CN116590308A - Potato drought tolerance related heat shock protein gene HSP101 and application thereof - Google Patents
Potato drought tolerance related heat shock protein gene HSP101 and application thereof Download PDFInfo
- Publication number
- CN116590308A CN116590308A CN202310518401.5A CN202310518401A CN116590308A CN 116590308 A CN116590308 A CN 116590308A CN 202310518401 A CN202310518401 A CN 202310518401A CN 116590308 A CN116590308 A CN 116590308A
- Authority
- CN
- China
- Prior art keywords
- potato
- drought tolerance
- drought
- heat shock
- hsp101
- 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.)
- Granted
Links
- 244000061456 Solanum tuberosum Species 0.000 title claims abstract description 49
- 235000002595 Solanum tuberosum Nutrition 0.000 title claims abstract description 48
- 230000024346 drought recovery Effects 0.000 title claims abstract description 32
- 108010004889 Heat-Shock Proteins Proteins 0.000 title claims abstract description 22
- 101150043687 CLPB1 gene Proteins 0.000 title claims abstract description 14
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 21
- 235000012015 potatoes Nutrition 0.000 claims abstract description 9
- 238000009395 breeding Methods 0.000 claims abstract description 8
- 230000001488 breeding effect Effects 0.000 claims abstract description 8
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 7
- 230000006872 improvement Effects 0.000 claims abstract description 7
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 7
- 239000002773 nucleotide Substances 0.000 claims abstract description 5
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 5
- 239000013604 expression vector Substances 0.000 claims description 4
- 238000003259 recombinant expression Methods 0.000 claims description 4
- 238000000034 method Methods 0.000 abstract description 7
- 230000007246 mechanism Effects 0.000 abstract description 4
- 210000000805 cytoplasm Anatomy 0.000 abstract description 3
- 238000004458 analytical method Methods 0.000 description 14
- 230000008641 drought stress Effects 0.000 description 14
- 241000196324 Embryophyta Species 0.000 description 12
- 238000011529 RT qPCR Methods 0.000 description 10
- 230000014509 gene expression Effects 0.000 description 10
- 230000012010 growth Effects 0.000 description 8
- 230000009261 transgenic effect Effects 0.000 description 8
- JLIDBLDQVAYHNE-YKALOCIXSA-N (+)-Abscisic acid Chemical compound OC(=O)/C=C(/C)\C=C\[C@@]1(O)C(C)=CC(=O)CC1(C)C JLIDBLDQVAYHNE-YKALOCIXSA-N 0.000 description 7
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 6
- 229930195725 Mannitol Natural products 0.000 description 6
- 239000000594 mannitol Substances 0.000 description 6
- 235000010355 mannitol Nutrition 0.000 description 6
- 102000002812 Heat-Shock Proteins Human genes 0.000 description 5
- 230000002018 overexpression Effects 0.000 description 5
- 239000013598 vector Substances 0.000 description 5
- 241000227653 Lycopersicon Species 0.000 description 4
- 235000007688 Lycopersicon esculentum Nutrition 0.000 description 4
- 239000012634 fragment Substances 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 230000001105 regulatory effect Effects 0.000 description 4
- 230000004044 response Effects 0.000 description 4
- 239000002689 soil Substances 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 241000589158 Agrobacterium Species 0.000 description 3
- 101710163595 Chaperone protein DnaK Proteins 0.000 description 3
- 101710178376 Heat shock 70 kDa protein Proteins 0.000 description 3
- 101710152018 Heat shock cognate 70 kDa protein Proteins 0.000 description 3
- 240000007594 Oryza sativa Species 0.000 description 3
- 235000007164 Oryza sativa Nutrition 0.000 description 3
- 239000008118 PEG 6000 Substances 0.000 description 3
- 229920002584 Polyethylene Glycol 6000 Polymers 0.000 description 3
- 108700019146 Transgenes Proteins 0.000 description 3
- 230000033228 biological regulation Effects 0.000 description 3
- 238000010367 cloning Methods 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- MTHSVFCYNBDYFN-UHFFFAOYSA-N diethylene glycol Chemical compound OCCOCCO MTHSVFCYNBDYFN-UHFFFAOYSA-N 0.000 description 3
- 230000002452 interceptive effect Effects 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 235000009566 rice Nutrition 0.000 description 3
- 150000003839 salts Chemical class 0.000 description 3
- 230000017260 vegetative to reproductive phase transition of meristem Effects 0.000 description 3
- 235000002566 Capsicum Nutrition 0.000 description 2
- 240000008574 Capsicum frutescens Species 0.000 description 2
- 229920000742 Cotton Polymers 0.000 description 2
- 108700026244 Open Reading Frames Proteins 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 239000001390 capsicum minimum Substances 0.000 description 2
- 239000000679 carrageenan Substances 0.000 description 2
- 229920001525 carrageenan Polymers 0.000 description 2
- 229940113118 carrageenan Drugs 0.000 description 2
- 235000010418 carrageenan Nutrition 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 230000001086 cytosolic effect Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000002708 enhancing effect Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000001965 increasing effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 150000003384 small molecules Chemical class 0.000 description 2
