CN116479013A - BpWRKY70 gene for improving saline-alkali tolerance of white birch, protein coded by BpWRKY70 gene and application of BpWRKY70 gene - Google Patents
BpWRKY70 gene for improving saline-alkali tolerance of white birch, protein coded by BpWRKY70 gene and application of BpWRKY70 gene Download PDFInfo
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- 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
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Abstract
The invention provides a BpWRKY70 gene for improving the salt and alkali tolerance of white birch, and a coded protein and application thereof, belonging to the technical field of genetic engineering, wherein the nucleotide sequence of the BpWRKY70 gene of white birch is shown as SEQ ID No. 1. The BpWRKY70 gene provided by the invention can be used for cultivating saline-alkali tolerant transgenic white birch plants, and research results provide basis for improving the saline-alkali tolerant varieties of white birch, and have important scientific significance for revealing excellent genes of forest genetic engineering breeding. The BpWRKY70 gene of the white birch has obvious saline-alkali tolerance and does not influence the growth of plants, so that the gene has very important application prospect in the growth of transgenic plants, especially transgenic forests.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a BpWRKY70 gene for improving the saline-alkali tolerance of white birch, and a coded protein and application thereof.
Background
Plants are subjected to various stress during their growth and development, with biotic and abiotic stresses affecting all climatic stages of plant development. Biotic stresses include those from pathogenic bacteria, fungi, viruses, and the like. Abiotic stresses include drought, cold, heat, salt and oxidative stresses, and the like. Adaptation to these stresses and responses to different environmental stresses is critical for plant survival and for the continued generation. In order to resist adversity stress, plants respond to different environments by generating a series of responses, transcription factors play an important role in the response processes, the expression of target genes is regulated by specifically combining with cis-acting elements of downstream target gene promoter regions, the transcription factors, the promoters and interactions thereof are critical to transcriptional regulation, and the stress resistance of the plants is improved through transcriptional regulation processes.
The WRKY transcription factor family is considered to be one of the largest transcription factor families in plants, and has a key role in regulating plant stress response. WRKY proteins are classified according to the number of WRKY domains and the differences that exist in the zinc finger motifs. Generally WRKY proteins fall into three classes, namely class I, class II and class III. Group I proteins contain two WRKY domains, while the remaining two groups of proteins each contain one. Most members of the WRKY transcription factor have nuclear localization signals, serine/threonine rich regions, leucine zippers, kinase domains, glutamine rich regions, proline rich regions and other structures. These different structures confer different transcriptional regulatory functions to the WRKY transcription factor. It was found that WRKYTF is expected to function as a key regulatory protein by precisely binding to the W-box (TTGAC (C/T)) that regulates gene expression. Numerous studies have shown that the WRKY transcription factor family plays an important role in regulating abiotic stresses such as drought, salt, extreme temperatures. And can respond to various signal paths, such as abscisic acid, jasmonic acid, salicylic acid, mitogen-activated protein kinase (MAPK), active oxygen removal and the like.
Betula alba (Betula platyphylla Suk.) is one of the most important and precious broad-leaf tree species, widely distributed in the cold temperate zone and the cold zone forest of the northern hemisphere. The birch has hard wood texture, white and fine texture and wide application in furniture, building materials, papermaking and the like. The betula alba is a high-quality material for researching the stress-resistant mechanism of woody plants due to the excellent cold resistance, drought resistance and barren soil resistance. Therefore, the research on the white birch is continuous and intensive, the genetic quality of the white birch is continuously improved, and a new forest variety with high quality and high resistance is cultivated. Along with the continuous change of the global environment, plant survival faces serious challenges, soil salinization is aggravated, plant growth is not facilitated, and development and application of forestry are severely restricted. In a gene regulation network for plant stress resistance, a transcription factor is a molecular switch for regulating the expression of stress response genes, and compared with a functional gene, the transcription factor can regulate the expression of a plurality of related genes in stress, and is considered as a good candidate for improving plant stress resistance by using genetic engineering. In the invention, the WRKY transcription factor is utilized to enhance the salt and alkali tolerance of the white birch, so that the method is a more efficient method for improving the salt tolerance of plants.
Disclosure of Invention
In view of the above, the invention aims to provide a BpWRKY70 gene for improving the saline-alkali tolerance of white birch, and a coded protein and application thereof, and the BpWRKY70 gene provided by the invention improves the saline-alkali stress tolerance of white birch.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a BpWRKY70 gene for improving the salt and alkali tolerance of white birch, and the nucleotide sequence of the BpWRKY70 gene is shown as SEQ ID No. 1.
The invention also provides application of the BpWRKY70 gene in the technical scheme in the saline-alkali stress tolerance of white birch.
The invention also provides application of the BpWRKY70 gene in reducing the hydrogen peroxide content of the white birch leaves.
The invention also provides application of the BpWRKY70 gene in reducing the superoxide anion content of the white birch leaves.
The invention also provides application of the BpWRKY70 gene in improving the activity of birch superoxide dismutase.
