CN117487849B - Application of PdeERF gene in regulation and control of drought resistance of plants - Google Patents
Application of PdeERF gene in regulation and control of drought resistance of plants Download PDFInfo
- Publication number
- CN117487849B CN117487849B CN202311385703.6A CN202311385703A CN117487849B CN 117487849 B CN117487849 B CN 117487849B CN 202311385703 A CN202311385703 A CN 202311385703A CN 117487849 B CN117487849 B CN 117487849B
- Authority
- CN
- China
- Prior art keywords
- plants
- drought
- pdeerf
- gene
- leaf
- 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
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 67
- 230000033228 biological regulation Effects 0.000 title description 3
- 241000196324 Embryophyta Species 0.000 claims abstract description 255
- 230000002018 overexpression Effects 0.000 claims abstract description 152
- 230000008641 drought stress Effects 0.000 claims abstract description 111
- 241000219000 Populus Species 0.000 claims abstract description 40
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 40
- 239000002028 Biomass Substances 0.000 claims abstract description 19
- WSMYVTOQOOLQHP-UHFFFAOYSA-N Malondialdehyde Chemical compound O=CCC=O WSMYVTOQOOLQHP-UHFFFAOYSA-N 0.000 claims abstract description 19
- 229930002875 chlorophyll Natural products 0.000 claims abstract description 19
- 235000019804 chlorophyll Nutrition 0.000 claims abstract description 19
- ATNHDLDRLWWWCB-AENOIHSZSA-M chlorophyll a Chemical compound C1([C@@H](C(=O)OC)C(=O)C2=C3C)=C2N2C3=CC(C(CC)=C3C)=[N+]4C3=CC3=C(C=C)C(C)=C5N3[Mg-2]42[N+]2=C1[C@@H](CCC(=O)OC\C=C(/C)CCC[C@H](C)CCC[C@H](C)CCCC(C)C)[C@H](C)C2=C5 ATNHDLDRLWWWCB-AENOIHSZSA-M 0.000 claims abstract description 19
- 229940118019 malondialdehyde Drugs 0.000 claims abstract description 19
- 230000009261 transgenic effect Effects 0.000 claims abstract description 19
- 230000000694 effects Effects 0.000 claims abstract description 16
- 239000013604 expression vector Substances 0.000 claims abstract description 14
- 238000009825 accumulation Methods 0.000 claims abstract description 9
- 230000001105 regulatory effect Effects 0.000 claims abstract description 9
- 239000002773 nucleotide Substances 0.000 claims abstract description 8
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 8
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims abstract description 6
- 239000001301 oxygen Substances 0.000 claims abstract description 6
- 229910052760 oxygen Inorganic materials 0.000 claims abstract description 6
- 230000014509 gene expression Effects 0.000 claims description 20
- 238000000034 method Methods 0.000 claims description 17
- 230000019516 nonphotochemical quenching Effects 0.000 claims description 16
- 230000000243 photosynthetic effect Effects 0.000 claims description 12
- 241000589155 Agrobacterium tumefaciens Species 0.000 claims description 7
- 238000010791 quenching Methods 0.000 claims description 7
- 230000000171 quenching effect Effects 0.000 claims description 7
- 230000005068 transpiration Effects 0.000 claims description 7
- 238000009395 breeding Methods 0.000 claims description 6
- 238000006862 quantum yield reaction Methods 0.000 claims description 6
- 230000027756 respiratory electron transport chain Effects 0.000 claims description 6
- 230000007613 environmental effect Effects 0.000 claims description 5
- 230000003334 potential effect Effects 0.000 claims description 5
- 239000013598 vector Substances 0.000 claims description 5
- 230000001276 controlling effect Effects 0.000 claims description 4
- 238000011156 evaluation Methods 0.000 claims description 4
- 238000003208 gene overexpression Methods 0.000 claims description 4
- 230000001131 transforming effect Effects 0.000 claims description 4
- 238000012216 screening Methods 0.000 claims description 3
- 230000002015 leaf growth Effects 0.000 claims 1
- 230000012010 growth Effects 0.000 abstract description 26
- 239000007789 gas Substances 0.000 abstract description 8
- 241000589158 Agrobacterium Species 0.000 abstract description 4
- 238000012360 testing method Methods 0.000 description 63
- 238000011282 treatment Methods 0.000 description 31
- 230000007423 decrease Effects 0.000 description 29
- 239000011148 porous material Substances 0.000 description 24
- 230000002829 reductive effect Effects 0.000 description 23
- 230000008859 change Effects 0.000 description 19
- 230000001965 increasing effect Effects 0.000 description 15
- 239000002689 soil Substances 0.000 description 15
- 230000003247 decreasing effect Effects 0.000 description 13
- 239000001963 growth medium Substances 0.000 description 13
- 230000005764 inhibitory process Effects 0.000 description 13
- 239000000463 material Substances 0.000 description 12
- 208000005156 Dehydration Diseases 0.000 description 11
- 230000018044 dehydration Effects 0.000 description 11
- 238000006297 dehydration reaction Methods 0.000 description 11
- 230000002035 prolonged effect Effects 0.000 description 11
- 238000005259 measurement Methods 0.000 description 10
- 230000000630 rising effect Effects 0.000 description 9
- 239000007788 liquid Substances 0.000 description 8
- 238000012258 culturing Methods 0.000 description 7
- 238000010186 staining Methods 0.000 description 7
- 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 6
- 230000009467 reduction Effects 0.000 description 6
- 230000035882 stress Effects 0.000 description 6
- 208000024891 symptom Diseases 0.000 description 6
- 206010020649 Hyperkeratosis Diseases 0.000 description 5
- 238000011529 RT qPCR Methods 0.000 description 5
- 238000007621 cluster analysis Methods 0.000 description 5
- 238000005520 cutting process Methods 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 238000011084 recovery Methods 0.000 description 5
- 238000012163 sequencing technique Methods 0.000 description 5
- 210000001519 tissue Anatomy 0.000 description 5
- 238000004383 yellowing Methods 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 108091030071 RNAI Proteins 0.000 description 4
- 108091023040 Transcription factor Proteins 0.000 description 4
- 102000040945 Transcription factor Human genes 0.000 description 4
- 230000003321 amplification Effects 0.000 description 4
- 238000000137 annealing Methods 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 239000002299 complementary DNA Substances 0.000 description 4
- 238000004925 denaturation Methods 0.000 description 4
- 230000036425 denaturation Effects 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 230000018109 developmental process Effects 0.000 description 4
- 230000009368 gene silencing by RNA Effects 0.000 description 4
- 230000001939 inductive effect Effects 0.000 description 4
- 208000015181 infectious disease Diseases 0.000 description 4
- 238000003199 nucleic acid amplification method Methods 0.000 description 4
- 238000012257 pre-denaturation Methods 0.000 description 4
- 238000004321 preservation Methods 0.000 description 4
- 230000009466 transformation Effects 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 3
- 239000003242 anti bacterial agent Substances 0.000 description 3
- 229940088710 antibiotic agent Drugs 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 230000001488 breeding effect Effects 0.000 description 3
- 230000015556 catabolic process Effects 0.000 description 3
- 238000006731 degradation reaction Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 230000002068 genetic effect Effects 0.000 description 3
- 238000005286 illumination Methods 0.000 description 3
- 230000003834 intracellular effect Effects 0.000 description 3
- 108020004999 messenger RNA Proteins 0.000 description 3
- 235000016709 nutrition Nutrition 0.000 description 3
- 230000035764 nutrition Effects 0.000 description 3
- 239000003642 reactive oxygen metabolite Substances 0.000 description 3
- 230000004083 survival effect Effects 0.000 description 3
- RVBUGGBMJDPOST-UHFFFAOYSA-N 2-thiobarbituric acid Chemical compound O=C1CC(=O)NC(=S)N1 RVBUGGBMJDPOST-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 108020004414 DNA Proteins 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 230000002354 daily effect Effects 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 230000001976 improved effect Effects 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 238000009630 liquid culture Methods 0.000 description 2
- 238000002844 melting Methods 0.000 description 2
- 230000008018 melting Effects 0.000 description 2
- 230000000877 morphologic effect Effects 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000002441 reversible effect Effects 0.000 description 2
- 239000008399 tap water Substances 0.000 description 2
- 235000020679 tap water Nutrition 0.000 description 2
- 210000003462 vein Anatomy 0.000 description 2
- YHVQIDWAIRCSOQ-UHFFFAOYSA-N 1-nitrotetrazol-2-ium chloride Chemical compound [Cl-].[O-][N+](=O)N1C=[NH+]N=N1 YHVQIDWAIRCSOQ-UHFFFAOYSA-N 0.000 description 1
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 108010027344 Basic Helix-Loop-Helix Transcription Factors Proteins 0.000 description 1
- 102000018720 Basic Helix-Loop-Helix Transcription Factors Human genes 0.000 description 1
- 108010001572 Basic-Leucine Zipper Transcription Factors Proteins 0.000 description 1
- 102000000806 Basic-Leucine Zipper Transcription Factors Human genes 0.000 description 1
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 1
- 108091062157 Cis-regulatory element Proteins 0.000 description 1
- LMKYZBGVKHTLTN-NKWVEPMBSA-N D-nopaline Chemical compound NC(=N)NCCC[C@@H](C(O)=O)N[C@@H](C(O)=O)CCC(O)=O LMKYZBGVKHTLTN-NKWVEPMBSA-N 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 101000618525 Homo sapiens Membrane transport protein XK Proteins 0.000 description 1
- 101001109463 Homo sapiens NACHT, LRR and PYD domains-containing protein 1 Proteins 0.000 description 1
- 101000636213 Homo sapiens Transcriptional activator Myb Proteins 0.000 description 1
- 102100022472 Membrane transport protein XK Human genes 0.000 description 1
- LGCMKPRGGJRYGM-UHFFFAOYSA-N Osalmid Chemical compound C1=CC(O)=CC=C1NC(=O)C1=CC=CC=C1O LGCMKPRGGJRYGM-UHFFFAOYSA-N 0.000 description 1
- 241000220317 Rosa Species 0.000 description 1
- OUUQCZGPVNCOIJ-UHFFFAOYSA-M Superoxide Chemical compound [O-][O] OUUQCZGPVNCOIJ-UHFFFAOYSA-M 0.000 description 1
- 102100030780 Transcriptional activator Myb Human genes 0.000 description 1
- 102100029469 WD repeat and HMG-box DNA-binding protein 1 Human genes 0.000 description 1
- 101710097421 WD repeat and HMG-box DNA-binding protein 1 Proteins 0.000 description 1
- 230000036579 abiotic stress Effects 0.000 description 1
- 230000006578 abscission Effects 0.000 description 1
- 239000002250 absorbent Substances 0.000 description 1
- 230000002745 absorbent Effects 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 238000009412 basement excavation Methods 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 238000005842 biochemical reaction Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000028446 budding cell bud growth Effects 0.000 description 1
- 238000010804 cDNA synthesis Methods 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 230000010485 coping Effects 0.000 description 1
- 230000004300 dark adaptation Effects 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000007598 dipping method Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 210000002615 epidermis Anatomy 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 230000001744 histochemical effect Effects 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000011890 leaf development Effects 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 239000005416 organic matter Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 230000029553 photosynthesis Effects 0.000 description 1
- 238000010672 photosynthesis Methods 0.000 description 1
- 238000003976 plant breeding Methods 0.000 description 1
- 230000008635 plant growth Effects 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 238000002331 protein detection Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 108010054624 red fluorescent protein Proteins 0.000 description 1
- 230000029058 respiratory gaseous exchange Effects 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 239000012883 rooting culture medium Substances 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 238000005728 strengthening Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000005303 weighing Methods 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
- C12N15/79—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)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
- C12N15/8205—Agrobacterium mediated transformation
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (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 application discloses an application of PdeERF gene in regulating drought resistance of plants. The PdeERF is derived from populus americana, the nucleotide sequence of which is shown as SEQ ID NO. 1, and the application constructs an expression vector containing PdeERF53 gene, converts the expression vector into agrobacterium, and infects poplar with agrobacterium to obtain transgenic plant with drought resistance. The application evaluates drought resistance of transgenic plants, including plant seedling height, ground diameter, growth phenotype of leaves, biomass accumulation of plants, leaf gas exchange parameters and chlorophyll fluorescence parameters of plants, relative chlorophyll content, relative leaf conductivity, leaf water content, malondialdehyde content of leaves and root systems, root system activity, leaf active oxygen content and the like, and the final result shows that over-expression PdeERF gene obviously improves the tolerance of poplar to drought stress.
