CN114686514B - Application of NtNAC083 gene in negative regulation of tobacco cold stress response - Google Patents
Application of NtNAC083 gene in negative regulation of tobacco cold stress response Download PDFInfo
- Publication number
- CN114686514B CN114686514B CN202210156602.0A CN202210156602A CN114686514B CN 114686514 B CN114686514 B CN 114686514B CN 202210156602 A CN202210156602 A CN 202210156602A CN 114686514 B CN114686514 B CN 114686514B
- Authority
- CN
- China
- Prior art keywords
- ntnac083
- gene
- tobacco
- cold stress
- cold
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 235000002637 Nicotiana tabacum Nutrition 0.000 title claims abstract description 53
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 45
- 241000208125 Nicotiana Species 0.000 title claims abstract description 44
- 230000008645 cold stress Effects 0.000 title claims abstract description 32
- 230000004044 response Effects 0.000 title claims abstract description 17
- 230000003828 downregulation Effects 0.000 title abstract description 7
- 241000196324 Embryophyta Species 0.000 claims abstract description 36
- 230000002018 overexpression Effects 0.000 claims abstract description 29
- 230000014509 gene expression Effects 0.000 claims abstract description 24
- 244000061176 Nicotiana tabacum Species 0.000 claims abstract description 9
- 238000000034 method Methods 0.000 claims description 20
- 239000013604 expression vector Substances 0.000 claims description 9
- 230000009261 transgenic effect Effects 0.000 claims description 8
- 238000004458 analytical method Methods 0.000 claims description 6
- 241000589158 Agrobacterium Species 0.000 claims description 5
- 239000013598 vector Substances 0.000 claims description 5
- 230000001131 transforming effect Effects 0.000 claims description 4
- 239000002299 complementary DNA Substances 0.000 claims description 3
- 238000001976 enzyme digestion Methods 0.000 claims description 3
- 239000012634 fragment Substances 0.000 claims description 3
- 238000001802 infusion Methods 0.000 claims description 3
- 230000001404 mediated effect Effects 0.000 claims description 3
- 239000013612 plasmid Substances 0.000 claims description 3
- 108091008146 restriction endonucleases Proteins 0.000 claims description 3
- 238000012163 sequencing technique Methods 0.000 claims description 3
- 238000012795 verification Methods 0.000 claims description 3
- 238000010276 construction Methods 0.000 claims 1
- 101710189714 Major cell-binding factor Proteins 0.000 abstract description 7
- 230000003938 response to stress Effects 0.000 abstract description 5
- 101100079509 Oncidium hybrid cultivar NCED gene Proteins 0.000 abstract description 4
- 238000010353 genetic engineering Methods 0.000 abstract description 4
- 230000009456 molecular mechanism Effects 0.000 abstract description 4
- 230000001105 regulatory effect Effects 0.000 abstract description 4
- 230000033228 biological regulation Effects 0.000 abstract description 2
- 230000035882 stress Effects 0.000 description 14
- 239000000463 material Substances 0.000 description 11
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 5
- 108091023040 Transcription factor Proteins 0.000 description 5
- 102000040945 Transcription factor Human genes 0.000 description 5
- 230000008859 change Effects 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 150000002500 ions Chemical class 0.000 description 5
- 102000016938 Catalase Human genes 0.000 description 4
- 108010053835 Catalase Proteins 0.000 description 4
- 239000003642 reactive oxygen metabolite Substances 0.000 description 4
- 241000219194 Arabidopsis Species 0.000 description 3
- 108020004414 DNA Proteins 0.000 description 3
- 101150043797 NCED3 gene Proteins 0.000 description 3
- 102000019197 Superoxide Dismutase Human genes 0.000 description 3
- 108010012715 Superoxide dismutase Proteins 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000003753 real-time PCR Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 150000003839 salts Chemical class 0.000 description 3
- 102100030000 Recombining binding protein suppressor of hairless Human genes 0.000 description 2
- 241000209140 Triticum Species 0.000 description 2
- 235000021307 Triticum Nutrition 0.000 description 2
- SXEHKFHPFVVDIR-UHFFFAOYSA-N [4-(4-hydrazinylphenyl)phenyl]hydrazine Chemical compound C1=CC(NN)=CC=C1C1=CC=C(NN)C=C1 SXEHKFHPFVVDIR-UHFFFAOYSA-N 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 230000025488 response to cold Effects 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 230000017260 vegetative to reproductive phase transition of meristem Effects 0.000 description 2
- 101100186131 Arabidopsis thaliana NAC053 gene Proteins 0.000 description 1
- 101100132482 Arabidopsis thaliana NAC072 gene Proteins 0.000 description 1
- WSMYVTOQOOLQHP-UHFFFAOYSA-N Malondialdehyde Chemical compound O=CCC=O WSMYVTOQOOLQHP-UHFFFAOYSA-N 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 238000012271 agricultural production Methods 0.000 description 1
