3b gene according to the first aspect of the invention, or a protein according to the second aspect of the invention, or a recombinant expression vector or host bacterium according to the third aspect of the invention, for increasing the tolerance of a plant to stress by scavenging active oxygen.
An eighth aspect of the present invention provides a primer pair, wherein the primer pair is: 5'-ATTTATCGCTCACCATGTCGGAC-3' and 5'-AGCTGTAAGGAAACACGGACACA-3'; or the primer pair is as follows: 5'-ATGTGCTTGTTTTGTGGTCAATAATA-3' and 5'-ACACAGTCGACACTCACATTTTATTAG-3'; or the primer pair is as follows: 5' -CCGGAATTCATGTCGGACAAGTGCG-3 'and 5' -CCCAAGCTTCTGATCGCAGGTGCAG-3'。
According to the invention, full-length cDNA of the AcMT3b gene is cloned from the kiwi fruit for the first time, gene expression analysis shows that the expression abundance of the AcMT3b gene in leaves is high, the expression of the AcMT3b gene can be regulated and controlled by the pseudomonas syringae infected kiwi fruit, and the change of the expression of the AcMT3b gene is closely related to the development and the after-ripening softening of the kiwi fruit, so that the gene participates in the stress response process of the pseudomonas syringae, can influence the development and the after-ripening softening of the kiwi fruit, and can be used as an important target gene for transgenic breeding of the kiwi fruit. The gene is transferred into prokaryotic expression strain, and the transgenic strain is used for expressing heavy metal ions (Cu 2+ 、Zn 2+ ) The obvious improvement of growth under NaCl stress, hydrogen peroxide stress and high and low temperature stress shows that the protein coded by the AcMT3a gene can improve the tolerance of plants (such as kiwi fruits and the like) to adversity through scavenging active oxygen. Therefore, the gene can be used as an important gene resource in kiwi fruits and other plants or microorganismsThe stress resistance and heavy metal stress resistance gene engineering of the plant are applied.
Drawings
FIG. 1 shows the analysis of tissue-specific expression of the AcMT3b gene (leaves, seeds, bark, fruit, female, male, root).
FIG. 2 is a transcriptome data analysis of AcMT3b gene in different treated kiwi fruit tissue samples.
FIG. 3 shows the prokaryotic expression analysis of AcMT3b (the bands indicated by the arrows are specifically expressed target proteins after induction by IPTG).
FIGS. 4A-4F show the growth of transgenic E.coli on the medium (A: OD under heavy metal copper ion stress 600 Measuring; b: OD under Hydrogen peroxide stress 600 Measuring; c: OD under sodium chloride stress 600 Measuring; d: OD under heavy metal zinc ion stress 600 Measuring; e: OD under stress at 20 ℃ 600 Measuring; f: OD under stress at 45 ℃ 600 And (3) measuring).
Detailed Description
The invention will be further described with reference to specific embodiments in order to provide a better understanding of the invention. The specific techniques or conditions are not identified in the examples and are performed according to techniques or conditions described in the literature in this field or according to the product specifications. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
EXAMPLE 1 cloning of the Actinidia chinensis AcMT3b Gene
Specific primers were designed as follows:
f (5' end): 5'-ATTTATCGCTCACCATGTCGGAC-3';
r (3' end): 5'-AGCTGTAAGGAAACACGGACACA-3'.
PCR amplification was performed using "red sun" kiwi leaf cDNA as template (obtained by reverse transcription with random primers), F and R as primers. The reaction system is as follows: cDNA template 1. Mu.L, 10 XPCRBuffer 2. Mu.L, taq plus DNAPolymerase (5U. Mu.L) -1 )0.2μL、dNTP(2.5mmol·L -1 ) 1.6. Mu.L, upstream and downstream primers (10. Mu. Mol.L) -1 ) 1 mu L, ddH each 2 O13.2. Mu.L, total 20. Mu.L. The amplification procedure was: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, annealing at 58℃for 30s, elongation at 72℃for 60s,30 cycles; extending at 72℃for 10min.