Description
Technical Field
The application relates to the technical field of plant drought resistance, in particular to application of PdeERF gene in regulating and controlling plant drought resistance.
Background
Drought is one of the major factors responsible for plant maldevelopment, and to date, more than one third of the world is located in arid and semiarid regions where water is deficient and drought-induced crop yield losses may outweigh all other losses. Drought stress can affect plant growth, climate, water and nutrient relationship, photosynthesis, assimilation distribution and respiration, reduce leaf size, inhibit stem extension and root proliferation, disturb water relationship of plants, and reduce water utilization efficiency.
Drought stress is one of the main factors limiting sustainable development of agriculture and forestry production in China. At present, the development of the breeding work of new varieties of drought-resistant woods is urgent, but the traditional woods have a long period, difficult breeding and other difficulties. Therefore, the cultivation of the drought-resistant tree novel germplasm by using the genetic engineering technology has wide development prospect.
Disclosure of Invention
In the context of long-term drought stress, plants have evolved various mechanisms to cope with drought stress. The transcription factor is protein molecule capable of combining with cis-acting element of gene promoter in eukaryote, and through strengthening or inhibiting mode, the target gene is ensured to express in specific strength and specific time and space, and the expression of downstream gene is regulated and controlled. When plants are stressed by adversity, transcription factors are involved in the regulation of many important processes, such as activating intracellular related enzyme systems, participating in hormone secretion, affecting cellular metabolic activities and signal transduction, and can also be used as a special messenger molecule to participate in various physiological and biochemical reactions. Currently, the transcription factor family related to stress resistance is mainly AP2/ERF, WRKY, MYB, NAC, bZIP, bHLH and the like as proved by transgenesis. Wherein the AP2/ERF transcription factor is widely existed in plants, accounting for about 8% of the total number of known plant transcription factors, and can directly or indirectly regulate the expression of downstream genes, thereby regulating the growth and development of plants and coping with abiotic stress.
In a first aspect, the embodiment of the application provides application of PdeERF gene in regulating drought resistance of plants. In some embodiments, the PdeERF gene is (a 1) or (a 2) as follows: (a1) A nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO. 1; (a2) A nucleic acid molecule having more than 95% homology to (a 1) and being associated with drought resistance in plants.
According to the embodiment of the application, the PdeERF gene is taken as a research object, the expression mode of the gene in different tissues of poplar is analyzed, the over-expression and inhibition expression vector of the gene is constructed, genetic transformation and drought stress tests are carried out on the poplar, the characters of PdeERF transgenic plants under drought stress, including the seedling height, ground diameter, growth phenotype of leaves, biomass accumulation of the plants, net photosynthetic rate (Pn) of the plants, transpiration rate (Tr), stomatal conductance (Gs), intercellular CO 2 concentration/environmental CO 2 concentration (Ci/Ca), instantaneous water utilization efficiency (R WUEi) and other leaf gas exchange parameters, the maximum photochemical efficiency (Fv/Fm) of the plant, PS II potential activity (Fv/Fo), PS II effective quantum photochemical yield (Fv '/Fm'), actual photochemical efficiency (PHPS II), photochemical quenching (qP), non-photochemical quenching (NPQ), apparent Electron Transfer Rate (ETR) and other fluorescent parameters are analyzed, the relative content of chlorophyll of the plants, the relative content of the leaf of drought resistance, the leaf moisture content of the leaf of the root system, the leaf moisture content of the leaf of the plant and the oxygen content of the leaf are regulated and controlled, and the activity of the gene of the plant is further evaluated, and the gene of the plant is further is provided, and the activity of the gene is assessed (35 of the poplar is further improved.
In the embodiment of the application, through the observation comparison of phenotype and related physiological indexes between PdeERF transgenic plants and wild plants, the function of PdeERF gene for regulating and controlling the drought resistance of poplar is preliminarily known, and a new method is created for fine variety breeding of poplar.
The embodiment of the application digs the function of PdeERF gene for regulating and controlling the drought resistance of the poplar, which is helpful for improving the drought resistance of the poplar, provides gene resources for cultivating new varieties of the drought-resistant poplar, and lays a foundation for deeply clarifying the molecular mechanism of the drought resistance formation of the poplar. Meanwhile, poplar is used as woody model plant, and the excavation and functional identification of excellent gene resources of poplar also provide reference and reference for related researches of other plant species.
In certain embodiments, the PdeERF gene is used to modulate one or more of the above plant traits. In certain embodiments, the plant is a poplar. In certain preferred embodiments, the plant is a nanlin 895 poplar.
In a second aspect, the embodiments provide the use of a recombinant vector comprising PdeERF gene or an expression cassette comprising PdeERF gene to cultivate transgenic plants with enhanced drought resistance by overexpressing PdeERF gene, wherein the nucleotide sequence of PdeERF gene is as shown in SEQ ID NO. 1.
In a third aspect, embodiments also provide a drought-resistant plant breeding method comprising the steps of: transforming PdeERF gene into plant to obtain transgenic positive plant; in the gene positive plants, the final drought-resistant plants are screened out according to the expression quantity of PdeERF genes in leaf and root tissues and the drought resistance evaluation result.
In some embodiments, the step of transforming the PdeERF gene into a plant comprises: constructing PdeERF53 gene over-expression vector; using electric shock method to make the over-expression carrier into agrobacterium tumefaciens; and infecting plants by using agrobacterium tumefaciens to obtain the drought-resistant plants.
In a fourth aspect, embodiments also provide the use of the breeding method of the third aspect for cultivating drought-resistant plants.
In a fifth aspect, the examples also provide a primer pair for amplifying the PdeERF gene described above, comprising PdeERF-OE-F as shown in SEQ ID NO. 2 and PdeERF-OE-R as shown in SEQ ID NO. 3.
Drawings
Fig. 1 is a graph showing the absolute water content change of soil under drought stress according to an embodiment of the present application.
FIG. 2 is a graph showing leaf area of 5 th to 7 th leaves of normally watered plants of OE, RE and WT and relative water loss rate results of dehydration treatment thereof provided by examples of the present application; wherein (a) is the leaf area result and (b) is the relative water loss result.
FIG. 3 is a graph of the phenotype change of OE, RE and WT plant leaves before and after dehydration treatment provided by an example of the application.
FIG. 4 is a graph showing the effect of drought stress on leaf stomata characteristics provided by an embodiment of the present application; wherein, (a) is a result of a density of pores per unit area, (b) is a result of a pore area, (c) is a result of a pore length, (d) is a result of a pore width, and (e) is a result of a pore opening.
Fig. 5 is a graph showing changes in leaf stomata morphology under drought stress provided by the example of the present application.
FIG. 6 shows a phenotype diagram of WT, OE and RE plants under drought stress provided by an embodiment of the application; wherein FIG. 6A is a phenotype plot of WT, OE, RE plants under normal watering and different time drought stress, respectively; FIG. 6B is a phenotype plot of OE plants under normal watering and different time drought stress; FIG. 6C is a phenotype diagram of RE plants under normal watering and different time drought stress; FIG. 6D is a phenotype plot of WT plants under normal watering and different time drought stress.
FIG. 7 shows shoot height and ground diameter growth results for WT, OE and RE plants under drought stress provided by examples of the present application; wherein, (a) is the result of high growth amount of seedlings, and (b) is the result of ground diameter growth amount.
FIG. 8 is a graph showing net photosynthetic rate results for WT, OE, and RE plants under drought stress provided by an example of the present application.
FIG. 9 shows the transpiration rate results for WT, OE and RE plants under drought stress provided by examples of the application.
FIG. 10 shows stomatal conductance results of WT, OE and RE plants under drought stress provided by examples of the application.
FIG. 11 is a graph showing the results of intracellular CO 2 concentration/environmental CO 2 concentration for WT, OE, and RE plants under drought stress provided by examples of the present application.
FIG. 12 is a graph showing the results of instantaneous moisture utilization of WT, OE and RE plants under drought stress provided by examples of the present application.
FIG. 13 shows the results of maximum photochemical efficiencies of WT, OE and RE plants under drought stress provided by examples of the application.
FIG. 14 shows the PS II potential activity results of WT, OE and RE plants under drought stress provided by examples of the application.
FIG. 15 shows the result of effective photochemical quantum yield of PS II in WT, OE and RE plants under drought stress provided by examples of the present application.
FIG. 16 shows the actual photochemical efficiency results for WT, OE and RE plants under drought stress provided by examples of the application.
FIG. 17 shows the photochemical quenching results of WT, OE and RE plants under drought stress provided by examples of the application.
FIG. 18 shows the results of non-photochemical quenching of WT, OE and RE plants under drought stress provided by examples of the application.
FIG. 19 is a graph showing apparent electron transfer rate results for WT, OE and RE plants under drought stress provided by examples of the application.
FIG. 20 shows the results of relative leaf chlorophyll content (SPAD values) of WT, OE and RE plants under drought stress provided by examples of the present application.
FIG. 21 is a graph showing the relative conductivity results of WT, OE and RE plant leaves under drought stress provided by examples of the application.