- 230000009418 agronomic effect Effects 0.000 description 1
- -1 azulene tetrazolium Chemical compound 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000010220 ion permeability Effects 0.000 description 1
- 230000003859 lipid peroxidation Effects 0.000 description 1
- 229940118019 malondialdehyde Drugs 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000006461 physiological response Effects 0.000 description 1
- 230000008640 plant stress response Effects 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000003827 upregulation Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- 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)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
Landscapes
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention provides an application of an NtNAC083 gene in negative regulation of tobacco cold stress response, and belongs to the technical field of gene regulation. The invention discovers that the NtNAC083 gene can be used as a negative regulatory factor to participate in the response of plants to low-temperature cold stress, and the tolerance of tobacco plants to low temperature is effectively reduced through the discovery of the cold phenotype of overground parts of an over-expression strain; further revealing by physiological and molecular experimental data that the NtNAC083 gene may regulate plant response to low temperature by modulating expression of the cold stress critical genes CBF1 or NCED 3. The obtained product expands effective solving means for researching low-temperature stress response molecular mechanism of tobacco and cultivating low-temperature-resistant and early-flowering-resistant new varieties by a genetic engineering method.
Description
Technical Field
The invention belongs to the technical field of gene regulation and control, and particularly relates to application of an NtNAC083 gene in negative regulation and control of tobacco cold stress response.
Background
Temperature plays an important role in limiting plant distribution areas and affecting crop yield. In recent years, under the continuous change of global climate environment, low temperature has become one of the main environmental factors endangering agricultural production, and low temperature stress mainly causes the flowering period of plants to change, thereby influencing crop yield and quality. In the south China, the phenomenon of tobacco early flowering induced by low-temperature stress is serious, the yield and quality of tobacco leaves are seriously affected, and huge economic losses are caused for tobacco farmers. At present, losses caused by early differentiation of flower buds of tobacco leaves due to low-temperature stress are mainly some agronomic measures (such as topping and branching), but a great deal of manpower, material resources and financial resources are generally consumed in implementation of the measures. Therefore, developing a molecular mechanism research of low-temperature stress response of plants (tobacco) and cultivating a new variety with low temperature resistance and early bloom resistance by a genetic engineering method is the most effective way for solving the problem.
NAC transcription factors are present in higher plants and widely distributed, and have various biological functions, not only are they commonly involved in the growth and development processes of plants, but also exert different effects in the stress response processes of plants. For example: overexpression of the anc 072/RD26, NTL4 (anc 053) and anc 055 transcription factors enhances the tolerance of arabidopsis to drought, high salt and temperature stresses; overexpression of the MLNAC5 transcription factor in Arabidopsis increases drought and low temperature resistance of Arabidopsis; under high salt, drought and low temperature stresses, wheat TaNAC2 transcription factor expression is significantly increased, and tolerance of the knockout material wheat to salt, drought and low temperature stresses is enhanced by activating several stress response genes; however, little research is currently done on the function of tobacco NAC transcription factors.
Disclosure of Invention
The invention provides an application of an NtNAC083 gene in negative regulation of tobacco cold stress response, and the application of the gene in research of molecular mechanism of low-temperature stress response of tobacco expands effective solving means for cultivating a new variety with low temperature resistance and early bloom resistance by a genetic engineering method.
In order to achieve the aim, the invention provides an application of the NtNAC083 gene in negative regulation of tobacco cold stress response.
Preferably, the NtNAC083 gene negatively regulates tobacco response to cold stress by modulating expression of a cold stress critical gene CBF1 or NCED 3.
Preferably, the NtNAC083 gene negatively regulates tobacco tolerance to cold stress by affecting propylene glycol and ion permeability.
