CN117487849A - Application of PdeeRF53 gene in regulation and control of drought resistance of plants - Google Patents
Application of PdeeRF53 gene in regulation and control of drought resistance of plants Download PDFInfo
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- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- 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
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Abstract
The application discloses application of a PdeERF53 gene in regulating drought resistance of plants. The PdeERF53 is from populus americana, the nucleotide sequence of the PdeERF53 is shown as SEQ ID NO. 1, and the application obtains a transgenic plant with drought resistance by constructing an expression vector containing a PdeERF53 gene, transforming the expression vector into agrobacterium and infecting poplar with the agrobacterium. The application evaluates drought resistance of transgenic plants, including seedling height, ground diameter and growth phenotype of leaves of the plants, biomass accumulation of the plants, leaf gas exchange parameters and chlorophyll fluorescence parameters of the 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 of the plants, and finally results show that the tolerance of poplar to drought stress is remarkably improved by over-expression of PdeERF53 genes.
Description
Technical Field
The application relates to the technical field of plant drought resistance, in particular to application of a PdeERF53 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 confirmed 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, embodiments of the present application provide for the use of PdeERF53 genes in regulating drought resistance in plants. In some embodiments, the PdeERF53 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 PdeeRF53 gene is taken as a research object, the expression mode of the PdeeRF53 gene in different tissues of poplar is analyzed, the overexpression and inhibition expression vector is constructed, genetic transformation and drought stress tests are carried out on the poplar, the characters of the PdeeRF53 transgenic plant under drought stress, including the growth phenotype of the seedling height, ground diameter and leaf of the plant, biomass accumulation of the plant, net photosynthetic rate (Pn), transpiration rate (Tr), stomatal conductance (Gs) and intercellular CO of the plant are analyzed 2 Concentration/ambient CO 2 Concentration (Ci/Ca), instantaneous water use efficiency (R) WUEi ) The gas exchange parameters of the leaf, the maximum photochemical efficiency (Fv/Fm), the potential activity of PS II (Fv/Fo), the effective photochemical quantum yield of PS II (Fv '/Fm'), the actual photochemical efficiency (phi PS II), the fluorescence parameters of chlorophyll such as photochemical quenching (qP), non-photochemical quenching (NPQ), apparent Electron Transfer Rate (ETR), the relative chlorophyll content of the plant, the relative conductivity of the leaf of the plant, the water content of the leaf, the Malondialdehyde (MDA) content of the leaf and root system of the plant, the root system activity of the plant and the activity of the plant The content of sexual oxygen (ROS) is evaluated, so that the function of PdeERF53 gene in regulating drought resistance of poplar is evaluated, and excellent gene resources are further provided for drought resistance breeding of poplar.
In the embodiment of the application, through the observation comparison of phenotype and related physiological indexes between the PdeERF53 transgenic plant and the wild plant, the function of regulating and controlling the drought resistance of the poplar by the PdeERF53 gene is preliminarily known, and a new method is created for fine variety breeding of the poplar.
The embodiment of the application digs the function of PdeERF53 gene for regulating and controlling the drought resistance of the poplar, is beneficial to 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 PdeERF53 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 containing a pdererf 53 gene or an expression cassette containing a pdererf 53 gene for growing transgenic plants with enhanced drought resistance by over-expressing the pdererf 53 gene, wherein the nucleotide sequence of the pdererf 53 gene is shown in SEQ ID No. 1.
In a third aspect, embodiments also provide a drought-resistant plant breeding method comprising the steps of: transforming the PdeERF53 gene into a plant to obtain a transgenic positive plant; in the gene positive plants, the final drought-resistant plants are screened out according to the expression quantity of the PdeERF53 gene in leaf and root tissues and the drought resistance evaluation result.
In some embodiments, the step of transforming the PdeERF53 gene into a plant comprises: constructing an overexpression vector of the PdeeRF53 gene; 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 embodiments also provide a primer pair for amplifying the aforementioned PdeERF53 gene, comprising PdeERF53-OE-F as shown in SEQ ID NO. 2 and PdeERF53-OE-R as shown in SEQ ID NO. 3.