- 230000035882 stress Effects 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- UHVMMEOXYDMDKI-JKYCWFKZSA-L zinc;1-(5-cyanopyridin-2-yl)-3-[(1s,2s)-2-(6-fluoro-2-hydroxy-3-propanoylphenyl)cyclopropyl]urea;diacetate Chemical compound [Zn+2].CC([O-])=O.CC([O-])=O.CCC(=O)C1=CC=C(F)C([C@H]2[C@H](C2)NC(=O)NC=2N=CC(=CC=2)C#N)=C1O UHVMMEOXYDMDKI-JKYCWFKZSA-L 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 102100024341 10 kDa heat shock protein, mitochondrial Human genes 0.000 description 1
- 101150047137 ABF1 gene Proteins 0.000 description 1
- 101150017339 ABI5 gene Proteins 0.000 description 1
- 241000219194 Arabidopsis Species 0.000 description 1
- 241000219195 Arabidopsis thaliana Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 101100489922 Caenorhabditis elegans abf-2 gene Proteins 0.000 description 1
- 240000004160 Capsicum annuum Species 0.000 description 1
- 235000002567 Capsicum annuum Nutrition 0.000 description 1
- 108010059013 Chaperonin 10 Proteins 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 108700024394 Exon Proteins 0.000 description 1
- 101150076784 HSP100 gene Proteins 0.000 description 1
- 101100025200 Homo sapiens MSC gene Proteins 0.000 description 1
- 108091092195 Intron Proteins 0.000 description 1
- WSMYVTOQOOLQHP-UHFFFAOYSA-N Malondialdehyde Chemical compound O=CCC=O WSMYVTOQOOLQHP-UHFFFAOYSA-N 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 102000006404 Mitochondrial Proteins Human genes 0.000 description 1
- 108010058682 Mitochondrial Proteins Proteins 0.000 description 1
- 108010006519 Molecular Chaperones Proteins 0.000 description 1
- 102000005431 Molecular Chaperones Human genes 0.000 description 1
- 102100038169 Musculin Human genes 0.000 description 1
- 241000208125 Nicotiana Species 0.000 description 1
- 241000228653 Nicotiana attenuata Species 0.000 description 1
- 235000002637 Nicotiana tabacum Nutrition 0.000 description 1
- 101100438011 Oryza sativa subsp. japonica BZIP12 gene Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 108091030071 RNAI Proteins 0.000 description 1
- 102000018120 Recombinases Human genes 0.000 description 1
- 108010091086 Recombinases Proteins 0.000 description 1
- 235000004789 Rosa xanthina Nutrition 0.000 description 1
- 241000109329 Rosa xanthina Species 0.000 description 1
- 108010088928 Small Heat-Shock Proteins Proteins 0.000 description 1
- 240000003768 Solanum lycopersicum Species 0.000 description 1
- 235000002560 Solanum lycopersicum Nutrition 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- 230000021736 acetylation Effects 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 238000009412 basement excavation Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000001511 capsicum annuum Substances 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 238000002856 computational phylogenetic analysis Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- FCRACOPGPMPSHN-UHFFFAOYSA-N desoxyabscisic acid Natural products OC(=O)C=C(C)C=CC1C(C)=CC(=O)CC1(C)C FCRACOPGPMPSHN-UHFFFAOYSA-N 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000010195 expression analysis Methods 0.000 description 1
- 230000008124 floral development Effects 0.000 description 1
- 230000030279 gene silencing Effects 0.000 description 1
- 230000009368 gene silencing by RNA Effects 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 230000035784 germination Effects 0.000 description 1
- 230000008642 heat stress Effects 0.000 description 1
- 230000013632 homeostatic process Effects 0.000 description 1
- 230000015784 hyperosmotic salinity response Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 229940118019 malondialdehyde Drugs 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 230000009013 pigment accumulation Effects 0.000 description 1
- 230000008635 plant growth Effects 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 239000003642 reactive oxygen metabolite Substances 0.000 description 1
- 230000003938 response to stress Effects 0.000 description 1
- 230000025469 response to water deprivation Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 239000002195 soluble material Substances 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000011426 transformation method Methods 0.000 description 1
- 230000005068 transpiration Effects 0.000 description 1
Classifications
-
- 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
- C12N15/79—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
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)
- Biophysics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Botany (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Plant Pathology (AREA)
- Gastroenterology & Hepatology (AREA)
- Microbiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a potato drought tolerance related heat shock protein gene HSP101, the nucleotide sequence of which is shown as SEQ ID No.1, and the amino acid sequence of the encoded protein is shown as SEQ ID No. 2. The gene is located in cytoplasm, and interference of the gene can obviously reduce drought tolerance of potato. The method provides genetic materials and theoretical basis for analyzing drought tolerance mechanisms and drought tolerance improvement breeding of the potatoes.