Preferably, the sequence of the NtNAC083 gene is as shown in SEQ ID NO: 1. The gene sequence is a known sequence, and the number of the gene sequence on Ncbi is XM_016602248.1.
Preferably, the conditions of the cold stress are treatment at 4 ℃ for 30 days.
The invention also provides a method for improving the cold stress response capability of tobacco, which comprises the following steps:
and constructing an over-expression vector of a tobacco gene NtNAC083, and transforming the over-expression vector into tobacco through agrobacterium mediation to obtain a transgenic plant for improving the cold stress response capability of the tobacco.
Preferably, the method for constructing the overexpression vector of the tobacco gene NtNAC083 comprises the following steps:
the following primers were designed based on the full length sequence of NtNAC 083:
NtNAC083-F：CACTGTTGATACATATGGTTGGGAAAATTAGCTCGG；NtNAC083-R：TGTTGATTCAGAATTCTCACTGAAATTGAAAAGCTGGA；
using common tobacco K326 cDNA as a template, carrying out NtNAC083 full-length sequence amplification by PrimeSTAR HS DNA high-fidelity enzyme, linearizing AN over-expression vector pRI101-AN by using NEB restriction enzymes NdeI and EcoRI-HF, constructing a target gene fragment into pRI101-AN by using AN infusion method, extracting recombinant plasmids, carrying out enzyme digestion verification and sequencing, and obtaining the over-expression vector.
Preferably, after constructing an overexpression vector of a tobacco gene NtNAC083, converting ordinary tobacco K326 by using an agrobacterium-mediated leaf disc method, and analyzing the expression quantity of the obtained transgenic plant NtNAC083 gene to obtain a transgenic plant NtNAC083-OE for improving the cold stress response capability of tobacco.
Preferably, the conditions of cold stress are treatment at 4 ℃ for 30 days.
Compared with the prior art, the invention has the advantages and positive effects that:
the invention discovers that the NtNAC083 gene can be used as a negative regulatory factor to participate in the response of plants to low-temperature cold stress, and the tolerance of tobacco plants to low temperature is effectively reduced through the discovery of the cold phenotype of overground parts of an over-expression strain; further NtNAC083 gene by physiological data it is possible to regulate plant response to low temperature by modulating expression of the cold stress critical genes CBF1 or NCED 3. The obtained product expands effective solving means for researching low-temperature stress response molecular mechanism of tobacco and cultivating low-temperature-resistant and early-flowering-resistant new varieties by a genetic engineering method.
Drawings
Fig. 1 shows analysis of NtNAC083 gene expression pattern and identification of over-expression strain, with significant differences (p < 0.05);
FIG. 2 shows that the control K326 and the NtNAC083-OE provided in the examples of the present invention differ significantly (p < 0.05) in the case of low temperature treatment and in the case of DAT/NBT staining;
FIG. 3 shows the enzyme activities of SOP and CAT for control K326 and NtNAC083-OE provided in the examples of the present invention with respect to ion leakage and malondialdehyde content before and after low temperature treatment; FW, fresh weight of leaf. Three biological replicates were performed for each experiment. * Significant differences (p < 0.05);
FIG. 4 shows the root length phenotype of control K326 and NtNAC083-OE provided by the examples of the present invention before and after low temperature treatment, with significant differences (p < 0.05);
fig. 5 shows the analysis of cold stress related gene expression provided in the examples of the present invention, the difference was significant (p < 0.05).
Detailed Description
The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1 expression pattern
To verify whether NtNAC083 is involved in tobacco cold stress response, wild-type tobacco K326 was treated at 4 ℃ for 0, 1, 3, 6, 12, 24, 36, 48h, RNA was extracted at different time points, and the expression level thereof under cold stress was analyzed using real-time fluorescent quantitative PCR technique, and the results are shown in fig. 1A.
NtNAC083 was strongly induced by cold stress and expressed approximately 500-fold higher than control (treatment 0 h) at 1h, and up-regulation of expression was also evident at 24-36h, 173-fold and 279-fold higher than control, respectively. This result suggests that NtNAC083 plays an important role when tobacco is subjected to cold stress.
Further, analysis of the expression pattern of NtNAC083 in tobacco plants showed that the real-time fluorescent quantitative PCR results showed that the NtNAC083 gene was expressed in roots, stems, leaves, flowers, and that the relative expression levels of flowers, roots, upper leaves were higher, the lower leaves, and terminal buds were expressed less frequently, and the expression level of the middle leaves was lowest (fig. 1B).