Drawings
Fig. 1 is a graph of absolute moisture content change of soil under drought stress provided in the examples of the present application.
FIG. 2 is a graph showing leaf area of 5 th to 7 th leaves of OE, RE and WT normally watered plants and relative water loss results of dehydration treatment thereof provided in 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 phenotypic changes before and after dehydration of OE, RE and WT plant leaves provided in the examples of the present application.
FIG. 4 is a graph showing the effect of drought stress on leaf stomata characteristics provided in the examples 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 in the examples of the present application.
FIG. 6 shows a phenotype diagram under drought stress for WT, OE and RE plants provided by examples of the present 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 is a plot of 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 as provided by the examples of this application.
FIG. 9 is a graph showing transpiration rate results for WT, OE and RE plants under drought stress as provided in the examples herein.
FIG. 10 is a stomatal conductance result of WT, OE and RE plants under drought stress as provided in the examples of the present application.
FIG. 11 is an illustration of intercellular CO of WT, OE and RE plants under drought stress provided by examples of the present application 2 Concentration/ambient CO 2 Concentration results.
FIG. 12 is a graph showing the results of instantaneous moisture utilization of WT, OE and RE plants under drought stress as provided by the examples herein.
FIG. 13 is a graph showing the results of maximum photochemical efficiency for WT, OE and RE plants under drought stress as provided by the examples herein.
FIG. 14 shows the PS II potential activity results of WT, OE and RE plants under drought stress provided by the examples of the present application.
FIG. 15 shows the result of effective photochemical quantum yield for PS II in WT, OE and RE plants under drought stress as provided by the examples herein.
FIG. 16 shows actual photochemical efficiency results for WT, OE and RE plants under drought stress as provided by examples of the present application.
FIG. 17 is a graph showing the results of photochemical quenching of WT, OE and RE plants under drought stress as provided in the examples herein.
FIG. 18 is a non-photochemical quenching result of WT, OE and RE plants under drought stress as provided in the examples herein.
FIG. 19 is a graph showing apparent electron transfer rate results for WT, OE and RE plants under drought stress as provided by the examples herein.
FIG. 20 shows the results of relative leaf chlorophyll content (SPAD values) of WT, OE and RE plants under drought stress as provided by the examples of the present application.
FIG. 21 is a graph showing relative conductivity results for WT, OE and RE plant leaves under drought stress as provided by the examples herein.
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 is a graph showing malondialdehyde content results of WT, OE and RE plant leaves and root systems under drought stress provided by examples of the present 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 result for WT, OE and RE plants under drought stress as provided by the examples herein.
FIG. 25 is an active oxygen content of WT, OE and RE plants under drought stress provided by examples of the present application; wherein (a) is the hydrogen peroxide content result and (b) is superoxide anion O 2- Content results.
FIG. 26 shows DAB staining results of OE, RE and WT plants following drought stress as provided in the examples of the present application.
FIG. 27 shows NBT staining results of OE, RE and WT plants following drought stress as provided in the examples of the present application.
FIG. 28 is a cluster analysis of drought resistance of WT, OE and RE plants as provided by examples of the present application.
Detailed Description
In order to make the objects, technical solutions and advantages of the present application more apparent, the present application will be further described in detail with reference to examples. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the present application. Reagents not specifically and individually described in this 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 of PdeeRF53 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: the PdeeRF53 over-expression vector is pKGW-RR-MGW, and the inhibition expression vector is pK7 GWIGG 2 (II).
(2) Transformation of PdeERF53 Gene into poplar
Referring to the genomic sequence of populus americana (https:// phytozome-next. Jgi. Doe. Gov/info/pdeltoideswv94_v2_1), primers were designed to amplify the full length sequence of CDS and RNAi sequence of PdeERF53 gene, for construction of overexpression vector (OE) and suppression expression vector (RE), respectively. The primer for amplifying the CDS full-length sequence of the PdeERF53 gene is PdeERF53-OE-F (SEQ ID NO: 2)/PdeERF 53-OE-R (SEQ ID NO: 3), and the primer for amplifying the RNAi sequence of the PdeERF53 gene is PdeERF53-RE-F (SEQ ID NO: 4)/PdeERF 53-RE-R (SEQ ID NO: 5).