FIG. 22 is a graph showing the moisture content results of WT, OE and RE plant leaves under drought stress provided by examples of the present application; wherein (a) is a relative moisture content result and (b) is an absolute moisture content result.
FIG. 23 shows malondialdehyde content results for WT, OE and RE plant leaves and root systems under drought stress provided by examples of the application; wherein, (a) is the vane malondialdehyde content result and (b) is the fine root malondialdehyde content result.
FIG. 24 is a root system vigor results for WT, OE and RE plants under drought stress provided by examples of the present application.
FIG. 25 shows the active oxygen content of WT, OE and RE plants under drought stress provided by examples of the application; wherein (a) is the hydrogen peroxide content result and (b) is the superoxide anion O 2- content result.
FIG. 26 shows DAB staining results of OE, RE and WT plants following drought stress provided by examples of the application.
FIG. 27 shows NBT staining results of OE, RE and WT plants following drought stress provided by examples of the application.
FIG. 28 is a graph showing the results of cluster analysis of drought resistance of WT, OE and RE plants provided by an embodiment of the application.
Detailed Description
In order to make the objects, technical solutions and advantages of the present application more apparent, the present application will be described in further detail with reference to the following examples. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the application. The reagents not specifically and individually described in the present application are all conventional reagents and are commercially available; methods which are not specifically described in detail are all routine experimental methods and are known from the prior art.
1. Construction PdeERF of transgenic poplar
(1) Material preparation
The genetic transformation material was wild-type nanlin 895 poplar (p.× euramericana 'Nanlin 895'). Strains: coli competent DH 5. Alpha. (Beijing engine biology Co., ltd.); the Agrobacterium tumefaciens competence (Agrobacterium tumefaciens) was nopaline type EHA105 (Shanghai Biotechnology Co., ltd.). And (3) a carrier: pdeERF53 the over-expression vector was pKGW-RR-MGW and the inhibition vector was pK7GWIWG (II).
(2) Transformation of PdeERF Gene into poplar
Referring to the genomic sequence of populus americana (https:// phytozome-next. Jgi. Doe. Gov/info/PdeltoidesWV 94_v2_1), the primers were designed to amplify the full length CDS sequence and RNAi sequence of the PdeERF gene, for construction of an over-expression vector (OE) and an expression-repressing vector (RE), respectively. The primer for amplifying the full-length CDS sequence of PdeERF gene is PdeERF-53-OE-F (SEQ ID NO: 2)/PdeERF-OE-R (SEQ ID NO: 3), and the primer for amplifying the RNAi sequence of PdeERF gene is PdeERF-53-RE-F (SEQ ID NO: 4)/PdeERF-RE-R (SEQ ID NO: 5).
Leaf blades of Nanlin 895 poplar are used as materials, mRNA is extracted, target gene fragments are cloned, an over-expression vector (OE) and an RNAi inhibition expression vector (RE) of PdeERF genes are respectively constructed, and agrobacterium EHA105 is transformed, and the specific test steps are as follows:
(1) Extracting mRNA from poplar leaves by adopting a CTAB method, and reversely transcribing the mRNA into cDNA by adopting a reverse transcription kit Hifair III 1st Strand cDNA Synthesis Super Mix (Shanghai auxiliary holy Corp);
(2) cDNA is used as a template, and a primer PdeERF-OE-F/PdeERF-OE-R is adopted to clone the CDS full-length sequence (1392 bp, the nucleotide sequence is shown as SEQ ID NO: 1) of PdeERF gene; using cDNA as template, v adopting primer PdeERF-RE-F/PdeERF-RE-R to clone RNAi interference fragment of PdeERF gene (204 bp, nucleotide sequence is shown as SEQ ID NO: 6);
(3) The gateway clone method is adopted respectively in BP close TM II Enzyme Mix andThe full length of PdeERF gene is cloned to an over-expression vector pKGW-RR-MGW (doi: 10.1111/nph.13973) through BP and LR recombination reaction under the catalysis of LR clone TM II Enzyme Mix; cloning the RNAi-interfering fragment of PdeERF gene into the inhibition expression vector pK7GWIWG (II) (http:// www.kelei-biology.com/m/view.phpand=983);
(4) The over-expression vector and the inhibition expression vector carrying PdeERF gene are respectively transformed into agrobacterium tumefaciens EHA105 by an electric shock method for infection of Nanlin 895 poplar;
(5) Young leaves of strong and healthy nanlin 895 poplar growing for about 30 days are selected as explants, and agrobacterium EHA105 carrying PdeERF gene overexpression and expression inhibition vectors are used for genetic transformation respectively. The specific experimental steps are as follows:
Activating and culturing strains: taking strains stored at the temperature of minus 80 ℃, melting on ice, dipping a small amount of bacterial liquid by using a sterilized gun head, streaking on LB culture medium (LB+Spe+Rif) containing corresponding antibiotics, and inversely culturing at the temperature of 28 ℃ for 2-3 d until single colony is generated; small shaking bacteria liquid: picking single colony into a sterile test tube containing 3ml of SOB liquid culture medium of antibiotics, and performing dark culture for 12-24 hours at the temperature of 28 ℃ at 200 r/min; large shaking bacteria liquid: sucking 0.3-1.0 ml of bacterial liquid into 50ml of SOB liquid culture medium containing antibiotics, and carrying out dark culture at the temperature of 28 ℃ for 8-12 h at 200r/min until OD600 = 0.5-0.8; drawing materials and infecting: selecting a tissue culture seedling for rooting culture for 30d, taking the 3 rd to 5 th leaves which are dark green from top to bottom and flat, wherein the sizes, shapes and colors of the leaves are basically consistent, cutting the edges of the leaves, and cutting the leaves into cubes of 2 to 3cm 2; centrifuging the obtained bacterial liquid at room temperature for 10min at 5000r/min, discarding supernatant, re-suspending the precipitate with 50-75 ml of re-suspension (WPM is added without agar) until the OD600 = 0.5-0.8, and carrying out infection for 10-15 min; co-cultivation: taking out the leaf from the infection liquid, placing the leaf on sterile filter paper to suck the residual infection liquid on the surface, spreading the leaf back down on a culture medium (WPM+100 mu M AS), and culturing in dark at 24 ℃ for 2d; inducing callus: transferring the co-cultured leaves to a culture medium (WPM+1 mg/L2, 4-D+0.5mg/LKT+300mg/L Cef+50mg/L Kan+300mg/L TMT) for inducing callus differentiation, and performing dark culture at 24 ℃ with 1 change of the culture medium every 15D to obtain the leaves with callus; inducing bud growth: cutting the callus (about 1cm 2) and transferring into culture medium (WPM+0.02 mg/L TDZ+300mg/L Cef+50mg/L Kan+300mg/L TMT) for inducing bud differentiation, culturing at 24deg.C, and changing the culture medium 1 time every 15d until adventitious buds grow on the surface of the callus; induction of bud elongation: when the bud length of the leaf blade is 1cm, cutting adventitious bud, transferring into bud elongation culture medium, culturing (WPM+300 mg/L Cef+50mg/L Kan), culturing at 24deg.C, and changing culture medium for 1 time every 15 d; and (3) induction rooting: cutting out resistant seedlings with stem length greater than 3cm in bud elongation culture medium, placing in rooting culture medium for culture (WPM+250 mg/L Cef+50mg/L Kan), and culturing at 24deg.C with 1 culture medium change every 30 d.
2. Identification of transgenic positive seedlings
Screening PdeERF of the transgenic positive seedlings by DNA-PCR detection and red fluorescent protein detection, wherein the screening steps are as follows:
The resulting DNA of the transgenic resistant plants were subjected to PCR amplification with primers (shown in Table 1 below, wherein the 1 st primer pair is an internal reference primer) to determine whether it was a positive transgenic seedling.
TABLE 1
The PCR detection is carried out by taking PdeERF gene overexpression and inhibition expression vector plasmid as positive control, taking leaf DNA of wild type Nanlin 895 poplar as negative control and ddH 2 O as blank control, and the PCR reaction system comprises 10 mu l: the forward and reverse primers were each 0.5. Mu.l, 2X Hieff TM PCR MASTER Mix enzyme 5. Mu.l, template 3. Mu.l, the remainder ddH 2 O;
PCR procedure for amplification of DsRed-F1/AtUBQ10-R1 primer: pre-denaturation at 94℃for 5min, denaturation at 94℃for 30s, annealing at 59℃for 30s, extension at 72℃for 90s for 30 cycles, extension at 72℃for 5min, and preservation at 4 ℃; (2) PCR procedure for amplification of DsRed-R2/DsRed-F3 primer: pre-denaturation at 94℃for 5min, denaturation at 94℃for 30s, annealing at 62℃for 30s, extension at 72℃for 30s, 30 cycles total, extension at 72℃for 5min, preservation at 4 ℃;
Attb1/Attb2 primer amplification PCR procedure: pre-denaturation at 94℃for 5min, denaturation at 94℃for 30s, annealing at 65℃for 30s, extension at 72℃for 30s (inhibition of expression) or 90s (overexpression), 30 cycles total, extension at 72℃for 5min, preservation at 4 ℃;
PdeERF53 PCR procedure for amplification of the F5/AtUBQ10-R5 primer: pre-denaturation at 94℃for 5min, denaturation at 94℃for 30s, annealing at 58℃for 30s, extension at 72℃for 90s, 30 cycles total, extension at 72℃for 5min, and preservation at 4 ℃.
3. Transgenic line selection
According to the sequence information of PdeERF genes, primers are designed by utilizing NCBI online tool Primer-BLAST (http:// www.ncbi.nlm.nih.gov/tools/Primer-BLAST /) for a real-time fluorescence quantitative PCR (qRT-PCR) test, and the relative expression quantity of PdeERF in leaves and root systems of transgenic positive plants is detected. Action 7 (Podel. 01G 470900) is used as an internal reference gene, pdeERF is used as a target gene, and specific primer information is as follows:
internal reference primer: action-qF: as shown in SEQ ID NO. 7; action-qR: as shown in SEQ ID NO. 8;
Primers for detection of over-expression (OE)/Repressed Expression (RE) transgenic plants: pdeERF 53A 53-F1: as shown in SEQ ID NO. 17; pdeERF 53A 53-R1: as shown in SEQ ID NO. 18.
Using Hieff TM qPCRGREEN MASTER Mix (next holy life, shanghai) in Light/>96 (Roche, switzerland) qRT-PCR reactions were completed; the qRT-PCR system was 10. Mu.L, with 5. Mu.L of polymerase, 0.5. Mu.L of forward and reverse primers each, 1. Mu.L of template cDNA, and 3. Mu.L of ddH 2 O. The specificity of the PCR reaction was analyzed using a 3-step PCR amplification standard procedure, using the dissolution profile of the amplified product, 3 biological replicates, 2 technical replicates. The relative expression level of the gene is calculated by adopting a method of-2 ΔΔCT and calculated by Light/>96SW1.1 software analysis is completed, qRT-PCR reaction program, preculture (1 cycle) is performed at 95 ℃ for 600s;95℃for 10s, 59℃for 20s, 72℃for 30s (40 cycles); melting (1 cycle) at 95℃for 10s, 65℃for 60s, and 97℃for 1s.
