AcMT2a gene and its application

Abstract

The present invention provides an AcMT2a gene as shown in SEQ ID NO: 1, and its encoding gene and application. The present invention cloned the full-length cDNA of the AcMT2a gene from kiwifruit for the first time. The gene is highly expressed in bark, female flowers, roots, male flowers and fruits, participates in the stress response process of Pseudomonas syringae, and can affect the development and after-ripening and softening of kiwifruit fruits, and can be used as an important target gene for kiwifruit transgenic breeding. The gene is transferred into a prokaryotic expression strain, and the growth of the transgenic strain under heavy metal ion, NaCl stress, hydrogen peroxide stress and high temperature stress is significantly improved, and the growth under low temperature stress is significantly inhibited, which also shows that the protein encoded by the gene can improve the tolerance of plants to adversity by removing active oxygen. Therefore, the gene can be used as an important gene resource and can be used in the genetic engineering of kiwifruit and other plants or microorganisms to resist stress and heavy metal stress.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and specifically relates to an AcMT2a gene and an application thereof. Background Art

[0002] Kiwifruit belongs to the genus Actinidia of the family Actinidia. It is rich in nutrients and has a high vitamin C content. It can be eaten fresh or processed. It has high economic value and is known as the king of fruits. In recent years, although the kiwifruit industry has achieved rapid development, the growth and development of plants has been seriously affected by soil heavy metal pollution and abiotic stress (Huang Wei, Wan Mingchang, Qiao Rong. Development status and countermeasures of kiwifruit industry in Guizhou. Guizhou Agricultural Sciences, 2012, 40 (4): 184-186.).

[0003] We identified an AcMT2a gene from kiwifruit, which is closely related to the development and ripening and softening of kiwifruit fruit, and is involved in the kiwifruit's response to Pseudomonas syringae stress. It can be used as an important target gene for kiwifruit transgenic breeding. This gene can also improve the growth of prokaryotic strains under heavy metal stress, sodium chloride stress, hydrogen peroxide stress and high temperature stress. It can be used as an important genetic resource and can be applied in the genetic engineering of kiwifruit and other plants or microorganisms for stress resistance and heavy metal stress resistance. Summary of the invention

[0004] The purpose of the present invention is to overcome the deficiencies in the prior art and to provide an AcMT2a gene and an application thereof.

[0005] The first aspect of the present invention is to provide a kiwifruit AcMT2a gene, whose nucleotide sequence is shown in SEQ ID NO:1.

[0006] The second aspect of the present invention is to provide a protein encoded by the kiwifruit AcMT2a gene according to the first aspect of the present invention, the amino acid sequence of which is shown in SEQ ID NO:2.

[0007] The third aspect of the present invention is to provide a recombinant vector or host bacteria containing the kiwifruit AcMT2a gene coding region described in the first aspect of the present invention.

[0008] The original vector may be a vector commonly used in the field of gene recombination, such as a virus, a plasmid, etc. The present invention is not limited to this. In a specific embodiment of the present invention, the original vector is a pET28a vector plasmid or a pMD18-T vector plasmid, but it should be understood that the present invention may also use other plasmids, or viruses, etc.

[0009] Preferably, the original vector is a PET28a expression vector, and the kiwifruit AcMT2a gene coding region is located between the EcoR I and SalI restriction endonuclease sites of the PET28a expression vector.

[0010] The fourth aspect of the present invention is to provide the use of the AcMT2a gene as described in the first aspect of the present invention, or the protein as described in the second aspect of the present invention, or the recombinant expression vector as described in the third aspect of the present invention in improving the resistance of prokaryotic expression strains to copper metal stress, and / or zinc metal stress, and / or NaCl stress, and / or hydrogen peroxide stress, and / or high temperature stress; or the use of the AcMT2a gene as described in the first aspect of the present invention, or the protein as described in the second aspect of the present invention, or the recombinant expression vector as described in the third aspect of the present invention in reducing the resistance of prokaryotic expression strains to low temperature stress.

[0011] The prokaryotic expression strain may be a prokaryotic expression strain commonly used in the field of gene recombination, which is not limited in the present invention, such as E. coli BL21 (DE3), E. coli Rosetta and E. coli OrigamiB (DE3), etc. In a specific embodiment of the present invention, the prokaryotic expression strain is Escherichia coli E. coli BL21 (DE3).