Taking leaves of Nanlin 895 poplar as a material, extracting mRNA, cloning target gene fragments, respectively constructing an overexpression vector (OE) and an RNAi inhibition expression vector (RE) of a PdeeRF53 gene, and transforming agrobacterium EHA105, wherein 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 holo Co.);
(2) Cloning CDS full-length sequence (1392 bp, nucleotide sequence shown as SEQ ID NO: 1) of PdeERF53 gene by using cDNA as template and adopting primer PdeERF53-OE-F/PdeERF 53-OE-R; cloning RNAi interference fragment of PdeeRF53 gene (204 bp, nucleotide sequence shown as SEQ ID NO: 6) by using cDNA as template and v using primer PdeeRF53-RE-F/PdeeRF 53-RE-R;
(3) The gateway clone method is adopted respectively inBP Clonase TM II Enzyme Mix andLR Clonase TM II, cloning the full length of the PdeeRF53 gene to an overexpression vector pKGW-RR-MGW (doi: 10.1111/nph.13973) through the recombination reaction of BP and LR under the catalysis of Enzyme Mix; cloning an RNAi-interfering fragment of pdererf 53 gene into the inhibition expression vector pK7 gwwg 2 (II) (http:// www.kelei-biology.com/m/view.phpain=983);
(4) Respectively transforming the overexpression vector and the inhibition expression vector carrying the PdeERF53 gene into agrobacterium tumefaciens EHA105 by an electric shock method for infection of the Nanlin 895 poplar;
(5) Young leaves of strong and healthy Nanlin 895 poplar growing for about 30 days are selected as explants, and agrobacterium tumefaciens EHA105 carrying PdeERF53 gene overexpression and expression vector inhibition 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 tissue culture seedling for rooting culture for 30d, taking the 3 rd to 5 th leaves which are dark green and flat from top to bottom, wherein the sizes, shapes and colors of the leaves are basically consistent, cutting the edges of the leaves, and cutting the leaves into 2 to 3cm 2 Is a square of (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: callus (about 1cm in size) 2 ) Cutting off and transferring to 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 culture medium every 15d for 1 time until adventitious bud starts growing on the surface of 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: length of stem in shoot elongation MediumResistant seedlings larger than 3cm were excised and placed in rooting medium (WPM+250 mg/L Cef+50mg/L Kan), and cultivated at 24℃with 1 medium change every 30 d.
2. Identification of transgenic positive seedlings
Screening the positive seedlings of the PdeERF53 transgenes 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 expression vector plasmid for overexpression and inhibition of PdeeRF53 gene is used as positive control, the leaf DNA of wild Nanlin 895 poplar is used as negative control, and ddH is used 2 O is used as a blank control for PCR detection, and a PCR reaction system comprises 10 μl: forward and reverse primers were each 0.5. Mu.l, 2 XHieff TM PCR Master Mix enzyme 5. Mu.l, template 3. Mu.l, balance 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 ℃;
PCR procedure for amplification of Attb1/Attb2 primers: 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 ℃;
PCR procedure for PdeeRF53-F5/AtUBQ10-R5 primer amplification: 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 the PdeeRF53 gene, 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 amount of the PdeeRF53 in the leaves and root systems of transgenic positive plants is detected. Action 7 (pol.01G470900) is taken as an internal reference gene, pdeeRF53 is taken 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: pdererf 53-F1: as shown in SEQ ID NO. 17; pdererf 53-R1: as shown in SEQ ID NO. 18.
