CN116590331A - Application of SmD3-b in regulating drought and salt stress resistance of plants - Google Patents
Application of SmD3-b in regulating drought and salt stress resistance of plants Download PDFInfo
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- CN116590331A CN116590331A CN202310604456.8A CN202310604456A CN116590331A CN 116590331 A CN116590331 A CN 116590331A CN 202310604456 A CN202310604456 A CN 202310604456A CN 116590331 A CN116590331 A CN 116590331A
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- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
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- C12N15/8271—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
- 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 application discloses an application of SmD3-b in regulating drought and salt stress resistance of plants, belonging to the field of plant genetic engineering. The Sm protein gene derived from grape, namely SmD3-b, is constructed and then is introduced into tobacco to carry out a plant resistance test, and the gene is found to improve drought resistance and salt resistance of the tobacco, expand cognition of the gene on plant resistance, provide theoretical basis for obtaining high-resistance plants and have great application value.
Description
Technical Field
The application relates to the technical field of plant genetic engineering, in particular to application of SmD3-b in regulating and controlling drought and salt stress resistance of plants.
Background
The grape has high economic benefit and plays a very important role in agricultural production and economic development in China. The grape planting area in areas such as Xinjiang and Ningxia in China is large, but the water resources in the areas are deficient, many vineyards have no good irrigation conditions, and the growth and the development and the yield of the grapes are affected by the lack of water. In addition, soil salinization greatly limits the growth and development of grapes, resulting in yield and harvest reduction. Therefore, the digging of the drought-enduring and salt-enduring genes of the grape has important significance for creating new varieties of the drought-enduring and salt-enduring grape and promoting the sustainable development of the grape industry.
Understanding how plants sense and cope with stress is crucial to improving resistance of plants through rational breeding and genetic engineering strategies. Most studies on plant stress regulation gene expression have focused on gene expression regulation at the transcriptional level; in contrast, little is known about the regulation of stress-induced gene expression at post-transcriptional levels. Splicing of pre-mRNA is an important step between transcription and translation of most eukaryotic mRNA, accomplished by the spliceosome. The spliceosome consists of a ribonucleoprotein small body (small nuclear ribonucleoprotein, snRNP) and hundreds of non-snRNP proteins that recognize the splice sites of the RNA precursor and catalyze the splicing reaction. Some spliceosome components have been shown to be involved in responses to various abiotic stresses, and overexpression of certain spliceosomes or other splicing factors may increase plant tolerance to stresses. Thus, modulation of expression of these spliceosome components is an effective way to increase plant adaptability to drought and salt stress.
The snRNP consists of one or two small ribonucleic acids (snRNAs), sm or Like-Sm (Sm/Lsm) core proteins and several snRNP-related proteins. Sm proteins are a highly conserved family of RNA binding proteins, including B/B', D1, D2, D3, E, F and G. Studies have shown that the resulting pleiotropic phenotype after Arabidopsis SmD3-b mutation includes delayed flowering, reduced root growth, partial defects in veins, abnormal numbers of trichomes branches and altered numbers of flower organs (Knock-out mutations of Arabidopsis SmD3-b induce pleotropic phenotypes through altered transcript splicing, doi:10.1016/j. Plantasci.2011.01.011. Epub 20111028.). At present, whether SmD3-b has drought resistance in plants has not been studied. In addition, since the functions of the genes of the isogenic family are not completely identical in different species, it is not clear whether SmD3-b from grape regulates plant salt stress resistance.
Disclosure of Invention
In view of the above prior art, it is an object of the present application to provide the use of SmD3-b from grape in regulating drought and salt stress resistance in plants.
In order to achieve the above purpose, the application adopts the following technical scheme:
in a first aspect of the application there is provided the use of the grape SmD3-b gene in any one of (1) to (2) as follows:
(1) Improving drought resistance and salt resistance of plants;
(2) Cultivating plant variety with improved drought tolerance and salt tolerance.
Further, the grape SmD3-b gene is a nucleic acid molecule shown in the following i) or ii):
i) The nucleotide sequence is a nucleic acid molecule shown as SEQ ID NO. 1;
ii) a nucleic acid molecule other than i) encoding the amino acid sequence shown in SEQ ID NO. 2.