[0012] The fifth aspect of the present invention is to provide the use of the AcMT2a gene as described in the first aspect of the present invention, or the protein as described in the second aspect of the present invention, or the recombinant expression vector or host bacteria as described in the third aspect of the present invention in regulating the development and / or ripening and softening of kiwifruit.

[0013] The sixth aspect of the present invention is to provide the use of the AcMT2a gene as described in the first aspect of the present invention, or the protein as described in the second aspect of the present invention, or the recombinant expression vector or host bacteria as described in the third aspect of the present invention in regulating the stress response of kiwifruit to Pseudomonas syringae.

[0014] The seventh aspect of the present invention is to provide the use of the AcMT2a gene as described in the first aspect of the present invention, or the protein as described in the second aspect of the present invention, or the recombinant expression vector or host bacteria as described in the third aspect of the present invention in improving the tolerance of plants to adversity by removing reactive oxygen.

[0015] The eighth aspect of the present invention is to provide a primer pair, wherein the primer pair is: 5'-TCTCTCACAACTCTCTCTCTCTC-3' and 5'-TGCTTTGTAGAAGGCTGGTAATTAG-3'; or the primer pair is: 5'-GAAAAATGTCTTGCTGCGGAG-3' and R: 5'-TACATCTGGCACCACAACGAC-3'; or the primer pair is: 5'-CCG GAATTC ATGTCTTGCTGCGGAGG-3' and 5'-ACGC GTCGAC TCATTTGCAGGTGCAAG-3'.

[0016] The present invention cloned the full-length cDNA of the AcMT2a gene from kiwifruit for the first time. Gene expression analysis showed that the AcMT2a gene was highly expressed in bark, female flowers, roots, male flowers and fruits. Pseudomonas syringae infection of kiwifruit can regulate the expression of the AcMT2a gene, and the expression change of the AcMT2a gene is closely related to the development and ripening and softening of kiwifruit fruits, indicating that the gene is involved in the response process to Pseudomonas syringae stress and can affect the development and ripening and softening of kiwifruit fruits. It can be used as an important target gene for kiwifruit transgenic breeding. The gene was transferred into a prokaryotic expression strain, and the transgenic strain was exposed to heavy metal ions (Cu 2+ 、Zn 2+ ), NaCl stress, hydrogen peroxide stress and high temperature stress, and its growth was significantly improved under low temperature stress, and its growth was significantly inhibited under low temperature stress, indicating that the protein encoded by the AcMT2a gene can improve the tolerance of plants (such as kiwifruit) to adversity by removing reactive oxygen. Therefore, this gene can be used as an important gene resource and can be used in the genetic engineering of kiwifruit and other plants or microorganisms to resist stress and heavy metal stress. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is tissue-specific expression analysis of the AcMT2a gene (leaves, seeds, bark, fruits, female flowers, male flowers, and roots).

[0018] Figure 2 Transcriptome data analysis of AcMT2a gene in kiwifruit tissue samples treated with different methods.

[0019] Figure 3 This is the prokaryotic expression analysis of AcMT2a (the band indicated by the arrow is the specifically expressed target protein produced after IPTG induction).

[0020] Figure 4 A-4F shows the growth of transgenic Escherichia coli on culture medium (A: OD under heavy metal copper ion stress 600Determination; B: OD under hydrogen peroxide stress 600 Determination; C: OD under sodium chloride stress 600 Determination; D: OD under heavy metal zinc ion stress 600 Determination; E: OD under 20℃ stress 600 Determination; F: OD under 45℃ stress 600 determination). DETAILED DESCRIPTION

[0021] The present invention will be further described below with reference to the accompanying drawings in conjunction with specific embodiments to better understand the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. Where the manufacturer of the reagents or instruments used is not indicated, they are all conventional products that can be obtained commercially.

[0022] Example 1. Cloning of the kiwifruit AcMT2a gene

[0023] The specific primers were designed as follows:

[0024] F (5′ end): 5′-TCTCTCACAACTCTCTCTCTCTC-3′;

[0025] R (3′ end): 5′-TGCTTTGTAGAAGGCTGGTAATTAG-3′.