Description
Technical Field
The invention relates to the technical field of plant genetic engineering and potato breeding, in particular to a potato drought tolerance related heat shock protein gene HSP101 and application thereof.
Background
Potatoes belong to shallow root plants, which are very sensitive to drought. Drought can strongly inhibit the growth and development process, which in turn leads to reduced potato tuber yield and reduced quality. To cope with drought stress, potatoes have evolved a series of strategies to resist drought. At the biochemical level, potato plants, when subjected to drought stress, have increased accumulation of soluble materials enabling plant root cells to take up more water from the soil. In the physiological aspect, the potato reduces transpiration and water loss to the greatest extent by reducing the pore conductivity, the number of leaves and the leaf area, thereby improving the water utilization rate of plants. On the molecular level, the drought-related genes of the potatoes react rapidly to induce expression when being subjected to drought stimulation, so that the tolerance of plants is improved. For example, drought results in the synthesis of abscisic acid, thereby inducing the expression of drought-related genes such as ABI5, ABF1, and ABF 2.
Heat shock proteins (Heat Shock Proteins, HSPs) are a class of heat stress proteins that are widely available from bacteria to mammals. The HSPs family plays an important role in drought stress response mechanisms in a variety of plants. In tomato, under drought stress conditions, silencing of HSP70 can lead to high damage and leakage of plant cell membranes, reduced relative water content, reduced pigment accumulation and reduced antioxidant enzyme activity, suggesting that HSP70 genes play a key role in tomato response and adaptation to drought. In rice, oshspa 50.2 is a positive regulator of rice drought tolerance, may be involved in the regulation of reactive oxygen species homeostasis under drought stress, and may act as a molecular chaperone in mediating drought stress signaling in stress responses. In addition, rice oshs17.7 may also help to protect plasma membrane structures against drought stress and subsequent osmotic regulation (Sato et al, 2008). In capsicum, transcription of cahsp25.9 is induced by heat, salt and drought stress, which can positively regulate heat, salt and drought tolerance of plants. In addition, cahsp25.9 may regulate drought stress tolerance by reducing the accumulation of reactive oxygen ROS and regulating the expression of stress-related genes; caHSP16.4 as a small heat shock protein gene in capsicum can also be involved in the regulation of heat and drought tolerance of plants. In corn ZmHsf08 may enhance its tolerance to salt and drought stress by enhancing ROS levels and malondialdehyde content in plants. In cotton, ghhsp24.7 can mediate mitochondrial protein acetylation to regulate stomatal conductance under cotton drought stress. Thus, the plant HSP family plays an important role in drought stress resistance and simultaneously regulates the growth and development process of plants. In potatoes, sths26.5 is presumed to be involved in regulating tuber germination. The cytoplasmic small molecule heat shock protein HSP17.5 is expressed in large quantity in the development stage of roses, which shows that the cytoplasmic small molecule heat shock protein HSP17.5 plays an important role in flower development. In the temperature range of 12 ℃ to 27 ℃, expression of HSP70 in arabidopsis thaliana is positively correlated with flowering time; RNAi-HSP90 transgenic lines delay Arabidopsis flowering under normal growth conditions and induce expression changes in flowering-related genes.