In a second aspect of the present application, there is provided the use of a protein encoded by the grape SmD3-b gene in any one of the following (1) - (2):
(1) Improving drought resistance and salt resistance of plants;
(2) The product for improving drought resistance and salt resistance of plants is prepared.
Further, the protein encoded by the grape SmD3-b gene is a protein shown in the following (A1) or (A2):
(A1) A protein consisting of an amino acid sequence shown as SEQ ID NO.2 in a sequence table;
(A2) A fusion protein obtained by ligating the N-terminal and/or C-terminal of the protein defined in (A1) with a protein tag.
In a third aspect of the present application, there is provided the use of an expression cassette, recombinant expression vector or recombinant bacterium comprising the grape SmD3-b gene in any one of (1) to (2) as follows:
(1) Improving drought resistance and salt resistance of plants;
(2) Cultivating plant variety with improved drought tolerance and salt tolerance.
In a fourth aspect of the application, there is provided a method of increasing drought and salt stress tolerance in a plant comprising the steps of:
transferring the SmD3-b gene of the grape into a target plant;
or up-regulating the expression of the grape SmD3-b gene or a homologous gene thereof in the plant genome;
the grape SmD3-b gene is a nucleic acid molecule shown in the following i) or ii):
i) The nucleotide sequence is a nucleic acid molecule shown as SEQ ID NO. 1;
ii) a nucleic acid molecule other than i) encoding the amino acid sequence shown in SEQ ID NO. 2.
In a fifth aspect of the present application, there is provided a method of growing transgenic plants having improved drought and salt tolerance comprising the steps of:
transferring the SmD3-b gene of the grape into a wild type target plant to obtain a plant with drought resistance and salt tolerance superior to those of the wild type;
the grape SmD3-b gene is a nucleic acid molecule shown in the following i) or ii):
i) The nucleotide sequence is a nucleic acid molecule shown as SEQ ID NO. 1;
ii) a nucleic acid molecule other than i) encoding the amino acid sequence shown in SEQ ID NO. 2.
Further, the grape SmD3-b gene is transferred into the target plant through pHB-gfp expression vector.
Further, the plant of interest is grape or tobacco.
The application has the beneficial effects that:
aiming at the current situation that the research on drought tolerance and salt tolerance functions of plant spliceosome proteins is weak, the application clones a Sm protein gene, namely SmD3-b, from grapes. Transgenic experiments prove that SmD3-b participates in drought tolerance and salt tolerance, expands cognition of people on plant resistance, provides theoretical basis for obtaining high-resistance plants, and has great application value.
Drawings
FIG. 1 shows the expression of the grape SmD3-b gene of the present application after mimicking drought stress (mannitol) and salt stress (sodium chloride).
FIG. 2 shows the growth of transgenic grape SmD3-b gene on tobacco under drought and salt stress; in the figure, #1 and #2 represent two transgenic tobacco lines, respectively, after overexpression of SmD3-b.
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 previously mentioned, stress response is an important direction in plant research. Some spliceosome protein genes are differentially expressed during abiotic, biotic stress and different stages of growth and development. Drought and salt stress have great influence on grape growth and development, and are one of the main limiting factors for the healthy development of the grape industry. However, little is known about how drought and salt stress affect the expression pattern of spliceosome protein genes in plants, and whether spliceosome protein genes play a role in plant drought and salt stress responses remains lacking transgenic evidence support.
In view of this, the present application has conducted cloning and functional studies on the SmD3-b gene derived from grape. Firstly, cloning a grape SmD3-b gene from grape; then detecting the expression condition of the grape SmD3-b gene at different drought and salt treatment time points, and finding that the grape SmD3-b gene can respond to drought and salt stress to generate differential expression; furthermore, the application introduces the SmD3-b gene of the grape into tobacco, and discovers that the growth condition of tobacco plants with the SmD3-b gene being overexpressed is obviously better than that of wild tobacco plants under the condition of simulating drought and salt stress.
The above results indicate that: the SmD3-b gene of the grape can exert drought resistance and salt tolerance in plants, and is a novel gene related to drought resistance and salt stress resistance in the grape.