Using Hieff TM qPCRGreen Master Mix (next holy, shanghai) in Light +.>96 (Roche, switzerland) qRT-PCR reactions were completed; the qRT-PCR system was 10. Mu.L, wherein the polymerase 5. Mu.L, forward and reverse primers were each 0.5. Mu.L, template cDNA 1. Mu.L, ddH 2 O3. Mu.L. 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 adopts-2 ΔΔCT Calculated by the method from Light +.>96SW1.1 software analysis was completed, qRT-PCR procedure, preculture (1 cycle) 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 PdeERF53 gene was measured for 38 transgenic lines of PdeERF53 gene, and it was found that the expression level of PdeERF53 gene in different lines was significantly different 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 PdeeRF53 gene
Rooting tissue culture seedlings of strong WT, OE25, OE26, OE27, RE19, RE20 and RE24 with 6 weeks of growth are selected, transplanted into a nutrition pot (16 cm×14cm×12 cm), and the culture medium is nutrition soil (pH 6.0) containing N, P2% -5% 2 O 5 And K 2 O, organic matter content (dry weight) exceeds 20%. After 4 weeks of transplanting, all plants were watered 1 time per week with 1/2Hoagland nutrient solution and 1 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) DEG C, the illumination period is 16h illumination and 8h darkness every day, and 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 very 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 views were randomly selected for each slide under a 40-fold objective, the number, length (length of dumbbell-shaped guard cells) and width (widest value perpendicular to dumbbell-shaped guard cells) of the air holes in each view 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 as follows: 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, the proportion of the number of normal leaves on the plant to the total number of leaves of the whole plant is as follows: OE27 (34.74%) > OE25 (31.61%) > OE26 (28.02%) > WT (23.99%) > RE20 (9.38%) > RE19 (7.43%) > RE24 (6.45%). After 7d recovery from rehydration (DS 21), the OE plant leaves changed from yellow to green with new leaf expansion, neither WT nor RE plants had new leaf expansion and the leaves were in curled and shrunken state. 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 as follows: OE27 (39.24%) > OE26 (37.51%) > OE25 (29.31%) > WT (28.62%) > RE19 (6.38%) > RE24 (3.04%) > RE20 (2.22%). Compared with WT, the time for the OE strain to generate the victim 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 of the PdeERF53 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 the PdeERF53 gene.
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 measurement, the standard LI-COR leaf chamber and red and blue light source (6400-02 LED light source) are adopted, and the illumination intensity is set to 1500 mu mol.m -2 ·s -1 The air flow rate was 500. Mu. Mol.s -1 . The assay content mainly comprises net photosynthetic rate (P n ) Transpiration rate (T) r ) Air hole conductivity (G) s ) Intercellular CO 2 Concentration/ambient CO 2 Concentration (Ci/Ca), and corresponding saturated water pressure, leaf surface temperature, etc. Instantaneous water contentUtilization (R) WUEi ) The calculation formula of (2) is as follows: r is R WUEi ＝P n /G s 。
(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 A) is provided; 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. By 14d, each line P n The drought resistance coefficient sequencing of (2) is as follows: OE27>OE26>OE25>WT>RE19>RE20>RE24 (shown in Table 4 below). During rehydration, OE and WT P n The value is markedly increased, where P of OE26 and OE27 n Values returned to control levels, with OE25 and WT slightly below control; p of RE n The value still continues to drop during reconstitution and reaches a minimum at 21 d. At the end of the test, each strain P n The drought resistance coefficient sequencing of (2) is as follows: OE27 (104.31%)>OE26(99.86％)>OE25(75.48％)>WT(51.97％)>RE20(19.76％)>RE19(16.31％)>RE24 (7.48%). Thus, compared with WT, OE still maintains higher photosynthetic capacity under drought stress and recovery rate is fast, while RE's photosynthetic capacity is severely inhibited and recovery rate is slow.