The expression level of the PdeERF gene was measured for 38 transgenic lines of PdeERF53 genes, and it was found that the expression level of PdeERF gene was significantly different in different lines in leaves and roots. Strains OE25, OE26, OE27, RE19, RE20 and RE24 with significant changes in expression levels in leaves and roots were selected for subsequent drought resistance identification experiments. Wherein, the expression quantity of the OE strain (OE 25, OE26 and OE 27) in the leaf is 12.63 times, 24.07 times and 28.13 times of the wild type Nanlin 895 poplar (WT) respectively; the expression quantity in the fine roots is 3.35 times, 15.92 times and 15.90 times of that of the wild type nanlin 895 poplar respectively; the expression amount in the RE strain (RE 19, RE20 and RE 24) leaves is 0.42 times, 0.30 times and 0.36 times of that of the wild type southern forest 895 poplar respectively; the expression amount in the fine roots is 0.22 times, 0.25 times and 0.34 times that of the wild type nanlin 895 poplar, respectively.
4. Drought resistance evaluation test of PdeERF gene
Rooting tissue culture seedlings of the strong WT, OE25, OE26, OE27, RE19, RE20 and RE24 which are 6 weeks old are selected and transplanted into a nutrition pot (16 cm multiplied by 14cm multiplied by 12 cm), wherein the culture medium is nutrition soil (pH 6.0) and contains N, P 2O5 and K 2 O which are 2% -5%, and the organic matter content (dry weight) is more than 20%. After 4 weeks of transplanting, all plants were watered 1 time per week with 1/2Hoagland nutrient solution and1 tap water and conventional management was performed. All transplanting seedlings are placed in a culture room for culture, the temperature of the culture room is (25+/-2), the illumination period is 16h illumination and 8h darkness each day, the light intensity is 400 mu mol.m -2·s-1, the light source is a white cold light lamp, and the indoor air relative humidity is 70% -80%.
When the average height of all plants reaches 40-50 cm, selecting 5 th-7 th fully-unfolded blades at the top end of the plant which is normally watered by each plant line part to measure the area of the blades, carrying out natural dehydration treatment, and measuring the relative water loss rate of the blades. Meanwhile, drought stress tests are carried out on the rest plants of each plant line, and the drought resistance of the plants is evaluated. The drought stress test method is as follows: plant material (transgenic lines) of the aforementioned robust, consistent growth was selected and randomly distributed into 2 treatments for 21 d: (1) control treatment (CK): watering a small amount of soil in a nutrition pot every day, and keeping the water content of the soil to be 70% -75% of the maximum field water holding capacity; (2) drought treatment (DS): the soil is watered thoroughly before drought begins, then the soil is allowed to dry naturally, the soil moisture content (measuring depth is 6 cm) is measured by a soil moisture meter (TZS-2X-G, thunb tuo-plaun agricultural science and technology) every 2d, and after drought treatment, the water is recovered for 7d, and the total treatment is 21d.
Drought stress test materials were randomly divided into 2 groups: 12 strains/line/group, 4 strains/repeat, random block arrangement, 3 repeats. The first set of materials is used for observation of phenotypes, growth, biomass, leaf gas exchange parameters, chlorophyll fluorescence, stomatal parameters, chlorophyll (SPAD) values; the second group of materials are used for collecting the leaves and the fine roots and analyzing physiological indexes such as relative conductivity of the leaves, water content of the leaves, malondialdehyde content of the leaves and the roots, activity of the roots, active oxygen content of the leaves and the like.
A first group of materials: plant phenotypes were observed daily; test nos. 0d (DS 0), 7d (DS 7), 14d (DS 14) and 21d (DS 21), leaf chlorophyll content, leaf stomata parameters, leaf gas exchange parameters and chlorophyll fluorescence of all materials were determined; the 0d (DS 0) and 21d (DS 21) of the test measure the seedling height and the ground diameter of all materials, and calculate the growth quantity of the seedling height and the ground diameter of the plants during the test; at the end of the test (21 d), leaf, stem and root samples were collected for biomass determination. A second group of materials: and (3) collecting leaves and fine roots for physiological index analysis at the 0d, 7d and 14d of the test. The 4 th to 6 th fully unfolded leaves at the top end of the plant are collected by the leaf sample, and the main vein is removed; when the fine root sample is collected, distilled water is firstly used for cleaning, and then filter paper is used for sucking the water on the surface; the leaf and the fine root samples are frozen by liquid nitrogen and stored in a refrigerator at-80 ℃ for measuring physiological indexes, wherein the relative conductivity of the leaf and the water content of the leaf are measured by adopting fresh leaves.
5. Determination of leaf area and relative leaf loss
The leaf area of the fully developed leaf of the 5 th to 7 th leaves on the top of transgenic plants (OE, RE) and WT plants under the normal watering (CK) condition is measured by adopting an intelligent leaf area measuring instrument (YMJ-CHA 3; thunb tuo-pu cloud agricultural science and technology company), repeated/processed for 3 times, averaged and standard deviation is calculated. When the leaves of OE, RE and WT plants are dehydrated under the normal watering (CK) condition, 5 th to 7 th leaves which are fully unfolded from top to bottom are taken and spread on dry filter paper for natural dehydration, a one-thousandth balance is used for weighing, the weight reading of the leaves is recorded as WE (0) (0 min), then the weight reading of the leaves is measured every 20min, the n-th reading is recorded as WE (n), the test is repeated for 3 times, the average value is taken, the standard error is calculated, and after a plurality of time points are recorded, the relative water loss rate of the leaves is calculated according to a formula, namely the ratio of the leaf relative water loss to the water loss of [ WE (0) -WE (n) ]/WE (0).
FIG. 2 shows leaf area of 5 th to 7 th leaves of OE, RE and WT normally watered plants (FIG. 2 a) and their relative water loss rate of the dehydration treatment (FIG. 2 b); FIG. 3 shows the phenotypic changes before and after dehydration of the leaves of OE, RE and WT plants. As shown in the figure, under normal watering conditions, 5 th to 7 th leaves of each strain are fresh green, the leaf quality is soft and glossy (figure 3), and the difference of leaf areas among different strains is not obvious (p <0.05; figure 2); after dehydration treatment, the leaf phenotype and relative water loss rate of each strain were significantly different, with only slight yellowing and shrinkage of the leaf of each strain of OE, the leaf quality remained soft and glossy, while the leaf of each strain of RE was significantly dry and shrinkage and loss of gloss, indicating that RE lost more water than OE per leaf area (p >0.05; FIG. 2; FIG. 3). The relative water loss rate difference of the leaves among the strains is small in the early stage of dehydration treatment (< 2 h); after 2h dehydration, the RE plants had a faster water loss and the OE had a relatively slower water loss compared to WT; after dehydration for 5 hours, the leaf relative water loss difference between each strain is obvious, and the leaf phenotype is also obvious; relative water loss rates of OE25, OE26 and OE27 at 5h of dehydration compared to WT were 0.58, 0.81 and 0.78 times WT, RE19, RE20 and RE24 were 1.05, 1.03 and 1.04 times WT, respectively. It can be seen that the leaf loss of RE plants is relatively fast compared to WT, while OE plants are relatively slow under drought treatment.
6. Soil moisture content determination
Fig. 1 shows a graph of the absolute moisture content of soil under drought stress, as shown in fig. 1, during the test, the absolute moisture content of soil under drought stress continuously decreases with the extension of drought time, reaching the lowest point at 14d, only 2.37%, and extremely significantly lower than 44.23% of the soil moisture content at 0 d. After rehydration, the soil moisture content quickly rose back to 40.98% and was restored to the control treatment level.
7. Blade air hole parameter measurement
The pore morphology feature parameters were obtained by nail polish blotting. 3 plants with consistent growth vigor are randomly selected from each plant line in the test of 0d, 7d and 14d, colorless nail polish is uniformly smeared on the 2 nd to 3 rd vein parts of the lower epidermis of the 5 th leaf which is completely unfolded, after the nail polish is naturally dried, nail polish layers are torn by forceps and then attached to glass slides, and cover slips are covered to prepare temporary glass slides which serve as samples. The air holes were observed with an optical microscope (Leica DM2500 LED), 3 clear fields were randomly selected for each slide under a 40-fold objective, the number, length (length of dumbbell-shaped guard cells) and width (the widest value perpendicular to the dumbbell-shaped guard cells) of the air holes in each field were measured with LAS X (Leica Application Suite X) software, and finally the air hole opening was calculated and the area of the air holes was used to represent the air hole opening. Air pore area=pi ab, air pore opening=1/4 pi ab; where a=pore length, b=pore width.
Fig. 4 shows the effect of drought stress on leaf stomata characteristics, and fig. 5 shows the change in leaf stomata morphology under drought stress. As can be seen from the graph, the difference between the pore density and pore size of the blades of different strains under normal conditions is not obvious (p < 0.05); when drought stress is 7d, the difference between the pore size and the opening degree of each strain and the contrast treatment (CK) is not obvious; along with the prolongation of drought time, the water content of soil is continuously reduced, the pore length of each strain is not obviously changed, the pore width and the pore opening degree are reduced to different degrees, and the difference between different strains is larger; at 14d, the pore width and opening of each strain was significantly lower than the control, with WT, OE25, OE26, OE27, RE19, RE20, RE24 reduced to 11.05%, 35.49%, 20.20%, 27.74%, 14.24%, 8.85% and 3.01% of their control treatments (CK), respectively; the pore opening was reduced to 14.71%, 34.53%, 24.61%, 30.48%, 10.38%, 9.04% and 3.39% of its Control (CK), respectively. Thus, compared with WT, RE plants have larger pore width variation, smaller pore opening, and OE plants have smaller pore width variation, larger pore opening, wherein the pore widths of OE25 and OE27 are significantly higher than WT, and the pore openings of OE25 and OE26 are significantly higher than WT.
8. Phenotypic observation
During drought treatment, plants were observed daily for morphological features including leaf yellowing, wilting and abscission, and changes in phenotype were noted by photographing at test days 0d, 7d, 14d and 21 d. At the end of the test (DS 21), the number of surviving plants of the drought-treated plants was counted and the survival rate was calculated. Survival = number of surviving plants/total number of treated plants x 100%.