The sequence of the grape SmD3-b gene is shown as SEQ ID NO.1, and the specific steps are as follows:
ATGAGCAGAAGCTTGGGAATACCTGTGAAGCTTCTACACGAAGCCGCGGGTCATGTGGTGACTGTGGAACTGAAAAGCGGTGAGCTTTACAGAGGAAACATGATCGAGTGCGAAGATAATTGGAACTGTCAGCTTGAAAGCATCACCTTCACCGGCAAGGATGGGAAGGTTTCACAGCTTGAGCATGTTTTTATCCGAGGCAGCAAAGTCAGGTTTATGGTCATTCCAGACATGCTGAAGAATGCTCCAATGTTCAAGCGTCTTGATGCTAGAATCAAGGGCAAGGGCTCAGCACTTGGAGTTGGCCGGGGTAGGGCCGTTGCAATGCGTGCTAGAGCTCAGGCAGCTGGTCGTGGAGCTCCACCTGGTAGGGGTGTTGTGCCACCAGTACGGAGATGA
the amino acid sequence of the protein coded by the grape SmD3-b gene is shown as SEQ ID NO.2, and the specific steps are as follows:
msrslgipvk llheaaghvv tvelksgely rgnmiecedn wncqlesitf tgkdgkvsql ehvfirgskv rfmvipdmlk napmfkrlda rikgkgsalg vgrgravamr araqaagrga ppgrgvvppv rr。
the full-length sequence of the grape SmD3-b related nucleotide or the fragment thereof can be obtained by a PCR amplification method, a recombination method or an artificial synthesis method. For the PCR amplification method, primers can be designed according to the nucleotide sequences disclosed in the present application, and amplified to obtain the sequences by using a commercially available cDNA library or a cDNA library prepared according to a conventional method known to those skilled in the art as a template.
When the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods. In addition, mutations can be introduced into the nucleotide sequences of the present application by chemical synthesis.
In the present application, the expression pattern of grape SmD3-b, i.e., the presence or absence and amount of RNA transcripts of grape SmD3-b in cell tissues, can be analyzed by a real-time fluorescent quantitative PCR method.
Based on the above-found SmD3-b gene, the scope of the present application also includes DNA fragments homologous to the SmD3-b gene.
These DNA fragments homologous to the SmD3-b gene include alleles, homologous genes, mutant genes and derivative genes corresponding to the nucleotide sequence of the application (SEQ ID NO. 1), which are all within the scope of the protection of the application.
The nucleotide sequence of the SmD3-b gene of the application can be easily mutated by a person skilled in the art using known methods, such as directed evolution and point mutation. Those artificially modified nucleotides having 70% or more identity with the nucleotide sequence of the SmD3-b gene of the present application are derived from the nucleotide sequence of the present application and are equivalent to the sequence of the present application as long as the functions are functionally equivalent to the nucleotide sequence shown in SEQ ID NO. 1.
The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes nucleotide sequences having 75% or more, or 85% or more, or 90% or more, or 95% or more identity to the nucleotide sequence set forth in SEQ ID NO.1 of the present application. The identity of amino acid or nucleotide sequences can be determined 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).
The 70% or more identity may be 70%, 75%, 80%, 85%, 90% or 95% or more identity.
The tobacco is selected as a transgenic object because of the fast growth speed, short life cycle, simple and easy operation of the transformation method and high genetic transformation efficiency. However, the SmD3-b gene and the plant expression vector containing the gene can also be used for producing other transgenic plants with improved drought resistance and salt tolerance.
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 application are all conventional in the art and are commercially available. The experimental procedure, without specifying the detailed conditions, was carried out according to the conventional experimental procedure or according to the operating instructions recommended by the suppliers.
Example 1: cloning of grape SmD3-b
1. Plant material:
the material used in the application is 'Crisense seedless' grape tissue culture seedling, and the culture medium is MS culture medium containing sucrose (30 g/L) and plant agar (6 g/L).
Extraction and reverse transcription of RNA:
grape leaves were harvested for RNA extraction. Total RNA was extracted using RNAprep Pure polysaccharide polyphenol plant Total RNA extraction kit (DP 441) following the protocol described above using TaKaRa PrimeScript TM The RT reagent Kit (Perfect Real Time) reverse transcribes 1. Mu.g RNA into cDNA.