With the increasing greenhouse effect, extreme drought weather is frequent, and the potato tuber forming process is severely affected by these extreme environments, severely threatening the yield and quality of potato tubers. Therefore, the research on the action mechanism of the heat shock protein gene of the potato in response to drought stress, the excavation and analysis of the drought-enduring gene of the potato can provide scientific research basis for improving and breeding the drought-enduring of the potato.
Disclosure of Invention
Based on the above, the invention aims to provide a potato drought tolerance related heat shock protein gene HSP101 and application thereof.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a potato drought tolerance related heat shock protein gene HSP101, the nucleotide sequence of which is shown as SEQ ID No.1, and the amino acid sequence of the encoded protein is shown as SEQ ID No. 2.
The invention also provides application of the potato drought tolerance related heat shock protein gene HSP101 in potato drought tolerance improvement breeding, and interference of the heat shock protein gene HSP101 can reduce the drought tolerance of potatoes.
The invention also provides a recombinant expression vector of the potato drought tolerance related heat shock protein gene HSP 101.
The invention also provides application of the recombinant expression vector of the potato drought tolerance related heat shock protein gene HSP101 in potato drought tolerance improvement breeding.
The invention has the beneficial effects that: the invention provides a potato drought tolerance related heat shock protein gene HSP10, the nucleotide sequence of which is shown as SEQ ID No.1, and the amino acid sequence of the encoded protein is shown as SEQ ID No. 2. The gene is located in cytoplasm, and interference of the gene can obviously reduce drought tolerance of potato. The method provides genetic materials and theoretical basis for analyzing drought tolerance mechanisms and drought tolerance improvement breeding of the potatoes.
Drawings
FIG. 1 is a sequence profile of StHSP101, wherein 1A is a agarose gel electrophoresis result diagram of StHSP101 after CDS amplification, 1B is StHSP101 profile, 1C is StHSP101 conserved domain analysis, and 1D, 1E are phylogenetic tree analysis;
FIG. 2 is an analysis of StHSP101 expression patterns, wherein 2A is identified by qRT-PCR of 20% PEG6000 simulated drought treatment induced StHSP101 expression; 2B is qRT-PCR identification of 20 mu M ABA induced StHSP101 expression;
FIG. 3 shows qRT-PCR identification of StHSP101 transgenic lines, wherein 3A is the qRT-PCR identification of interference lines and 3B is the qRT-PCR identification of over-expressed lines;
FIG. 4 is a phenotypic analysis of StHSP101 transgenic lines under mannitol simulated drought conditions, wherein 4A growth profile analysis, 4B stem length analysis, 4C above ground fresh weight analysis, 4D branching number analysis, 4E below ground fresh weight analysis, and 4F root number analysis;
FIG. 5 is a phenotypic analysis of StHSP101 transgenic lines under drought conditions, where 5A is the phenotype before drought treatment, 5B is the phenotype after drought treatment 10d, and 5C is the phenotype after water-up after drought treatment 14 d.
Detailed Description
The technical scheme of the invention will be further described in detail below with reference to specific embodiments. It is to be understood that the following examples are illustrative only and are not to be construed as limiting the scope of the invention. All techniques implemented based on the above description of the invention are intended to be included within the scope of the invention. It should be noted that, the experimental materials in the examples of the present invention are all commercially available, and the experimental methods without specific conditions are usually performed according to conventional conditions or according to the recommended conditions of the experimental material manufacturer. In addition, the potato E3 in the present invention is the hubei potato No. 3.
EXAMPLE 1 cloning and sequence characterization of StHSP101
The CDS sequence of the StHSP101 is amplified by taking the cDNA of a potato E3 leaf as a template, the size of a target fragment is detected to be 2000-3000bp (figure 1A) by 1% agarose gel electrophoresis, the target fragment is consistent with the size of a gene predicted by a database, the accession number is PGSC0003DMG400024644, sequencing and analysis results show that the gene is positioned in cytoplasm, the gene open reading frame is 2739bp (174-2912 bp), the gene open reading frame contains 6 introns and 7 exons, the nucleotide sequence is shown as SEQ ID No.1, the code 912 amino acids (figure 1B) are shown as SEQ ID No. 2.StHSP101 was subjected to a conserved domain analysis using SMART (FIG. 1C), and the results showed that the 200-345, 598-740 amino acid sequence region of StHSP101 had AAA conserved domain typical of HSP100 gene family (SMART 00382). The amino acid sequences of the homologous proteins were used to construct the evolutionary tree (FIGS. 1D, 1E). The results show that the amino acid sequence of potato StHSP101 is close to the relatedness of tomato (Solanum lycopersicum), capsicum annuum and tobacco (Nicotiana attenuata), especially the highest homology and closest relatedness to tomato.