FIG. 6 shows a phenotype plot under drought stress for WT, OE and RE plants, wherein FIG. 6A is a phenotype plot for WT, OE, RE plants under normal watering (CK) and different time drought stress (DS 7, DS14, DS 21), respectively; FIG. 6B is a phenotype plot of OE plants under normal watering and different time drought stress; FIG. 6C is a phenotype diagram of RE plants under normal watering and different time drought stress; FIG. 6D is a phenotype plot of WT plants under normal watering and different time drought stress. As shown in fig. 6, at the end of the experiment (DS 21), all normally watered plants and drought plants survived, with 100% survival, but with significant differences in phenotypic changes between different lines; normally watering plants (CK) grow vigorously during the test period, obvious leaf falling, yellowing, wilting and death phenomena are avoided, and the morphology of drought treatment (DS) plants is obviously changed; the time for the victim symptoms of different strains to appear is obviously different; most RE plants (RE 19, RE20 and RE 24) show leaf wilting symptoms in drought for 4-5 days, while OE plants (OE 25, OE26 and OE 27) show leaf wilting symptoms in drought for 7-8 days, and WT shows symptoms in drought for 4-6 days. At drought 7d (DS 7), there was a clear difference in morphological features of the different strains. Wilting of the whole plant leaves of the RE plants occurs, and the lower leaves wither seriously; the lower leaves of the WT plants start to wither, and the upper leaves show wilting and yellowing; the leaves of the OE plant do not obviously wither, and only the lower leaves show slight yellowing and wilting. At this time, the proportion of the number of normal leaves on the plant to the total number of leaves of the whole plant is ordered as ：OE27(82.15％)>OE25(74.85％)>OE26(72.56％)>WT(57.18％)>RE19(36.97％)>RE24(35.28％)>RE20(29.33％).
As can be seen from fig. 6, as the drought treatment time is prolonged, the RE plant leaves wilt, yellow and fall off in a large amount until drought 14d (DS 14), only the top 3-5 leaves remain, and the leaf edges have a curling phenomenon; the WT blade is largely detached, but the degree is lighter than RE; the OE plants had only serious damage to the lower leaves and the upper leaves remained normal. At this time, after the normal leaf number on the plants accounts for 7d of total leaf number of the whole plants (DS 21) after the water recovery of ：OE27(34.74％)>OE25(31.61％)>OE26(28.02％)>WT(23.99％)>RE20(9.38％)>RE19(7.43％)>RE24(6.45％)., the leaves of the OE plants are changed from yellow to green and the new leaves are developed, and the WT and RE plants have no new leaf development and the leaves are in curled and shrunken states. At the moment, the sequencing of the proportion of the number of normal leaves on the plants to the total number of leaves of the whole plant is ：OE27(39.24％)>OE26(37.51％)>OE25(29.31％)>WT(28.62％)>RE19(6.38％)>RE24(3.04％)>RE20(2.22％)., compared with the WT, the time for the OE strain to generate the victimization symptom is late, the degree is light, and the recovery is quick after rehydration; RE strain has early and repeated occurrence of victim symptoms, and is slow to recover after rehydration. Therefore, the over-expression PdeERF gene can obviously reduce the damage degree of the plant under drought stress and obviously improve the drought resistance of the plant.
9. Growth assessment
And (3) measuring the seedling height and the ground diameter of the plant respectively in the 0d and 21d test, and calculating the growth amount of the seedlings during the test. Seedling height the height of the seedling base to the top bud base was measured to an accuracy of 0.1cm. The diameter of the position 1cm above the root and stem of the seedling is measured to be 0.01cm. And the drought resistance coefficient is adopted to evaluate the influence of drought stress on each observation index of the plant, wherein the drought resistance coefficient is =drought treatment/control treatment multiplied by 100%.
FIG. 7 shows the results of shoot height and ground diameter growth of WT, OE and RE plants under drought stress, from which it can be seen that drought stress inhibits shoot height and ground diameter growth of all plants, with greater inhibition of shoot height than ground diameter. At the end of the test (DS 21), the seedling height growth was significantly reduced (excluding OE 26) for all strain drought plants compared to the control treatment (CK), while the reduction in ground path growth was not significant (excluding RE 20). At this time, the drought resistance coefficients of the high growth amount of each plant seedling are as follows: OE26> OE27> OE25> WT > RE24> RE20> RE19; drought resistance coefficients of the ground diameter growth amount are respectively as follows: OE26> OE25> OE27> WT > RE19> RE24> RE20 (as shown in table 2 below); the average drought resistance coefficients of the OE, WT and RE seedlings are 60.23%, 40.84% and 35.51%, and the average drought resistance coefficients of the ground diameters are 68.88%, 63.24% and 60.26%, respectively. Therefore, the drought resistance coefficient of the seedling height and the ground diameter growth of the OE strain under drought stress is obviously larger than that of the WT and RE strains, which indicates that the influence of the drought stress on the growth of the WT and RE strains is larger, the inhibition degree of the OE is relatively smaller, and the growth of the plant under the drought stress can be obviously improved by over-expressing PdeERF genes.
TABLE 2
10. Biomass mass
After the test is finished (DS 21), the plants of WT, OE25, OE26, OE27, RE19, RE20 and RE24 of the control treatment (CK) and the drought treatment (DS) are harvested, the soil is carefully washed by tap water, then the water is wiped by filter paper and absorbent paper, the plants are decomposed into three parts of roots, stems and leaves, the three parts are deactivated for 2 hours in a 105 ℃ oven, then the plants are baked to constant weight at 60 ℃, the dry weight of the roots, the stems and the leaves is respectively weighed by an electronic day, and the influence of the drought treatment on the growth of different strains is compared. The root-cap ratio is calculated by the following formula: root cap ratio = root dry weight/(dry weight of stem + She Ganchong). The calculation formula of the total biomass is as follows: total biomass = root dry weight + stem dry weight + She Ganchong.
Table 3 shows the biomass accumulation results for all strains under drought stress. It can be seen from the table that RE and WT are more severely inhibited than OE. At the end of the test (DS 21), leaf and total biomass accumulation was significantly lower for all drought plants (DS) than for control plants (CK), and also for the stems and root system, but not significantly different from CK. At this time, the drought resistance coefficient sequencing of the biomass of the blade and the root system is as follows: OE26> OE27> OE25> WT > RE20> RE19> RE24; the drought resistance coefficient sequencing of the stem biomass is as follows: OE27> OE25> OE26> WT > RE19> RE20> RE24; the drought resistance coefficient sequencing of the root-cap ratio is as follows: RE20> RE24> RE19> WT > OE27> OE26> OE25; the drought resistance coefficient of the total biomass is ordered as follows: OE26> OE25> OE27> WT > RE19> RE20> RE24 (as shown in table 3 below); average drought resistance coefficients for OE, WT and RE leaf biomass were 47.82%, 14.45% and 8.28%, respectively; the average drought resistance coefficients of the stem segments are 78.55%, 73.48% and 48.6% respectively; the average drought resistance coefficient of the root system is 91.46%, 82.77% and 50.41% respectively; the root cap ratio average drought resistance coefficients are 153.18%, 222.66% and 229.93% respectively; the average drought resistance coefficient of the total biomass is 63.10%, 40.85% and 25.05% respectively. Therefore, the drought resistance coefficient of the OE leaves, stem segments, root systems and total biomass is obviously larger than that of the WT and the RE, the drought resistance coefficient of the root-cap ratio is obviously smaller than that of the WT and the RE, and the influence of drought stress on biomass accumulation of the WT and the RE is larger and the inhibition effect on the OE is relatively smaller.
TABLE 3 Table 3
11. Blade gas exchange parameters
Leaf gas exchange parameters of each of the plant line Control (CK) and Drought (DS) plants were measured using an LI-6400 photosynthetic determinator (LI-COR inc., lincoln, NE, USA) at test nos. 0d, 7d, 14d and 21d, 3 replicates/treatments for 9 a.m.: 00-11: 00. before the test starts, the 5 th leaf which is fully unfolded at the top end of the plant is marked as a measurement leaf of a photosynthetic index, and when the later leaf falls seriously, the healthy leaf which is unfolded at the upper part is used as the measurement leaf. In the measurement, a standard LI-COR leaf chamber and a red-blue light source (6400-02 LED light source) were used, the illumination intensity was set to 1500. Mu. Mol.m -2·s-1, and the air flow rate was set to 500. Mu. Mol.s -1. The measurement content mainly comprises the net photosynthetic rate (P n), the transpiration rate (T r), the stomatal conductance (G s), the intercellular CO 2 concentration/environmental CO 2 concentration (Ci/Ca), and the corresponding saturated water pressure, leaf surface temperature and other ambient environmental conditions. The calculation formula of the instantaneous water utilization rate (R WUEi) is as follows: r WUEi＝Pn/Gs.
(1) Net photosynthetic rate
FIG. 8 shows the results of net photosynthetic rates of WT, OE and RE plants under drought stress. As shown, drought stress significantly reduced the net photosynthetic rate of all plant leaves (P n; P < 0.05); during the test period, the Pn values of all control plants did not change significantly, while the Pn values of drought plants gradually decreased with prolonged drought time. By 7d, the Pn value of RE drought plants was significantly lower than its CK, while the decrease in WT and OE was relatively small. The maximum drop in RE occurs at 0-7 d, while the maximum drop in OE and WT occurs at 7-14 d. At 14d, the drought resistance coefficient ranking of each strain P n was: OE27> OE26> OE25> WT > RE19> RE20> RE24 (as shown in table 4 below). During rehydration, the P n values for OE and WT were significantly increased, with the P n values for OE26 and OE27 returning to control levels, OE25 and WT slightly below the control; the P n value of RE continues to drop during reconstitution and reaches a minimum at 21 d. At the end of the test, the drought resistance coefficient of each strain P n is sequenced to ：OE27(104.31％)>OE26(99.86％)>OE25(75.48％)>WT(51.97％)>RE20(19.76％)>RE19(16.31％)>RE24(7.48％)., so that compared with the WT, the OE can still maintain higher photosynthetic capacity under drought stress, and the recovery speed is high, while the photosynthetic capacity of RE is severely inhibited, and the recovery speed is low.
TABLE 4 Table 4
(2) Rate of transpiration
FIG. 9 shows the transpiration rate results for WT, OE and RE plants under drought stress, as shown by the drought stress significantly decreasing the transpiration rate of all plant lines leaves (T r; p < 0.05). During the test period, the T r value of the control plant is not changed obviously, while the T r value of the drought plant is gradually reduced along with the prolonged drought time. By 7d, the T r values were significantly reduced for all drought plants, significantly below their CK. By 14d, T r continues to drop, with relatively less OE drop than WT, and greater RE drop. At this time, the T r drought resistance coefficients of the respective strains are ranked as follows: OE25> OE27> OE26> WT > RE19> RE24> RE20 (as shown in table 4). During rehydration, the value of T r for OE increases significantly, the value of T r for WT increases less, the value of T r for RE continues to decrease (except for RE 20) and reaches a minimum at 21 d. At the end of the test, the T r drought resistance coefficients of the strains are ordered as ：OE27(74.38％)>OE26(73.19％)>OE25(71.97％)>WT(24.56％)>RE19(9.21％)>RE20(7.49％)>RE24(5.19％).