A200 bp nucleotide fragment obtained is connected to a PMD18-T vector (TAKARA company) for sequencing, the sequence of the fragment is shown as SEQ ID No. 1, the full length of the fragment is 359 nucleotides, and the fragment comprises an open reading frame (ORF, 15-209 nucleotide sequences from the 5' end of sequence 1), a 5' -UTR of 14 nucleotides (1-14 nucleotide sequences from the 5' end of sequence 1) and a 3' -UTR of 150 nucleotides (210-359 nucleotide sequences from the 5' end of sequence 1), and the fragment encodes a protein with 64 amino acids (sequence 2 in the sequence table) and a molecular weight of about 6.6 kDa. This gene was designated as AcMT3b gene, and the above-mentioned PMD18-T recombinant vector containing the nucleotide sequence of SEQ ID No. 1 was designated as PMD18-AcMT3b.
EXAMPLE 2 Kiwi AcMT3b Gene expression Pattern analysis
<1> Kiwi berry AcMT3b Gene tissue-specific expression
The cDNA randomly reverse transcribed from the RNA of the leaves, seeds, bark, fruits, female flowers, male flowers and roots of red-yang kiwi fruits is used as a template, and the AcMT3b gene specific primer (F: 5'-ATGTGCTTGTTTTGTGGTCAATAATA-3'; R: 5'-ACACAGTCGACACTCACATTTTATTAG-3') is used for carrying out real-time fluorescence quantitative PCR, wherein the reaction system comprises 2 mu L template, 10 mu L2 XSYBR Premix and 10 mu mol.L -1 0.3. Mu.L each of the upstream and downstream primers of (a); the amplification procedure was 95℃pre-denatured for 30s;94℃for 5s,60℃for 20s,72℃for 20s,45 cycles. The primer amplification efficiency and corresponding Qr values were calculated using the LightCycler 4.05 software, respectively. The results showed that the gene was expressed in 7 tissues of "red sun" kiwi but higher in leaves than in the other 6 tissues (FIG. 1)
<2> effect of different treatments on AcMT3b Gene expression on Kiwi fruit
The change of the gene space-time expression mode of the species under different development periods and adversity stress can be obtained by a high-throughput sequencing technology. The experiment utilizes Kiwifruit Genome Database (http:// kiwifriitgenome. Org /) to obtain RPKM values of AcMT3b in kiwi fruit tissue samples under different treatments, and analyzes the expression mode.
Samples aa_0DPI, aa_2DPI, aa_14DPI' actinidia arguta leaves were inoculated with pseudomonas syringae for 0, 2 and 14 days before leaf collection.
Samples ae_0DPI, ae_2DPI, ae_14DPI 'maohua' kiwi fruit leaves were inoculated with pseudomonas syringae for 0, 2 and 14 days before the leaves were collected.
Samples ah_0DPI, ah_2DPI, ah_14DPI 'red sun' kiwi leaf were inoculated with pseudomonas syringae for 0 day, 2 days and 14 days before leaf collection.
Samples AJ_0DPI, AJ_2DPI, AJ_14DPI 'golden' kiwi leaves were inoculated with Pseudomonas syringae for 0, 2 and 14 days before the leaves were collected.
Samples HealtyControl, I, I24 and I48 were not inoculated with Pseudomonas syringae, and Pseudomonas syringae were inoculated for 3 hours, 24 hours and 48 hours, and the whole kiwi seedlings were collected.
Samples ASM, ASMI3, ASMI24, ASMI48 refer to Chinese goosebeery seedlings after 15 days of treatment with the plant activator, namely, benzothiadiazine (ASM), and the whole Chinese goosebeery seedlings were collected after 3 hours, 24 hours and 48 hours of untreated and pseudomonas syringae inoculation.
Samples dap20_image, dap120_ matureGreen, DAP127 _slope collected fruits of red kiwi 20 days, 120 days, and 127 days after pollination.
Samples 147DAFB, 168DAFB, 175DAFB, 223DAFB collected the 'Hort16A' kiwi fruit 147 days, 168 days, 175 days and 223 days after flowers.
Samples 231DAFB_CT, 231DAFB_1DAT, 231DAFB_2DAT, 231DAFB_4DAT 'Hort16A' kiwi fruit untreated, ethylene treated for 1 day, ethylene treated for 2 days, and ethylene treated for 4 days after flower harvesting.