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 significant decrease in transpiration rate (T r ；p<0.05). During the test period, T of the control plants r The values were not significantly changed, but T in drought plants r The value gradually decreases with increasing drought time. At 7d, whatT with drought plants r The values are all significantly reduced, significantly below their CK. At 14d, T r Continuous decline, OE reduced relatively less compared to WT, while RE reduced more. At this time, T of each strain r The drought resistance coefficient sequencing is as follows: OE25 >OE27>OE26>WT>RE19>RE24>RE20 (as shown in Table 4). T of OE during rehydration r The value is obviously increased, T of WT r The value rise amplitude is small, T of RE r The values continue to drop (except RE 20) and reach a minimum at 21 d. At the end of the test, T of each strain r The drought resistance coefficient sequencing is as follows: 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 inhibition of stomatal conductance by drought stress on all lines (G s ；p<0.05 Similar to the trend of Pn and Tr. During the test period, G of all control plants s The values were not significantly changed, but G in drought plants s The value gradually decreases with increasing drought time. G by 7d, all drought plants s The values are all significantly lower than their CK, with RE being the most reduced, and the OE being reduced relatively less for the WT times. At 14d, G s Continuously decline, each strain G s The drought resistance coefficient sequencing of (2) is as follows: OE25>OE27＝WT>OE26>RE19>RE24>RE20 (as shown in Table 4). G of OE and WT during rehydration s The value gradually rises, where G of OE25 and OE26 s The value was restored to the control level, OE27 was slightly below control, WT rise was small, and RE G s The value still continues to drop and reaches a minimum at 21 d. G of each line at the end of the test s The drought resistance coefficient sequencing is as follows: 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 of
FIG. 11 shows intercellular CO as WT, OE and RE plants under drought stress 2 Concentration/ambient CO 2 The concentration results, as shown, show that drought stress significantly affected the intercellular CO of all strain leaves 2 Concentration/ambient CO 2 Concentration (Ci/Ca; p)<0.05 The trend of Ci/Ca changes slightly 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 at the end of the test are ordered as follows: 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, R of all control plants WUEi The values were not significantly changed, but R was found in drought plants WUEi The value gradually increases with the increase of drought time. By 7d, R of all plants WUEi The variation amplitude is small. By 14d, R of OE compared to WT WUEi The change amplitude is larger, the change amplitude of RE is relatively smaller, and each strain R WUEi The drought resistance coefficient sequencing is as follows: OE27>OE26>OE25>WT>RE20>RE24>RE19 (as shown in Table 4). After 7d rehydration, the different strains showed different behavior, R of RE WUEi Continuously rising to reach the highest value; r of OE WUEi Greatly reduced and restored to the control level; WT R WUEi The reduction was small and did not return to the control level. Each strain R at the end of the test WUEi The drought resistance coefficient sequencing of (2) is as follows: RE24 (397.91%)>RE20(286.46％)>WT(230.36％)>RE19(212.92％)>OE26(174.28％)>OE27(174.28％)>OE25 (94.29%). Throughout the duration of the test period,the maximum rise amplitude for OE and WT occurs at 7-14 d, while the maximum rise amplitude for RE occurs 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 /F o ) Maximum photochemical quantum yield (F) v /F m ) Etc. Subsequently, the same leaf was photoactivated for 30min with activation light on, main assay: PS II efficient photochemical Quantum yield (F) v '/F m '), the actual photochemical efficiency of the photoreaction center PS II (ΦPS II), the transfer rate of photosynthetic electrons of the photoreaction center PS II (ETR), the photochemical quenching coefficient (qP), the non-photochemical quenching coefficient (NPQ), and the like. Wherein qp= (F m '-F s )/(F m '-F o ')，NPQ＝F m /F m '-1。
(1) Maximum photochemical efficiency
FIG. 13 shows the results of maximum photochemical efficiency for WT, OE and RE plants under drought stress, as shown by the significant reduction in maximum photochemical efficiency for individual lines for drought stress (F v /F m ；p<0.05). F of all control plants during the test period v /F m No significant change in the values occurred, whereas F was found in drought plants v /F m The value gradually decreases with increasing drought time. F of each line by 7d v /F m The amplitude of the variation is not obvious; f of all strains by 14d v /F m Significantly reduced, RE has significantly greater amplitude than 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). F of WT and OE during rehydration v /F m Gradually return to the control level, while F of RE v /F m The continuous decrease reaches a minimum. At the end of the test, the drought resistance coefficients of the strains are ordered as follows: 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 result of the PS II potential activity of WT, OE and RE plants under drought stress, as shown by the significant inhibition of PS II potential activity of individual lines by drought stress (F v /F o ；p<0.05 And F) v /F m The trend of change is similar. F of all control plants during the test period v /F o No significant change in the values occurred, whereas F was found in drought plants v /F o The value gradually decreases with increasing drought time. By 7d, each line F v /F o The change in (2) is 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). F of OE during rehydration v /F o The value 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; f of RE v /F o The values continued to decrease (except for RE 20). At the end of the test, the drought resistance coefficients of the strains are ordered as follows: 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 the PS II effective photochemical quantum yield for WT, OE and RE plants under drought stress, as shown by the PS II effective photochemical quantum yield for each strain (F v' /F m' ) Drought stress displayInhibition gradually decreased with increasing drought time (p<0.05 Trend of change with F v /F m Similarly. At 7d, RE is reduced by a greater amount than WT, significantly lower than its CK value; f of each line by 14d v' /F m' The sustained decrease, OE, decreased less and RE decreased more than 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 of all strains v' /F m' Gradual rise (excluding RE 24), where the rise in OE is of greater magnitude, gradually reverting 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 coefficients of the strains are ordered as follows: 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 follows: 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 coefficients of the strains are ordered as follows: 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 coefficients of the strains are ordered as follows: 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 coefficients of the strains are ordered as follows: 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 coefficients of the strains are ordered as follows: 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 of each strain RMP is ordered as: 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 coefficient of MDA content in each plant leaf is as follows: RE19 (138.28%) > RE20 (134.93%) > RE24 (134.18%) > OE27 (116.85%) > WT (112.93%) > OE26 (109.26%) > OE25 (103.01%); the drought resistance coefficient sequence of the root system is as follows: 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 plant root system activity is ordered as follows: 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
The type of Reactive Oxygen Species (ROS) commonly found in plants is mainly H 2 O 2 And O 2- The method comprises the steps of carrying out a first treatment on the surface of the Diaminobenzidine (DAB) may be H 2 O 2 Oxidation, resulting in brown precipitate; nitrotetrazolium chloride blue (NBT) capable of reacting with O 2- In combination, NBT is reduced to a blue polymer, so that the damage degree of the plant can be judged according to the dyeing condition, and further the stress resistance of the plant can be judged. At test days 0d, 7d and 14d, fresh leaves of control treated (CK) and drought treated (DS) plants of each strain were harvested and histochemically treated with DAB and NBT, respectivelyStaining, observing H in tissue 2 O 2 And O 2- Is a cumulative situation of (2); determination of H by Nanjing's kit (Nanjing's institute of biological engineering) 2 O 2 And O 2- Is contained in the composition.
FIG. 25 shows H for WT, OE and RE plants under drought stress 2 O 2 And O 2- Content results, as shown in the figure, under normal conditions, H in the plant 2 O 2 And O 2- The content is low, and the difference between plants is not obvious.
FIG. 26 shows DAB staining results for OE, RE and WT plants after drought stress; FIG. 27 shows NBT staining results for OE, RE and WT plants after drought stress, as shown, H 2 O 2 And O 2- The content of H increases with the increase of drought degree 2 O 2 The content of (C) is consistent with DAB dyeing result, O 2- The content of (C) is consistent with the NBT staining result. H of WT and OE drought plants by 7d 2 O 2 The difference between the content and the control is not obvious (except OE 25), and the H of RE drought plants 2 O 2 The content is obviously increased compared with the control, and at the moment, each strain H 2 O 2 The drought resistance coefficient sequencing of the content is as follows: RE20 (178.96%)>RE24(161.64％)>RE19(157.76％)>OE25(145.45％)>WT(134.21％)>OE26(108.66％)>OE27 (107.89%); likewise, all lines drought plants O 2- The content of (C) was slightly increased at 7d, but the difference between the control was not significant (except RE 20), at which time each strain O 2- The drought resistance coefficient sequencing of the content is as follows: RE20 (140.97%)>RE24(119.43％)>OE25(117.66％)>WT(116.74％)>RE19(111.59％)>OE26(110.13％)>OE27 (107.87%). H of drought plants of each line by 14d 2 O 2 And O 2- The content is obviously increased, but the difference of the rising amplitude of different strains is obvious, and the rising amplitude of RE strain is the largest, the rising amplitude of OE strain is the smallest after WT. At this time, each strain H 2 O 2 The drought resistance coefficient sequencing of the content is as follows: RE20 (301.18%)>RE24(290.39％)>WT(235.24％)>RE19(233.99％)>OE25(181.51％)>OE27(145.83％)>OE26 (138.93%); each strain O 2- The drought resistance coefficient sequencing of the content is as follows: RE20 (173.11%)>RE24(168.28％)>RE19(164.70％)>WT(139.98％)>OE25(132.84％)>OE26(124.06％)>OE27 (120.72%). It can be seen that, in the late drought stress stage, the H of OE plants 2 O 2 And O 2- The content is obviously lower than that of the WT and RE plants, which indicates that the ROS accumulated in the OE plants under drought stress are less and the damage of the plants 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 the over-expression of the PdeERF53 gene obviously improves the drought stress tolerance of poplar, and the inhibition of the expression of the PdeERF53 gene obviously reduces the drought resistance of poplar.