[0026] PCR amplification was performed using cDNA from the bark of "Hongyang" kiwifruit as a template (obtained by reverse transcription with random primers) and primers F and R. The reaction system was: cDNA template 1 μL, 10×PCRBuffer 2 μL, Taq plus DNA Polymerase (5 U·μL -1 )0.2μL, dNTP(2.5mmol·L -1 )1.6μL, upstream and downstream primers (10μmol·L -1 ) 1 μL each, ddH2O 13.2 μL, total 20 μL. Amplification program: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 60 s, 30 cycles; 72℃ extension for 10 min.

[0027] The obtained nucleotide fragment of about 320 bp was connected to the PMD18-T vector (TAKARA) for sequencing. The fragment sequence is shown in SEQ ID No: 1, with a total length of 325 nucleotides, including an open reading frame (ORF, nucleotide sequence of positions 35-289 from the 5' end of sequence 1), 34 nucleotides of 5'-UTR (nucleotide sequence of positions 1-34 from the 5' end of sequence 1) and 36 nucleotides of 3'-UTR (nucleotide sequence of positions 290-325 from the 5' end of sequence 1), encoding a protein of 84 amino acids (SEQ ID No: 2 in the sequence table) with a molecular weight of about 8.5 kDa. The gene was named AcMT2a gene, and the PMD18-T recombinant vector containing the nucleotides of SEQ ID No: 1 was named PMD18-AcMT2a.

[0028] Example 2. Analysis of the expression pattern of AcMT2a gene in kiwifruit

[0029] <1> Tissue-specific expression of AcMT2a gene in kiwifruit

[0030] Real-time fluorescence quantitative PCR was performed using cDNA obtained by reverse transcription of total RNA from bark, male flowers, roots, female flowers, fruits, seeds, and leaves of "Hongyang" kiwifruit as templates and using AcMT2a gene-specific primers F: 5'-GAAAAATGTCTTGCTGCGGAG-3' and R: 5'-TACATCTGGCACCACAACGAC-3'. The reaction system was 2 μL template, 10 μL 2×SYBR Premix, and 10 μmol·L -1 0.3 μL of upstream and downstream primers were added; the amplification program was 95℃ pre-denaturation for 30s; 94℃ for 5s, 60℃ for 20s, 72℃ for 20s, 45 cycles. The primer amplification efficiency and the corresponding Qr value were calculated using LightCycler 4.05 software. The results showed that the gene was expressed in all 7 tissues of "Hongyang" kiwifruit, with the highest expression in the bark of "Hongyang" kiwifruit, followed by male flowers, and the lowest in leaves ( Figure 1 ).

[0031] <2> Effects of different treatments on kiwifruit on the expression of AcMT2a gene

[0032] High-throughput sequencing technology can be used to obtain changes in the spatiotemporal expression patterns of genes in species at different developmental stages and under adverse stress. In this experiment, transcriptome data from the Kiwifruit Genome Database (http: / / kiwifruitgenome.org / ) were used to obtain the RPKM values ​​of AcMT2 in kiwifruit tissue samples under different treatments and analyze its expression pattern.

[0033] Samples HY0, HY12, HY24, HY48 and HY96 refer to the barks of 'Hongyang' kiwifruit inoculated with Pseudomonas syringae, which can cause kiwifruit bacterial canker, and the barks were collected 0 hours, 12 hours, 24 hours, 48 ​​hours and 96 hours after inoculation, respectively.

[0034] Samples Aa_0DPI, Aa_2DPI, Aa_14DPI of 'Ruanzao' kiwifruit leaves were collected 0 days, 2 days and 14 days after inoculation with Pseudomonas syringae.

[0035] Samples Ae_0DPI, Ae_2DPI, and Ae_14DPI of 'hairflower' kiwifruit leaves were collected 0 days, 2 days, and 14 days after inoculation with Pseudomonas syringae.

[0036] Samples AH_0DPI, AH_2DPI, AH_14DPI'Hongyang' kiwifruit leaves were collected 0 days, 2 days and 14 days after inoculation with Pseudomonas syringae.