The method for constructing the overexpression vector of the NtNAC083 gene comprises the following steps of designing a primer according to the full-length sequence of the NtNAC 083:
NtNAC083-F：CACTGTTGATACATATGGTTGGGAAAATTAGCTCGG；NtNAC083-R：TGTTGATTCAGAATTCTCACTGAAATTGAAAAGCTGGA；
the method comprises the steps of using common tobacco K326 cDNA as a template, carrying out NtNAC083 full-length sequence amplification by PrimeSTAR HS DNA high-fidelity enzyme, linearizing AN over-expression vector pRI101-AN by NEB restriction enzymes NdeI and EcoRI-HF, constructing a target gene fragment into pRI101-AN by using AN infusion method, extracting recombinant plasmids for enzyme digestion verification and sequencing to obtain the over-expression vector, transforming common tobacco K326 by using AN agrobacterium-mediated leaf disc method, successfully screening to obtain transgenic tobacco T0 generation plants, obtaining 8 positive seedlings, analyzing the expression quantity of NtNAC083 genes in 8 strains obtained by screening, and carrying out analysis on the expression quantity of the genes in the 8 strains, wherein the expression quantity of the genes is up-regulated to be 19.65 times and 17.53 times that of a control, and the two strains are selected for subsequent phenotypic analysis.
Example 2 Cold phenotype of aerial parts of an overexpressing Strain
And selecting and planting over-expression and wild (K326) plants, and carrying out cold (4 ℃) stress treatment when the plants grow to a 5-6 leaf period. Phenotypes of NtNAC083-OE transgenic plants and K326 control plants after cold stress treatment were observed (fig. 2A), and plants were analyzed for low temperature tolerance.
In the cold treatment process, compared with a control, the NtNAC083-OE strain has serious wilting and water loss symptoms of leaves, and only new leaves still keep a normal growth state; observations of cold-treated early stage leaves revealed severe freeze-spotting of the NtNAC083-OE material (fig. 2A). Further, the cold-treated leaves were stained by Diaminobenzidine (DAB) and azulene tetrazolium (NBT) to observe the change in ROS accumulation in the leaves, and as shown in FIG. 2C/D, the ROS content of leaves of tobacco NtNAC083-OE material was much higher than that of the control. This demonstrates that the overexpression of the NtNAC083 gene reduces the tolerance of tobacco plants to low temperatures, i.e. that the NtNAC083 gene is involved as a negative regulator in the plant's response to cold stress at low temperatures.
Example 3 physiological data
The change of external conditions often causes the plant to generate a certain physiological response, and the low-temperature stress can also influence the physiological change of the plant as well as other stress factors. In plants, the extent of damage to the membrane system in adverse (cold) conditions can be seen by the level of propylene glycol (MDA) and ion leakage levels.
The MDA and ion leakage rates of the NtNAC083 overexpressing plants and the control before and after the low temperature treatment were analyzed, and the results are shown in fig. 3A/B, the MDA content in the tobacco of the overexpressing lines NtNAC083-OE5, ntNAC083-OE8 and the control K326 was not significantly different before the cold treatment, the MDA content in the tobacco of the NtNAC083-OE5 and the NtNAC083-OE8 after the cold stress was significantly increased, and the variation amplitude was significantly larger than that of the control, indicating that the cold tolerance of the plants of the NtNAC083-OE5 and the NtNAC083-OE8 was weak.
After cold treatment, control K326 and NtNAC083-OE5 and NtNAC083-OE8 tobacco leaf ion leakage was significantly increased compared to that before treatment, and the plants of NtNAC083-OE5 and NtNAC083-OE8 increased more (FIG. 3B); the NtNAC083 is illustrated to regulate tobacco resistance to low temperatures by affecting MDA and ion leakage rates.
SOD and CAT in plants are important members of a biomembrane protective enzyme system, and cooperate to reduce the damage of defensive Reactive Oxygen Species (ROS) or other peroxy radicals to the membrane system, inhibit membrane lipid peroxidation and reduce the damage of low-temperature stress to cells. Under low temperature stress, the enzyme activities of superoxide dismutase (SOD) and Catalase (CAT) in the NtNAC083-OE5 and NtNAC083-OE8 over-expression materials are obviously reduced compared with those of a control, which indicates that the defense system of the over-expression material plant is seriously damaged under the low temperature stress, and the damage caused by low temperature cannot be adapted or saved.