(3) Air hole conductivity
FIG. 10 shows stomatal conductance results for WT, OE and RE plants under drought stress, as shown by the inhibition of stomatal conductance by drought stress on all lines (G s; p < 0.05), similar to the trend of change in Pn and Tr. During the test period, the G s values of all control plants did not change significantly, while the G s values of drought plants gradually decreased with prolonged drought time. By 7d, all drought plants had G s values significantly lower than their CK, with maximum decrease in RE, WT times, and relatively less decrease in OE. By 14d, G s continuously decreased, and drought resistance coefficients of each strain G s were ranked as: OE25> oe27=wt > OE26> RE19> RE24> RE20 (as shown in table 4). During rehydration, the values of G s for OE and WT gradually increased, with the values of G s for OE25 and OE26 returning to the control level, OE27 slightly below the control, with the WT increasing slightly, while the value of G s for RE still continues to decrease and reaches a minimum at 21 d. At the end of the test, the G s drought resistance coefficients of the strains are ordered as ：OE25(81.06％)>OE27(74.40％)>OE26(73.53％)>WT(24.49％)>RE19(9.00％)>RE20(7.32％)>RE24(1.85％).
(4) Intercellular CO 2 concentration/ambient CO 2 concentration
FIG. 11 shows the results of the concentration of intracellular CO 2/ambient CO 2 in WT, OE and RE plants under drought stress, as shown, drought stress significantly affected the concentration of extracellular CO 2/ambient CO 2 (Ci/Ca; p < 0.05) in all plant lines leaves, with the trend of Ci/Ca slightly different from Pn, gs and Tr. During the test period, the Ci/Ca values of all control plants did not change significantly, while the Ci/Ca values of drought plants tended to decrease and then increase with increasing drought time. At 7d, the Ci/Ca values of RE drought plants decreased by a larger amount, and OE decreased by a smaller amount, compared to WT. At 14d, the Ci/Ca values of all the strains gradually rise, and the drought resistance coefficients of all the strains are sequenced as follows: RE24> OE26> WT > OE27> OE25> RE20> RE19 (as shown in Table 4). During rehydration, the Ci/Ca values of all strains gradually decrease, and the drought resistance coefficients of all strains are ordered at the end of the test ：WT(90.20％)>RE20(89.12％)>OE27(88.11％)>OE25(87.37％)>RE19(86.55％)>OE26(85.58％)>RE24(83.03％).
(5) Instantaneous water utilization
FIG. 12 shows the results of instantaneous water use of WT, OE and RE plants under drought stress, as shown by the significant improvement in instantaneous water use of all plants by drought stress (R WUEi; p < 0.05). During the test period, the R WUEi values of all control plants did not change significantly, while the R WUEi values of drought plants gradually increased with prolonged drought time. By 7d, the R WUEi variation was small for all plants. At 14d, compared with the WT, the variation amplitude of R WUEi of OE is larger, the variation amplitude of RE is relatively smaller, and at the moment, the drought resistance coefficients of the strains R WUEi are ordered as follows: OE27> OE26> OE25> WT > RE20> RE24> RE19 (as shown in table 4). After 7d rehydration, the performances of different strains are different, and R WUEi of RE continuously rises to reach the highest value; r WUEi of OE is greatly reduced and restored to the control level; the R WUEi of WT had less degradation and did not return to the control level. The drought resistance coefficient ranking for each strain R WUEi at the end of the test was ：RE24(397.91％)>RE20(286.46％)>WT(230.36％)>RE19(212.92％)>OE26(174.28％)>OE27(174.28％)>OE25(94.29％). for the whole test period, with the maximum rise amplitude for OE and WT occurring at 7-14 d and the maximum rise amplitude for RE at 14-21 d.
12. Chlorophyll fluorescence parameter
Test nos. 0d, 7d, 14d and 21d, chlorophyll fluorescence parameters of individual strain control treated (CK) and drought treated (DS) plants were measured using an LI-6400 fluorescence system tester (LI-COR inc., lincoln, NE, USA) respectively, 3 replicates/treatments. Before the test starts, marking the 5 th leaf which is fully unfolded at the top end of the plant as a measurement leaf of a fluorescence index, and taking the healthy leaf which is unfolded at the upper part as the measurement leaf when the later leaf falls seriously. Dark adaptation of plant leaves for 30min before measurement, main measurement: initial fluorescence (F o), variable fluorescence (F v), maximum fluorescence (F m), latent activity of photoreaction center PS ii (F v/Fo), maximum photochemical quantum yield (F v/Fm), and the like. Subsequently, the same leaf was photoactivated for 30min with activation light on, main assay: the effective photochemical quantum yield (F v'/Fm'), the actual photochemical efficiency (phi PS II) of the photoreaction center PS II, the transfer rate (ETR) of photosynthetic electrons of the photoreaction center PS II, the photochemical quenching coefficient (qP), the non-photochemical quenching coefficient (NPQ) and the like of PS II. Wherein qp= (F m'-Fs)/(Fm'-Fo'),NPQ＝Fm/Fm' -1).
(1) Maximum photochemical efficiency
FIG. 13 shows the results of maximum photochemical efficiencies for WT, OE and RE plants under drought stress, as shown by the significant reduction in maximum photochemical efficiency for each strain by drought stress (F v/Fm; p < 0.05). During the test period, the F v/Fm values of all control plants did not change significantly, while the F v/Fm values of drought plants gradually decreased with prolonged drought time. The magnitude of the change in F v/Fm for each strain was not apparent by 7 d; by 14d, F v/Fm was significantly reduced for all lines, with RE being significantly more reduced than for WT and OE. At this time, the drought resistance coefficient of each strain is ranked as follows: OE26> OE27> WT > OE25> RE20> RE24> RE19 (as shown in table 5 below). During rehydration, F v/Fm of WT and OE gradually recovered to control levels, while F v/Fm of RE continued to decrease to a minimum. At the end of the test, the drought resistance coefficient of each strain is ordered as ：OE26(97.82％)>OE27(97.70％)>OE25(97.67％)>WT(94.07％)>RE20(74.49％)>RE24(71.76％)>RE19(58.17％).
TABLE 5
(2) PS II potential Activity
FIG. 14 shows the results of the potential PS II activity of WT, OE and RE plants under drought stress, as shown by the significant inhibition of PS II potential activity of each strain by drought stress (F v/Fo; p < 0.05), similar to the trend of F v/Fm. During the test period, the F v/Fo values of all control plants did not change significantly, while the F v/Fo values of drought plants gradually decreased with prolonged drought time. By 7d, the change in each strain F v/Fo was still not apparent; by 14d, the decrease in amplitude increases, and RE decreases significantly more than WT and OE. At this time, the drought resistance coefficient of each strain is ranked as follows: OE26> OE27> OE25> WT > RE20> RE24> RE19 (as shown in table 5). During rehydration, the F v/Fo value of OE is obviously increased and gradually restored to the control level; the rise amplitude of the WT is small and does not return to the control level; the F v/Fo values of RE continue to decrease (except RE 20). At the end of the test, the drought resistance coefficient of each strain is ordered as ：OE27(100.09％)>OE26(97.29％)>OE25(89.40％)>WT(77.16％)>RE20(62.26％)>RE24(46.33％)>RE19(43.79％).
(3) Efficient photochemical quantum yield of PS II
FIG. 15 shows that the PS II effective photochemical quantum yield of WT, OE and RE plants under drought stress is significantly inhibited by drought stress (F v'/Fm') for each strain, with a trend similar to F v/Fm with increasing drought time (p < 0.05). At 7d, RE is reduced by a greater amount than WT, significantly lower than its CK value; by 14d, F v'/Fm' was continuously reduced for each strain, with less OE drop and greater RE drop compared to WT. At this time, the drought resistance coefficient of each strain is ranked as follows: OE25> WT > OE27> OE26> RE24> RE19> RE20 (as shown in table 5). During rehydration (DS 21), F v'/Fm' was gradually increased for all strains (except RE 24), with the magnitude of the increase in OE being greater, gradually returning to the control level; the rise amplitude of WT and RE is small, still below their CK value. At the end of the test, the drought resistance coefficient of each strain is ordered as ：OE25(97.05％)>OE27(95.37％)>OE26(94.11％)>WT(75.50％)>RE20(63.42％)>RE19(62.13％)>RE24(35.48％).
(4) Actual photochemical efficiency
FIG. 16 shows the actual photochemical efficiency results for WT, OE and RE plants under drought stress, as shown by the fact that drought stress resulted in a significant decrease in actual photochemical efficiency (ΦPSII) for each strain (p < 0.05). During the test, the PhiPS II values of all control plants are not obviously changed, while the PhiPS II values of drought plants are gradually reduced along with the prolongation of the stress time; at 7d, the phi PS II values of all drought plants are significantly lower than their CK values; by 14d, a significant difference between the different strains began to appear; the decrease in OE is relatively small and the decrease in RE is large compared to WT. At this time, the drought resistance coefficient of each strain is ranked as follows: OE25> OE26> OE27> WT > RE24> RE19> RE20 (as shown in table 5). During rehydration, the values of OE and WT rise significantly, OE returns to the control level, WT is slightly below the control, and RE still continues to decrease to a minimum value. At the end of the test, the drought resistance coefficients of the strains are ordered as ：OE25(96.27％)>OE26(92.48％)>OE27(87.94％)>WT(72.14％)>RE19(30.43％)>RE20(27.39％)>RE24(24.20％).
(5) Photochemical quenching
FIG. 17 shows the results of photochemical quenching of WT, OE and RE plants under drought stress, as shown by the fact that drought stress significantly affected photochemical quenching of the individual lines (qP; p < 0.05). During the test period, the qP values of all control plants did not change significantly, while the qP values of drought plants gradually decreased over time. By 7d, qP values were lower for all drought plants than their CK, RE was reduced to a greater extent and OE was reduced to a lesser extent compared to WT. By 14d, the qP value for each strain was continuously reduced to a minimum. At this time, the drought resistance coefficient of each strain is ranked as follows: OE26> OE25> WT > OE27> RE24> RE19> RE20 (as shown in table 5). During rehydration, the qP values of all strains gradually rise, the rising amplitude of OE is larger compared with that of WT, and the control level is gradually restored; RE strains have a smaller rise, still significantly lower than their CK value. At the end of the test, the drought resistance coefficient of each strain is ordered as ：OE25(101.61％)>OE26(99.27％)>WT(92.92％)>OE27(80.57％)>RE19(55.98％)>RE24(53.63％)>RE20(51.41％).