[0037] Samples AJ_0DPI, AJ_2DPI, and AJ_14DPI of 'Jinyan' kiwifruit leaves were collected 0 days, 2 days, and 14 days after inoculation with Pseudomonas syringae.

[0038] The whole kiwifruit seedlings were collected from samples HealtyControl, I3, I24, and I48 that were not inoculated with Pseudomonas syringae, and those inoculated with Pseudomonas syringae for 3 hours, 24 hours, and 48 hours.

[0039] Samples ASM, ASMI3, ASMI24, and ASMI48 refer to Actinidia kiwifruit seedlings treated with the plant activator benzothiazide (ASM) for 15 days, and the whole kiwifruit seedlings were collected after being untreated or inoculated with Pseudomonas syringae for 3 hours, 24 hours, and 48 hours.

[0040] Samples DAP20_immature, DAP120_matureGreen, and DAP127_ripe were collected from Hongyang kiwifruit 20 days, 120 days, and 127 days after pollination.

[0041] Samples 147DAFB, 168DAFB, 175DAFB, and 223DAFB were collected from 'Hort16A' kiwifruit fruits at 147 days, 168 days, 175 days, and 223 days after flowering.

[0042] Samples 231DAFB_CT, 231DAFB_1DAT, 231DAFB_2DAT, and 231DAFB_4DAT were collected from the 'Hort16A' kiwifruit fruits 231 days after anthesis, which were untreated, treated with ethylene for 1 day, treated with ethylene for 2 days, and treated with ethylene for 4 days.

[0043] Samples Sanuki_Gold_Control, Sanuki_Gold_5CW4, and Sanuki_Gold_20CW4 refer to fruits collected from mature 'Sanuki_Gold' kiwifruit, stored at 5 degrees for 4 weeks, and stored at 20 degrees for 4 weeks.

[0044] AH_leaf, AH_root, AH_stem are the leaves, roots and stems of Hongyang kiwifruit.

[0045] The results are as follows Figure 2 As shown. After inoculation with Pseudomonas syringae, the expression of AcMT2 in the bark of "Hongyang" kiwifruit and the tissue culture seedlings of Actinidia chinensis was significantly upregulated in the early stage, and then decreased and returned to the initial level; while the AcMT2a gene was significantly upregulated in the leaves of "Jinyan" kiwifruit, downregulated in the leaves of "Hongyang" kiwifruit and "Maohua" kiwifruit, and had little change in the leaves of "Ruanzao" kiwifruit; and after treatment with the plant disease resistance chemical inducer benzothiazide (ASM), its expression level decreased significantly. As the fruit develops, AcMT2a shows a significant downregulation in the fruits of both "Hongyang" and "Hort16A" varieties; but in the ripening and softening stage of kiwifruit, ethylene and low temperature, two regulatory means to promote fruit ripening, can upregulate the expression of AcMT2a gene. The results showed that the AcMT2a gene was closely related to the development and ripening and softening of kiwifruit, and was involved in the response of kiwifruit to Pseudomonas syringae stress.

[0046] Example 3. Prokaryotic expression and functional verification of AcMT2a gene

[0047] The prokaryotic expression vector of the AcMT2a gene was constructed using the PET28a expression vector (the expression vector in this embodiment is only an example, and the present invention can also use other expression plasmids and viral vectors, etc.), and the E. coli expression strain E. coli BL21 (DE3) was used to induce the recombinant protein, and the effect of the recombinant protein on the growth of the BL21 strain was determined. The specific method is as follows:

[0048] <1> Obtaining the recombinant vector containing the coding region of AcMT2a gene