TABLE 7
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The foregoing is merely a preferred embodiment of the present application, but the scope of the present application is not limited thereto, and any changes or substitutions easily contemplated by those skilled in the art within the technical scope of the present application should be covered by the scope of the present application.
Claims (10)
- Application of PdeeRF53 gene in regulating drought resistance of plant, wherein the PdeeRF53 gene is as follows (a 1) or (a 2):(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.
- 2. The use according to claim 1, wherein the pdererf 53 gene modulates plant characteristics in drought resistance of plants comprising at least one of the following (1) to (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 of plants under drought stress 2 Concentration/ambient CO 2 Concentration, instantaneous water utilization efficiency;(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 plant species in which the pdererf 53 gene is regulated is poplar.
- 4. The application of the recombinant vector containing the PdeERF53 gene or the expression cassette containing the PdeERF53 gene is characterized in that the application is that transgenic plants with enhanced drought resistance are cultivated by over-expressing the PdeERF53 gene, wherein the nucleotide sequence of the PdeERF53 gene is shown as SEQ ID NO. 1.
- 5. A method of drought-resistant plant breeding comprising the steps of:transforming the PdeERF53 gene into a plant to obtain a transgenic positive plant;and in the gene positive plants, the final drought-resistant plants are screened out according to the expression quantity of the PdeERF53 gene in the leaves and root systems and the drought resistance evaluation result.
- 6. The breeding method according to claim 5, wherein the pdererf 53 gene is as follows (a 1) or (a 2):(a1) A nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO. 1;(a2) A nucleic acid molecule derived from populus americana and having more than 95% homology to (a 1) and associated with drought resistance in plants.
- 7. The method of breeding according to claim 5, wherein the step of transforming PdeERF53 gene into a plant comprises:Constructing an overexpression vector of the PdeeRF53 gene;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.
- 8. The method according to claim 5, wherein the drought-resistant plant is poplar.
- 9. Use of a breeding method according to any one of claims 5 to 9 for the cultivation of drought-tolerant plants.
- 10. A primer pair for amplifying PdeERF53 gene, the primer pair comprising:PdeeRF53-OE-F: the nucleotide sequence is shown as SEQ ID NO. 2;PdeeRF53-OE-R: the nucleotide sequence is shown as SEQ ID NO. 3.
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| 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 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
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| EN-JUNG HSIE等: "Functional characterization of an abiotic stress-inducible transcription factor AtERF53 in Arabidopsis thaliana", PLANT MOL BIOL, vol. 82, no. 3, 30 June 2013 (2013-06-30), pages 223 - 237 * |
| MEI-CHUN CHENG等: "Arabidopsis RGLG2, functioning as a RING E3 ligase, interacts with AtERF53 and negatively regulates the plant drought stress response", PLANT PHYSIOL, vol. 158, no. 1, 31 January 2012 (2012-01-31), pages 363 - 375 * |
| 无: "PREDICTED: Populus trichocarpa ethylene-responsive transcription factor ERF054 (LOC18108574), transcript variant X2, mRNA", NCBI, 8 December 2022 (2022-12-08), pages 1 - 2 * |
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