CN118389580A - Application of ABH gene in regulation of plant salt stress resistance - Google Patents
Application of ABH gene in regulation of plant salt stress resistance Download PDFInfo
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Abstract
The invention discloses application of an ABH gene in regulating and controlling plant salt stress resistance, and belongs to the technical field of plant genetic engineering. The gene VITIS VINIFERA uncharacterized LOC100251931 with unknown functions on the chromosome 6 of the grape is subjected to intensive research, and the gene is found to be capable of negatively regulating and controlling the tolerance of the grape to salt stress, can be used as a novel grape salt tolerance related gene, provides a theoretical basis for breeding a grape salt tolerance variety, and provides a novel thought for a cultivation technology for relieving grape salt damage.
Description
Technical Field
The invention relates to the technical field of plant genetic engineering, in particular to application of an ABH gene in regulating and controlling plant salt stress resistance.
Background
Grape (VITIS VINIFERA L.) is one of the fruits with the largest cultivation area and long cultivation history, belonging to Vitaceae, vitis genus deciduous vine. The cultivation range is wide, the processed products are rich, and huge economic and ecological benefits are created for society. With global climate change, soil salinization becomes an important limiting factor for restricting the sustainable development of the grape industry. When the grape is subjected to salt stress, the osmotic potential is improved, so that the water absorption of plants is inhibited, and the yield and quality of the grape are finally affected due to ion toxicity caused by excessively high accumulation of Na +、Mg2+、Cl- and the like. Therefore, increasing tolerance of grapes to salt stress has become an urgent problem to be solved in grape cultivation.
When the grape faces salt stress, molecular level regulation is a fundamental and key step, and a molecular regulation mechanism under the state of salt stress of the grape is explored, so that a solid theoretical basis can be provided for revealing the salt tolerance mechanism of the grape, and candidate genes are provided for breeding of salt-tolerant varieties. In recent years, many researchers have discovered many transcription factors, such as VvMYC2, of grapes responding to salt stress through transcriptome sequencing technology, so that the germination rate of transgenic arabidopsis thaliana under salt stress is improved. VvWRKY2, vlWRKY, vvWRKY, vvNAC, 17 increase the tolerance of transgenic tobacco or arabidopsis to salt stress. In addition to transcription factors, genes in grapes that respond to salt stress by different pathways are also increasingly being mined. For example: over-expression VvSNAT 1 enhances melatonin synthesis and ROS scavenging capacity of arabidopsis, thereby increasing tolerance to salt stress. VvKCS 11A improves salt tolerance of Arabidopsis by regulating accumulation and membrane stability of ion transporter and channel, permeation regulating substance.
However, the research on the salt tolerance mechanism of grape is not enough at present, and at the molecular level, a new gene related to grape stress tolerance still needs to be further excavated.
Disclosure of Invention
In view of the above prior art, it is an object of the present invention to provide the use of ABH gene in regulating salt stress resistance of plants. According to the research of the invention, the ABH gene can be silenced in the grape, so that the resistance of the grape to salt stress can be improved, and a new candidate gene is provided for the breeding of the grape salt-resistant variety.
In order to achieve the above purpose, the invention adopts the following technical scheme:
In a first aspect of the present invention, there is provided the use of the ABH gene as a negative regulatory gene in (1) or (2) as follows:
(1) Improving the resistance of the plant to salt stress;
(2) Cultivating a salt-tolerant plant variety;
the ABH gene is a DNA molecule shown in the following i) or ii) or iii):
i) The nucleotide sequence is a DNA molecule shown in SEQ ID NO. 1;
ii) a DNA molecule other than i) encoding the amino acid sequence shown in SEQ ID NO. 2;
iii) A DNA molecule which has 90% or more identity to the DNA fragment defined in i) or ii) and which encodes a protein functionally equivalent to the protein shown in SEQ ID NO. 2.
The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. Homology can be assessed using computer software, e.g., using the BLAST algorithm (Altschul et al.1990.Journal of Molecular Biology 215:403-410;Karlin and Altschul.1993.Proceedings of the National Academy of Sciences 90:5873-5877).
In the above nucleic acid molecule, the 90% or more homology may be at least 90%, 92%, 93%, 95%, 96%, 98% or 99% homology.
