CN106480065B - A kind of apple adversity gene MdoCKX7 and its application - Google Patents
A kind of apple adversity gene MdoCKX7 and its application Download PDFInfo
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Abstract
A kind of apple MdoCKX7 gene and its application, the nucleotide sequence of the MdoCKX7 gene is as shown in SEQ.ID.NO.1, using the transgenic technology of strong promoter driving principle, the overexpression carrier of MdoCKX7 gene is transferred in arabidopsis, obtains transgenic arabidopsis.First passage plant gene engineering technology of the present invention improve plant anti-salt, drought resistance and other beneficial to the production traits, obtain a kind of DNA fragmentation of anti contravariance related gene complete coding region section cloned and isolated from apple, and the function of the gene is demonstrated, it finally found that transgenic plant salt resistance and drought-resistant ability using MdoCKX7 gene in arabidopsis after overexpression significantly improve using its function.
Description
Technical Field
The invention relates to a stress resistance gene and application thereof, in particular to an apple stress resistance related gene MdocKX7 and application thereof.
Background
Fruit trees are generally planted in hilly and mountainous areas, saline-alkali soil and other soil environment conditions, and are influenced by drought, water shortage and salt stress in the aspect of cultivation. At present, due to the complexity of the genetic background of fruit trees, the screening work of stress-resistant genes of the fruit trees is relatively lagged; in addition, the resistance traits of plants are generally controlled by multiple genes, which further increases the difficulty of resistance research of fruit trees. Therefore, the research of a fruit tree stress resistance gene which can improve the resistance under drought and saline-alkali stress conditions and plays a key role in the resistance and yield of fruit trees is urgently needed.
Disclosure of Invention
The invention aims to solve the technical problem of providing an apple stress resistance gene MdockX7 and application thereof in drought-resistant and salt-resistant transgenic arabidopsis thaliana.
The technical scheme adopted by the invention for solving the technical problems is that an apple stress resistance gene is a DNA fragment of a complete coding section of a salt-resistant related gene separated and cloned from an apple and is named as MdMoCKX7, the nucleotide sequence of the apple stress resistance gene is shown in SEQ ID No.1, and the protein amino acid sequence is shown in SEQ ID No. 2.
The technical scheme adopted by the invention for further solving the technical problems is that the preparation method of the MdockX7 gene of the apple comprises the following steps:
(1) extracting RNA and reverse transcription from the tissue culture leaves of the Gala apples;
(2) obtaining the full-length cDNA sequence: designing degenerate primers MdockX7-F and MdockX7-R according to the MIEL1 gene conserved amino acid sequence in Arabidopsis found by NCBI, and then carrying out PCR amplification by taking the Gala genome cDNA synthesized by reverse transcription as a template to obtain a cDNA full-length sequence; wherein,
the MdocKX7-F sequence is shown in SEQ ID No.3,
the MdocKX7-R sequence is shown in SEQ.ID.NO. 4;
(3) and (3) recovering a PCR product, connecting a vector, transforming and sequencing to obtain an MdockX7 gene, wherein the Open Reading Frame (ORF) of the MdMoCKX7 gene is 1542bp, and 513 amino acids are coded.
Further, in the step (3), the PCR amplification reaction system is:
the PCR reaction program is pre-denaturation at 94 ℃ for 5 min; the cycle parameters are 94 ℃ denaturation 30s, 56 ℃ annealing 30s and 72 ℃ extension 90s, and 32 cycles are carried out; fully extending for 10min at 72 ℃.
The technical scheme adopted for further solving the technical problems is that an apple MdockX7 gene is applied to transgenic arabidopsis thaliana, and an overexpression vector of the MdMoCKX7 gene is transferred into arabidopsis thaliana by utilizing a transgenic technology of a strong promoter (cauliflower mosaic virus 35S promoter) driving principle, so that a transgenic plant is obtained. Experiments prove that the mortality rate of transgenic arabidopsis thaliana with the overexpression MdMoCKX7 gene is obviously reduced compared with that of a wild type under the stress of salt and drought, and the MdMoCKX7 gene plays an important role in stress resistance of plants.