Clone validation of SmD 3-b:
primers were designed according to NCBI accession number XM_ 002270496.4:
SmD3-b-F：5′-ATGAGCAGAAGCTTGGGAATACC-3′(SEQ ID NO.3)；
SmD3-b-F：5′-TCATCTCCGTACTGGTGGCAC-3′(SEQ ID NO.4)。
PCR reactions were performed using PrimeSTAR Max Premix high fidelity enzyme following the protocol, and the PCR products were ligated intoT1 Simple Cloning Vector (Beijing full gold Biotechnology Co., ltd.) vector, transformed E.coli DH 5. Alpha. And screened positive single colonies by colony PCR, sent to the Probiotech Co., ltd. For sequencing. The nucleotide sequence of the cloned grape SmD3-b is shown as SEQ ID NO.1, and has one base difference with XM_ 002270496.4.
Example 2: expression of the grape SmD3-b Gene at different drought and salt treatment time points
1. Plant material:
subculturing for 1 month and uniformly growing 'Crisense seedless' grape tissue culture seedlings, respectively soaking the tissue culture seedlings in 300mM mannitol solution and 200mM sodium chloride solution, then respectively sampling at different time points, and quick-freezing and preserving with liquid nitrogen.
Extraction and reverse transcription of RNA:
as in example 1.
Expression level variation of SmD3-b Gene at different treatment time points:
specific primers are designed to carry out real-time fluorescence quantitative PCR analysis on the change rule of the expression quantity of SmD3-b in different treatment time points, and the designed primers are as follows:
qSmD3-b-F：5′-GCAAGGATGGGAAGGTTTCA-3′(SEQ ID NO.5)；
qSmD3-b-R：5′-AACATTGGAGCATTCTTCAGCAT-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 SmD3-b in a sample to be tested:
the cDNA was used as a template for fluorescent quantitative analysis using specific primers for SmD3-b and ACTIN, respectively, and the reaction was performed in a real-time fluorescent quantitative PCR apparatus (CFX connect Real Time PCR Detection System, bio-Rad) using a 20. Mu.L system (10. Mu. L SYBR Premix Ex Taq, 0.6. Mu.M each for the upstream and downstream primers, 1. Mu.L for the cDNA template, and 7.8. Mu.L for water) as follows: 95 ℃ for 30s;95℃for 5s,60℃for 10s,40 cycles.
5. By 2 -△△Ct The relative quantitative analysis of SmD3-b was performed by the method:
the results show that: as drought and salt stress treatment time was prolonged, smD3-b expression levels tended to increase and then decrease, indicating that SmD3-b could differentially express in response to drought and salt stress (FIG. 1).
Example 3: drought and salt tolerance function detection of grape SmD3-b gene transgenic tobacco
The super-expression vector for SmD3-b was constructed using the pHB-gfp vector (described in the literature "MicroRNA171c-targeted SCL6-II, SCL6-III, and SCL6-IV genes regulate shoot branching in Arabidopsis, doi.org/10.1093/mp/ssq 042"), the primers were as follows:
pHBSmD3-b-F：5′-accagtctctctctcaagcttATGAGCAGAAGCTTGGGAATACC-3′(SEQ ID NO.9)；
pHBSmD3-b-R：5′-gcccttgctcaccatggatccTCTCCGTACTGGTGGCACAAC-3′(SEQ ID NO.10)。
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 high fidelity enzymes as follows: 98 ℃ for 10s,55 ℃ for 5s,72 ℃ for 10s 34 cycles; extending at 72℃for 5mins.
The PCR products were separated by 1.5% agarose gel electrophoresis and purified using a SanPrep column DNA gel recovery kit (B518131, bio) according to standard procedures.
The pHB-gfp vector plasmid was digested and the digestion reaction system was 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-gfp and SmD3-b were ligated and the recombinant plasmid was transferred into E.coli DH 5. Alpha. According to the standard procedure of ClonExpress II One Step Cloning Kit kit. Positive clones were screened by colony PCR and sent to the sequencing industry. Single colonies sequenced correctly were shaken overnight and plasmids were extracted according to the procedures described in the SanPrep column plasmid DNA miniprep extraction kit (Bio) standard instructions. Thus obtaining the super-expression vector plasmid containing SmD3-b.