Samples Sanuki_gold_ Control, sanuki _gold_5CW4, sanuki_gold_20CW4 refer to the collection of ripe 'Sanuki_gold' kiwi fruits, 4 weeks at 5 degrees and 4 weeks at 20 degrees.
Leaf, root, stem of ah_leaf, ah_root, ah_stem red kiwi fruit.
The results are shown in FIG. 2. After the pseudomonas syringae is inoculated, the AcMT3b genes in the red-yang kiwi fruit bark and the Chinese kiwi fruit tissue culture seedlings are obviously down-regulated; the expression level of the actinidia chinensis leaves AcMT2 in four varieties (soft date, red sun, erigeron breviscapus and golden gorgeous) is obviously up-regulated; however, the expression level of the chemical inducer of plant disease resistance, namely, the benzothiadiazine (ASM) is obviously reduced after the treatment. Along with the development of fruits, acMT3b shows obvious up-regulation expression in fruits of two varieties of red sun and Hort 16A; however, in the post-ripening softening stage of kiwi fruits, two regulation means of promoting post-ripening of fruits, namely ethylene and low temperature, can down regulate the expression of AcMT3b genes. The result shows that the AcMT3b gene is closely related to the development and post-maturation softening of kiwi fruits and participates in the stress response process of kiwi fruits to pseudomonas syringae.
Example 3 functional verification of prokaryotic expression of AcMT3b Gene
The prokaryotic expression vector of the AcMT3b gene is constructed by using the PET28a expression vector (the expression vector is only used as an example in the embodiment, other expression plasmids, virus vectors and the like can be adopted in the invention), meanwhile, the E.coli BL21 (DE 3) is adopted to induce recombinant protein, and the influence of the recombinant protein on the growth of the BL21 strain is measured by the following specific method:
<1> acquisition of recombinant vector containing coding region of AcMT3b Gene
Designing AcMT3b gene coding region primer
F:5'-CCGGAATTC(EcoRI cleavage site) ATGTCGGACAAGTGCG-3',
R:5'-CCCAAGCTTC(HindIII cleavage site) TGATCGCAGGTGCAG-3',
PCR amplification was performed using pMD18-AcMT3b as a template, and the reaction system was 1. Mu.L, 10 XPCRBuffer 2. Mu.L, and Taqplus DNAPolymerase (5U. Mu.L) for cDNA template -1 )0.2μL、dNTP(2.5mmol·L -1 ) 1.6. Mu.L, upstream and downstream primers (10. Mu. Mol.L) -1 ) 1 mu L, ddH each 2 O13.2. Mu.L, total 20. Mu.L. The amplification procedure was: pre-denaturation at 95 ℃ for 4min; denaturation at 95℃for 45s, annealing at 58℃for 30s, 30 cycles total; extending at 72℃for 8min. The amplified product was ligated with PET28a expression vector by double cleavage with EcoRI and HindIII to give a heavy productGroup carrier. Sequencing and identifying the recombinant vector, and naming the recombinant expression vector with the correct 15 th-209 th nucleotide sequence and the correct reading frame containing the sequence 1 in the sequence table as PET28a-AcMT3b.
<2> prokaryotic expression of AcMT3b Gene
The recombinant vector PET28a-AcMT3b is introduced into E.coli BL21 (DE 3) to obtain recombinant expression bacteria, the recombinant bacteria with correct identification are cultured in LB culture medium containing 50 mug/mL ampicillin until OD600 = 0.4-0.6, IPTG (isopropyl-beta-D-thiogalactoside) is added to a final concentration of 1mM, induced culture is carried out for 4 hours at 30 ℃, no-load plasmid PET28a is used as a reference, bacterial cells are collected centrifugally, and bacterial cell proteins are subjected to 15% SDS-PAGE electrophoresis detection. The results show that the AcMT3b gene realizes high-efficiency heterologous expression under the induction of IPTG, the recombinant protein contains the AcMT3b, and the apparent molecular weight of the expressed protein is similar to the theoretical molecular weight and is about 18kDa (figure 3).