[0049] Design of primers for the coding region of AcMT2a gene

[0050] F:5'-CCG GAATTC (EcoRI restriction site)ATGTCTTGCTGCGGAGG-3',

[0051] R:5'-ACGC GTCGAC (SalI restriction site) TCATTTGCAGGTGCAAG-3',

[0052] PCR amplification was performed using pMD18-AcMT2a as a template. The reaction system consisted of 1 μL cDNA template, 2 μL 10× PCR Buffer, and 5 U μL Taqplus DNA Polymerase. -1 )0.2μL, dNTP(2.5mmol·L -1 )1.6μL, upstream and downstream primers (10μmol·L -1 ) 1 μL each, ddH2O 13.2 μL, total 20 μL. The amplification procedure is: 95℃ pre-denaturation for 4 min; 94℃ denaturation for 45 s, 68℃ annealing for 30 s, 30 cycles in total; 72℃ extension for 8 min. The amplified product was connected to the PET28a expression vector by EcoR I and SalI double restriction digestion to obtain a recombinant vector. The recombinant vector was sequenced and identified, and the recombinant expression vector that was identified as correct and contained the nucleotide sequence of position 35-289 of sequence 1 in the sequence list and had an accurate reading frame was named PET28a-AcMT2a.

[0053] <2> Prokaryotic expression of AcMT2a gene

[0054] The obtained recombinant vector PET28a-AcMT2a was introduced into E. coli BL21 (DE3) to obtain recombinant expression bacteria. The correctly identified recombinant bacteria were cultured in LB medium containing 50μg / mL ampicillin to OD600 = 0.4-0.6, IPTG (isopropyl-β-D-thiogalactoside) was added to a final concentration of 1mM, and induced at 30°C for 4h. The empty plasmid PET28a was used as a control, and the bacteria were collected by centrifugation. The bacterial protein was detected by 15% SDS-PAGE electrophoresis. The results showed that under IPTG induction, the AcMT2a gene achieved efficient heterologous expression, the recombinant protein contained the target protein, and the molecular weight of the protein was close to the theoretical molecular weight, about 16kDa ( Figure 3 ).

[0055] <3> Effects of AcMT2a recombinant protein on bacterial growth

[0056] The strains containing PET28a-AcMT2a and PET28a were cultured to the same OD600, IPTG was added to a final concentration of 1 mM, and the heavy metal ion (Cu) 2+ , 500μM; Zn 2+ The OD600 values ​​were measured after culturing for 0h, 0.5h, 1h, 1.5h, 2h, 2.5h, 3h and 3.5h under the stress of sodium (500μM), salt (NaCl, 500mM), hydrogen peroxide (H2O2, 1mM), low temperature (20℃) and high temperature (45℃). Figure 4 The results showed that, except for low temperature stress, the growth of strains containing PET28a-AcMT2a was higher than that of strains containing PET28a under other stresses, indicating that the protein encoded by the AcMT2a gene can improve the growth of strains under heavy metal copper ion, zinc ion, sodium chloride, hydrogen peroxide stress and high temperature stress, and also indicating that the protein encoded by the AcMT3a gene can improve the tolerance of plants (such as kiwifruit, etc.) to adversity by removing reactive oxygen. The AcMT2a gene was introduced into other prokaryotic expression bacteria (E. coli Rosetta and E. coli OrigamiB (DE3) etc.) for the above test. The results were the same as those of E. coli. The protein encoded by the AcMT2a gene can improve the growth of other prokaryotic strains under heavy metal stress, sodium chloride stress, hydrogen peroxide stress and high and low temperature stress.

[0057] The specific embodiments of the present invention are described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions made to the present invention are also within the scope of the present invention. Therefore, the equalization changes and modifications made without departing from the spirit and scope of the present invention should be included in the scope of the present invention.

Claims

1. Use of kiwifruit AcMT2a gene or protein encoded by kiwifruit AcMT2a gene or recombinant expression vector containing kiwifruit AcMT2a gene coding region in improving the growth of prokaryotic expression strains under copper metal stress and / or zinc metal stress and / or NaCl stress and / or hydrogen peroxide stress and / or high temperature stress; The nucleotide sequence of the kiwifruit AcMT2a gene is shown in SEQ ID NO: 1; The amino acid sequence of the protein encoded by the kiwifruit AcMT2a gene is shown in SEQ ID NO:

2.

2. The use according to claim 1, characterized in that: The original vector of the recombinant expression vector is the PET28a expression vector, and the kiwifruit AcMT2a gene coding region is located between the EcoR I and SalI restriction endonuclease sites of the PET28a expression vector.

3. The use according to claim 1 or 2, characterized in that: The prokaryotic expression strain is Escherichia coli E. coli BL21, E. coli Rosetta or E. coli OrigamiB.

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