Example 2StHSP101 expression Pattern analysis
Taking potato E3 as an experimental material, respectively carrying out 20 mu M ABA and 20% PEG6000 treatment for 0h, 0.5h, 1h, 3h, 6h, 12h and 24h, taking leaf extract RNA, carrying out differential expression analysis on StHSP101 at different time points by utilizing qRT-PCR technology, and the result shows that under the 20% PEG6000 treatment, the relative expression quantity of StHSP101 gene rises at 24h (figure 2A), which shows that StHSP101 is strongly induced by PEG, in order to further study whether StP 101 participates in potato drought stress response through an ABA signal path, carrying out qRT-PCR detection on stem and leaf samples before and after treatment by using 20 mu M ABA for treating E3 tissue culture seedlings, and the experimental result shows that the change trend of transcription abundance of StHSP101 is basically consistent with that of drought treatment results, and the highest at 24h (figure 2B). The above results indicate that StHSP101 may be involved in potato drought stress response through ABA signaling pathway.
Example 3StHSP101 transgenic line screening and identification
By constructing a StHSP101 overexpression vector and an interference vector, taking E3 as a receptor, and adopting an agrobacterium transformation method to carry out genetic transformation to construct a StHSP101 overexpression strain and a StHSP101 interference strain. The construction method of the StHSP101 excessive vector and the interference vector comprises the following steps: according to the CDS sequence of the target gene, primers required by Cloning are designed by using primer design software Vector NTI 10, PCR amplification is carried out by using high-fidelity polymerase MegaFiTM Fidelity 2 ×PCR Master mix of abm company, after target gene fragments are obtained, the purified PCR target fragments are recombined with enzyme-digested Vector by using recombinase Pro Ligation-Free Cloning Kit of abm company, E.coli is transformed and sequenced, and finally agrobacterium transformation experiments are carried out by using agrobacterium competent cell LBA4404 of WEIDI company, and the over-expression strain and interference strain are obtained through PCR identification. The conversion efficiency is identified by adopting qRT-PCR, the detection result of the qRT-PCR of the over-expression strain shows that 14 out of 17 positive over-expression strains are obviously up-regulated (figure 3B), the detection result of the qRT-PCR of the interference strain shows that 19 out of 19 positive interference strains are obviously down-regulated compared with the WT (figure 3A), and the interference efficiency is higher. In order to verify the accuracy of the results, the results were repeated twice, and the results were consistent.
Example 4 phenotypic identification
StHSP101 enhances the tolerance of potato test-tube plantlet to mannitol
Wild Type (WT), interference transgene (RNAi), excess strain (OE) were grown in MS+3% sucrose+0.42% carrageenan medium containing 0.3mol/L mannitol, control was grown in normal MS+3% sucrose+0.42% carrageenan medium, and long-day (22 ℃ C./18 ℃ C., 16h/8 h) cultivation was performed, after 14d cultivation, it was found that during mannitol treatment, excess plant growth was relatively better compared to WT, while interference strain growth was significantly weaker than wild type (FIG. 4A). Measurements of stem length, ground fresh weight, number of branches, underground fresh weight and number of roots showed that WT and transgenic lines were not significantly different under normal conditions from each other, and that after mannitol treatment, the interfering transgenic lines showed lower stem length, ground fresh weight, number of branches and number of roots than the wild type (fig. 4B, C, D, F), but under normal conditions the underground fresh weight, interfering lines were significantly lower than the wild type, and this phenotype was exacerbated after mannitol treatment (fig. 4E). The above results demonstrate that under normal conditions, stHSP101 may increase the resistance of potato seedlings and that under mannitol-simulated drought conditions, interference with StHSP101 significantly decreases the drought tolerance of potato seedlings.