In a second aspect of the present invention, there is provided the use of a protein encoded by the ABH gene as a negative regulator in (1) or (2) as follows:
(1) Improving the resistance of the plant to salt stress;
(2) And preparing a product for improving the salt tolerance of plants.
In the application, the amino acid sequence of the protein coded by the ABH gene is shown as SEQ ID NO. 2.
In a third aspect of the present invention, there is provided the use of a recombinant expression vector, transgenic cell line or genetically engineered bacterium comprising an ABH gene in (1) or (2) as follows:
(1) Improving the resistance of the plant to salt stress;
(2) Cultivating salt-tolerant plant varieties.
In the above application, the recombinant expression vector may be constructed using existing plant expression vectors or prokaryotic expression vectors. Such as pHB, pCXUN, pCAMBIA1300, pTA7001, pTA7002, pBin, PET-30a, PMAL-C2X, pGEX-4T or other derived vectors.
In the above application, the plant is preferably grape.
In the application, the ABH gene or the protein coded by the ABH gene is used as a target, and the ABH gene is silenced or the protein coded by the ABH gene is inactivated, so that the resistance of the grape to salt stress is improved, and the application of the grape in soil salinization improvement is widened.
In a fourth aspect of the invention, there is provided a method of increasing tolerance of a grape to salt stress comprising the steps of:
ABH gene in grape was knocked out or silenced.
In the above method, gene knockout refers to a phenomenon in which a specific target gene is inactivated by homologous recombination. Gene knockout is the inactivation of a particular target gene by a change in DNA sequence.
Gene silencing refers to the phenomenon of causing genes to be either not expressed or to be underexpressed without damaging the original DNA. Gene silencing is premised on the fact that the DNA sequence is not altered, so that the gene is not expressed or is underexpressed. Gene silencing can occur at two levels, one at the transcriptional level due to DNA methylation, heterochromatin, and positional effects, etc.; another is post-transcriptional gene silencing, i.e., inactivation of a gene by specific inhibition of the target RNA at the post-transcriptional level of the gene, including antisense RNA, co-suppression (co-suppression), RNA interference (RNAi), and microrna (miRNA) -mediated translational inhibition, among others.
In a fifth aspect of the present invention, there is provided a method of breeding a salt-tolerant grape variety, comprising the steps of:
knocking out or silencing an ABH gene in a wild grape plant to obtain a transgenic grape plant, wherein the transgenic grape plant has higher resistance to salt stress than the wild grape plant;
And (3) taking the grape transgenic plant as a parent to carry out selfing or hybridization with other grape plants to obtain the grape variety with improved salt resistance.
The invention has the beneficial effects that:
The gene VITIS VINIFERA uncharacterized LOC100251931 with unknown functions on the chromosome 6 of the grape is subjected to intensive research, and the gene is found to be capable of negatively regulating and controlling the tolerance of the grape to salt stress, can be used as a novel grape salt tolerance related gene, provides a theoretical basis for breeding a grape salt tolerance variety, and provides a novel thought for a cultivation technology for relieving grape salt damage.
Drawings
Fig. 1: the expression condition of the grape ABH gene of the invention at different time points after salt stress.
Fig. 2: resistance to salt stress after ABH gene silencing of the grape of the invention; wherein, the left graph shows the growth conditions of ABH-silenced (ABH-RNAi) grape plants and control grape plants under salt stress conditions; the right panel shows root weight and aerial part weight (average of multiple measurements) of ABH-silenced (ABH-RNAi) grape plants and control grape plants under salt stress conditions.
Detailed Description
It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
As mentioned above, the physiological disorder of grape caused by salinized soil is more and more obvious, and the normal growth and development of grape, fruit yield and quality are seriously affected. Therefore, there is a need to mine new genes related to stress tolerance of grapes.
