Key gene rcERF110 for regulating stress resistance of rosa chinensis and application thereof

Abstract

The application discloses a key gene for regulating the stress resistance of Rosa chinensis RcERF110 and application, the nucleotide sequence of which is shown as SEQ ID No. 1, the amino acid sequence is SEQ ID NO. 2, the expression mode of the gene RcERF110 is significantly induced by drought stress and high-temperature stress. Through a genetic transformation experiment, it is found that overexpression of the gene in a model plant tobacco can significantly enhance the drought resistance and high-temperature resistance of the transgenic plants. Further gene silencing experiments in Rosa chinensis prove that inhibiting the expression of the gene RcERF110 can significantly reduce the dehydration tolerance of petal tissues and weaken the heat stress resistance of the plants. Not only does the application provide a new theoretical basis for analyzing the molecular mechanism of the response of plants in the Rosaceae family to abiotic stress, but also lays an important foundation for creating drought-resistant and heat-tolerant Rosa chinensis germplasm through molecular breeding technology, and has important application value for genetic improvement of the stress resistance of horticultural plants.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of engineering technology of Rosa chinensis, and particularly relates to a key gene for regulating the stress resistance of Rosa chinensis RcERF110 and application thereof. BACKGROUND

[0002] Drought stress and high temperature stress are the two most common abiotic stresses in plant growth and development. At the physiological level, cell and tissue damage in plants leads to reduced growth and development, and plant cell membranes, osmotic adjustment substance accumulation, and photosynthetic characteristics are all affected, which severely reduces plant quality and yield, causing economic losses. Drought has a serious impact on important organs such as roots and leaves of plants. In order to cope with harsh environments, plants produce various strategies such as stomatal closure, enhanced water transport, synthesis and deposition of cuticular wax to resist drought stress. Drought and high temperature stress are related and often occur simultaneously or sequentially, which affects plant geographical distribution and reduces species diversity to some extent. Drought causes plants to lack water, which affects photosynthesis, respiration, physiological metabolism, hormone accumulation, membrane damage, and other processes, leading to inhibited plant growth and development and reduced survival rate. High temperature stress affects plants during the reproductive stage (Pang Y et al. 2024; Si F et al. 2014; Ji YZ et al. 2023; Fan C et al. 2024; Zhang C et al. 2023). At this time, plants are most sensitive to temperature, and high temperature stress can cause plants to have reduced flower buds, flower drop, and fruit sterility. For example, high temperature can reduce soybean pollen vitality, leading to reduced yield. Plant seeds are affected by temperature during germination, and high temperature can reduce seed germination rate, seedling activity, and abnormal development, and reduce the number of radicles and plumules. Under the influence of high temperature stress, plant height, tiller number, and total biomass all decrease, and morphological characteristics change, with leaf wilting and shedding, root and stem growth inhibition, and branch and fruit injury (Cao W et al. 2024; Tong N et al. 2024; Jing L et al. 2024; Poletto T et al. 2019).

[0003] Abiotic stress has inhibitory effect on the growth and development of plants, and changes the production and distribution of hormones, and the AP2 / ERF family responds to stress signals to cope with stress response. The AP2 / ERF family can also play a role in various abiotic stresses by activating some downstream functional genes that respond to stress (Shao Wenjing, 2020; Chen Yushu, 2023;). It has been reported that the dehydration response element binding protein (DREB) of the DREB family is an important transcription factor for regulating plant drought, and the overexpression of certain transcription factors in rice and Arabidopsis can improve drought and salt tolerance, and the expression of certain transcription factors can lead to the tolerance of Arabidopsis to high salt and drought (Salman S et al. 2024; Pratap B S et al. 2022; Song Yating, 2024). After plants are subjected to high temperature stress, the expression of AP2 / ERF family transcription factors is induced, which improves the high temperature tolerance of plants during seed and vegetative growth periods (Seong Im P et al. 2021). Certain transcription factors of the AP2 / ERF subfamily can be expressed under the induction of dehydration, high salt, and high temperature without ABA, and the constitutive activation form of ectopic expression can enhance the resistance of plants to drought, high salt, and heat. There are relevant reports on Arabidopsis, corn, rice, apple, and chrysanthemum (Kun L et al. 2021, Xinping D et al. 2021).

[0004] Rosa chinensis Rosa chinensis has a long cultivation history, a wide cultivation range, and rich varieties, and is widely used in landscaping engineering, but the environment is not suitable, which can lead to the reduction of the ornamental value and economic value of Rosa chinensis, and even the death of Rosa chinensis. Therefore, it is urgent to improve the drought tolerance and heat tolerance of Rosa chinensis by gene editing technology, so as to provide a practical scheme for breeding of drought-tolerant and heat-tolerant varieties of Rosa chinensis. SUMMARY

[0005] The purpose of the present application is to provide a key gene for regulating the stress resistance of Rosa chinensis RcERF110 and its application in view of the current situation of the deletion of key elements of the molecular regulation network of plant stress resistance. RcERF110 The key gene of the present application is derived from the ancient Rosa chinensis variety 'Yueyue powder' in China. (Rosa chinensis 'Old Blush') It is one of the ERF family members of Rosa chinensis. AP2 / ERF is a superfamily of transcription factors existing in plants, and the AP2 / ERF family plays an important role in plant signal transduction, growth and development, and stress response.

[0006] To achieve the above purpose, the technical scheme designed by the present application is as follows:

[0007] The present application provides a key gene for regulating the stress resistance of Rosa chinensis RcERF110 , and the nucleotide sequence thereof is shown as SEQ ID No. 1.

[0008] The application also provides a key gene as above RcERF110 The encoded protein RcERF110 The amino acid sequence of which is shown in SEQ ID No. 2.

[0009] The application also provides a primer pair for obtaining the key gene as above RcERF110 The primer pair is as follows:

[0010] RcERF110 Forward primer: 5'-GGGGTACCATGTGCCTTCTGAAGGTGGCG-3',

[0011] RcERF110 Reverse primer: 5'-:CGGGATCCTTAGCCGGAAGACGAATGATTG-3'.

[0012] The method for obtaining the key gene as above RcERF110 is as follows:

[0013] The cDNA of the leaf of Rosa chinensis 'Yueyue Fen' is used as a template and the primer pair for the key gene as above is used for PCR amplification, and the purified key gene is obtained; wherein, RcERF110 RcERF110 The PCR system is as follows:

[0014]

[0015]

[0016] The conditions of PCR are as follows:

[0017]

[0018] The application also provides a primer pair for obtaining the silencing region fragment of the key gene as above RcERF110 The primer pair is as follows: RcERF110i

[0019] RcERF110i Forward primer: 5'-GCTCTAGACAGCCACTACAACACCAAC-3',

[0020] RcERF110i Reverse primer: 5'-GGGGTACCGCCCGATGAACCGCCC-3';

[0021] The method for obtaining the silencing region fragment of the key gene as above of RcERF110 is as follows: RcERF110i The cDNA of the leaf of Rosa chinensis 'Yueyue Fen' is used as a template and the primer pair for the key gene as above is used for PCR amplification, and the purified key gene is obtained; wherein,

[0022] RcERF110 The conditions of PCR are as follows: RcERF110i ​​​​PCR amplification is carried out by using the primer pair, and the silencing region fragment is obtained by purification RcERF110i ; the PCR amplification method and system are the same as the above method and system.

[0023] The application further provides a recombinant expression vector containing the key gene RcERF110 above.

[0024] The construction method of the recombinant expression vector above comprises the following steps:

[0025] The target fragment is introduced into the cloning vector pMD ® 18T, and then the cloning vector containing the target gene RcERF110-pMD ® 18T and pCAMBIA2300s are subjected to double enzyme digestion, and then the obtained target fragment is connected to the pCAMBIA2300s vector subjected to enzyme digestion, so that the recombinant expression vector is obtained. BamHⅠ XbaI pCAMBIA2300s pCAMBIA2300s-RcERF110 .

[0026] The application further provides a recombinant silencing vector containing the silencing region fragment above. RcERF110i The plant silencing vector is pTRV2.

[0027] The construction method of the recombinant silencing vector above comprises the following steps:

[0028] The key gene RcERF110 silencing region fragment RcERF110i is introduced into the cloning vector pMD ® 18T, and then the cloning vector containing the gene silencing fragment RcERF110i-pMD ® 18T is respectively subjected to enzyme digestion, and then the obtained target fragment is connected to the pTRV2 vector subjected to enzyme digestion, so that the recombinant silencing vector is obtained. XbaⅠ KpnⅠ RcERF110i pTRV2- RcERF110i .

[0029] The application further provides a recombinant host cell, wherein the host cell is Agrobacterium GV3101, and the host cell contains the recombinant expression vector or the recombinant silencing vector above.

[0030] The application of one of the following in regulating drought resistance of plants comprises:

[0031] (1) the key gene​​​​​​RcERF110 ;

[0032] (2) the recombinant expression vector as described above;

[0033] (3) the host cell as described above.

[0034] As a preferred solution, the plant is tobacco or rose.

[0035] The application of one of the following in cultivating a plant variety resistant to drought includes:

[0036] (1) the key gene as described above RcERF110 ;

[0037] (2) the recombinant expression vector as described above;

[0038] (3) the host cell as described above.

[0039] The idea of the present application is as follows:

[0040] The present application systematically analyzes the regulation mechanism of the key gene RcERF110 on the non-biological stress tolerance of plants by integrating the forward genetics and reverse genetics strategies. RcERF110 The experimental results show that the overexpression type tobacco exhibits significantly enhanced stress resistance under drought and high temperature (45℃) stress conditions. RcERF110 The overexpression type tobacco exhibits significantly enhanced stress resistance under drought and high temperature stress conditions, while the rose petals treated by virus-induced gene silencing exhibit opposite phenotypes under dehydration and heat stress. RcERF110 The present application verifies that the key gene RcERF110 is induced by high temperature and drought, and overexpression of the gene in tobacco enhances the drought and high temperature tolerance of the transgenic plants, while silencing of the gene in rose exhibits weakened resistance to drought and high temperature stress.

[0041] (1) Based on the transcriptome data of rose, the full-length coding region sequence of the key gene RcERF110 is cloned by RT-PCR technology;

[0042] (2) Recombinant expression vector construction and genetic transformation: the open reading frame of the gene ​ is directionally connected downstream of the CaMV35S constitutive promoter to construct a plant overexpression vector ​ , and T3 generation homozygous transgenic tobacco lines are obtained by the leaf disc transformation method mediated by Agrobacterium;

[0043] (3) Virus-induced gene silencing system construction: according to the gene ​ Design specific fragments of conserved domains, construct ​ Silencing vectors, VIGS treatment of Rosa hybrida petal in vitro by using Agrobacterium-mediated transient transformation system;

[0044] (4) Function verification: molecular characterization of transgenic plants by qRT-PCR technology, combined with drought / high temperature stress phenotype analysis, physiological index determination (conductivity, MDA level, H2O2 content, etc.), systematic analysis of gene ​ The molecular mechanism of enhancing plant stress tolerance by regulating downstream target gene network. The research results clarify the key role of the gene in plant stress signal transduction, and provide key gene resources for crop stress resistance genetic improvement.

[0045] The beneficial effects of the present application are:

[0046] The gene identified in the present application ​ Has a key role in regulating plant stress resistance. By constructing a CaMV 35S promoter-based overexpression vector ​ - ​ , using Agrobacterium-mediated genetic transformation technology to obtain transgenic tobacco lines with stable inheritance, their drought stress tolerance and heat stress resistance are significantly enhanced compared with wild type control. At the same time, the ​ - ​ Vector constructed based on virus-induced gene silencing system is transformed into Rosa hybrida petal by Agrobacterium infiltration method, the interference group shows the characteristics of increased dehydration stress sensitivity and aggravated heat damage phenotype. Molecular mechanism research shows that the gene ​ By regulating the downstream stress response gene network, maintain cell osmotic balance and redox homeostasis. This research result not only reveals the key regulatory function of gene ​ In plant abiotic stress response, but also provides an important target gene for molecular design breeding of Rosaceae crops. It has important application value for precise regulation of plant stress tolerance traits by CRISPR / Cas9 and other gene editing technologies, and creation of stress-resistant high-quality varieties.

[0047] In summary: the gene ​ Is isolated and identified in the present application, and has biological function in the resistance of Rosa hybrida, and can enhance the drought resistance and heat resistance of plants, reduce the damage of plants, and improve the quality and ornamental of plants by gene editing technology; it has important practical significance for improving the drought resistance and heat resistance of plants by gene editing technology in the future. BRIEF DESCRIPTION OF DRAWINGS

[0048] ​ The original plant overexpression vector ​ Structural schematic diagram (vector size is 11630bp);

[0049] ​ As a silencing vector for primitive plants ​ Structural diagram (carrier size is 9663bp);

[0050] ​ For the different stresses of the rose 'Monthly Powder' ​ A diagram showing gene expression.

[0051] In the figure, A represents the gene in the leaves at different time points after high-temperature treatment with 'Monthly Powder'. ​ A diagram illustrating the situation;

[0052] B represents the gene concentration in leaves at different time points after 'Yueyuefen' was subjected to water stress treatment. ​ A diagram illustrating the situation.

[0053] ​ For genes ​ Spatiotemporal expression analysis diagram in the rose 'Monthly Pink';

[0054] In the diagram, A represents gene. ​ A diagram illustrating the different tissue parts of the rose 'Monthly Pink';

[0055] B represents the genes in leaves at different time points after high-temperature treatment with 'Monthly Powder'. ​ A diagram illustrating the situation.

[0056] ​ wild tobacco and ​ Graph illustrating drought resistance in genetically modified tobacco;

[0057] In the diagram, A represents wild type and ​ Phenotypic diagrams of transgenic tobacco before, after, and after rehydration in drought treatment;

[0058] B represents wild type and ​ In genetically modified tobacco ​ Image of gene expression detection results;

[0059] C represents wild type and ​ Figures showing the relative H2O2 content in leaves of genetically modified tobacco before and after drought treatment;

[0060] D represents wild type and ​ Figures showing the MDA content in leaves of genetically modified tobacco before and after drought treatment.

[0061] E represents wild type and ​ Relative electrical conductivity of leaves in genetically modified tobacco before and after drought treatment;

[0062] ​ wild tobacco and ​ Graph illustrating the heat resistance of genetically modified tobacco;

[0063] In the diagram, A represents wild type and ​ Phenotypic images of genetically modified tobacco before, after, and after high-temperature treatment;

[0064] B represents wild type and ​ Relative electrical conductivity of leaves in genetically modified tobacco before and after high-temperature treatment;

[0065] C represents wild type and ​ Figures showing the H2O2 content in leaves of genetically modified tobacco before and after high-temperature treatment;

[0066] D represents wild type and ​ Figures showing the MDA content in leaves of genetically modified tobacco before and after high-temperature treatment;

[0067] E represents wild type and ​ Relative electrical conductivity of leaves in genetically modified tobacco before and after high-temperature treatment.

[0068] ​ For the roses to fall silent ​ Analysis of the dehydration stress tolerance of petals;

[0069] In the diagram, A represents the rose control and... ​ Phenotypic images of silent petals before, after, and after dehydration and rehydration;

[0070] B represents rose comparison and ​ In the silent petals ​ Image of gene testing results;

[0071] C represents rose comparison and ​ Figures showing the H2O2 content of silent petals after dehydration stress treatment and rehydration.

[0072] D represents rose comparison and ​ Figures showing the MDA content of silent petals after dehydration stress treatment and rehydration.

[0073] E represents rose comparison and ​ Relative effective area of ​​petal discs before and after dehydration stress treatment in silent petals;

[0074] ​ For the roses to fall silent ​ Analysis diagram of petal heat resistance;

[0075] In the diagram, A represents the rose control and... ​ Phenotypic images of silent petals after treatment at normal and high temperatures;

[0076] B represents rose comparison and ​ DAB staining results of petal discs after treatment at normal and high temperatures in silent petals;

[0077] C is the control of Rosa chinensis and ​ Silencing petal in ​ Gene detection results chart;

[0078] D is the control of Rosa chinensis and ​ Silencing petal in the petal disc after normal temperature and high temperature treatment H2O2 content chart;

[0079] E is the control of Rosa chinensis and ​ Silencing petal dehydration stress treatment and rehydration petal MDA content detection chart;

[0080] F is the control of Rosa chinensis and ​ Silencing petal dehydration stress treatment before and after treatment petal disc relative conductivity detection chart.

[0081] ​ A is the expression vector ​ Construction chart (insert size is 1356bp);

[0082] ​ B is the silencing vector ​ Construction chart (insert size is 354bp). DETAILED DESCRIPTION

[0083] The present application will be further described in conjunction with specific embodiments, so that those skilled in the art can understand.

[0084] Example 1 Isolation of genes ​ and detect the gene in Rosa chinensis 'Yueyue powder' dehydration stress and high temperature treatment at different time points of gene expression in leaves

[0085] 1) Isolation of genes ​

[0086] The gene is isolated from Rosa chinensis 'Yueyue powder' ​ , the sequence is shown as SEQ ID No. 1, the encoded protein RcERF110, and the amino acid sequence is shown as SEQ ID No. 2.

[0087] The desired fragment is amplified using Rosa chinensis 'Yueyue powder' leaf cDNA as template, and the specific steps are as follows:

[0088] According to the sequence shown in SEQ ID No. 1 ​ Design ​ The primer pair of the gene is:

[0089] ​ Forward primer: 5'-GGGGTACCATGTGCCTTCTGAAGGTGGCG-3',

[0090] ​ Reverse primer: 5'-CGGGATCCTTAGCCGGAAGACGAATGATTG-3'.

[0091] And the RNA reverse transcription of cDNA as template and the above ​ Gene primer pair PCR, wherein the PCR amplification conditions are as follows:

[0092] Table 1 PCR amplification system

[0093]

[0094] Table 2 PCR amplification conditions

[0095]

[0096] Purification of the amplified product, namely the key gene ​ ;

[0097] The amplified gene ​ is connected into pMD ® 18-T vector, screening positive clones and sequencing, obtaining the full-length gene; the clone is named ​ ® 18 - ​ Plasmid.

[0098] 2) Gene ​ Expression level detection of the gene in the leaves of Rosa hybrida under water stress and high temperature treatment at different time points

[0099] Rosa hybrida water stress treatment: after the tissue culture seedlings of Rosa hybrida 'Yueyuefen' were transplanted and grew for one month, 15 healthy plants were removed from the soil and washed, then placed in clean water for 1 day, and then the plants were taken out, the water was absorbed with filter paper, and then placed on clean filter paper for water loss treatment. The upper leaf tissues of the plants were collected at 0h, 0.5h, 1h, 2h, 4h, 6h, 8h and 10h of water loss treatment.

[0100] Rosa hybrida high temperature treatment: after the tissue culture seedlings of Rosa hybrida 'Yueyuefen' were transplanted and grew for two months, 30 healthy plants were subjected to high temperature (42℃) stress treatment, and the upper leaf tissues of the plants were collected at 0h, 0.5h, 1h, 3h, 6h, 9h, 12h and 24h of high temperature treatment.

[0101] The total RNA of Rosa hybrida was extracted by Huaiyueyang Qucik plant microRNA rapid extraction kit (ZH011-50PLUS), and the reverse transcription was performed using Aikuerui reverse transcription kit (AG11705).

[0102] The expression of the gene was detected by qRT-PCR method. ​ The qRT-PCR reaction was performed by using Aigene fluorescence quantitative PCR kit (AG11718), and the instrument model was ABI QuantStudio 3 (Applied Biosystems, CA, USA). Each sample was repeated three times, the relative expression was calculated by using method, and the original data was statistically analyzed by using SPSS software.

[0103] The specific primer sequences of the qRT-PCR reaction were as follows:

[0104] qRcERF110-F: 5'-CTTATGGAGGTGGCTATGCTGATTT-3';

[0105] qRcERF110-R: 5'-GGCAGTGGTGACAGATTTCGT-3'.

[0106] The gene was selected as the internal reference gene, and the primer sequence was as follows: ​ qGAPDH-F: 5'-CTTATGGAGGTGGCTATGCTGATTT-3';

[0107] qGAPDH-R: 5'-GGCAGTGGTGACAGATTTCGT-3'.

[0108] It was found by real-time fluorescence quantitative PCR detection that the expression level of the gene in the leaves of roses appeared a peak after 3h of high temperature (42℃) stress treatment, and then gradually decreased (A) appeared a peak again at 9h, and then decreased again; in addition, the expression level of the gene was in an induced up-regulated state after drought stress treatment, and the expression of the gene appeared a peak at 6h (B).

[0109] RcERF110 In summary, the gene was induced to express by water loss stress and high temperature stress, and may be involved in the response process of water loss stress and high temperature stress of plants. Figure 3 Figure 3 Example 2 Construction of overexpression vector of gene The cloned gene fragment was overexpressed in tobacco, and the biological function of the gene was verified from the phenotype observation and physiological index determination of transgenic tobacco. The specific steps were as follows:

[0110] RcERF110

[0111] RcERF110

[0112] RcERF110

[0113] ​​​​​​​​1) Construction of overexpression vectors:

[0114] First, the positive clone pMD obtained in Example 1 was... ® 18- RcERF110 Plasmids and overexpression vector pCAMBIA2300s ( Figure 1 plasmids used BamH I and Xba I Double enzyme digestion. After digestion, use a solution containing... RcERF110 Gene restriction enzyme fragments and restriction enzymes pCAMBIA2300s The vector was used for ligation reaction and transformed into *E. coli* DH5α. Positive clones were screened by PCR amplification to obtain the transformation vector, which was named... pCAMBIA2300s-RcERF110 ( Figure 9 A).

[0115] 2) Genetic transformation of tobacco

[0116] This embodiment uses Agrobacterium-mediated method to treat tobacco ( Nicotiana tabacum L. Genetic transformation is performed to convert the recombinant plasmid into a genetically modified plasmid. pCAMBIA2300s - RcERF110 After transforming Agrobacterium GV3101, tobacco leaf explants were selected for genetic transformation. After Agrobacterium infection (OD600 = 0.8–1.0) and co-culture (25 ± 1℃, dark culture for 48 h), the explants were transferred to MS selection medium containing 100 mg / L kanamycin and 400 mg / L cefotaxime for resistant shoot induction. Through multiple rounds of selective culture (subcultured every 21 days), kanamycin-resistant adventitious shoots were obtained. After obtaining complete plants through in vitro rooting culture, the tissue culture seedlings underwent gradual acclimatization treatment:

[0117] Seedlings were first hardened off in an artificial climate chamber for 7 days, then transplanted into a sterile substrate for cultivation. RT-qPCR was used to analyze the T0 generation plants. RcERF110 Gene expression analysis was conducted to screen for positive transformation lines with significantly elevated transcription levels (p<0.01), and the transformation was successfully constructed. RcERF110 Overexpression transgenic tobacco lines ( RcERF110 - OE ).

[0118] Example 3 Gene RcERF110 The construction and transformation of silent carriers

[0119] Silencing genes in rose petals using VIGS technology RcERF110 The potential role of this gene in rose petal dehydration and high-temperature stress was determined using phenotypic observation and physiological index measurement. The specific steps were:

[0120] 1) Construction of silent carriers:

[0121] First, the gene obtained in Example 1 RcERF110 Using the full-length CDS fragment as a template, gene silencing fragments were utilized. RcERF110i Primer pairs are used for PCR amplification to obtain the desired gene silencing fragment.

[0122] RcERF110i Forward primer: 5'-GCTCTAGACAGCCACTACAACACCAAC-3',

[0123] RcERF110i Reverse primer: 5'-GGGGTACCGCCCGATGAACCGCCC-3';

[0124] The PCR amplification system and amplification conditions are the same as those in Example 1 above. RcERF110 Amplification system and conditions for the full-length CDS fragment.

[0125] Genes RcERF110 Silent fragments RcERF110i Importing the cloning vector pMD ® In 18T, then containing RcERF110i Cloning vector RcERF110i-pMD ® 18T and pTRV2 ( Figure 2 At the same time Xba I and Kpn I Perform double enzyme digestion. After digestion, the resulting gene-silenced fragment... RcERF110i The pTRV2 vector, digested with enzymes, was ligated and transformed into *E. coli* DH5α. Positive clones were detected by PCR to obtain the transformation vector, which was named... pTRV2-RcERF110i ( Figure 9 B).

[0126] 2) Instant transformation of rose petals:

[0127] Using Agrobacterium-mediated transient genetic transformation of rose petals, pTRV2- RcERF110 i The gene was introduced into rose petals, and after infection and co-culture, it was obtained. RcERF110 Silent rose petals.

[0128] The main steps of the instantaneous transformation of rose petals described above are as follows:

[0129] Virus-induced gene silencing (VIGS) technology was used to analyze the gene function of rose petals. The specific procedure was as follows: The petals carrying pTRV1, pTRV2, and... pTRV2-RcERF110s Agrobacterium strains containing the recombinant vector were cultured at 28°C in LB liquid medium (containing 50 mg / L rifampin and 100 mg / L kanamycin) with shaking until OD.600 ≥1.8. Collect bacterial cells by centrifugation at 5000×g for 10 min, resuspend in induction medium containing 10 mM MgCl2, 200 mM acetosyringone, and 10 mM MES buffer (pH 5.6), and record the OD values ​​of each bacterial culture. 600 Corrected to 0.9 ± 0.05. Then pTRV1 was compared with pTRV2 and pTRV2 respectively. pTRV2 - RcERF110s The bacterial suspensions were mixed at a 1:1 volume ratio and left to stand at room temperature (25±1℃) in the dark for 4 hours to activate the bacterial type III secretion system.

[0130] The middle petals of roses in their early blooming stage were selected, and 1 cm diameter petal discs were prepared using a sterile punch. The petal discs were thoroughly saturated with bacterial suspension through vacuum permeation (0.08 MPa, 10 min). After removing residual liquid from the surface with sterile filter paper, the samples were placed in petri dishes containing sterile distilled water. Low-temperature induction was first performed: incubation at 8℃ in the dark for 48 h to promote Agrobacterium T-DNA transfer; then, the samples were transferred to a 24℃ artificial climate chamber for gene silencing induction, with a photoperiod of 16 h / 8 h (day / night), and continued incubation for 24 h. Throughout the experiment, a blank vector control group (pTRV1+pTRV2) and an experimental group (pTRV1+...) were included. pTRV2 - RcERF110s Parallel processing is performed.

[0131] Example 4: Genetically Modified Tobacco RcERF110 Drought and heat resistance testing

[0132] 1) Drought resistance testing of genetically modified tobacco

[0133] Wild-type seedlings (WT) at 7 weeks of age and those obtained in Example 2 RcERF110 Overexpression type (OE) tobacco plants ( RcERF110 - OE Tobacco plants were used as experimental materials for a soil drought stress experiment. Before the experiment, the plants were saturated with irrigation, followed by a 25-day drought treatment (growing chamber conditions: day / night temperature 25℃ / 20℃, photoperiod 16h / 8h, relative humidity 45%±5%). Phenotypic characteristics were observed and physiological indicators were measured before and after the drought treatment, followed by 30 hours of rehydration. Cell membrane permeability was measured using the conductivity method, and malondialdehyde (MDA) content was determined using the thiobarbituric acid (TBA) colorimetric method. The methods for measuring conductivity and MDA content were referenced in Li Hesheng's (2000) "Principles and Techniques of Plant Physiological and Biochemical Experiments." H2O2 content was measured using a Nanjing Jiancheng H2O2 test kit (item number A064-1-1). Soil drought treatment was applied to the tobacco plants.

[0134] Results showed that: compared with RcERF110 transgenic tobacco ( RcERF110 - OE ), the control group tobacco leaf after drought treatment wilting, rehydration leaf more serious yellowing injury ( Figure 5 A); after treatment control group leaf H2O2 and MDA content increased ( Figure 5 C, D), relative conductivity increased significantly ( Figure 5 E), indicating that overexpression RcERF110 gene transgenic tobacco ( RcERF110 - OE ) to reduce the extent of injury, drought tolerance enhanced.

[0135] 2) transgenic tobacco heat tolerance detection

[0136] This example with 7 weeks of wild type (WT) and RcERF110 overexpression type tobacco plants ( RcERF110 - OE ) as experimental material, in the artificial climate chamber to carry out high temperature stress tolerance analysis. The plants were placed in high temperature treatment group (45℃±0.5℃ continuous stress, relative humidity 60%±5%, photoperiod 16 h / 8 h) for 84 h. Respectively, before treatment (0 h) and after treatment (84 h) to collect the fourth fully expanded leaf, observation and treatment before and after the conductivity, H2O2 and MDA content determination, then normal temperature (25℃) recovery 1 day after taking pictures record plant state.

[0137] As Figure 6 shown: after 84 h of high temperature stress, WT plants showed significant phenotype damage: normal temperature recovery 30 h after a large area of yellowing phenomenon ( Figure 6 A). Physiological index analysis showed that, after treatment WT plant leaf H2O2 content, MDA content and relative conductivity were significantly increased ( Figure 6 C-E). This result shows that the overexpression of the gene RcERF110 significantly inhibited the high temperature induced reactive oxygen species burst and membrane lipid peroxidation damage, prove that the gene through enhancing the cell redox homeostasis and membrane system stability, improve the tobacco to the continuous high temperature stress tolerance. All experiments were set three independent biological repeats, data using one-way ANOVA analysis (ANOVA) for statistical verification.

[0138] Example 5 transient silencing of petunia flower petal RcERF110 dehydration tolerance and heat tolerance detection

[0139] 1) petunia gene silencing disc dehydration tolerance detection

[0140] The gene obtained in Example 3 above. RcERF110 Silent rose petal discs were placed on filter paper and dehydrated for 8 hours, then rehydrated by immersion in distilled water. Photos were taken at 0 hours, 8 hours, 3 hours, and 6 hours after dehydration. The relative effective area of ​​the petal discs was calculated, and the H2O2 and MDA content of the petals was measured at 0 hours and 8 hours after dehydration.

[0141] pass VIGS Technology silences rose genes RcERF110 The study found that the flower petals of the gene-silenced type exhibited a more severe wilting phenotype after 8 hours of dehydration treatment than the control, and their recovery ability was significantly weakened after 3 hours of rehydration. Figure 7 A). Compared with the control, the accumulation of H2O2 and MDA in the petals of the silent group was significantly increased ( Figure 7 (C, D), while the relative effective area of ​​the petal disc decreased compared to the control group ( Figure 7 E). These results indicate that genes RcERF110 The silencing of this gene weakens the dehydration tolerance of the petals, suggesting that the gene may positively participate in the adaptive response to dehydration stress in roses by regulating the antioxidant defense system and membrane integrity.

[0142] 2) Detection of heat resistance of rose gene-silenced discs

[0143] Similarly, the genes obtained in Example 3 above... RcERF110 Silent rose petal discs were subjected to high-temperature stress treatment at 42℃ for 50 h before being photographed. The rose discs were then subjected to DAB staining and hydrogen peroxide content detection (DAB staining method referenced Luo et al., 2016, and H2O2 content detection was performed using the Nanjing Jiancheng test kit (A007-1-1).

[0144] The experimental results showed that the petals in the experimental group exhibited a significant yellowing phenotype. Figure 8 A), while the phenotypic damage in the control group was relatively mild. DAB staining showed that the silencing group had a deeper degree of H2O2 accumulation than the control group (A), while the control group had a relatively milder degree of phenotypic damage. Figure 8 B), whose H2O2 and MDA content and relative conductivity were significantly higher than those of the control group ( Figure 8 DF). Gene. RcERF110 Silence exacerbates oxidative damage and membrane system disruption under high-temperature stress, weakening the heat resistance of rose petals. In conclusion, silence in roses... RcERF110 Genes can reduce the heat resistance of rose petals.

[0145] All other parts not described in detail are existing technologies. Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Use of one of the following in enhancing drought or heat tolerance in plants, characterized in that: It comprises: (1) Key genes RcERF110 the nucleotide sequence of which is represented by SEQ ID No. 1 ; (2) a recombinant expression vector containing the above-mentioned key gene RcERF110 a plant expression vector pCAMBIA2300s; (3) a recombinant host cell, which contains the above-mentioned recombinant expression vector, and the host cell is Agrobacterium GV3101, Wherein, the plant is tobacco.

2. Use of one of the following in the breeding of plant varieties which are resistant to drought and heat, characterized in that: It comprises: (1) Key genes RcERF110 the nucleotide sequence of which is represented by SEQ ID No. 1 ; (2) a recombinant expression vector containing the above-mentioned key gene RcERF110 a plant expression vector pCAMBIA2300s; (3) a recombinant host cell, which contains the above-mentioned recombinant expression vector, and the host cell is Agrobacterium GV3101, Wherein, the plant is tobacco.

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