The SmD3-b super-expression vector plasmid is transferred into the tobacco by using a leaf disc method. The method comprises the following steps: LB shaking bacteria, collecting thallus, placing in 50ml sterile water containing 75 μmol/L acetosyringone to form suspension, adjusting suspension OD 600 Cutting sterile tobacco leaves into small pieces with the numerical value of 0.6, putting the small pieces into the adjusted suspension, infecting for 8min, inoculating to symbiotic culture medium (MS+6g/L agar+30g/L sucrose+2mg/L6-BA+0.5 mg/L NAA), dark culturing for 2-3 days, transferring to screening culture medium (MS+6g/L agar+30g/L sucrose+2mg/L6-BA+0.5 mg/L NAA+15mg/L Hyg), and changing culture medium once every half month until the resistant buds are differentiated; bud length to be resistantUpdating the screening culture medium when the plant grows to 1-2cm, and transferring the plant into a rooting culture medium (1/2MS+6g/L agar+30 g/L sucrose+1 mg/L IBA+15mg/L Hyg) for rooting after the plant grows stably. And transferring the rooting seedling into soil, and harvesting seeds. Screening positive transgenic lines until homozygous by glufosinate, and using homozygous seeds for drought resistance identification.
Seeds of homozygous transgenic tobacco were tested, with seeds of wild type tobacco as controls. The seed surface was sterilized, rinsed with 70% ethanol for 30 seconds, sterilized with 2% NaClO solution for 10 minutes, rinsed with sterile water and then spread in 1/2MS medium. Culturing at 26deg.C for 16h under light/8 h in dark, germinating, picking tobacco seedlings with consistent size, transferring into 1/2MS solid culture medium containing 100mM mannitol, and 1/2MS solid culture medium containing 100mM sodium chloride, respectively, and growing for 8 days.
The results are shown in fig. 2, which shows that: under simulated drought and salt stress conditions, the growth conditions of SmD3-b overexpressed tobaccos (# 1, # 2) are obviously higher than that of Wild Type (WT), i.e. SmD3-b can positively regulate drought tolerance and salt tolerance.
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 (9)
1. Use of the grape SmD3-b gene in any one of the following (1) - (2):
(1) Improving drought resistance and salt resistance of plants;
(2) Cultivating plant variety with improved drought tolerance and salt tolerance.
2. The use according to claim 1, characterized in that the grape SmD3-b gene is a nucleic acid molecule as shown in i) or ii) below:
i) The nucleotide sequence is a nucleic acid molecule shown as SEQ ID NO. 1;
ii) a nucleic acid molecule other than i) encoding the amino acid sequence shown in SEQ ID NO. 2.
3. Use of a protein encoded by the grape SmD3-b gene in any one of the following (1) - (2):
(1) Improving drought resistance and salt resistance of plants;
(2) The product for improving drought resistance and salt resistance of plants is prepared.
4. The use according to claim 3, wherein the protein encoded by the grape SmD3-b gene is a protein represented by (A1) or (A2) as follows:
(A1) A protein consisting of an amino acid sequence shown as SEQ ID NO.2 in a sequence table;
(A2) A fusion protein obtained by ligating the N-terminal and/or C-terminal of the protein defined in (A1) with a protein tag.
5. Use of an expression cassette, recombinant expression vector or recombinant bacterium comprising the grape SmD3-b gene in any one of the following (1) - (2):
(1) Improving drought resistance and salt resistance of plants;
(2) Cultivating plant variety with improved drought tolerance and salt tolerance.
6. A method of increasing drought and salt stress tolerance in a plant comprising the steps of:
transferring the SmD3-b gene of the grape into a target plant;
or up-regulating the expression of the grape SmD3-b gene or a homologous gene thereof in the plant genome;
the grape SmD3-b gene is a nucleic acid molecule shown in the following i) or ii):
i) The nucleotide sequence is a nucleic acid molecule shown as SEQ ID NO. 1;
ii) a nucleic acid molecule other than i) encoding the amino acid sequence shown in SEQ ID NO. 2.
7. A method of growing transgenic plants with improved drought and salt tolerance comprising the steps of:
transferring the SmD3-b gene of the grape into a wild type target plant to obtain a plant with drought resistance and salt tolerance superior to those of the wild type;
the grape SmD3-b gene is a nucleic acid molecule shown in the following i) or ii):
i) The nucleotide sequence is a nucleic acid molecule shown as SEQ ID NO. 1;
ii) a nucleic acid molecule other than i) encoding the amino acid sequence shown in SEQ ID NO. 2.
8. The method according to any one of claims 6 to 7, wherein the grape SmD3-b gene is transferred into the plant of interest by means of a pHB-gfp expression vector.
9. The method of claim 8, wherein the plant of interest is grape or tobacco.
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