In summary, the invention improves the salt resistance, drought resistance and other beneficial production traits of plants for the first time through a plant gene engineering technology, separates and clones a DNA fragment of a complete coding section of a stress resistance related gene from a Gala apple tissue culture seedling, verifies the function of the gene, and finally discovers that the salt resistance and drought resistance of a transgenic plant are obviously improved after overexpression is adopted by utilizing the function.
Drawings
FIG. 1 is a phenotypic observation of MdockX7 transgenic Arabidopsis. Wherein (A) root systems of wild type Arabidopsis (WT) and transgenic Arabidopsis (L1, L5, L6) which grow for 10 days are observed. (B-C) As compared with wild type Arabidopsis thaliana, the transgenic Arabidopsis thaliana has increased length of main root and significantly increased number of lateral roots. Bars is 10 mm.
FIG. 2 shows that MdockX7 transgenic Arabidopsis thaliana has reduced sensitivity to cytokinins. Wherein, (A) is root observation of wild type and transgenic arabidopsis treated by cytokinin (ZT) with different concentrations; (B-C) statistical analysis of main root length and lateral root number of wild type and transgenic arabidopsis thaliana treated by cytokinin with different concentrations; bars is 10 mm.
FIG. 3 shows the effect of overexpression of MdockX7 gene in Arabidopsis thaliana on salt resistance in Arabidopsis thaliana. Wherein (A) growing Arabidopsis thaliana on MS culture medium for 6 days, selecting seedlings with consistent growth vigor, transferring to MS culture medium containing 100mM NaCl or 200mM NaCl, continuously culturing for 10 days, and observing the growth condition of the seedlings; (B) the survival rate of Arabidopsis thaliana was counted for 10 days in MS medium containing 200mM NaCl.
FIG. 4 shows the effect of overexpression of MdockX7 gene in Arabidopsis thaliana on Arabidopsis thaliana drought resistance. Wherein (A) selecting wild type and transgenic arabidopsis thaliana with consistent growth vigor for drought treatment, and observing the growth condition of arabidopsis thaliana after the drought treatment is carried out for 20 days; observing the growth condition of the arabidopsis after rehydrating the arabidopsis for 2 days; (B) and (4) drought-treating the arabidopsis thaliana, and counting the survival rate of the plants.
Detailed Description
The invention is further described below with reference to the figures and examples.
Example 1: cloning of apple MdMoCKX7 Gene
Gala tissue culture leaf RNA extraction and reverse transcription
1. Extraction of total RNA using CTAB method:
(1) taking 1.5g of Gala apple tissue culture seedlings treated by 200mM NaCl salt for 24 hours, putting the Gala apple tissue culture seedlings into a precooled mortar, adding liquid nitrogen, grinding, and transferring into a precooled 50ml centrifuge tube;
(2) rapidly adding 10ml extraction buffer solution (in which PVP and mercaptoethanol are added at present) preheated to 65 deg.C, gently mixing, and water-bathing at 65 deg.C for 0.5 hr, wherein the composition of the extraction buffer solution is shown in Table 1;
TABLE 1 extraction buffer
| CTAB | 20％(w/v) |
| Tris-HCI | 0.1mol/l |
| EDTA | 25mmol/l |
| NaCI | 2mol/l |
| Mercaptoethanol | 2％(w/v) |
| PVP | 2％(w/v) |
| RNase free ddH2O | The volume is fixed to 100ml |
(3) Adding a mixture of water-saturated phenol/chloroform/isoamyl alcohol (25:24:1) with the same volume as the tube liquid obtained in the previous step, oscillating in ice bath for 0.5 hour, and centrifuging at 4 ℃ and 12,000rpm for 20 minutes; transferring the supernatant to a new 50ml centrifuge tube;
(4) adding precooled LiCl (10 mol/L) of 1/3 supernatant, standing at-20 ℃ for 3 hours, centrifuging at 12,000rpm for 30 minutes, and discarding the supernatant;
(5) adding 500 mul SSTE buffer solution to fully suspend and precipitate, and then subpackaging the mixture into 2 centrifugal tubes with the volume of 1.5ml averagely, wherein the composition of the SSTE buffer solution is shown in Table 2;
TABLE 2 SSTE buffer
| NaCI | 1mol/l |
| SDS | 0.5％(w/v) |
| EDTA | 10mmol/l |
| RNase free ddH2O | The volume is up to 10ml |
(6) Adding water saturated phenol/chloroform/isoamyl alcohol (25:24:1) mixture in the same volume as the suspension, shaking in ice bath for 10min, centrifuging at 12,000rpm for 10min at 4 deg.c, and transferring the supernatant to new 1.5ml centrifuge tube;
(7) adding chloroform/isoamyl alcohol (24:1) mixture with the same volume as the supernatant respectively, oscillating for 10 minutes in ice bath, centrifuging for 10 minutes at 12,000rpm at 4 ℃, and transferring the supernatant to a new 1.5ml centrifuge tube;
(8) adding precooled absolute ethyl alcohol with the volume 2.5 times of the volume of the supernatant, and standing for 1-2 hours at the temperature of-20 ℃;
(9) centrifuging at 12,000rpm for 20 min at 4 deg.C, washing with 70% ethanol for 2 times;
(10) centrifuging at 14,000rpm for 10min at 4 deg.C, and air drying the precipitate on a super clean bench; add 20. mu.l DEPC water to dissolve the RNA.
(11) The samples were stored at-80 ℃ for further use, or immediately subjected to the following reverse transcription experiments.
2. Synthesis of reverse transcribed cDNA first Strand
(1) The mixture of Table 3 (in Table 3, RNA extracted in step 1; if RNA stored at-80 ℃ C., it must be allowed to melt slowly on ice) was prepared in a 0.2ml microcentrifuge tube:
TABLE 3-mixture
| RNA | 2μg |
| Oligo(dT)Primer(50μM) | 1μl |
| dNTP Mixture(10mM each) | 1μl |
| RNase fｒee ddH2O | Up to 10μl |
(2) Gently mixing with a gun head, placing the microcentrifuge tube on a PCR instrument (65 ℃ for 5 minutes) for denaturation and annealing reaction, and then quenching on ice;
(3) the reverse transcription reaction solution of Table 4 was continuously prepared in the above-mentioned microcentrifuge tube.
TABLE 4 reverse transcription reaction solution
| The above-mentioned denatured and annealed reaction solution | 10μl |
| 5×PrimeScriptTM Buffer | 4μl |
| RNase Inhibitor(40U/μl) | 0.5μl |
| PrimeScriptTM RTase(200U/μl) | 1μl |
| RNase free ddH2O | 4.5μl |
| Total | 20μl |
(4) The reverse transcription reaction was performed on a PCR instrument under the following conditions: 30 ℃ for 10 minutes, 42 ℃ for 60 minutes, 70 ℃ for 15 minutes, and 4 ℃. The synthesized reverse transcription product cDNA was used for the subsequent related experiments.
3. Tailing of cDNA Ends
5 'RACE amplification was performed using TdT-terminal transferase as a template, and poly (C) tail added to the 3' end of the cDNA reverse transcribed and synthesized in step 2.
The PCR reagents of Table 5 were sequentially added to a 1.5ml centrifuge tube to a final volume of 50. mu.l.
TABLE 5PCR reaction reagents
| Purification of cDNA products | 25μl |
| 5×TdT Buffer | 10μl |
| 0.1％BSA | 5μl |
| 10mM dCTP (final concentration 0.5mM) | 2.5μl |
| TdT | 15U |
| ddH2O | Up to 50μl |
(1) Reacting at 37 ℃ for 30 minutes;
(2) add 50. mu.l (equal volume) of phenol/chloroform/isoamyl alcohol (25:24:1) and mix well; centrifuging, and transferring the upper layer (water layer) to another new 1.5ml centrifuge tube; (3) adding 50 μ l (equal volume) of chloroform/isoamyl alcohol (24:1), and mixing well; centrifuging, and transferring the upper layer (water layer) to another new 1.5ml centrifuge tube;
(4) adding 5 μ l (1/10 vol) of 3M NaAC (pH5.2), adding 125 μ l (2.5 times vol) of pre-cooled anhydrous ethanol, and standing at-20 deg.C for 30-60 min;
(5) centrifuging at 12,000rpm for 15 min at 4 deg.C, washing with 70% ethanol for 2 times;
(6) centrifuging at 12,000rpm for 10min at 4 deg.C, and air drying on a super clean bench; the DNA was dissolved in 20. mu.l of sterile water and stored at 4 ℃. The resulting tailed cDNA was used as template for the following 5' RACE amplification.
II, obtaining the full-Length cDNA sequence
According to the conserved amino acid sequence of the MIEL1 gene in Arabidopsis thaliana found by NCBI, degenerate primers (MdockX7-F and MdockX7-R) are designed, and cDNA synthesized by reverse transcription is used as a template for PCR amplification.
MdocKX 7-F: 5'-GACTACCGCTCTCTCTCTATACT-3' and the sequence is shown in SEQ ID NO. 3;
MdocKX 7-R: 5'-GAAACTAATTACGCCAATAAG-3' and the sequence is shown in SEQ ID NO. 4;
among them, the PCR amplification system is shown in Table 6.
TABLE 6 PCR amplification System
| 10 XPCR buffer (containing Mg)2+) | 2.5μl |
| dNTP(2.5mM/l) | 2.0μl |
| Primer 1 (10. mu.M/l) | 1.0μl |
| Primer 2 (10. mu.M/l) | 1.0μl |
| cDNA or genomic DNA template | 1.0μl |
| Taq enzyme | 0.2μl |
| ddH2O | Up to 25.0μl |
PCR reaction procedure: pre-denaturation at 94 ℃ for 5 min; the cycle parameters are 94 ℃ denaturation for 30 seconds, 56 ℃ annealing for 30 seconds and 72 ℃ extension for 90 seconds, and 32 cycles are carried out; fully extended for 10 minutes at 72 ℃. After the PCR reaction was completed, 1.0% agarose gel electrophoresis was performed to detect whether there was a band of an appropriate size, and the PCR product was recovered (recovered according to the Takara "Agarose gel DNA Purification Kit", Inc.), Vector ligation (3.0. mu.l of the PCR-recovered product was ligated with pMD18-T Vector, the procedure was as described in pMD18-T Vector), transformation (ligation product transformed into E.coli competent cell DH 5. alpha., inversely culturing for 12-20 hours at 37 ℃ on an LB plate culture medium coated with IPTG/X-Gal on the surface; the white colonies were picked up, culturing overnight in LB liquid culture medium), sequencing (placing 1ml of shaken bacteria into a 1.5ml centrifuge tube, sealing, after the sequence determination is carried out by Beijing Liu He Hua Dagen science and technology GmbH, the MdockX7 gene is obtained, the nucleotide sequence is shown in SEQ.ID.NO. 1; the amino acid sequence is shown in SEQ.ID.NO. 2.
Example 2: acquisition of transgenic Arabidopsis
1. Respectively disinfecting the obtained arabidopsis seeds with 70% alcohol for 3min, disinfecting with 4% sodium hypochlorite for 8-10 min (shaking for multiple times), washing with sterilized water for 5 times, and sucking dry water; sowing the seeds on a seed germination culture medium (directly spreading the seeds on the surface), performing light culture (at 25-28 ℃, 16h long day or 8h short day for 10d) until seedlings grow out, and transplanting the seedlings on a matrix for culture until the seedlings bloom.
2. Selecting a monoclonal colony of the agrobacterium tumefaciens, inoculating the colony in 10mL YEP liquid culture medium (containing 50mg/L hygromycin), performing shaking culture at 28 ℃ and 200rpm until OD600 is 0.6-0.8 (about 48 h); adding one mL of the bacterial liquid into 20mL of YEP liquid culture medium, performing shaking culture at 28 ℃ and 200rpm until OD600 is 0.6-0.8 (about 5 h); then, the cells were collected by centrifugation, and suspended and diluted 20-fold with an invader (containing 0.05g/ml sucrose and 0.03-0.05% Silweet) for further use.
3. Soaking arabidopsis inflorescence into the staining solution for 15-20 s, collecting fruit pods, and collecting 50 mg.L-1And after Hyg resistance screening, PCR detection is carried out to obtain a positive transgenic plant, T3 generation homozygote is obtained through continuous 3 generation screening, seeds are collected, and phenotype analysis is carried out.
Example 3: transgenic Arabidopsis thaliana root phenotype observation
1. MdockX7 transgenic Arabidopsis thaliana affecting root system development
Selecting wild type arabidopsis (WT) and transgenic arabidopsis (L1, L5, L6) which germinate for 4-10 days and grow in a consistent manner, vertically culturing for 6 days on an MS culture medium, observing the development condition of plant root systems, and counting the length of main roots and the number of lateral roots. As can be seen from fig. 1, ectopic expression of mdackx 7 gene in arabidopsis significantly promoted lateral root development, and transgenic arabidopsis also exhibited a phenotype of major root elongation.
2. Effect of MdockX7 transgenic Arabidopsis on cytokinin sensitivity
Treating wild type and transgenic Arabidopsis thaliana by cytokinin (0, 0.1, 1 and 10uM) with different concentrations, observing root development conditions of Arabidopsis thaliana, and counting the length of main roots and the number of lateral roots. As can be seen from FIG. 2, ectopic expression of the MdocKX7 gene in Arabidopsis significantly reduced the sensitivity to cytokinins.
Example 4: verification of stress resistance function of MdockX7 gene in arabidopsis thaliana
1. Effect of overexpression of MdockX7 Gene in Arabidopsis on salt resistance of Arabidopsis
Growing arabidopsis on an MS culture medium for 6 days, selecting seedlings with consistent growth vigor, transferring the seedlings to the MS culture medium containing 100mM NaCl or 200mM NaCl, continuously culturing for 10 days, observing the growth condition of the seedlings, and counting the survival rate. As can be seen from fig. 3, the transgenic arabidopsis thaliana exhibited a better growth state and a higher survival rate than the wild type under the salt stress condition.
2. Influence of overexpression of MdockX7 gene in Arabidopsis thaliana on drought resistance of Arabidopsis thaliana
Selecting wild type and transgenic arabidopsis thaliana with consistent growth vigor for drought treatment, and observing the growth condition of the arabidopsis thaliana after the drought treatment is carried out for 20 days; and (4) rehydrating the arabidopsis thaliana for 2 days, observing the growth condition of the arabidopsis thaliana, drought-treating the arabidopsis thaliana, and counting the survival rate of the plant. As can be seen from FIG. 4, the survival rate of the transgenic Arabidopsis thaliana after drought stress treatment is significantly higher than that of the wild type.
In conclusion, the result of the stress resistance function verification of the MdockX7 gene in Arabidopsis fully indicates that the MdMAX1 gene is a stress resistance related gene, and the salt resistance and drought resistance of a plant can be improved by the over-expression of the gene.
Claims (1)
1. An application of an apple MdockX7.2 gene in transgenic arabidopsis thaliana is characterized in that the application is to improve the salt resistance and drought stress resistance of the transgenic arabidopsis thaliana, and the nucleotide sequence of the gene is shown as SEQ No. ID. No. 1.
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