EXAMPLE 4 root phenotype
The root is an important organ of the plant, the growth state of the root has a decisive effect on the overground part, and as shown in figure 4, under the normal growth condition, the plant overexpressing the NtNAC083-OE5 and the plant overexpressing the NtNAC083-OE8 have no obvious difference from the root length of the control, and are about 9 cm. Under low temperature stress, the root length of seedlings of the NtNAC083-OE5 and NtNAC083-OE8 over-expressed materials is obviously inhibited and reduced to 5cm, the root length of a control is not substantially inhibited, meanwhile, the fresh weight of the seedlings is analyzed, the fresh weight of the over-expressed materials is obviously lower than that of the control, and the result shows that the over-expression of the NtNAC083 gene seriously influences the growth and development of the roots, so that the low temperature tolerance of the roots is reduced.
Example 5 Effect of NtNAC083 on Cold stress related Gene expression in tobacco
Low temperature stress has been shown to induce expression of related genes in plants, for example: CBF1 and NCED3, thereby affecting the cold resistance or cold resistance of plants. The present study further analyzed the expression levels of two genes in the control material and the over-expression at low temperature by real-time fluorescent quantitative PCR, and the results are shown in fig. 5, in which the expression levels of CBF1 and NCED3 genes in control K326 are significantly up-regulated after low-temperature induction, but the expression of both genes in the over-expression material is suppressed, indicating that the expression of cold stress genes CBF1 and NCED3 is suppressed due to the overexpression of NtNAC083 in the over-expression material, resulting in a decrease in cold resistance of plants. These results indicate that the NtNAC083 gene may regulate plant response to low temperatures by modulating expression of the cold stress critical genes CBF1 or NCED 3.
Sequence listing
<110> institute of tobacco in China agricultural sciences (institute of tobacco in Qingzhou of China tobacco head)
Application of <120> NtNAC083 gene in negative regulation of tobacco cold stress response
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1080
<212> DNA
<213> NtNAC083 Gene
<400> 1
atggttggga aaattagctc ggatcttcct cctggattta ggtttcatcc tactgatgaa 60
gaattaatca tgtattatct tcgatatcaa gctacctcga ggccatgtcc cgtttcaatt 120
atccccgaag ttgatctata caaattcgat ccctgggaat tgcctggtta gtatgcaata 180
tatactcgaa aaattgttta aaattcgaag aatataatac acttttgaat gtttcttgaa 240
atattatgat gattatgttt tatgaaatgt agagaaagct gaatttggag aaaatgaatg 300
gtatttcttc acccctcgtg ataggaaata tccaaatggt gttaggccaa atagagcagc 360
tgtttcaggt tattggaagg ctacaggaac tgataaagct atttatagtg catctaaata 420
tgttggtgta aaaaaagctc ttgtttttta taaaggaaaa cctccaaaag gtgtcaagac 480
tgattggatt atgcatgaat atcgtttaaa tgaatcaaga tctcaaccta acaagcaaag 540
tggctctatg agggtaagac ctttcagttc caaattagtc ggggtcagtt atacgaatcg 600
tttgtatata tccattcgac gaattacact aatgtttttt tttccttctt ggatattttt 660
cagttagatg attgggtact ttgtagaatt tataagaaga aaaatatggg aaaaagtatg 720
gagatgatga aagctgaaga agaagaatca gaggctcaaa ttgagattac caataatgat 780
attgaagttg ttggtgctac tactagtgga ccacaaacaa acaaattgcc aaggaattgt 840
tcattgtcac atctactaga attggattat tttgggtcaa ttccacaatt gctaggtgac 900
aattcatacc atacaagttt tgatattgac caaagttaca cgatgaatag tgttaataca 960
aatgtggttc atcaaatgga gaaatctcag ctaggggaag tgtcacacca acataatagc 1020
caaaataata attataatat cttcttcaac cagcagccag tttttgtaaa tccagctttt 1080
Claims (6)
1.NtNAC083Use of overexpression of a gene for reducing cold stress tolerance in tobacco, characterized in that theNtNAC083The sequence of the gene is shown in SEQ ID NO: 1.