(6) Non-photochemical quenching
Figure 18 shows the result of non-photochemical quenching of WT, OE and RE plants under drought stress, as shown by the significant increase in non-photochemical quenching (NPQ) of individual lines under drought stress (p < 0.05). During the test period, the NPQ values of all control plants did not change significantly, while the NPQ values of drought plants gradually increased with prolonged stress time. By 7d, the differences between the lines were not significant, the OE rise was slightly higher and the RE rise slightly lower than for WT. By 14d, the NPQ of OE gradually decreases, while the NPQ of RE and WT still continues to rise, reaching a maximum. At this time, the drought resistance coefficient of each strain is ranked as follows: WT > OE25> OE27> OE26> RE24> RE19> RE20 (as shown in table 5). During rehydration, NPQ was gradually decreased for all strains, with greater decrease in OE and WT and less decrease in RE strains. At the end of the test, the drought resistance coefficient of each strain is ordered as ：RE24(283.15％)>RE20(268.14％)>OE26(195.78％)>WT(189.97％)>OE25(185.41％)>RE19(179.30％)>OE27(151.59％).
(7) Apparent electron transfer rate
Figure 19 shows apparent electron transfer rates for WT, OE and RE plants under drought stress, as shown, drought stress resulted in a significant decrease in apparent Electron Transfer Rate (ETR) for each strain (p < 0.05). During the test period, the ETR values of all control plants were unchanged and not significantly, while the ETR values of drought plants gradually decreased over time. By 7d, ETR values were significantly reduced for all plants; by 14d, the decrease continues, with less OE and greater RE than the WT. At this time, the drought resistance coefficient of each strain is ranked as follows: OE25> OE26> WT > OE27> RE19> RE24> RE20 (as shown in table 5). During rehydration, the ETR values of OE and WT increased significantly, OE26 and OE27 recovered to control levels, OE25 and WT were slightly below control, and the ETR values of RE continued to decrease to a minimum (excluding RE 19). At the end of the test, the drought resistance coefficient of each strain is ordered as ：OE26(99.58％)>OE25(96.73％)>WT(88.59％)>OE27(86.73％)>RE19(58.38％)>RE20(30.17％)>RE24(19.53％).
13. Physiological index
(1) Chlorophyll relative content
Test 0d, 7d, 14d and 21d, respectively, the 5 th fully developed leaf of each of the Control (CK) and Drought (DS) plants was selected as a measurement leaf, the relative chlorophyll content (SPAD value) was determined with a chlorophyll meter SPAD-502Plus (Minolta Co, japan), 5 points were measured for each leaf, and the average was taken as the relative chlorophyll content for that leaf, and 3 replicates/treatments were performed.
FIG. 20 shows the results of leaf chlorophyll relative content of WT, OE and RE plants under drought stress, as shown by the significant reduction of chlorophyll relative content of individual lines by drought stress (SPAD; p < 0.05). During the test period, the SPAD value of the control plant did not change significantly, while the SPAD value of the drought plant was gradually decreased with prolonged stress time. By 7d, the SPAD values of OE and WT decrease less, while the SPAD values of RE decrease significantly below their CK values. By 14d, OE is reduced less and RE is reduced more than WT. At this time, the drought resistance coefficient of each strain is ranked as follows: OE26> OE27> OE25> RE20> WT > RE19> RE24 (as shown in table 6 below). After rehydration recovery, the SPAD values of WT and OE gradually rise, OE returns to the control level, the WT rise is small, slightly lower than the control, while the SPAD value of RE still continues to decrease, significantly lower than its control value. At the end of the test, the drought resistance coefficient of each strain is ordered as ：OE26(100.03％)>OE27(97.48％)>OE25(87.27％)>WT(74.50％)>RE20(73.88％)>RE19(65.65％)>RE24(64.98％).
TABLE 6
(2) Relative conductivity of blade
At test nos. 0d, 7d and 14d, fresh leaves of control treated (CK) and drought treated (DS) plants of each strain were collected, respectively, and relative conductivity (RMP) was determined using 3173 portable conductivity meter (Jenco Instruments, inc, USA) for 3 replicates/treatments.
FIG. 21 shows the relative conductivity results for the leaves of WT, OE and RE plants under drought stress, as shown by the significant increase in leaf relative conductivity (RMP) for all plants (p < 0.05). During the test period, the RMP values of all control plants did not change significantly, whereas the RMP values of drought plants gradually increased with prolonged stress time. By 7d, the RMP value of OE varies less than that of WT, and the rising amplitude of RE is greater than that of CK. At this time, the drought resistance coefficient ranking of each strain RMP is ：RE19(125.62％)>RE20(124.05％)>RE24(123.84％)>OE27(110.54％)>OE25(102.23％)>WT(100.16％)>OE26(99.85％)( as shown in table 6). At 14d, compared with the WT, RE is larger in rising amplitude, OE is smaller in rising amplitude, and drought resistance coefficients of each strain RMP are ordered as follows: RE19> RE20> RE24> WT > OE25> OE27> OE26. It can be seen that drought stress has a greater effect on RE lines and, by WT, OE lines are affected to a lesser extent.
(3) Blade moisture content
Test nos. 0d, 7d and 14d, fresh leaves of each of the line control treated (CK) and drought treated (DS) plants were collected for determination of Relative Water Content (RWC) and Absolute Water Content (AWC), 3 replicates/treatments, respectively.
FIG. 22 shows the results of moisture content of leaves of WT, OE and RE plants under drought stress, as shown by similar trends of variation of relative leaf moisture content (RWC) and absolute moisture content (AWC) under drought stress, which are all gradually decreasing, and the difference between different strains is significant (p < 0.05). By 7d, the RWC differences were not significant for each strain; by 14d, the differences between the strains were significant, with relatively less OE degradation compared to WT, greater RE degradation, significantly lower than its CK value. At this time, the drought resistance coefficient of each strain RWC is ordered as follows: OE25> OE27> OE26> WT > RE19> RE20> RE24 (as shown in table 6). For AWC, OE and WT decrease less in magnitude, while RE decreases more magnitude, significantly below its CK value, to 7 d. By 14d, the AWC for each strain was continuously reduced to a minimum. At this time, the drought resistance coefficient of each strain AWC is ranked as: OE27> OE25> OE26> RE19> RE24> WT > RE20 (as shown in table 6).
(4) Malondialdehyde content
At test nos. 0d, 7d and 14d, leaves and roots of control treated (CK) and drought treated (DS) plants of each strain were collected, respectively, and Malondialdehyde (MDA) content was measured using thiobarbituric acid (TBA) method, 3 replicates/treatments.
FIG. 23 shows the malondialdehyde content results for leaves and roots of WT, OE and RE plants under drought stress, as shown, drought stress significantly increased malondialdehyde content for leaves and roots of each strain (MDA; p < 0.05). During the test period, the MDA content of all control plants did not change significantly, while the MDA content of drought plants gradually increased with time. At 7d, the variation amplitude of MDA content in the leaves and root systems of OE and WT is smaller, while the rising amplitude of RE is larger and is obviously higher than the CK value. At this time, the drought resistance coefficients of the roots with the MDA content of ：RE19(138.28％)>RE20(134.93％)>RE24(134.18％)>OE27(116.85％)>WT(112.93％)>OE26(109.26％)>OE25(103.01％); in each plant leaf are sequenced to ：RE24(196.47％)>RE20(176.20％)>RE19(170.54％)>OE26(127.36％)>WT(122.24％)>OE27(106.43％)>OE25(105.85％)( as shown in Table 6. By 14d, MDA content in each plant leaf and root system continuously rises to reach the maximum value, and is obviously higher than the control level. RE rise is greater and OE rise is less than WT. At this time, the drought resistance coefficient of MDA content of each plant leaf is ordered as follows: RE24> RE20> RE19> WT > OE26> OE27> OE25; the drought resistance coefficient sequencing of the MDA content of the root system is as follows: RE24> RE20> RE19> WT > OE27> OE26> OE25. Thus, RE strain suffers from a larger degree under drought stress, WT is centered, and OE strain suffers from a minimum degree.
(5) Root system vitality
Test nos. 0d, 7d and 14d, fine roots of control treated (CK) and drought treated (DS) plants of each strain were collected, and root activity was measured using root activity detection kit (TTC), 3 repetitions/treatments.
FIG. 24 shows root system vigor results for WT, OE and RE plants under drought stress, as shown by the significant reduction in root system vigor for each strain (p < 0.05) by drought stress. As drought time is prolonged, root activity of each strain is continuously reduced, and reaches a minimum value at 14 d. By 7d, the decrease in OE was small compared to WT, the decrease in RE was large, and the CK value was significantly lower. At this time, the drought resistance coefficient of each strain root system activity is ranked ：OE27(97.89％)>OE26(94.95％)>OE25(92.44％)>WT(82.23％)>RE20(54.00％)>RE19(53.40％)>RE24(52.81％)( as shown in table 6). By 14d, the root system activity of each strain is continuously reduced, the amplitude reduction of OE is smaller and the amplitude reduction of RE is larger than that of WT, so that the minimum value is reached. At this time, the drought resistance coefficient of each strain is ranked as follows: OE27> OE25> OE26> WT > RE19> RE24> RE20, whereby it can be seen that drought stress is more damaging to RE and less damaging to WT and OE.
(6) Active oxygen content determination
Reactive Oxygen Species (ROS) common in plants are mainly H 2O2 and O 2-; diaminobenzidine (DAB) may be oxidized by H 2O2 to give a brown precipitate; the nitrotetrazolium chloride (NBT) can be combined with O 2- to reduce NBT into a blue polymer, so that the damage degree of plants and the stress resistance of the plants can be judged according to the dyeing condition. At test 0d, 7d and 14d, fresh leaves of Control (CK) and Drought (DS) plants of each strain are collected, DAB and NBT are respectively adopted for histochemical staining, and accumulation conditions of H 2O2 and O 2- in tissues are observed; the contents of H 2O2 and O 2- were determined using Nanjing's built kit (Nanjing's built institute of biological engineering).
FIG. 25 shows the results for H 2O2 and O 2- content of WT, OE and RE plants under drought stress, as shown by the lower H 2O2 and O 2- content in plants under normal conditions, with insignificant differences between plants.