<2> Effect of AcMT3b recombinant protein on Strain growth
Strains containing PET28a-AcMT3b and PET28a were grown to the same OD 600 The strain containing PET28a-AcMT3b and PET28a was assayed for heavy metal ion (Cu 2+ ，500μM；Zn 2+ 500. Mu.M), salts (NaCl, 500 mM), hydrogen peroxide (H) 2 O 2 1 mM), low temperature (20 ℃) and high temperature (45 ℃) stress for 0h, 0.5h, 1h, 1.5h, 2h, 2.5h, 3h and 3.5h, respectively, and then determining the OD 600 Values. The results are shown in fig. 4: the growth of the strain containing PET28a-AcMT3b is higher than that of the strain containing PET28a, which shows that the protein coded by the AcMT3b gene can improve the growth of the strain under heavy metal copper ions, zinc ions, sodium chloride, hydrogen peroxide stress and high and low temperature stress (figures 4A-4F), and also shows that the protein coded by the AcMT3a gene can improve the tolerance of plants (such as kiwi fruits and the like) to stress by scavenging active oxygen. The results of the above experiments conducted by introducing the AcMT3b gene into other prokaryotic expression bacteria, and the proteins coded by the AcMT3b gene can improve the growth of other prokaryotic expression bacteria (E.coli Rosetta, E.coli OrigamiB (DE 3) and the like) under heavy metal stress, sodium chloride stress, hydrogen peroxide stress and high and low temperature stress.
The above description of the specific embodiments of the present invention has been given by way of example only, and the present invention is not limited to the above described specific embodiments. Any equivalent modifications and substitutions for the present invention will occur to those skilled in the art, and are also within the scope of the present invention. Accordingly, equivalent changes and modifications are intended to be included within the scope of the present invention without departing from the spirit and scope thereof.
Claims (10)
1. A kiwi fruit AcMT3b gene is characterized in that the nucleotide sequence is shown as SEQ ID NO. 1.
2. The protein coded by the actinidia AcMT3b gene of claim 1, wherein the amino acid sequence of the protein is shown as SEQ ID NO. 2.
3. A recombinant vector or host bacterium comprising the coding region of the actinidia AcMT3b gene of claim 1.
4. A recombinant expression vector according to claim 3, wherein the original vector is a PET28a expression vector and the kiwi AcMT3b gene coding region is located between the EcoR I and HindIII restriction enzyme sites of the PET28a expression vector.
5. Use of the actinidia AcMT3b gene of claim 1, or the protein of claim 2, or the recombinant expression vector of claim 3 or 4, to increase copper metal stress, and/or zinc metal stress, and/or NaCl stress, and/or hydrogen peroxide stress, and/or high temperature stress, and/or low temperature stress resistance of a prokaryotic expression strain.
6. The use according to claim 5, wherein the prokaryotic expression strain is E.coli BL21 (DE 3), E.coli Rosetta or E.coli OrigamiB (DE 3).
7. Use of the actinidia AcMT3b gene of claim 1, or the protein of claim 2, or the recombinant expression vector or host bacterium of claim 3 or 4, for modulating the development, and/or post-ripening softening of actinidia fruits.
8. Use of the actinidia AcMT3b gene of claim 1, or the protein of claim 2, or the recombinant expression vector or host bacterium of claim 3 or 4, for modulating the stress response of actinidia to pseudomonas syringae.
9. Use of the actinidia AcMT3b gene of claim 1, or the protein of claim 2, or the recombinant expression vector or host bacterium of claim 3 or 4, for increasing plant tolerance to stress by scavenging active oxygen.
10. A primer pair, wherein the primer pair is: 5'-ATTTATCGCTCACCATGTCGGAC-3' and 5'-AGCTGTAAGGAAACACGGACACA-3'; or alternatively
The primer pair is as follows: 5'-ATGTGCTTGTTTTGTGGTCAATAATA-3' and 5'-ACACAGTCGACACTCACATTTTATTAG-3'; or alternatively
The primer pair is as follows: 5' -CCGGAATTCATGTCGGACAAGTGCG-3 'and 5' -CCCAAGCTTCTGATCGCAGGTGCAG-3'。
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