In view of this, the present invention has conducted intensive studies on genes related to salt stress tolerance in the genome of grape. VITIS VINIFERA uncharacterized LOC100251931 (NCBI Reference Sequence: XM_ 002285585.5) is a gene whose function is unknown on the chromosome 6 of Vitis vinifera. In order to study the functions, the invention firstly clones the gene from grape, and the cloned gene is named ABH gene. The nucleotide sequence of the coding region of the ABH gene is shown as SEQ ID NO.1, and is specifically as follows:
ATGGCTTGTCTGGTTGAGTCCTCCACAGTTGAAACCAGTGATGGCATCAAGCTCCATACAAGGGTCTTCAAACCAAGGGAGGAGATCAAGGACAACTTGGTGGTTGTTCTGGTGCATCCTTACTCAGTCTTGGGTGGTTGTCAGGCCCTTGTGAAGGGGATTGCTCTTGGGTTAGCAGAAAAAGGCTACAGAGCTGTGACCTTTGACATGAGAGGCGCCGGAAGATCCACAGGAAGGCCTTCTCTTACTGGGTTTTCAGAAATCAAGGATGTGGTTGCTGTCTGCAAATGGGTCTGTGACAATCTTTCTTCTGATAGGATTTTGTTGGTGGGTTCTTCTGCAGGAGCACCTATTGCAGGATCCGCGGTAAATCAGATTGAACAAGTGGTGGGCTATGTGAGTCTGGGGTACCCATTCGGCTTGATGGCCTCAATCCTCTTTGGCCGCCACCACAAAGCCATCCTGCAGTTCCCGAAACCTAAACTCTTTGTCATGGGGACTCAGGATGGGTTCACCAGCGTTAAGCAGCTGAGGAACAAGCTGAGCTCTGCAGCAGGCCATGTTGAGACCCACCTTATCGAAGGAGCAGGCCACTTCCAAATGGAAGGGCCAGCTTATGATGCCCAGATGGCAGATAAGATCACAGAATTCATCCCCAAACTG TAG.
the amino acid sequence of the protein coded by the ABH gene is shown as SEQ ID NO.2, and the amino acid sequence is specifically as follows:
MACLVESSTVETSDGIKLHTRVFKPREEIKDNLVVVLVHPYSVLGGCQALLKGIALGLAEKGYRAVTFDMRGAGRSTGRPSLTGFSEIKDVVAVCKWVCDNLSSDRILLVGSSAGAPIAGSAVNQIEQVVGYVSLGYPFGLMASILFGRHHKAILQFPKPKLFVMGTQDGFTSVKQLRNKLSSAAGHIETHLIEGAGHFQMEGPAYDAQMADKITEFIPKL.
The ABH gene is selected as a research object, and the expression level of the gene under the condition of salt stress is found to be changed, so that the gene is presumed to be possibly responsive to the salt stress resistance of the grape.
In order to further study the functions of the ABH gene, the invention constructs an ABH gene silencing vector, and introduces the ABH gene silencing vector into grapes to obtain transgenic plants with the ABH gene silencing. As a result, ABH gene-silenced grapes were found to grow significantly higher under salt stress than controls. The above results indicate that: the grape ABH gene can play a salt-resistant function in plants, is a novel salt-resistant gene related to resisting salt stress in grapes, and thus the invention is provided.
In order to enable those skilled in the art to more clearly understand the technical scheme of the present application, the technical scheme of the present application will be described in detail with reference to specific embodiments.
The test materials used in the examples of the present invention, which are not specifically described, are all conventional in the art and are commercially available. Specific experimental conditions and methods are not noted in the examples of the present invention, and generally conventional conditions, such as j. Sambrook et al, scientific press, 2002, guidelines for molecular cloning experiments (third edition); D.L. speket et al, scientific press, 2001, guidance of cell experiments; or according to manufacturer recommended conditions.
Example 1: cloning of the grape ABH Gene
1. Plant material:
The material used in the invention is 'SA15' grape stock seedling.
Extraction and reverse transcription of RNA:
Grape leaves were harvested for RNA extraction. Total RNA was extracted according to the instructions using RNAprep Pure polysaccharide polyphenol plant total RNA extraction kit (DP 441) and 1. Mu.g RNA was reverse transcribed to cDNA using TAKARA PRIMESCRIPT TM RT REAGENT KIT (PERFECT REAL TIME) kit.