FIG. 26 shows DAB staining results for OE, RE and WT plants after drought stress; FIG. 27 shows the NBT staining results of OE, RE and WT plants after drought stress, as shown by the increase in H 2O2 and O 2- levels with increasing drought levels, H 2O2 levels consistent with DAB staining results and O 2- levels consistent with NBT staining results. At 7d, the H 2O2 content of the WT and the OE drought plants are not obviously different from the control (except OE 25), the H 2O2 content of the RE drought plants is obviously increased compared with the control, at the moment, the drought resistance coefficient of the H 2O2 content of each plant is ：RE20(178.96％)>RE24(161.64％)>RE19(157.76％)>OE25(145.45％)>WT(134.21％)>OE26(108.66％)>OE27(107.89％);, the content of the O 2- of each drought plant slightly rises at 7d, but the difference between the drought plants and the control is not obvious (except RE 20), at the moment, the drought resistance coefficient of the O 2- content of each plant is ：RE20(140.97％)>RE24(119.43％)>OE25(117.66％)>WT(116.74％)>RE19(111.59％)>OE26(110.13％)>OE27(107.87％). to 14d, the H 2O2 content and the O 2- content of each drought plant of each plant obviously rise, the control shows obvious difference, but the rising amplitude difference of different plants is obvious, the rising amplitude of RE plants is the largest, the rising amplitude of the OE plants is the smallest, and the rising amplitude of the RE plants is the WT times. At this time, the drought resistance coefficient sequence of the H 2O2 content of each strain is ：RE20(301.18％)>RE24(290.39％)>WT(235.24％)>RE19(233.99％)>OE25(181.51％)>OE27(145.83％)>OE26(138.93％);, the drought resistance coefficient sequence of the O 2- content of each strain is ：RE20(173.11％)>RE24(168.28％)>RE19(164.70％)>WT(139.98％)>OE25(132.84％)>OE26(124.06％)>OE27(120.72％)., and in the later period of drought stress, the H 2O2 content and the O 2- content of the OE plant are obviously lower than those of the WT and RE plants, which indicates that the ROS accumulated in the OE plant body is less under the drought stress, and the damage of the plant is lighter.
14. Drought resistance evaluation
(1) Cluster analysis
And (3) carrying out cluster analysis (minimum distance method) by taking the drought resistance coefficients of leaf gas exchange parameters, chlorophyll fluorescence parameters and physiological indexes of OE, RE and WT plants at 14d and the drought resistance coefficients of seedling high growth, ground diameter growth and total biomass accumulation of each plant during the test period as indexes. FIG. 28 shows the results of cluster analysis of drought resistance of WT, OE and RE plants, as shown, OE strains (OE 25, OE26, OE 27) are clustered into one type, and drought resistance is stronger; RE strains (RE 19, RE20 and RE 24) are gathered into one type, and the drought resistance is poor; WT strains are a single class with drought resistance between OE and RE.
(2) Membership function analysis
And (3) taking the drought resistance coefficients of the leaf gas exchange parameters, chlorophyll fluorescence parameters and physiological indexes of OE, RE and WT in the 14d, and the drought resistance coefficients of the seedling high growth quantity, the ground diameter growth quantity and the total biomass accumulation of each plant in the test period as indexes, and adopting a membership function analysis method to evaluate the drought resistance of each plant line. The results show that the drought resistance sequences of the strains are as follows: OE27> OE26> OE25> WT > RE19> RE24> RE20 (as shown in Table 7 below), indicating that OE strains are the strongest in drought resistance, RE strains are the weakest in drought resistance, and WT drought resistance is between OE and RE, with results consistent with cluster analysis. The result shows that over-expression PdeERF gene obviously improves the drought stress tolerance of poplar, and inhibiting PdeERF gene expression obviously reduces the drought resistance of poplar.
TABLE 7
/>
The present application is not limited to the above-mentioned embodiments, and any changes or substitutions that can be easily understood by those skilled in the art within the technical scope of the present application are intended to be included in the scope of the present application.
Claims (7)
- The application of the Pdeerf53 gene in regulating and controlling the drought resistance of poplar is characterized in that the nucleotide sequence of the PdeERF gene is shown as SEQ ID NO. 1.
- 2. The use of claim 1, wherein the PdeERF gene modulates poplar characteristics in poplar drought resistance comprising at least one of the following (1) - (10):(1) Plant seedling height, ground diameter and leaf growth under drought stress;(2) Biomass accumulation of plants under drought stress;(3) Net photosynthetic rate, transpiration rate, stomatal conductance, intercellular CO 2 concentration/environmental CO 2 concentration, instantaneous water use efficiency of plants under drought stress;(4) Maximum photochemical efficiency of plants under drought stress, potential activity of PS II, effective photochemical quantum yield of PS II, actual photochemical efficiency, photochemical quenching, non-photochemical quenching, and apparent electron transfer rate;(5) Chlorophyll relative content of plants under drought stress;(6) Leaf relative conductivity of plants under drought stress;(7) Moisture content of leaves under drought stress;(8) Malondialdehyde content of plant leaves and root systems under drought stress;(9) Root system activity of plants under drought stress;(10) Active oxygen content of plants under drought stress.
- 3. The use according to claim 1, wherein the primer pair for amplifying PdeERF genes comprises PdeERF-OE-F as shown in SEQ ID NO.2 and PdeERF-OE-R as shown in SEQ ID NO. 3.
- 4. The application of the recombinant vector containing PdeERF gene or the expression cassette containing PdeERF gene is characterized in that the application is to cultivate transgenic poplar with enhanced drought resistance through over-expression of PdeERF gene, and the nucleotide sequence of PdeERF gene is shown as SEQ ID NO. 1.
- 5. A drought-resistant poplar breeding method is characterized by comprising the following steps:transforming PdeERF gene into poplar to obtain transgenic positive plant, wherein the nucleotide sequence of PdeERF gene is shown as SEQ ID NO. 1;and screening out a final drought-resistant plant according to the expression quantity of the PdeERF gene in the leaves and root systems and the drought resistance evaluation result.
- 6. The method of claim 5, wherein the step of transforming PdeERF gene into poplar comprises:constructing PdeERF53 gene over-expression vector;using electric shock method to make the over-expression carrier into agrobacterium tumefaciens;And (3) infecting plants by using agrobacterium tumefaciens to obtain the drought-resistant poplar.
- 7. Use of the breeding method according to any one of claims 5 or 6 for cultivating drought-resistant poplar.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311385703.6A CN117487849B (en) | 2023-10-23 | 2023-10-23 | Application of PdeERF gene in regulation and control of drought resistance of plants |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311385703.6A CN117487849B (en) | 2023-10-23 | 2023-10-23 | Application of PdeERF gene in regulation and control of drought resistance of plants |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN117487849A CN117487849A (en) | 2024-02-02 |
| CN117487849B true CN117487849B (en) | 2024-04-26 |
Family
ID=89677349
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311385703.6A Active CN117487849B (en) | 2023-10-23 | 2023-10-23 | Application of PdeERF gene in regulation and control of drought resistance of plants |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117487849B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112501182A (en) * | 2020-12-07 | 2021-03-16 | 山西农业大学 | Poplar ERF transcription factor gene and application thereof |
| CN113461793A (en) * | 2021-06-30 | 2021-10-01 | 华南农业大学 | Capsicum annuum ERF transcription factor CaERF102 and application thereof in increasing capsaicin content |
-
2023
- 2023-10-23 CN CN202311385703.6A patent/CN117487849B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112501182A (en) * | 2020-12-07 | 2021-03-16 | 山西农业大学 | Poplar ERF transcription factor gene and application thereof |
| CN113461793A (en) * | 2021-06-30 | 2021-10-01 | 华南农业大学 | Capsicum annuum ERF transcription factor CaERF102 and application thereof in increasing capsaicin content |
Non-Patent Citations (3)
| Title |
|---|
| Arabidopsis RGLG2, functioning as a RING E3 ligase, interacts with AtERF53 and negatively regulates the plant drought stress response;Mei-Chun Cheng等;Plant Physiol;20120131;第158卷(第1期);第363-375页 * |
| Functional characterization of an abiotic stress-inducible transcription factor AtERF53 in Arabidopsis thaliana;En-Jung Hsie等;Plant Mol Biol;20130630;第82卷(第3期);第223-237页 * |
| PREDICTED: Populus trichocarpa ethylene-responsive transcription factor ERF054 (LOC18108574), transcript variant X2, mRNA;无;NCBI;20221208;第1-2页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN117487849A (en) | 2024-02-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Shao et al. | Using CRISPR/Cas9 genome editing system to create MaGA20ox2 gene‐modified semi‐dwarf banana | |
| CN116751767B (en) | Application of populus euphratica PeDUB1 gene in improving drought resistance and salt resistance of plants | |
| CN108359674A (en) | Rice serine/threonine protein kitase gene OsSAPK8 coded sequences and its application | |
| CN110331145A (en) | The application of miR156 and its relevant biological material in regulation disease resistance of plant | |
| CN114854767B (en) | Trifolium pratense calmodulin-like protein TrCML6 gene and application thereof in drought resistance | |
| CN110804614B (en) | Cabbage type rape drought-resistant gene BnatZF1A, primer, expression vector and application thereof, and method for improving drought resistance | |
| CN111763251A (en) | Trifolium repens transcription factor TrNAC and coding sequence and application thereof | |
| CN117487849B (en) | Application of PdeERF gene in regulation and control of drought resistance of plants | |
| CN114214333B (en) | Gene for regulating and controlling plant leaf epidermal hair development and secondary wall thickness and application thereof | |
| CN106834345A (en) | A kind of method that polygenes superposition cotransformation improves rape synthesis resistance | |
| CN113462706B (en) | Gene for increasing tomato fruit weight and ventricular number and regulation and control method thereof | |
| CN115960916A (en) | Tea tree WRKY transcription factor gene and cold-resistant application thereof | |
| CN115873865A (en) | Application of soybean GmFAH1 gene in improving soybean drought resistance | |
| CN112501181A (en) | Rice stress resistance related gene OsTZF7 and encoding protein and application thereof | |
| CN104962563A (en) | BpMyB106 gene in Betula platyphylla and amino acid sequence and application thereof | |
| CN105132428A (en) | ZmLRT gene related to plant root system characters as well as relevant biological material and application thereof | |
| CN110205328B (en) | Plant stress resistance related gene TcAE and application thereof | |
| CN115490762B (en) | Chimonanthus praecox CpCBL8 gene, protein encoded by same and application thereof | |
| CN108948162A (en) | One cultivates peanut environment stress Gene A hDOG1L and its application | |
| CN116640799B (en) | Application of medicago sativa MtMET1 gene in regulation and control of plant stress tolerance | |
| Salsinha et al. | Anatomical characteristics of the upland local Indonesian rice cultivars as structural protection against drought | |
| CN107903312A (en) | A kind of paddy rice zinc finger protein and its encoding gene and application | |
| CN116731139B (en) | Application of PtoERF15 gene of populus tomentosa in regulation and control of drought resistance of poplar | |
| CN118064448A (en) | Application of waxy synthetic gene PpKCS in regulation and control of drought tolerance of bluegrass on grassland | |
| CN108586594B (en) | AmCBF1 transcription factor and application thereof in plant stress resistance |
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 |