StHSP101 enhancing drought resistance of Potato seedlings
Wild type, interference transgene, excess strain were planted in nutrient soil, after 15d of normal growth in long sunlight (22 ℃/18 ℃,16h/8 h), drought group was no longer watered, control group was watered normally, at which time each strain had consistent growth conditions in drought and control groups (fig. 5A). After 10d drought treatment, the soil moisture content in the drought group was significantly reduced, the soil was dried up, the growth of WT and excess lines was good, and the interference transgenic lines were significantly wilted (FIG. 5B). After 14d drought treatment, the drought group was watered, and both the wilted WT and the excess strain recovered their phenotype, while the interfering strain remained wilted (fig. 5C). The interference transgene in the whole process shows poorer drought tolerance under drought treatment conditions compared with wild type and excessive strains, and under control conditions, each strain has no obvious difference, and the results show that under drought conditions, the interference StHSP101 can obviously reduce the drought tolerance of potatoes.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (4)
1. The nucleotide sequence of the potato drought tolerance related heat shock protein gene HSP101 is shown as SEQ ID No.1, and the amino acid sequence of the encoded protein is shown as SEQ ID No. 2.
2. Use of the potato drought-tolerance-related heat shock protein gene HSP101 according to claim 1 for the improvement of potato drought tolerance breeding, characterized in that the interference heat shock protein gene HSP101 will reduce the drought tolerance of potatoes.
3. The recombinant expression vector of the potato drought tolerance-related heat shock protein gene HSP101 of claim 1.
4. Use of the recombinant expression vector of the potato drought tolerance-related heat shock protein gene HSP101 of claim 3 in potato drought tolerance improvement breeding.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310518401.5A CN116590308B (en) | 2023-05-09 | 2023-05-09 | Potato drought tolerance related heat shock protein gene HSP101 and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310518401.5A CN116590308B (en) | 2023-05-09 | 2023-05-09 | Potato drought tolerance related heat shock protein gene HSP101 and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116590308A true CN116590308A (en) | 2023-08-15 |
| CN116590308B CN116590308B (en) | 2024-03-29 |
Family
ID=87593017
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310518401.5A Active CN116590308B (en) | 2023-05-09 | 2023-05-09 | Potato drought tolerance related heat shock protein gene HSP101 and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116590308B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116904483A (en) * | 2023-08-25 | 2023-10-20 | 西部(重庆)科学城种质创制大科学中心 | Polypeptide gene StUCG5P for regulating potato tubers and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102643855A (en) * | 2012-01-16 | 2012-08-22 | 中国科学院昆明植物研究所 | Application of arabidopsis thaliana heat shock protein gene HSP101 in seed germination and preservation |
| CN106661556A (en) * | 2014-07-18 | 2017-05-10 | 菲利普莫里斯产品有限公司 | Tobacco protease genes |
| CN114621334A (en) * | 2022-03-29 | 2022-06-14 | 安徽省农业科学院园艺研究所 | Application of potato StABI5 gene in drought resistance regulation and method for regulating drought resistance of potatoes based on gene |
| US20220348949A1 (en) * | 2009-08-04 | 2022-11-03 | Evogene Ltd. | Polynucleotides and polypeptides for increasing desirable plant qualities |
-
2023
- 2023-05-09 CN CN202310518401.5A patent/CN116590308B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20220348949A1 (en) * | 2009-08-04 | 2022-11-03 | Evogene Ltd. | Polynucleotides and polypeptides for increasing desirable plant qualities |
| CN102643855A (en) * | 2012-01-16 | 2012-08-22 | 中国科学院昆明植物研究所 | Application of arabidopsis thaliana heat shock protein gene HSP101 in seed germination and preservation |
| CN106661556A (en) * | 2014-07-18 | 2017-05-10 | 菲利普莫里斯产品有限公司 | Tobacco protease genes |
| CN114621334A (en) * | 2022-03-29 | 2022-06-14 | 安徽省农业科学院园艺研究所 | Application of potato StABI5 gene in drought resistance regulation and method for regulating drought resistance of potatoes based on gene |
Non-Patent Citations (6)
| Title |
|---|
| HONGJU JIAN 等: ""Dynamic Translational Landscape Revealed by Genome-Wide Ribosome Profiling under Drought and Heat Stress in Potato"", 《PLANTS 2023》, vol. 12, no. 12, 6 June 2023 (2023-06-06), pages 10 * |
| JELENA RUDIĆ 等: ""Effects of Elevated Temperature and Salicylic Acid on Heat Shock Response and Growth of Potato Microplants"", 《HORTICULTURAE 2022》, vol. 8, no. 5, 24 April 2022 (2022-04-24), pages 10 * |
| KE WANG 等: ""Rapid Identification of High-Temperature Responsive Genes Using Large-Scale Yeast Functional Screening System in Potato"", 《PLANTS (BASEL)》, vol. 12, no. 21, 28 October 2023 (2023-10-28), pages 10 * |
| NCBI: ""PREDICTED: Solanum tuberosum chaperone protein ClpB1 (LOC102577597), mRNA"", 《GENBANK》, 5 January 2016 (2016-01-05), pages 006345071 * |
| 刘溶荣 等: ""马铃薯干旱相关microRNA的鉴定及其表达分析"", 《中国马铃薯》, vol. 37, no. 4, 31 August 2023 (2023-08-31), pages 289 - 305 * |
| 巩檑: ""近5年马铃薯基因组及重要性状基因研究进展"", 《宁夏农林科技》, vol. 64, no. 11, 30 November 2023 (2023-11-30), pages 6 - 11 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116904483A (en) * | 2023-08-25 | 2023-10-20 | 西部(重庆)科学城种质创制大科学中心 | Polypeptide gene StUCG5P for regulating potato tubers and application thereof |
| CN116904483B (en) * | 2023-08-25 | 2024-05-03 | 西部(重庆)科学城种质创制大科学中心 | Polypeptide gene StUCG P for regulating potato tuber and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116590308B (en) | 2024-03-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US9809827B2 (en) | Transgenic maize | |
| CN116284300B (en) | Extended protein gene StEXLB1 for enhancing drought resistance of potatoes and application thereof | |
| CN112876551B (en) | Transcription factor SpbHLH89 for regulating and controlling drought tolerance of tomato and application thereof | |
| CN106047893A (en) | Application of gene OsCOL16 to control of rice heading stage | |
| CN116590308B (en) | Potato drought tolerance related heat shock protein gene HSP101 and application thereof | |
| WO2023065966A1 (en) | Application of bfne gene in tomato plant type improvement and biological yield increase | |
| CN113717983A (en) | Longan gene DlGRAS34, protein and application thereof in regulating and controlling plant flowering | |
| CN111153975A (en) | Plant drought-resistant related protein TaNAC15, and coding gene and application thereof | |
| Pan et al. | Genome-wide identification of cold-tolerance genes and functional analysis of IbbHLH116 gene in sweet potato | |
| CN112342236B (en) | Application of rice histone methyltransferase in enhancing crop drought resistance and improving single plant yield | |
| CN110885842A (en) | Application of tomato TGase gene in improvement of abiotic stress resistance of tomato | |
| JP5787413B2 (en) | A method for producing a toothpick-forming plant in which the ability to produce tubers or the ability to form a toothpick is improved compared to a wild strain, and a toothpick-forming plant produced by the method | |
| CN112322600A (en) | Alfalfa salt-tolerant gene MsSnRK2.3 and encoding protein and application thereof | |
| CN106397556B (en) | Plant drought GAP-associated protein GAP ZmNAC111 and its encoding gene and application | |
| CN116042673A (en) | Application of AsNADP-ME1 gene in improving drought tolerance of plants | |
| CN104862319B (en) | Control arabidopsis gene AtTIE1 and its application of plant branching | |
| CN108752442B (en) | Stdof2 protein related to salt tolerance of colored potatoes as well as coding gene and application thereof | |
| CN116751812B (en) | Application of OsABI gene in enhancing nitrogen deficiency stress resistance of rice | |
| CN116121298B (en) | Application of inhibiting expression of HSRP1 gene in improving heat resistance of plants | |
| CN114561404B (en) | Apple MdSHN1 gene and application thereof in improving waterlogging tolerance of plants | |
| CN112831509B (en) | Tomato SlOST1 gene and application thereof | |
| CN102964438B (en) | Stress-resistance-related protein PpLEA3-23 of plant as well as coding gene and application of protein | |
| CN114214334B (en) | Application of gene EsH2A.3 from salt mustard in regulation and control of salt tolerance of plants | |
| CN111321158B (en) | Sweet potato beta-amylase gene IbBAM1a, and encoded protein and application thereof | |
| CN117402227A (en) | LEA gene and protein for regulating plant height and drought resistance 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 |