2. The use according to claim 1, wherein the conditions of cold stress are treatment at 4 ℃ for 30 days.
3. A method of reducing the cold stress response of tobacco comprising the steps of:
construction of claim 1NtNAC083The over-expression vector of the gene is transformed into tobacco through agrobacterium mediation to obtain a transgenic plant with reduced low temperature tolerance.
4. A method according to claim 3, wherein a tobacco gene is constructedNtNAC083The method of the over-expression vector of (2) is as follows:
according toNtNAC083The full length sequence was designed as follows:
NtNAC083-F：CACTGTTGATACATATGGTTGGGAAAATTAGCTCGG；NtNAC083-R：TGTTGATTCAGAATTCTCACTGAAATTGAAAAGCTGGA；
using common tobacco K326 cDNA as template, using PrimeSTAR HS DNA high-fidelity enzymeNtNAC083Amplifying the full-length sequence, linearizing AN overexpression vector pRI101-AN by using NEB restriction enzymes NdeI and EcoRI-HF, constructing a target gene fragment into pRI101-AN by using AN infusion method, extracting a recombinant plasmid, performing enzyme digestion verification and sequencing to obtain the overexpression vector.
5. A method according to claim 3, wherein a tobacco gene is constructedNtNAC083After the over-expression vector of (2), the agrobacterium-mediated leaf disc method is used for transforming the common tobacco K326 by the method of transforming the obtained transgenic plantNtNAC083Analysis of the gene expression level to obtain transgenic plants with reduced tolerance to low temperaturesNtNAC083-OE。
6. The method according to any one of claims 3 to 5, wherein the conditions of cold stress are treatment at 4 ℃ for 30 days.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210156602.0A CN114686514B (en) | 2022-02-21 | 2022-02-21 | Application of NtNAC083 gene in negative regulation of tobacco cold stress response |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210156602.0A CN114686514B (en) | 2022-02-21 | 2022-02-21 | Application of NtNAC083 gene in negative regulation of tobacco cold stress response |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114686514A CN114686514A (en) | 2022-07-01 |
| CN114686514B true CN114686514B (en) | 2023-11-03 |
Family
ID=82137457
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210156602.0A Active CN114686514B (en) | 2022-02-21 | 2022-02-21 | Application of NtNAC083 gene in negative regulation of tobacco cold stress response |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114686514B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115724930B (en) * | 2022-08-30 | 2023-09-05 | 广东省农业科学院作物研究所 | Tobacco NtMYB transcription factor and application thereof |
| CN116574742B (en) * | 2023-05-31 | 2024-02-20 | 武汉轻工大学 | HOVUSG4853900 gene related to highland barley freeze damage tolerance stress and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102924582A (en) * | 2012-11-22 | 2013-02-13 | 中国农业科学院作物科学研究所 | Plant-adversity-resistance related protein TaNAC67 as well as coding gene and application thereof |
| JP2016214218A (en) * | 2015-05-26 | 2016-12-22 | 国立大学法人埼玉大学 | Method for modifying quantitative character of secondary cell wall of plant by utilizing transcription inhibitory factor |
| CN109112130A (en) * | 2018-09-03 | 2019-01-01 | 中国农业科学院烟草研究所 | The special evoked promoter of the with high salt and aging of one kind, engineered vector and application |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8420890B2 (en) * | 2006-03-28 | 2013-04-16 | Cornell Research Foundation, Inc. | Use of NAP gene to manipulate leaf senescence in plants |
| US10865420B2 (en) * | 2013-06-11 | 2020-12-15 | Florida State University Research Foundation, Inc. | Materials and methods for controlling bundle sheath cell fate and function in plants |
-
2022
- 2022-02-21 CN CN202210156602.0A patent/CN114686514B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102924582A (en) * | 2012-11-22 | 2013-02-13 | 中国农业科学院作物科学研究所 | Plant-adversity-resistance related protein TaNAC67 as well as coding gene and application thereof |
| JP2016214218A (en) * | 2015-05-26 | 2016-12-22 | 国立大学法人埼玉大学 | Method for modifying quantitative character of secondary cell wall of plant by utilizing transcription inhibitory factor |