Clone verification of abh gene:
primers were designed based on the NCBI accession number XM_002285585.5 gene:
ABH-F：5′-ATGGCTTGTCTGGTTGAGTCCT-3′(SEQ ID NO.3)；
ABH-F：5′-CTACAGTTTGGGGATGAATTCTGTG-3′(SEQ ID NO.4)。
PCR reactions were performed using PRIMESTAR MAX PREMIX Hi-Fi enzyme following the protocol, and the PCR products were ligated into Simple Cloning Vector (Beijing full gold biotechnology Co., ltd.) vector, transformed E.coli DH 5. Alpha. And screened positive single colonies by colony PCR, and sent to the Biotechnology Co., ltd. For sequencing. The nucleotide sequence of the cloned grape ABH gene is shown as SEQ ID NO. 1.
Example 2: expression of the grape ABH gene at different salt treatment time points
1. Plant material:
1L of 100mM NaCl solution is irrigated to 'SA15' grape seedlings, the solution flows out from the bottom of the flowerpot, then the root systems are respectively taken at 1,2,3, 4 and 5 hours, the sampled root systems are marked as 0 hour before irrigation, 3 repeats are arranged, and liquid nitrogen quick freezing preservation is carried out.
Extraction and reverse transcription of RNA:
extracting RNA from root system, and reverse transcribing to obtain cDNA. The procedure of extraction and reverse transcription of RNA was the same as in example 1.
Expression level variation of abh gene at different salt treatment time points:
Designing a specific primer to perform real-time fluorescence quantitative PCR analysis on the expression quantity change rule of ABH in different salt treatment time points, wherein the designed specific primer is as follows:
qABH-F：5′-AATCAAGGATGTGGTTGCTGTCT-3′(SEQ ID NO.5)；
qABH-R：5′-AAGAGGATCGAGGCCATCAA-3′(SEQ ID NO.6)。
The internal reference gene is ACTIN, and the primers are as follows:
ACTIN-F：5′-TCCGTTGTCCAGAAGTCCTCTT-3′(SEQ ID NO.7)；
ACTIN-R：5′-GTCAGCAATACCAGGGAACATG-3′(SEQ ID NO.8)。
4. real-time fluorescent quantitative analysis of ABH gene in sample to be tested:
The cDNA was used as a template, and fluorescent quantitative analysis was performed using specific primers for ABH and ACTIN, respectively, and the reaction was performed in a real-time fluorescent quantitative PCR apparatus (CFX connection REAL TIME PCR Detection System, bio-Rad) using a 20. Mu.L System (10. Mu. L SYBR Premix Ex Taq, 1. Mu.L each for the upstream primer (10. Mu.M), 1. Mu.L for the cDNA template, and 20. Mu.L for the water make-up) as follows: 95 ℃ for 30s;95℃for 5s,60℃for 10s,40 cycles.
5. Relative quantitative analysis of ABH was performed using the 2 -△△Ct method:
The results are shown in fig. 1, and the results indicate that: as the salt treatment time was prolonged, the expression level of ABH gene showed a trend of decreasing followed by increasing, indicating that ABH gene could be differentially expressed in response to salt stress (fig. 1).
Example 3: salt resistance function detection of grape ABH gene silencing plants
Using the pHB vector (described in literature "MicroRNA171c-targeted SCL6-II,SCL6-III,and SCL6-IV genes regulate shoot branching in Arabidopsis,doi.org/10.1093/mp/ssq042"), a silencing vector for ABH gene was constructed, with the following primers:
ABH-1-F：5′-ccagtctctctctcaATGGCTTGTCTGGTTGAGTCCT-3′(SEQ ID NO.9)
ABH-1-R：5′-ttgtattttactagtCAGCAACCACATCCTTGATTTCT-3′(SEQ ID NO.10)
ABH-2-F：5′-aggatgtggttgctgACTAGTAAAATACAACACATGTTAATTGATACATT-3′(SEQ ID NO.11)
ABH-2-R：5′-aggatgtggttgctgGTTTATAGGTGCTTTAAAGTACTCAAATTG-3′(SEQ ID NO.12)
ABH-3-F：5′-aaagcacctataaacCAGCAACCACATCCTTGATTTCT-3′(SEQ ID NO.13)
ABH-3-R：5′-ctcctgcagctcgagATGGCTTGTCTGGTTGAGTCCT-3′(SEQ ID NO.14)
PCR reactions (10. Mu. LPRIMESTAR MASTER Mix, 1. Mu.L each of the upstream and downstream primers (10. Mu.M) in a 20. Mu.L reaction system, 1. Mu.L template, and 20. Mu.L made up with water) were performed using PRIMESTAR MAX PREMIX Hi-Fi enzyme as follows: 98 ℃ for 10s,55 ℃ for 5s,72 ℃ for 10s 34 cycles; extending at 72℃for 5mins.
The 3 PCR products were separated by 1.5% agarose gel electrophoresis and purified using SanPrep column DNA gel recovery kit (B518131, bio) according to standard procedures.
The pHB vector plasmid is subjected to enzyme digestion treatment, and the enzyme digestion reaction system is as follows:
The reaction solution was incubated at 37℃for 30 minutes, and then subjected to agarose gel electrophoresis followed by gel cutting for recovery.
The digested pHB and 3 ABH fragments were ligated and the recombinant plasmid was transferred into E.coli DH 5. Alpha. According to ClonExpress IIOne Step Cloning Kit kit standard procedures. Positive clones were screened by colony PCR and sent to the sequencing industry. The single colony with correct sequence was shaken overnight and plasmids were extracted according to the standard specification procedure of SanPrep column plasmid DNA miniprep kit (Bio). Thus, a plasmid containing the ABH silencing vector is obtained.
The ABH silencing vector plasmid was transferred into cabernet sauvignon by agrobacterium rhizogenes infection. The method comprises the following steps: cutting grape stem segments containing 1 bud eye, putting the lower incision into agrobacterium, immersing and penetrating agrobacterium rhizogenes (MSU 440) infection liquid containing a target carrier into the stem segments, performing sand culture cutting, and performing positive verification after rooting. The positive verified grape seedlings (VvABH-RNAi) are transplanted into flowerpots, and are irrigated with 100mM NaCl for 1 time after the grape seedlings grow normally, and irrigated with 1L each time for 4 times in 7 days.
Wild grape obtained by infection with the transferred empty plasmid was used as a control (EV).
Photographing after salt stress for 30 days to observe the growth condition of grape plants; and then taking out the whole grape plant, cutting the plant from the root parts, cleaning the root system, sucking the surface moisture by using toilet paper, and weighing the overground parts by using an electronic balance respectively.
The results are shown in fig. 2, which shows that: ABH-silenced (ABH-RNAi) grape plants grew significantly higher than the control (EV); the weight of the upper part of the land and the root system of the ABH-RNAi grape plant are higher than EV, namely the ABH gene can negatively regulate and control the salt resistance, the expression of the ABH gene is reduced, and the salt resistance of the plant can be improved.
The above description is only of the preferred embodiments of the present application and is not intended to limit the present application, but various modifications and variations can be made to the present application by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.
Claims (6)
- The use of abh gene as negative regulatory gene in (1) or (2) as follows:(1) Improving the resistance of the plant to salt stress;(2) Cultivating a salt-tolerant plant variety;the ABH gene is a DNA molecule shown in the following i) or ii) or iii):i) The nucleotide sequence is a DNA molecule shown in SEQ ID NO. 1;ii) a DNA molecule other than i) encoding the amino acid sequence shown in SEQ ID NO. 2;iii) A DNA molecule which has 90% or more identity to the DNA fragment defined in i) or ii) and which encodes a protein functionally equivalent to the protein shown in SEQ ID NO. 2.
- Use of a protein encoded by the abh gene as a negative regulator in (1) or (2) as follows:(1) Improving the resistance of the plant to salt stress;(2) And preparing a product for improving the salt tolerance of plants.
- 3. The use according to claim 2, wherein the amino acid sequence of the protein encoded by the ABH gene is shown in SEQ ID No. 2.
- 4. The application of the recombinant expression vector containing the ABH gene, a transgenic cell line or genetically engineered bacteria in the following (1) or (2):(1) Improving the resistance of the plant to salt stress;(2) Cultivating salt-tolerant plant varieties.
- 5. A method of increasing tolerance of a grape to salt stress comprising the steps of:ABH gene in grape was knocked out or silenced.
- 6. A method of breeding a salt-tolerant grape variety, comprising the steps of:knocking out or silencing an ABH gene in a wild grape plant to obtain a transgenic grape plant, wherein the transgenic grape plant has higher resistance to salt stress than the wild grape plant;And (3) taking the grape transgenic plant as a parent to carry out selfing or hybridization with other grape plants to obtain the grape variety with improved salt resistance.
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