The invention also provides an application of the BpWRKY70 gene in improving the activity of the betula peroxidase.
The invention also provides the protein coded by the BpWRKY70 gene in the technical scheme, and the amino acid sequence of the protein is shown as SEQ ID No. 2.
The invention also provides a method for cultivating the saline-alkali stress-resistant white birch, which comprises the following steps:
1) Connecting the BpWRKY70 gene in the technical scheme into a pROKII vector to obtain an over-expression vector;
2) Transferring the over-expression vector obtained in the step 1) into agrobacterium tumefaciens by adopting a conductivity method to obtain recombinant bacteria;
3) And (3) infecting the recombinant bacteria obtained in the step (2) by adopting a high-efficiency instantaneous infection technology to obtain a saline-alkali stress resistant white birch plant.
Preferably, the primers used for amplifying the BpWRKY70 gene are an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer is shown as SEQ ID No.3, and the nucleotide sequence of the downstream primer is shown as SEQ ID No. 4.
Preferably, the agrobacterium tumefaciens is agrobacterium tumefaciens EHA105.
The beneficial effects of the invention are as follows:
the invention provides a white birch BpWRKY70 gene for resisting saline-alkali stress, which can be used for cultivating a saline-alkali tolerant transgenic white birch plant, and research results provide basis for improving the saline-alkali tolerant varieties of white birch, and the white birch BpWRKY70 gene has obvious saline-alkali tolerant capability with important scientific significance for revealing excellent genes of forest genetic engineering breeding, and does not influence the growth of plants, so that the gene has very important application prospect in growing transgenic plants, in particular transgenic forests.
Drawings
FIG. 1 shows DAB staining results of BpWRKY70 transgenic birch under salt stress;
FIG. 2 shows the NBT staining results of BpWRKY70 transgenic birch under salt stress;
FIG. 3 shows the Evans blue staining results of BpWRKY70 transgenic birch under salt stress;
FIG. 4 is a measurement of superoxide dismutase (SOD) activity of BpWRKY70 transgenic birch under salt stress;
FIG. 5 is a measurement of Peroxidase (POD) activity of BpWRKY70 transgenic birch under salt stress;
FIG. 6 is a measurement of total protein concentration of BpWRKY70 transgenic birch under salt stress;
FIG. 7 is a BpWRKY70 transgenic Betula alba H under salt stress 2 O 2 Measuring the content;
FIG. 8 is a measurement of the relative conductivity of BpWRKY70 transgenic birch under salt stress.
Detailed Description
The invention provides a BpWRKY70 gene for improving the salt and alkali tolerance of white birch, wherein the nucleotide sequence of the BpWRKY70 gene is shown as SEQ ID No.1, and the BpWRKY70 gene is specifically as follows:
ATGGAGTCTTCTTGGCCGGAAAACTCATCCTCCAAGCCCAGAAAAGCCATTGAAGAGCTGATTCAAGGTCGCCAATTCGCAAATCAGCTCCGGGGACTACTCGGTAAGTCGATCGGAGATGATGGGTCAGTCCCAGCAAAAGATCTTGTTCTGAAAATCTTGAACTCCTTCACAAACACTCTTTCGATGTTGAATTCCGCCGAGTCCGAAGCCGACGTTTCTCAAATTCAGCCCAACACCCAAGCCAGTTCGCCGTGTTGGGATGCTCCCAAGTCGGAATCTTCCGGAGAAAGTTGCCGGAGTATCTCGACAGTCAAGGATCGGAGAGGATGTTATAAGAGAAGGAGGACTTCACTGACGACAACTAAAGACACCCCTACTCTGATCGATGACGGTCATGCATGGAGAAAATACGGACAAAAACTGATCCTTAATGCTAAATACCCAAGGCATTACTACAGGTGCACTCATAAATATGATCAGGGATGCCAAGCAGCGAAGCAGGTGCAAAGAATCCAAGAGGATCCGGCGATGCACAGGACTACATATATTGGGCAACACACGTGCAGAACGTTTCCAAAAGCCCCTGAATTAATCTTGGATTCTTCTCCCACAGACTCTCACTTTGTGCTCAGTTTTGACAACACCTTCACAAACAAACACAACCACCCGGTCCACCCCTTTCTCTCGTCATCCTTCCAATCAGTCAAACAGGAACACAAGGAAGAGATGCCCAGCGATGATGTCACCCACAACCAATCATCGTCGTCTGATTATCTCGTTTCCCCCGATCAGACGGCATTCGATTCATTCTACAACATGACTGTCTTATCATCACCGGTGGAGTCCGACGATAATGGGGTTTTGGTGGGCTCGGTCGACCACTTTGCCGATGACGATTTGATGGGTGCCTTTTCTTTTTAA。
the invention also provides application of the BpWRKY70 gene in the technical scheme in the saline-alkali stress tolerance of white birch.
The invention also provides application of the BpWRKY70 gene in reducing the hydrogen peroxide content of the white birch leaves.
The invention also provides application of the BpWRKY70 gene in reducing the superoxide anion content of the white birch leaves.
The invention also provides application of the BpWRKY70 gene in improving the activity of birch superoxide dismutase.
The invention also provides an application of the BpWRKY70 gene in improving the activity of the betula peroxidase.
The invention also provides a protein coded by the BpWRKY70 gene in the technical scheme, and the amino acid sequence of the protein is shown as SEQ ID No.2, and the protein is specifically as follows:
MESSWPENSSSKPRKAIEELIQGRQFANQLRGLLGKSIGDDGSVPAKDLVLKILNSFTNTLSMLNSAESEADVSQIQPNTQASSPCWDAPKSESSGESCRSISTVKDRRGCYKRRRTSLTTTKDTPTLIDGHAWRKYGQKLILNAKYPRHYYRCTHKYDQGCQAAKQVQRIQEDPAMHRTTYIGQHTCRTFPKAPELILDSSPTDSHFVLSFDNTFTNKHNHPVHPFLSSSFQSVKQEHKEEMPSDDVTHNQSSSSDYLVSPDQTAFDSFYNMTVLSSPVESDDNGVLVGSVDHFADDDLMGAFSF。
the invention also provides a method for cultivating the saline-alkali stress-resistant white birch, which comprises the following steps:
1) The birch BpWRKY70 gene in the technical scheme is connected into a pROKII vector to obtain an over-expression vector;
2) Transferring the over-expression vector obtained in the step 1) into agrobacterium tumefaciens by adopting a conductivity method to obtain recombinant bacteria;
3) And (3) infecting the recombinant bacteria obtained in the step (2) by adopting a high-efficiency instantaneous infection technology to obtain a salt stress-resistant white birch plant.
The BpWRKY70 gene of the technical scheme is connected into the pROKII vector to obtain a connection vector.
The invention preferably uses the birch cDNA as a template and uses the upstream and downstream primers to amplify to obtain the birch BpWRKY70 gene. The method for obtaining the cDNA of the white birch is not particularly limited, and can be obtained by a person skilled in the art according to conventional operation. The system and procedure for the amplification are not particularly limited, and those skilled in the art can operate conventionally. In the invention, sma I cleavage site is introduced into the upstream and downstream primers, and the nucleotide sequence of the upstream primer is shown as SEQ ID No.3, and is specifically as follows:
5’GACTCTAGAGGATCCCCGGGATGGAGTCTTCTTGGCC 3’；
the nucleotide sequence of the downstream primer is shown as SEQ ID No.4, and is specifically as follows:
5’ATTCGAGCTCGGTACCCGGGTTAAAAAGAAAAGGCACCC 3’。
in the invention, the pROKII vector is preferably subjected to SmaI digestion and then is subjected to BpWRKY70 gene with birch, and the digestion conditions are not particularly limited, so that the conventional method can be adopted by a person skilled in the art. The system and reaction conditions for the connection are not particularly limited by those skilled in the art, and those skilled in the art can employ conventional ones.
The invention transfers the obtained over-expression vector into agrobacterium tumefaciens by adopting a conductivity method to obtain recombinant bacteria. The present invention is not particularly limited to the conductivity method, and those skilled in the art can use a conventional conductivity method. In the present invention, the agrobacterium tumefaciens is preferably agrobacterium tumefaciens EHA105.
The invention uses high-efficiency instant infection technology to infect the white birch to obtain the salt stress-resistant white birch. The invention is not particularly limited to the high-efficiency instantaneous infection technology, and a person skilled in the art can adopt routine technology. In the present invention, the betula is preferably betula alba seedlings of 1 month.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Cloning of the 1 Betula alba BpWRKY70 Gene
1.1 cloning and obtaining a WRKY70 gene from northeast white birch, wherein the total length of the cDNA of the WRKY70 gene is 924bp, the gene sequence is shown as SEQ ID No.1, 307 amino acids are encoded, and the amino acid sequence is shown as SEQ ID No. 2.
2 instant BpWRKY70 gene white birch obtaining and stress tolerance identification
2.1 designing primers according to BpWRKY70 gene sequence, respectively introducing SmaI cleavage sites on the upper and lower streams, wherein the sequences of the primers are as follows:
F：5’GACTCTAGAGGATCCCCGGGATGGAGTCTTCTTGGCC 3’
R：5’ATTCGAGCTCGGTACCCGGGTTAAAAAGAAAAGGCACCC 3’
the coding region sequence of BpWRKY70 gene is obtained by PCR amplification of the white birch cDNA. The PCR reaction volume is 20 mu L, and the reaction system is as follows:
PCR reaction procedure:
after the amplified product is purified by agarose gel DNA purification kit (OMEGA), the specific operation steps are the same as the kit instruction, and the recovery quality of the product is detected by using 1% agarose gel electrophoresis.
2.2 construction of the overexpression vector pROKII-BpWRKY70
Coli containing pROKII empty plasmid was stored in laboratory, and pROKII plasmid was extracted using plasmid miniprep kit (Nanjinouzan Biotechnology Co., ltd.) and the specific procedure was as described in the kit. The products were checked for plasmid extraction quality using 1% agarose gel electrophoresis.
The pROKAI vector plasmid was digested with Sma I (Promega) endonuclease and recovered by purification, and the cleavage reaction system was as follows:
reaction conditions: and 4 hours at 25 ℃.
The enzyme-digested products were detected by 1% agarose gel electrophoresis, and the samples were purified and recovered using a purification recovery kit (OMEGA), and the specific procedures were as described in the kit instructions.
The gene was homologous to pROKII vector digested with SmaI (Promega). The connection system is as follows:
the ligation product transformed E.coli Top10 strain comprises the following steps:
(1) mu.L of the ligation solution was aspirated, and competent cells were added thereto, mixed well, and ice-bathed for 30min.
(2) Water bath at 42 ℃ for 60-90s and ice bath for 2min. 400. Mu.L of fresh LB liquid medium was added, and the culture was shake-cultured at 37℃and 220rpm for 1 hour.
(3) 4000rpm, and centrifuged for 1min. 300. Mu.L of supernatant was discarded. The cells were resuspended and plated on kan-resistant screening medium. Inverted culturing at 37deg.C for 12-24 hr.
Positive clones are identified, monoclonal clones are picked up and propagated in a liquid screening culture medium, bacterial liquid is taken as a template, and bacterial liquid PCR detection is carried out by using carrier primers, wherein the reaction system is as follows:
PCR reaction procedure:
the PCR products were detected by 1% agarose gel electrophoresis, positive clones were sequenced, correctly sequenced strains were propagated and plasmids were extracted for use.
After PCR verification and sequencing identification are correct, the recombinant plasmid (pROKII-BpWRKY 70) is transferred into the agrobacterium tumefaciens EHA105 strain by an electric shock method, and the specific operation steps are as follows:
(1) 1.0. Mu.g of recombinant plasmid was added to Agrobacterium competent cells, mixed well and transferred to a clean cuvette.
(2) After electric shock with 1750V voltage, 400 mu L of LB medium is added into the electric shock cup and mixed uniformly.
(3) The medium after mixing was transferred to a 1.5mL centrifuge tube and cultured with shaking at 28℃and 220rpm for 1 hour.
(4) 200. Mu.L of the bacterial liquid was aspirated and plated on the screening medium, and the medium was cultured upside down at 28℃for 2 days.
(5) Randomly picking bacterial spots for propagation and culture, and carrying out bacterial liquid PCR detection by using carrier primers. Detecting by 1% agarose gel electrophoresis, selecting positive clone as engineering strain for standby.
2.3 for further physiological investigation of the control of the inverse resistance of the transgenic BpWRKY70 Gene, the wild Betula alba was transiently infected for one month using the efficient transient infection technique with pROkII-35S as control and placed in tissue culture seedlings containing 200mM NaHCO, respectively 3 The stress treatment is carried out in the solution of (1), the plant after the stress is taken to be used for various chemical staining and physiological index detection, and the specific operation is as follows:
2.3.1 transient infection Betula alba tissue culture seedling
(1) The agrobacteria of pROKII-BpWRKY70 and pROKII-35S are streaked and separated on LB solid medium (containing 25mg/L Rif and 50 mg/LKan) and cultivated for 48 hours at 28 ℃ in an inversion way;
(2) Picking single colony carrying agrobacterium tumefaciens into 5ml LB liquid medium (containing 25mg/LRif and 50 mg/LKan), and shake culturing overnight at 28 ℃;
(3) Taking 1ml of the overnight cultured bacterial liquid, adding the bacterial liquid into 50ml of LB liquid medium, carrying out shaking culture at 28 ℃ and 220rpm until the OD600 is about 0.5 in the late logarithmic growth phase;
(4) Centrifuging at 5000rpm at normal temperature for 10min, removing supernatant, and collecting thallus
(5) 50ml of 1/2MS (AS containing 150. Mu.M) liquid medium was added, and the cells were repeatedly resuspended by pipetting;
(6) Shake culturing at 28 deg.C and 220rpm for 1 hr.
(7) And (3) putting the tissue culture seedlings of the betula alba into the prepared bacterial liquid, and slowly shaking for 12 hours at the rotating speed of 120rpm at the temperature of 22-25 ℃.
2.3.2 Biochemical staining and detection of physiological indicators of Betula alba plants after transient infection
(1) DAB staining:
and (3) respectively taking the birch leaves of the experimental group and the control group which are not subjected to stress treatment and stress treatment for 6 hours, placing the birch leaves into a centrifuge tube, adding DAB staining solution, and staining at room temperature overnight. After the dyeing is finished, 75% ethanol and 5% glycerol are boiled for decolorization.
(2) NBT staining:
and (3) respectively taking the white birch leaves of the experimental group and the control group which are not subjected to stress treatment and stress treatment for 6 hours, placing the white birch leaves into a centrifuge tube, adding NBT staining solution, and staining at room temperature overnight. After the dyeing is finished, 75% ethanol and 5% glycerol are boiled for decolorization.
(3) Evans blue staining:
and (3) respectively taking the birch leaves of the experimental group and the control group which are not subjected to stress treatment and stress treatment for 6 hours, placing the birch leaves into a centrifuge tube, adding Evans blue staining solution, vacuumizing for half an hour, and keeping the vacuum state for staining overnight. After the dyeing is finished, 75% ethanol and 5% glycerol are boiled for decolorization.
(4) Superoxide dismutase (SOD) activity assay (kit method):
accurately weighing plant tissues (0.2-0.5 g), and weighing the plant tissues according to the weight (g): volume (ml) =1: 4, adding four times of volume of homogenizing medium, shearing, homogenizing under ice water bath condition to obtain 20% homogenate, centrifuging at 3500rpm for 10min, collecting supernatant, and measuring, wherein the specific operation steps are shown in the following table 1:
TABLE 1 procedure
Mixing, standing at room temperature for ten minutes, and setting zero by double distilled water at a wavelength of 550nm in a 1cm optical path cuvette and reading.
And (3) calculating results:
x sample pretreatment dilution (3 times)/(homogenate concentration) x (gram wet tissue weight/ml)
* Namely the dilution factor in the reaction system
(5) Peroxidase (POD) Activity assay (kit method):
1) Pretreatment:
preparing a young leaf plant tissue homogenate with high water content: wiping water and impurities from plant tissues, accurately weighing the weight of the plant tissues, and according to the weight (g): volume (ml) =1: 9 (preferably, physiological saline or phosphate buffer: 0.1mol/LpH 7.7-7.4) was added in a ratio of 9 times the volume of the homogenized medium, and 10% of the homogenized tissue was prepared under the ice-water bath condition, centrifuged at 3500rpm for 10 minutes, and the supernatant was collected and assayed.
Preparation of dried plant tissue homogenates with a low water content: taking plant tissues, wiping water and impurities, shearing, putting into a mortar, adding liquid nitrogen, grinding into powder, transferring, accurately weighing, and (g) according to the weight: volume (ml) =1: 9 (preferably, physiological saline or phosphate buffer: 0.1mol/LpH =7 to 7.4) was added in a ratio of 9 times by volume, the mixture was vortexed and extracted for 3 to 5 minutes, and the mixture was centrifuged at 3500rpm for 10 minutes, and the supernatant was collected and assayed.
2) The specific operating steps are shown in table 2 below:
TABLE 2 procedure
Mixing, centrifuging at 11000rpm for 10min, collecting supernatant at 420nm, measuring with 1cm optical path, and measuring with distilled water.
And (3) calculating results:
definition: the amount of enzyme catalyzing 1ug of substrate per minute per mg of tissue protein at 37℃is defined as one enzyme activity unit.
The calculation formula is as follows:
(6) Determination of protein concentration (kit method):
1) Pretreatment:
weighing 0.1g of white birch plant tissue, grinding into powder under the liquid nitrogen condition, and weighing (g): volume (mL) =1: 9, adding physiological saline in proportion, centrifuging at 10000rpm for 10 minutes, and collecting supernatant. Then, the mixture was stirred with physiological saline at 1:9 to obtain 1% tissue homogenate as the test solution.
The specific operating steps are shown in table 3 below:
TABLE 3 procedure
Mixing well, standing for 10min, zeroing with distilled water at 595nm, and measuring absorbance of each tube.
And (3) calculating results:
(7)H 2 O 2 determination of content (kit method)
Accurately weighing the weight of the tissue according to the weight (g): volume (ml) =1: 9, adding 9 times of 0.9% physiological saline by volume, mechanically homogenizing under ice water bath condition, centrifuging at 1000rpm for 10 minutes, and taking 10% of supernatant for homogenizing to be detected. The specific operation steps are shown in the following table:
TABLE 4 procedure
| Blank pipe | Standard tube | Measuring tube | |
| Reagent one (ml) (37 ℃ C. Pre-temperature) | 1 | 1 | 1 |
| Double distilled water (ml) | 0.1 | ||
| 163mmolH 2 O 2 Sample application liquid (ml) | 0.1 | ||
| Sample to be measured (ml) | 0.1 | ||
| Reagent II (ml) | 1 | 1 | 1 |
Mixing, measuring absorbance of each tube, and recording, wherein the wavelength is 405nm, the light path is zeroed by double distilled water under the condition of 1 cm.
And (3) calculating results:
(8) Determination of relative conductivity:
taking 3-5 fresh leaves with consistent size after instant infection, flushing with double distilled water and ultrapure water for 3 times in sequence, sucking the surface moisture by filter paper, and placing the filter paper into a 50mL centrifuge tube. Adding 30mL of ultrapure water, pumping for 15min in a vacuum pump, measuring the conductivity value by using a conductivity meter, and recording as S1; then the centrifuge tube is put into a constant temperature water bath kettle at 90 ℃ for water bath for 20min, then cooled to room temperature, and the conductivity value is measured and recorded as: s2, performing operation.
And (3) calculating results: relative conductivity = S1/S2 × 100%
The results were:
cloning and sequence analysis of 1 Betula alba WRKY70 Gene
1.1 cloning of the Betula alba WRKY70 Gene
Extracting total RNA of wild type white birch, designing a primer for PCR amplification, recovering a gel to obtain a target fragment, connecting the target fragment into a T vector, sequencing, and obtaining a complete WRKY70 gene sequence of a coding region (CDS) as follows:
birch WRKY70 gene sequence (SEQ ID No. 1)
ATGGAGTCTTCTTGGCCGGAAAACTCATCCTCCAAGCCCAGAAAAGCCATTGAAGAGCTGATTCAAGGTCGCCAATTCGCAAATCAGCTCCGGGGACTACTCGGTAAGTCGATCGGAGATGATGGGTCAGTCCCAGCAAAAGATCTTGTTCTGAAAATCTTGAACTCCTTCACAAACACTCTTTCGATGTTGAATTCCGCCGAGTCCGAAGCCGACGTTTCTCAAATTCAGCCCAACACCCAAGCCAGTTCGCCGTGTTGGGATGCTCCCAAGTCGGAATCTTCCGGAGAAAGTTGCCGGAGTATCTCGACAGTCAAGGATCGGAGAGGATGTTATAAGAGAAGGAGGACTTCACTGACGACAACTAAAGACACCCCTACTCTGATCGATGACGGTCATGCATGGAGAAAATACGGACAAAAACTGATCCTTAATGCTAAATACCCAAGGCATTACTACAGGTGCACTCATAAATATGATCAGGGATGCCAAGCAGCGAAGCAGGTGCAAAGAATCCAAGAGGATCCGGCGATGCACAGGACTACATATATTGGGCAACACACGTGCAGAACGTTTCCAAAAGCCCCTGAATTAATCTTGGATTCTTCTCCCACAGACTCTCACTTTGTGCTCAGTTTTGACAACACCTTCACAAACAAACACAACCACCCGGTCCACCCCTTTCTCTCGTCATCCTTCCAATCAGTCAAACAGGAACACAAGGAAGAGATGCCCAGCGATGATGTCACCCACAACCAATCATCGTCGTCTGATTATCTCGTTTCCCCCGATCAGACGGCATTCGATTCATTCTACAACATGACTGTCTTATCATCACCGGTGGAGTCCGACGATAATGGGGTTTTGGTGGGCTCGGTCGACCACTTTGCCGATGACGATTTGATGGGTGCCTTTTCTTTTTAA。
The coding region of the obtained BpWRKY70 gene is 924bp in length and codes 307 amino acids. The amino acid sequence is as follows:
birch WRKY70 amino acid sequence (SEQ ID No. 2)
MESSWPENSSSKPRKAIEELIQGRQFANQLRGLLGKSIGDDGSVPAKDLVLKILNSFTNTLSMLNSAESEADVSQIQPNTQASSPCWDAPKSESSGESCRSISTVKDRRGCYKRRRTSLTTTKDTPTLIDGHAWRKYGQKLILNAKYPRHYYRCTHKYDQGCQAAKQVQRIQEDPAMHRTTYIGQHTCRTFPKAPELILDSSPTDSHFVLSFDNTFTNKHNHPVHPFLSSSFQSVKQEHKEEMPSDDVTHNQSSSSDYLVSPDQTAFDSFYNMTVLSSPVESDDNGVLVGSVDHFADDDLMGAFSF。
Obtaining and stress tolerance identification of 2-transfer WRKY70 gene white birch
Amplification primers were designed based on the WRKY70 gene sequence, and the amplified product was purified by agarose gel DNA purification kit (OMEGA) and ligated with pROKAI vector digested with SmaI (Promega). The connection product is transformed into an escherichia coli Top10 strain, and after the PCR verification and the sequencing identification are correct, the recombinant plasmid is transferred into an agrobacterium tumefaciens EHA105 strain by a conductivity method to obtain positive recombinant bacteria.
In order to further study the regulation of the reverse resistance of the transfer WRKY70 gene physiologically, a high-efficiency transient infection technique was used to transiently infect wild birch for one month with pROkII-35S as a control, and a tissue culture seedling containing 200mM NaHCO was placed 3 The solution of (2) is subjected to stress treatment, and the infected plants are used for detecting various biochemical staining and physiological indexes.
2.1DAB staining
H in cells 2 O 2 The released oxygen ions can oxidize DAB to form brown precipitate, and H in the cells can be judged according to the dyeing depth 2 O 2 The more severe the cell damage, the more severely the amount released, H 2 O 2 The more released. DAB staining was performed on the leaves of birch in the experimental group and the control group, which were not subjected to stress treatment and stress treatment for 6 hours, respectively, and the staining results were shown in FIG. 1.
Under the condition of non-stress growth (control), the leaf colors of the experimental group plants and the control group plants are lighter and have no obvious difference, which indicates H 2 O 2 The content is approximately the same; in NaHCO 3 Under abiotic stress conditions, the colors on leaves of plants in the experimental group and the control group are obviously changed. The color of the plant leaf transformed with the WRKY70 gene is lighter than that of the control group white birch leaf, which shows that H in the plant leaf of the experimental group 2 O 2 The content of H in leaf of white birch plant of control group 2 O 2 The low content indicates that the strain of the trans-WRKY 70 gene has low damage degree after being stressed. Experimental results prove that the WRKY70 gene plays a role in stress resistance in the white birch plant body.
2.2NBT staining
NBT staining results can be used to detect superoxide anions (O2) in plants - ) Can determine the content of superoxide anion (O2) in cells according to the degree of staining - ) The more severe the cell damage, the more superoxide anion (content O2 - ) The more. NBT staining was performed on the betula alba leaves of the experimental group and the control group which were not subjected to stress treatment for 6 hours, respectively, and the staining results are shown in FIG. 2.
Under non-stress growth conditionsThe leaves of the experimental and control plants were lighter in color and did not differ significantly from each other, indicating that superoxide anions (O2 - ) The content is approximately the same; in NaHCO 3 Under abiotic stress conditions, the colors on leaves of plants in the experimental group and the control group are obviously changed. The color of the plant leaf transformed with WRKY70 gene is lighter than that of the control group white birch leaf, which shows that superoxide anion (O2) - ) The content of superoxide anion (O2) in the leaf of the white birch plant of the control group is higher than that of the leaf of the white birch plant of the control group - ) The low content indicates that the strain of the trans-WRKY 70 gene has low damage degree after being stressed. Experimental results show that the WRKY70 gene can positively regulate and control the stress resistance function of plants.
2.3Evans blue staining
Evans blue staining solution can enter dead cells and be stained blue, and the number of the dead cells in the cells can be judged according to the degree of staining, so that the more severe the cells are damaged, the more the dead cells are. Evans blue staining was performed on the birch leaves of the experimental group and the control group which were not subjected to stress treatment for 6 hours, and the staining results are shown in FIG. 3.
Under the non-stress growth condition (control), the leaf colors of the experimental group plants and the control group plants are lighter, and have no obvious difference, so that the number of dead cells is approximately the same; in NaHCO 3 Under abiotic stress conditions, the colors on leaves of plants in the experimental group and the control group are obviously changed. The color of the plant leaf of the WRKY70 gene is lighter than that of the control group white birch leaf, which shows that the number of dead cells in the plant leaf of the experimental group is smaller than that of the control group white birch leaf, and the damage degree of the plant line of the WRKY70 gene after being stressed is low. Experimental results prove that the WRKY70 gene can improve the stress resistance of the white birch plants.
2.4 determination of superoxide dismutase (SOD) Activity (kit method)
SOD can catalyze the disproportionation reaction of superoxide anion free radical, resist the damage of active oxygen or other peroxide free radical to cell membrane system, thereby improving the stress resistance of plants, and the measurement result is shown in fig. 4.
In NaHCO 3 Under the abiotic stress condition, the SOD activity of the over-expressed and transiently infected plant of the WRKY70 gene is higher than that of a control, which indicates that the stress resistance of the over-expressed plant after the transient infection is stronger than that of the control plant. Experimental results show that the WRKY70 gene can positively regulate and control SOD activity.
2.5 measurement of Peroxidase (POD) Activity (kit method)
Under the catalysis of Peroxidase (POD), H 2 O 2 The oxidation of guaiacol to a tawny product, peroxidase, an important protective enzyme for reducing oxygen radical damage in plants, was closely related to the plant's ability to resist stress, and the experimental results are shown in fig. 5.
Under non-stress conditions (control), the POD activity of the experimental and control birch strains was approximately the same; in NaHCO 3 Under abiotic stress conditions, the POD activity of the white birch strain in the experimental group and the control group is changed. POD activity of the transient infection strain which overexpresses WRKY70 gene is higher than that of the control, which shows that the resistance of the plants in the experimental group to adversity stress is stronger than that of the control. The result shows that the expression quantity of the WRKY70 gene is positively related to the enzyme activity of the POD, and that the WRKY70 gene can improve the stress resistance of plants by regulating and controlling the antioxidant enzyme activity in the plants.
2.6 determination of Total protein concentration
The content of the soluble protein in the plant body is an important index for knowing the total metabolism of the plant body, the content of the protein in the plant body can reflect the adversity stress resistance capability of the plant, and under the adversity stress, the stronger the adversity stress resistance capability of the plant is, the higher the total protein content is, and the experimental result is shown in figure 6.
As shown in the figure, at NaHCO 3 Under abiotic stress conditions, the protein concentration of the white birch strain in the experimental group and the control group is changed. The protein concentration of the transient infection strain which overexpresses the WRKY70 gene is higher than that of the control, which indicates that the adversity stress resistance of the plants in the experimental group is stronger than that of the control. The results demonstrate that over-expression of the WRKY70 gene is positively correlated with stress resistance of plants.
2.7 H 2 O 2 Determination of content (kit method))
H 2 O 2 As active oxygen, it is ubiquitous in living organisms and is an important hub for conversion between active oxygen. Among the numerous oxidative metabolites, H 2 O 2 The cell aging and catabolism process can be accelerated, the principle is that the cell aging and catabolism process can damage cell membranes, directly or indirectly oxidize biomacromolecules, and under the stress of adversity, the stronger the plant has the capability of resisting adversity, H accumulated in vivo 2 O 2 The lower the content, the experimental results are shown in fig. 7.
As shown in the figure, at NaHCO 3 Under abiotic stress conditions, H of the white birch strain of the experimental group and the control group 2 O 2 The concentration was varied. H of transient invasive strains overexpressing the WRKY70 gene 2 O 2 The concentration is lower than that of the control, which indicates that the plants of the experimental group have stronger adversity stress resistance than the control. The result shows that the over-expression of the WRKY70 gene can improve the stress resistance of plants.
2.8 determination of relative conductivity
The relative conductivity of plant mesophyll is a basic index for reflecting the permeability of plant cell membranes, when the plant is affected by adverse conditions, the cell membranes are destroyed, the membrane permeability is increased, and thus the electrolyte in the cells is extravasated. The higher the relative conductivity of the plant, the weaker the stress resistance, and the experimental results are shown in fig. 8.
Under non-stress conditions (control), the relative conductivities of the experimental and control birch strains were approximately the same; in NaHCO 3 The relative conductivities of the experimental and control birch strains were varied under abiotic stress conditions. The relative conductivity of the transient infection strain which overexpresses the WRKY70 gene is lower than that of the contrast, which indicates that the stress resistance of the plants in the experimental group is stronger than that of the contrast, and the stress resistance of the plants can be improved by the overexpression of the WRKY70 gene.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. The BpWRKY70 gene for improving the saline-alkali tolerance of the white birch is characterized in that the nucleotide sequence of the BpWRKY70 gene is shown as SEQ ID No. 1.
2. The use of the BpWRKY70 gene of claim 1 in the tolerance of betula salina to saline-alkali stress.
3. The use of the BpWRKY70 gene according to claim 1 for reducing the hydrogen peroxide content of betula alba leaves.
4. The use of the BpWRKY70 gene of claim 1 for reducing the superoxide anion content of betula alba leaves.
5. The use of the BpWRKY70 gene of claim 1 for increasing the activity of birch superoxide dismutase.
6. The use of the BpWRKY70 gene of claim 1 for increasing the activity of betula peroxidase.
7. A protein encoded by the BpWRKY70 gene of claim 1, wherein the amino acid sequence of the protein is shown in SEQ ID No. 2.
8. The method for cultivating the saline-alkali stress resistant white birch is characterized by comprising the following steps of:
1) Ligating the BpWRKY70 gene of claim 1 into a pROKII vector to obtain an overexpression vector;
2) Transferring the over-expression vector obtained in the step 1) into agrobacterium tumefaciens by adopting a conductivity method to obtain recombinant bacteria;
3) And (3) infecting the recombinant bacteria obtained in the step (2) by adopting a high-efficiency instantaneous infection technology to obtain a saline-alkali stress resistant white birch plant.
9. The method according to claim 8, wherein the primers used for amplifying the BpWRKY70 gene are an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer is shown in SEQ ID No.3, and the nucleotide sequence of the downstream primer is shown in SEQ ID No. 4.
10. The method of claim 8, wherein the agrobacterium tumefaciens is agrobacterium tumefaciens EHA105.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| US20150337328A1 (en) * | 2014-05-23 | 2015-11-26 | Clemson University | Methods and Constructs for Conferring Enhanced Abiotic Stress Resistance in Plants |
| CN113388621A (en) * | 2021-07-09 | 2021-09-14 | 河南农业大学 | Rehmannia WRKY transcription factor RgWRKY37 gene and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20150337328A1 (en) * | 2014-05-23 | 2015-11-26 | Clemson University | Methods and Constructs for Conferring Enhanced Abiotic Stress Resistance in Plants |
| CN113388621A (en) * | 2021-07-09 | 2021-09-14 | 河南农业大学 | Rehmannia WRKY transcription factor RgWRKY37 gene and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| GENBANK DATABASE: "hypothetical protein FH972_017018 [Carpinus fangiana]，GenBank: KAE8099001.1", NCBI, 28 October 2019 (2019-10-28) * |
| 曹翔;彭贤瑞;范吉标;: "植物转录因子MYB基因家族研究进展", 河南农业, no. 24, 25 August 2020 (2020-08-25) * |
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