| CN109112130A (en) * | 2018-09-03 | 2019-01-01 | 中国农业科学院烟草研究所 | The special evoked promoter of the with high salt and aging of one kind, engineered vector and application |
Non-Patent Citations (5)
| Title |
|---|
| "GmWRKY27 interacts with GmMYB174 to reduce expression of GmNAC29 for stress tolerance in soybean plants";Fang Wang 等;《The Plant Journal》;第83卷;摘要、第225页右栏第1段、最后两段 * |
| "Isolation of multi-abiotic stress response genes to generate global warming defense forage crops";Netty Ermawati 等;《Journal of the Korean Society of Grassland and Forage Science》;第41卷(第4期);第242-249页 * |
| "New Insights into the Roles of Osmanthus Fragrans Heat-Shock Transcription Factors in Cold and Other Stress Responses";Jing Bin 等;《Horticulturae》;第8卷(第80期);第2.8节、第3.10节、第16页第3段 * |
| "Nicotiana tabacum cultivar TN90 unplaced genomic scaffold, Ntab-TN90 Ntab-TN90_scaffold44443, whole genome shotgun sequence，ACCESSION: NW_015907634.1";Sierro,N. 等;《GenBank》;第1-2页 * |
| "苹果矮化砧‘71-3-150’对冷胁迫的生理与转录组响应";王海波 等;《园艺学报》;第43卷(第8期);第1437-1451页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114686514A (en) | 2022-07-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Islam et al. | Functional identification of a rice trehalase gene involved in salt stress tolerance | |
| CN114686514B (en) | Application of NtNAC083 gene in negative regulation of tobacco cold stress response | |
| Gao et al. | Transgenic tomato overexpressing ath-miR399d improves growth under abiotic stress conditions | |
| Iqbal et al. | Physiological and transcriptome analysis of heteromorphic leaves and hydrophilic roots in response to soil drying in desert Populus euphratica | |
| Zhou et al. | The oil palm R2R3-MYB subfamily genes EgMYB111 and EgMYB157 improve multiple abiotic stress tolerance in transgenic Arabidopsis plants | |
| Liu et al. | MbMYBC1, a M. baccata MYB transcription factor, contribute to cold and drought stress tolerance in transgenic Arabidopsis | |
| Qin et al. | Down-regulation of GhADF1 in cotton (Gossypium hirsutum) improves plant drought tolerance and increases fiber yield | |
| Xu et al. | Overexpression of the transcription factor NtNAC2 confers drought tolerance in tobacco | |
| Wu et al. | LcMKK, a novel group A mitogen-activated protein kinase kinase gene in Lycium chinense, confers dehydration and drought tolerance in transgenic tobacco via scavenging ROS and modulating expression of stress-responsive genes | |
| Qiao et al. | Characterization of AhLea-3 and its enhancement of salt tolerance in transgenic peanut plants | |
| CN109371041B (en) | Rice gene OsHGN for increasing grain number per ear and application thereof | |
| CN116590308B (en) | Potato drought tolerance related heat shock protein gene HSP101 and application thereof | |
| CN103570813A (en) | Plant-stress-resistance related protein Gh01399, and coding gene and application thereof | |
| Tao et al. | Cloning and functional analysis of the BrCUC2 gene in Brassica rapa L | |
| CN114214333B (en) | Gene for regulating and controlling plant leaf epidermal hair development and secondary wall thickness and application thereof | |
| Kong et al. | Calcium-dependent protein kinase GhCDPK4 plays a role in drought and abscisic acid stress responses | |
| CN113214371B (en) | Loquat drought-resistant related EjWRKY17 gene and encoding protein and application thereof | |
| CN114907465A (en) | OsLEA9 protein related to cold resistance of rice in booting stage and related biological material and application thereof | |
| CN114292855A (en) | PagARR9 gene for regulating and controlling growth of xylem of poplar and application thereof | |
| CN114561420A (en) | Plant drought resistance related protein AGL27 and application of coding gene thereof | |
| CN117567578B (en) | Use of NtCDF transcription factor in improving cold tolerance of tobacco | |
| CN114990154B (en) | Application of GmFBX193 gene in negative regulation of soybean salt stress response | |
| CN117567577B (en) | Use of NtRVE transcription factor in improving low temperature stress resistance of tobacco | |
| CN110205325A (en) | The application of soybean VQ motif encoding gene GmVQ58 | |
| CN117003844B (en) | Potato tuber regulatory gene StRAP2.7b and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |