CN113493802A

CN113493802A - Application of chrysanthemum zinc finger protein BBX19 and related factors thereof in adjusting drought stress tolerance

Info

Publication number: CN113493802A
Application number: CN202010266514.7A
Authority: CN
Inventors: 洪波; 高俊平; 徐彦杰; 赵鑫; 帕丽努尔·艾外力
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2020-04-07
Filing date: 2020-04-07
Publication date: 2021-10-12
Anticipated expiration: 2040-04-07
Also published as: CN113493802B

Abstract

The invention provides application of chrysanthemum zinc finger protein BBX19 and related factors thereof in adjusting drought stress tolerance. In particular, the present invention provides a method for modulating drought stress tolerance in plants using CmBBX19 and/or CmBBX 19-related factors by modulating expression of CmBBX19 and/or its related factors. The invention also provides application of CmBBX19 and/or related factors thereof in improving the survival rate of plants in drought stress, reducing the transpiration rate, stomatal conductance, water loss and photosynthetic rate of the plants, breeding drought tolerance of the plants, regulating ABA-dependent pathway gene expression of the plants and the like. The invention defines the function of CmBBX19 and/or related factors thereof, so the application can be realized by regulating the expression of CmBBX19 and/or related factors, protein interaction between CmBBX19 and CmABF3 and the like.

Description

Application of chrysanthemum zinc finger protein BBX19 and related factors thereof in adjusting drought stress tolerance

Technical Field

The invention relates to the field of horticultural cultivation and production of chrysanthemum and regulation of chrysanthemum against abiotic stress, in particular to application of chrysanthemum zinc finger protein BBX19 and related factors thereof in regulation of drought stress tolerance.

Background

Drought stress is a global phenomenon that severely limits crop production and distribution. Plants have evolved complex morphological, physiological, cellular and molecular level systems in order to cope with drought stress. Among them, the plant hormone abscisic acid (ABA), which plays a key role in several aspects of plant growth and development and in adaptation to environmental stresses. Two independent but closely linked pathways mediating drought stress responses have been identified, the ABA-dependent and ABA-independent drought pathways.

The ABA signaling pathway includes three basic core components: 1) ABA receptor PYR/PYL/RCAR protein; 2) PP2C protein; 3) an SnRK protein. In the presence of ABA, PYR1/PYL/RCAR protein is combined, the activity of PP2C protein is inhibited, and the inhibition on SnRK2 is relieved. These activated SnRK2 proteins then phosphorylate downstream transcription factors, further triggering expression of ABA-responsive genes such as RAB18, RD29B and ADH 1.

Among the transcription factors phosphorylated by SnRK2 protein in the process of Arabidopsis ABA signal transduction, ABF2/AREB1, ABF3 and ABF4/AREB2 are key transcription factors involved in drought stress reaction. These transcription factors activate the expression of their downstream target genes by binding to ABA-responsive elements (ABRE, PyACGTGG/TG) in the promoter. ABF/AREB proteins have been reported in the literature to be bZIP transcription factors that can form heterodimers or homodimers in the nucleus. However, reports on the mechanisms by which they may interact with other transcription factors or be associated with abiotic stress tolerance are still very limited.

Another class of proteins that play a role in abiotic stress tolerance are members of the B-box (BBX) family, a subfamily of zinc finger proteins with one or two B-box domains at the N-terminus. The B-box domain is important for transcriptional regulation and protein-protein interactions, and BBX family members can be divided into five subfamilies according to the number of B-box and CCT domains. The BBX protein also plays a role in light-regulated development processes, such as seed germination, seedling photomorphogenesis, shade avoidance, and flowering photoperiod regulation. BBX18 inhibited the thermotolerance of arabidopsis, whereas BBX24 induced salt tolerance. In arabidopsis thaliana, heterologous overexpression of the BBX family member CmBBX22 in Chrysanthemum (Chrysanthemum morifolium) was demonstrated to improve drought tolerance and delay leaf senescence, and we previously also found that CmBBX24 has a dual role in regulating abiotic stress and flowering phase.

Several articles have reported that BBX proteins affect abiotic stress response mechanisms. In Arabidopsis BBX18 regulates the expression of a set of heat shock response genes, while in Chrysanthemum CmBBX24 regulates Gibberellin (GA) biosynthesis. Furthermore, when CmBBX22 is overexpressed in arabidopsis, it plays a role in drought resistance by transcriptionally activating downstream genes of the ABA signaling pathway, such as ABI3 and ABI 5. Another example of a relationship that has been shown in studies to indicate between BBX protein and ABA signaling elements is that BBX21 acts as a repressor during ABA-controlled seed germination through physical interaction with ABI5 and binding to the ABI5 promoter. However, in response to abiotic stress, particularly drought stress, it is currently unclear whether there is an interaction between the BBX protein and ABA signaling components.

Chrysanthemum is an important ornamental plant in the world. Drought, a major abiotic stress, limits the planting area and yield of chrysanthemum. Therefore, it is urgently needed to understand the molecular regulation mechanism of drought stress tolerance to breed chrysanthemum varieties with drought stress tolerance and drought tolerance.

Disclosure of Invention

In order to solve one or more of the above-mentioned problems in the prior art, the present inventors have long and intensively studied to find that CmBBX19, a chrysanthemum BBX family gene, is down-regulated after drought stress or ABA treatment, and regulates drought tolerance mainly by interacting with CmABF3, a major transcription factor in an ABA-dependent pathway, thereby completing the present invention.

The present application provides in a first aspect a method for modulating drought stress tolerance in a plant using a CmBBX19 and/or CmBBX 19-related factor, characterized in that said method is performed by modulating expression of a CmBBX19 and/or CmBBX 19-related factor, which is a related factor in an ABA-dependent pathway modulated by CmBBX 19.

Optionally, the modulating the plant drought stress tolerance is decreasing the plant drought stress tolerance and is performed by overexpressing the CmBBX19 and/or decreasing the expression of the CmBBX 19-related factor.

Preferably, the modulating of plant drought stress tolerance is increasing of plant drought stress tolerance and is performed by decreasing expression of CmBBX19 and/or increasing expression of the CmBBX 19-related factor.

It is further preferred that the modulation is a transcriptional level modulation and/or a translational level modulation.

It is further preferred that the CmBBX 19-related factor comprises a CmBBX19 interaction factor or a factor downstream of the interaction factor. Preferably, the CmBBX19 interaction factor is ABF3, especially CmABF 3; and/or the downstream factor is selected from the group consisting of RAB18, RD29B, ERD7 and LTI65, in particular from the group consisting of CmRAB18, CmRD29B, CmERD7 and CmLTI 65.

Further preferably, the method is performed by at least one of:

(1) knocking out or knocking down the CmBBX19 gene or overexpressing the CmBBX 19-related factor gene;

(2) RNA interference with the expression of the CmBBX19 gene or overexpression of the CmBBX 19-related factor gene;

(3) blocking protein interaction of CmBBX19 and CmABF 3;

wherein the CmBBX19 related gene comprises a CmBBX19 interacting factor gene or a downstream factor gene of the interacting factor.

Preferably, the CmBBX19 interacting factor gene is ABF3, more preferably CmABF 3; and/or the downstream factor gene is selected from the group consisting of RAB18, RD29B, ERD7 and LTI65, more preferably from the group consisting of CmRAB18, CmRD29B, CmERD7 and CmLTI 65.

Further preferably, the plant is a feverfew, preferably a Chrysanthemum, more preferably a ground-cover Chrysanthemum, and even more preferably a ground-cover Chrysanthemum variety (Chrysanthemum morifolium, cv.

The invention provides in a second aspect the use of the CmBBX19 gene and/or CmBBX19 related factor in: (1) restoring continued growth of top shoots of the plant after drought stress; (2) increasing the survival rate of plants under drought stress; (3) simultaneously, the transpiration rate, the stomatal conductance, the water loss and the photosynthetic rate of the plants are reduced; wherein the CmBBX 19-related factor comprises a CmBBX19 interaction factor or a factor downstream of the interaction factor; preferably, the CmBBX19 interaction factor is ABF3, especially CmABF 3; and/or the downstream factor is selected from the group consisting of RAB18, RD29B, ERD7 and LTI65, in particular from the group consisting of CmRAB18, CmRD29B, CmERD7 and CmLTI65 preferably said application is achieved by a method according to the first aspect of the invention.

In a third aspect, the invention provides a method for drought tolerant breeding of a plant, which is characterized in that the method is carried out by at least one of the following modes: (1) (ii) reduces expression of CmBBX 19; (2) increasing expression of a CmBBX 19-related factor in an ABA-dependent pathway, wherein the CmBBX 19-related factor comprises a CmBBX19 interacting factor or a factor downstream of the interacting factor; preferably, the CmBBX19 interaction factor is ABF3, especially CmABF 3; and/or the downstream factor is selected from the group consisting of RAB18, RD29B, ERD7 and LTI65, in particular from the group consisting of CmRAB18, CmRD29B, CmERD7 and CmLTI 65; (3) blocking the protein interaction of CmBBX19 with CmABF 3.

In a fourth aspect, the invention provides a method for modulating expression of a factor associated with a plant ABA-dependent pathway by modulating expression of the factor CmBBX 19; the ABA dependent pathway related factor is an ABA signal pathway related factor. Preferably, the ABA signal path correlation factor is a CmBBX19 correlation factor. More preferably, the CmBBX 19-related factor comprises a CmBBX19 interaction factor or a factor downstream of the interaction factor. It is further preferred that the CmBBX19 interaction factor is ABF3, in particular CmABF 3; and/or the downstream factor is selected from the group consisting of RAB18, RD29B, ERD7 and LTI65, in particular from the group consisting of CmRAB18, CmRD29B, CmERD7 and CmLTI 65.

Compared with the prior art, the invention has the following technical effects:

(1) the function and the effect of the CmBBX19 gene in the drought stress tolerance of the plant are determined, so that the expression of the CmBBX19 gene in the plant is regulated to regulate the drought stress tolerance of the plant.

(2) The role of CmBBX19 in an ABA dependent pathway is clarified, so that the expression of CmBBX19 can be regulated through ABA treatment, or further the expression of genes positioned at the downstream of the CmBBX19 interaction factor in the ABA pathway can be regulated, and the regulation of the functions of the CmBBX19 interaction factor and the downstream genes in the ABA pathway can be realized, so that the plant drought stress tolerance can be weakened or promoted.

(3) The effect of protein interaction between CmBBX19 and CmABF3 on plant drought stress tolerance is well defined and thus can be affected by enhancing or blocking protein interaction between them.

Drawings

FIG. 1 shows the expression of CmBBX19 in chrysanthemum. a. Transcription levels of CmBBX19 in different chrysanthemum organs based on real-time Quantitative (QRT) -PCR analysis. L, leaf; f, flower; s, stem and R, root. Sampling time was the level of transcript in drought-treated chrysanthemum leaves starting to receive light for 3 hours (ZT3) b. The relative water content of the soil at the time of sampling was 75% (well watered), 55% (mild drought), 25% (moderate drought), 10% (severe drought) and 75% (re watered for 1 day) respectively, c. the transcription level of CmBBX19 of chrysanthemum after 100. mu.M ABA treatment. Samples were harvested 1 hour after ABA treatment. Three independent experimental replicates were performed, with the reference gene CmUBI and error bars indicating Standard Deviation (SD). The differences were statistically significant (P <0.05) by Duncan's multiple range test.

Figure 2 is subcellular localization and transactivation of CmBBX 19. a. Subcellular localization of the CmBBX19-GFP fusion protein in chrysanthemum protoplasts was observed by confocal laser scanning microscopy, GFP being a green fluorescent protein. The left panel is a dark field showing green fluorescence, the middle panel is a bright field showing cell morphology, and the right panel is a merged image showing combinations. The scale bar is 10 μm. b. Analysis of the transactivation activity of the CmBBX19 protein in tobacco leaves. A plasmid combination of the dual LUC/REN reporter gene and an effector consisting of PBD-CmBBX19-VP16, PBD-VP16 or PBD was co-transformed into tobacco leaf lamina. LUC and REN activity was measured 3 days after infiltration. When the photographs show the firefly luciferase fluorescent signal, the corresponding effector is transferred into tobacco leaves. Three independent experimental replicates were performed and error bars indicate standard deviation.

FIG. 3 is tolerance to drought stress of CmBBX19 overexpressing (CmBBX19-OX) or CmBBX19 silencing (CmBBX19-RNAi) chrysanthemum transgenic plants. a. Expression of CmBBX19 was detected in Wild Type (WT) and transgenic plants by quantitative real-time (QRT) -PCR at sampling time ZT 3. . b. Comparison of phenotype of CmBBX19-OX or CmBBX19-RNAi plants with WT plants under drought stress conditions. . c. Survival of CmBBX19-OX, CmBBX19-RNAi and WT plants grown under drought stress conditions. Water loss rate of leaf blade of cmbbx19 transgenic line and WT plants. e-g.CmBBX19 transgenic lines and WT plants have transpiration rate (c), stomatal conductance (d) and photosynthetic rate (e) under normal growth and drought stress conditions.

FIG. 4 is the expression of abscisic acid (ABA) -dependent or independent pathway-related genes in CmBBX19-OX and CmBBX19-RNAi chrysanthemum plants. Real-time Quantitative (QRT) -PCR analysis was performed to assess the expression of each gene. CmUBI is used as an internal reference gene. Three independent experimental replicates were performed and error bars indicate standard deviation. According to the Duncan's multiple range test, the letters indicate significant differences (P < 0.05).

Figure 5 is the interaction of CmBBX19 with abscisic acid (ABA) signal component CmABF3 using yeast two-hybrid assays and bimolecular fluorescence complementation (BiFC). a. Yeast two-hybrid screen for CmBBX19 and CmABFs proteins. Yeast cells containing bait BD-CmBBX19 and AD-ABFs were obtained by yeast transformation. OD600 was adjusted to 0.1 with physiological saline and observed on non-selective medium lacking Leu and Trp (left panel) and selective medium lacking Leu, Trp, His and Ade (right panel). Negative controls contained empty AD or BD vectors. Schematic representation of domain analysis in CmABF3 and truncated CmABF 3. c. Yeast two-hybrid assay CmBBX19 and truncated CmABF3The interaction between them. Interaction of CmBBX19 and CmABF3 in bifc assay. Tobacco leaf and CmBBX19-YFP^NAnd CmABF3-YFP^CConstructs were co-infected and observed by confocal microscopy 3 days after infection. CmBBX19-YFP^N+YFP^CAnd YFP^N+CmABF3-YFP^CAs a negative control. The scale bar is 40 μm.

Figure 6 is the CmABF3 independent of CmBBX19 and CmRAB18 promoter binding. a.445bp CmRAB18 promoter. Triangles correspond to the hypothesized Abre motif, while rectangles correspond to the hypothesized GT1 consensus motif. The line below the promoter represents the fragment (-388/-339) used in the electrophoretic mobility modification assay (EMSA). b. The binding of CmABF3 and CmBBX19 to the CmRAB18 promoter was analyzed in a yeast single hybrid system. Empty vector (AD) was used as negative control. c. The binding of CmABF3 to the CmRAB18 promoter was analyzed using EMSA. Purified CmABF3 protein (3 μ g) was incubated with 50nm biotin-labeled probe. For competition detection, 100, 1000, 3000 or 5000 times the concentration of cold probe was added to the above experiment.

Figure 7 is the interaction of CmBBX19 with CmABF3 thereby inhibiting transcription of CmRAB 18. a. Electrophoretic mobility-based experimental analysis (EMSA) binding of CmABF3 and CmBBX19 to the CmRAB18 promoter. The purified protein (3. mu.g) was incubated with a 50nm biotin-labeled probe. For competition assays, purified CmBBX19 protein was added to the above experiments at 1 or 10 fold concentrations. b. Schematic representation of the dual reporter plasmid and the effector plasmid used in the dual luciferase reporter assay. c.CmABF3 or CmBBX19 interaction with CmRAB18 promoter as shown in the dual Luciferase (LUC) reporter system. A445 bp CmRAB18 promoter fragment was used. The LUC vector contains the Renilla luciferase (REN) gene, driven by the 35S promoter as a positive control. The samples were infected into tobacco leaf discs and the LUC and REN activities were measured 3 days after infection. The photographs show the firefly luciferase fluorescent signal when the corresponding effector and reporter are transferred into tobacco leaf lamina. Three independent experimental replicates were performed and error bars indicate standard deviation.

FIG. 8 shows drought stress tolerance of WT and CmBBX19-RNAi plants after CaLCuV-amiR-ABF3 infection. Expression of CmABF3 in WT and CmBBX19-RNAi plants after CaLCuV-amiR-ABF3 infection. b.20 days drought phenotype of CmABF 3-silenced WT and CmBBX19-RNAi plants under drought stress. CaLCuV-amiR-ABF3 infected WT and CmBBX19-RNAi plants at transpiration rate (c), stomatal conductance (d) and photosynthetic rate (e) under normal growth and drought stress.

Figure 9 is an amino acid sequence analysis of CmBBX 19.

FIG. 10 is a comparison of CmBBX19 expression in CmBBX19-RNAi and wild-type (WT) chrysanthemum with other members of BBX family subgroup IV.

FIG. 11 shows ABA content in leaves of CmBBX19 transgenic and WT plants

FIG. 12 shows the expression of abscisic acid (ABA) signaling pathway and Gibberellin (GA) biosynthesis-associated genes in CmBBX19-OX or CmBBX19-RNAi chrysanthemum plants.

FIG. 13 is the distribution of ABRE and G-box motifs in the gene promoter of the up-regulated LEA protein in CmBBX19-RNAi plants.

Figure 14 is a CmABF sequence analysis.

FIG. 15 is an analysis of the interaction of BBX19 with ABF in Arabidopsis thaliana.

FIG. 16 shows ABA-dependent pathway gene expression in WT and CmBBX19-RNAi plants after CaLCuV-amiR-ABF3 infection.

Figure 17 schematic representation of CmBBX19 involvement in drought stress responses, mainly through the abscisic acid (ABA) dependent pathway.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.

Examples

The invention will be further illustrated by the following examples, but it will be understood that the scope of the invention is not limited to these examples.

Materials and methods

Plant material and treatment

The Chrysanthemum used in this study was a ground cover Chrysanthemum variety (Chrysanthemum morifolium, cv. Fall Color). Transplanting 40-day-old tissue culture seedlings to a 9-cm-diameter tissue culture seedling containing 1: 1(v/v) peat: the vermiculite mixture is potted and grown in a culture room with the controlled temperature, the temperature is 23 +/-1 ℃, the relative humidity is 40 percent, the illumination of a fluorescent lamp is 100 mu mol m < -2 > s < -1 >, and the photoperiod is 16h illumination/8 h darkness.

Chrysanthemum plants were analysed for expression of CmBBX19 in different organs after 6 months of growth under long-day (16h light/8 h dark) conditions.

Plants of 60-day age were grown in specific environments (23 + -1 deg.C, 40% relative humidity, 16h light/8 h dark) for various degrees of drought stress treatment. Plants were given enough water at the beginning of the drought stress treatment and weighed (initial weight) and then weighed periodically throughout the treatment period. The relative water content of the soil was calculated as (final fresh weight-dry weight)/(initial weight-dry weight) × 100%.

For ABA treatment, roots of 40-day-old tissue culture seedlings were soaked in 100 μ M ABA solution. The corresponding aqueous solution was used as a control.

RNA extraction and qRT-PCR

Total RNA was extracted from the sample using TRIzol reagent (Takara) and first strand cDNA was synthesized according to the reverse transcriptase application instructions (Vazyme). The QRT-PCR reaction was performed using the ABI StepOnePelus Real-time PCR system (Applied biosystems, Foster City, USA). The CmUBI gene (GenBank accession No. EU862325) was used as an internal control. By using comparisons^ΔΔThe Ct method (Livak and Schmittgen, 2001) normalizes the copy number of the target gene to quantify the expression with reference to the CmUBI gene. In addition, the cDNA shown as the template is used, and the primer pair shown as SEQ ID NO.42 and 43 is used for cloning the CmBBX19 gene shown as SEQ ID NO. 44; cloning CmABF3 gene shown as SEQ ID NO.47 by using primer pairs shown as SEQ ID NO.45 and 46; the CmRAB18 gene shown in SEQ ID NO.50 was cloned with the primer pair shown in SEQ ID NO.48 and 49.

Subcellular localization

The ORF sequence of CmBBX19 without a stop codon was cloned into the Super promoter driven pSuper1300(GFP-C) vector. Mesophyll protoplasts were obtained from 40-day-old chrysanthemum according to the method of Yoo et al (2007), and 10. mu.g of pSuper prepared using the OMEGA plasmid Maxi Kit: the CmBBX19-GFP plasmid was transferred to approximately 2X 10 using polyethylene glycol (PEG) as described by Higuchi et al (2013)⁴In protoplasts. The converted mesophyll protoplasts are cultured for 16-20 h at 22 ℃, and then observed by an OLYMPUS FV1000 confocal laser scanning microscope. Confocal microscopy GFP images were obtained with 488nm excitation and 525nm emission.

Genetic transformation of Chrysanthemum

To construct the overexpression vector, the ORF region of CmBBX19 was amplified and cloned into the XbaI and SacI sites of the pBI121 vector (Chen et al, 2003). In constructing the RNAi vector, 319bp sense and antisense fragments of CmBBX19 containing XhoI/ClaI and XbaI/KpnI sites were amplified and cloned on both sides of the Pdk intron of pHANNIBAL vector to form an intron containing ` hairpin ` RNA (ihpRNA). The promoter and terminator of ihpRNA were cloned into the pART27 vector (Wesley et al, 2001). Respectively introducing the overexpression plasmid and the RNAi plasmid into an Agrobacterium EHA105 strain, and transforming the overexpression plasmid and the RNAi plasmid into chrysanthemum through Agrobacterium mediation (Hong et al, 2006) to obtain CmBBX19-OX and CmBBX19-RNAi strains.

Drought stress treatment

The drought treatment consisted of transplanting CmBBX19-OX, CmBBX19-RNAi and WT plants into 550 grams of 25 cm pots containing a mixture of turf and vermiculite (550 grams of 1:1 mixed turf and vermiculite contained), 3 plants per strain per pot. Normal watering followed by drought treatment for 30d, and then rehydration for 5 d. The relative water content of the soil before and after treatment is respectively 75% (before treatment), 8% (drought for 30d) and 75% (rehydration for 5 d). The survival rate and wilting index were recorded after the treatment was completed, and the phenotype was recorded by photographing. The wilting index was evaluated according to the following criteria: level 0, the plant type is neat and naturally extends; 1, the uppermost layer of blades are clasped, and the lower layer of blades begin to droop; 2, the uppermost layer of blades are clasped, and the lower layer of blade stems droop horizontally; grade 3, shrinking the whole leaf, allowing the lower leaf to droop, and allowing the leaf stalk to horizontally face downwards by 30-60 degrees; 4, folding and bending the uppermost layer of blades, and enabling the blade handles of the lower layer of blades to be approximately horizontal and downward by approximately 90 degrees; grade 5, the whole leaf is severely shrunken and wilted; grade 6, plants completely withered (Zhang et al, 2005).

8.0. + -. 0.2g of soil (now having a weight W1) around the roots of the plants was taken for determination of the Relative Water Content (RWC) of the soil. Drying for 24h at 65 ℃ and determining the weight as W2. The RWC is calculated by the formula RWC (%) - (W1-W2)/W1.

Dual luciferase reporter assay

To analyze the transactivation activity of CmBBX19, the ORF sequence of CmBBX19 without a stop codon was cloned into the 35S promoter-driven pBD-VP16 vector (Han et al, 2016). The Agrobacterium strain GV3101 carrying CmBBX19 and the reporter vector was mixed at 1/5 volumes (Han et al.2016) and co-infected into tobacco leaf discs.

To investigate the interaction of the CmBBX19/CmABF3 and the CmRAB18 promoter in plants, the CmRAB18 promoter was cloned into the pGreenII 0800-LUC vector and the ORF sequences of CmBBX19 and CmABF3 were cloned into the pGreenII 002962-SK vector (Hellens et al, 2005). Agrobacterium strain GV3101 containing CmBBX19 and CmABF3 and agrobacterium containing the CmRAB18 promoter driving LUC were mixed in a ratio of 1: 5, and co-infecting into the tobacco lamina.

The dual-luciferase reporter assay was performed using a kit from Promega (Gao et al, 2019). LUC images were taken using an ikon-L936 imaging system (Andor). LUC and REN activities were determined using GloMax 20/20 luminophore (Promega).

Yeast two-hybrid assay

The ORF sequence of CmBBX19 was amplified and cloned into the EcoRI/SalI site of the pGBKT7 vector (Louvet al, 1997). The ORF or fragment of CmABF/AREB was amplified and cloned into the EcoRI/SalI site of the pGADT7 vector (Chien et al, 1991). pGADT7 and pGBKT7 fusion constructs were transformed in combination, respectively, into the yeast Y2HGold strain. Using Matchmaker^TMGAL4 two-hybrid System (BD)Clontech) were subjected to a yeast two-hybrid test. Transformed yeast were grown in SD/-Trp-Leu medium and then spot-tested on SD/-Trp-Leu-His-Ade plates.

Bimolecular complementation assay

Vectors expressing CmBBX19-YFPN or CmABF3-YFPC or control vectors were transferred to agrobacterium strain GV3101, respectively, and cultured overnight, with OD600 adjusted to 1.0. The combinations co-infest tobacco leaves. Fluorescence of Yellow Fluorescent Protein (YFP) after 60h was imaged using an Olympus FV1000 confocal laser scanning microscope. The excitation wavelength of YFP is 488nm, and the emission wavelength is 525 nm.

Yeast single-hybrid assay

The promoter fragment of CmRAB18 was amplified from the chrysanthemum genomic DNA, cloned into the EcoRI/SalI site of pabali vector (Clontech), and then the ORF sequence of CmABF3 was amplified and cloned into the EcoRI/SalI site of pGADT7 vector (Clontech). Using Matchmaker^TMThe GAL4 single hybrid system (BD Clontech) performed a yeast single hybrid assay. Transformed yeast were grown on SD/-Ura medium and then spot-tested on SD/-Ura-Leu plates.

Protein expression and gel blocking assays

Gel retardation analysis was performed using a biotin-labeled probe and EMSA kit (Thermo Scientific) (Dai et al, 2012). The promoter fragment of CmRAB18 was first used as a probe, and the cold probe was an unlabeled DNA fragment of the same sequence. Expression of GST-CmBBX19 and GST-CmABF3 fusion proteins was induced by the E.coli strain Rosetta and incubated for 10h at 16 ℃. The glutathione sepharose resin is used for extracting and purifying the recombinant protein.

TABLE 1 primers used in the examples

Results and analysis

Expression of CmBBX19 is down-regulated by drought and ABA

In previous studies, we identified four genes encoding proteins of the BBX family IV subfamily in the chrysanthemum transcriptome database in response to water loss (Xu et al, 2013). Wherein, the predicted Open Reading Frame (ORF) of UN68402 is 639bp, and the expression is reduced after dehydration treatment. BLASTP results predict that the polypeptide contains structural features of the BBX group IV protein: the N-terminus has a highly conserved double B-box domain, but the C-terminus has no CCT domain (FIG. 9 a). Phylogenetic analysis showed that the predicted protein is a homologue to group IV of BBX of arabidopsis thaliana and is a homologue of AtBBX18 and AtBBX19 (fig. 9 b). Therefore, we named the gene as CmBBX19 according to the naming system suggested by Khanna et al (2009).

We examined the expression of CmBBX19 in all chrysanthemum organs, with relatively high expression in leaves, flowers and stems and low expression in roots (fig. 1 a). To determine the response of CmBBX19 to drought stress, we determined the expression level of CmBBX19 in mature leaves of WT plants under drought conditions using fluorescent quantitative PCR (qrt) -PCR. Relative to the water-replete conditions, the transcript level of CmBBX19 decreased significantly (2-fold) under severe drought and then returned to basal levels after 1 day of re-watering (fig. 1 b).

Since endogenous ABA levels rapidly increased under drought stress, next we tested whether ABA affected transcription of CmBBX19 during drought stress. As shown in figure 1c, CmBBX19 expression decreased 0.4 fold after 1 hour of exogenous ABA treatment, indicating that CmBBX19 may be a component of the ABA signaling pathway under drought stress.

To test whether CmBBX19 is a transcription factor, we transiently expressed the CmBBX 19-Green Fluorescent Protein (GFP) fusion protein in chrysanthemum protoplasts. The fusion protein was mainly localized to the nucleus but was also detected in the cytoplasm, whereas the GFP protein was present throughout the cell (FIG. 2 a).

Sequence analysis indicated that CmBBX19 contained a typical EAR motif at its C-terminus, suggesting that CmBBX19 might function as a transcriptional repressor (Wang et al, 2014). To investigate whether CmBBX19 has transcriptional repression activity, we performed a dual luciferase transactivation assay. As shown in fig. 2b, tobacco leaves expressing CmBBX19-VP16 exhibited lower relative luciferase activity compared to the control using VP16 alone, indicating that CmBBX19 is indeed a transcription repressing factor.

Modulation of drought stress tolerance by modulation of ABA signaling in CmBBX19

To determine whether CmBBX19 is involved in the regulation of drought stress tolerance, we obtained 19 CmBBX19 over-expressed chrysanthemum strains (CmBBX19-OX) and 30 CmBBX19 silencing strains (CmBBX 19-RNAi). Both lines CmBBX19-OX and CmBBX19-RNAi were compared to Wild Type (WT) plants and the expression level of CmBBX19 in transgenic plants was examined by qRT-PCR (fig. 3 a). We also tested the expression of other members of the chrysanthemum BBX group IV in the CmBBX19-RNAi line in transgenic lines, confirming that only CmBBX19 was silenced (fig. 10).

To verify the effect of expression of CmBBX19 on drought stress, we planted WT, over-expression and silencing lines in soil under normal irrigation conditions, 7 days later, drought treated for 25 days followed by 5 days of rehydration, and then calculated survival rates. Compared to CmBBX19-RNAi, CmBBX19-OX plants and WT showed more severe drought damage (FIG. 3 c). In one experiment, after 5 days of rehydration, WT survival was 67%, CmBBX19-RNAi plants were 100% and their apical shoots continued to grow, whereas CmBBX19-OX plants were only 33% in survival (fig. 3b), almost all surviving plants showed only weak growth of lateral or basal shoots. The result shows that the CmBBX19 participates in the drought resistance regulation mechanism of chrysanthemum.

To further understand the physiological mechanisms of drought resistance influenced by CmBBX19, we compared the transpiration rate, stomatal conductance, water loss, and photosynthetic rate of transgenic lines and WT plants under drought/dehydration conditions. Under drought/water loss conditions, the transpiration rate, stomatal conductance and photosynthetic rate of the CmBBX19-RNAi plants were significantly higher than those of the WT plants, while the water loss rate of the CmBBX19-RNAi plants was significantly lower than that of the WT (fig. 3d-g), whereas the CmBBX19-OX showed the opposite effect. These results are consistent with the results described above for CmBBX19 affecting drought tolerance in chrysanthemum.

Since exogenous ABA treatment also down-regulated the expression of CmBBX19, we explored whether CmBBX19 is involved in the ABA signaling pathway in response to drought stress. Specifically, we performed large-scale screening of Differentially Expressed Genes (DEGs) between leaves of CmBBX19-RNAi, CmBBX19-OX plants and WT plants using RNA sequencing (RNA-seq).

We first focused on the differential expression of genes in ABA-dependent and independent pathways in transgenic plants and WT plants under normal growth conditions. Compared with WT, ABA-dependent pathway genes such as CmRAB18, CmRD29B, CmERD7, CmLTI65 and the like were down-regulated in CmBBX19-OX plants and up-regulated in CmBBX19-RNAi plants, wherein ABA-independent pathway genes such as CmDREB2, CmDREB5 and the like were not changed in expression in both CmBBX19-OX and CmBBX19-RNAi plants (fig. 4).

Then, in order to study whether the drought tolerance regulated by CmBBX19 is related to ABA biosynthesis, related gene expression and ABA content in leaves were examined. In transgenic lines and WT, no significant difference was found between ABA content and expression of ABA biosynthesis-related genes (e.g., CmNCED and CmABA2) (FIG. 11; FIG. 12). We also examined whether the effect of CmBBX19 on drought tolerance is through the transcriptional regulation of key ABA signaling elements and downstream responsive genes. In transgenic lines and WT we found no significant differences in expression of ABA receptor genes, CmPYR/PYL/RCAR or core ABA signaling genes such as CmSnRK2, CmABI3, CmABI4, CmABI5, CmABF3 (figure 12). However, we detected significant differences in expression of genes encoding LEA-like protective proteins such as CmRAB18 and CmRD29B related genes of the downstream ABA signaling pathway (fig. 12).

One recent study showed that overexpression of another chrysanthemum BBX gene family member, CmBBX22, in arabidopsis thaliana can improve drought resistance by delaying leaf senescence (Liu et al, 2019). Therefore, we tested the expression of leaf senescence-associated genes such as CmNYC1 and CmNYE1 in transgenic plants and WT plants, and the results showed that CmBBX19 did not affect the expression of leaf senescence-associated genes (fig. 12 a). This suggests that CmBBX19 and CmBBX22 exert drought-resistant functions through different regulatory mechanisms.

From this we conclude that the impact of CmBBX19 on drought tolerance is mainly through the regulation of the accumulation of protective proteins in the ABA-dependent pathway, rather than through the alteration of key ABA signaling components at the level of ABA biosynthesis and transcription.

Expression of CmRAB18 is not directly regulated by CmBBX19, but by CmABF3

To investigate how CmBBX19 affected the expression of downstream genes, we analyzed the promoters of chrysanthemum genes associated with protective proteins. We found that the ABRE cis-element was enriched in the promoter of the LEA protein gene (fig. 13). However, in the CmBBX19 transgenic line, expression of the ABRE binding factor, CmABFs/AREBs, was not affected (fig. 4, S4), so we investigated whether CmBBX19 affects key ABA signaling elements at the post-transcriptional level. Based on sequence similarity and phylogenetic analysis, we identified 5 CmABF/AREB genes (CmABF1, CmABF2a, CmABF2b, CmABF3 and CmABI5) (fig. 14). In the yeast two-hybrid analysis, we used CmBBX19 as bait and fused 5 AREB-LIKE proteins to the Gal4 activation domain. Only yeasts expressing CmBBX19 and CmABF3 grew normally on selection medium, indicating that CmBBX19 specifically physically interacts with CmABF3, but not with CmABF1, CmABF2a, CmABF2b or CmABI5 (fig. 5a) (GenBank/EMBL accession number MN885646 to CmABF 3).

To verify the in vivo interaction of CmBBX19 with CmABF3, we performed a bimolecular fluorescence complementation (BiFC) assay, observing CmBBX19-YFP in tobacco leaf cells^N(CmBBX19 fused to the N-terminus of YFP) and CmABF3-YFP^C(CmABF3 fused to the C-terminus of YFP) a strong Yellow Fluorescent Protein (YFP) signal that was transiently co-expressed (FIG. 5 d). In contrast, in the negative control, CmBBX19-YFP^NAnd YFP^C,YFP^NAnd CmABF3-YFP^CNo detectable YFP signal was produced in the tobacco leaf. These results indicate that CmBBX19 can interact with CmABF3 in vivo.

According to previous studies, the ABF/AREB protein comprises conserved region 1(C1), conserved region 2(C2), conserved region 3(C3), conserved region 4(C4) and bZIP domain (Jakoby et al, 2002; Fujita et al, 2005; ZHao et al, 2019). To determine which conserved CmABF3 domain interacts with CmBBX19, we generated multiple truncated forms of CmABF3 (fig. 5b), and found that only the C1 domain interacted with CmBBX19 in yeast two-hybrid experiments. Previous studies have shown that the C1 domain of AREB1 has transactivation activity (Fujita et al, 2005), which contains a conserved RXXS/T site that SnRK2 protein kinase can phosphorylate in an ABA-dependent manner (Uno et al, 2000; Furihata et al, 2006). The results of the interaction of CmBBX19 with CmABF3 indicate that CmBBX19 may affect ABA signaling by inhibiting the transactivating activity of CmABF3, rather than by interfering with the promoter binding activity of CmABF 3.

We also isolated CmBBX19 and CmABF3 homologous genes from arabidopsis thaliana to test the preservation of BBX19 and ABF3 interactions in other species. The yeast two-hybrid and BiFC analysis results showed that neither CmBBX19 nor AtBBX19 interacted with AtABF3 (fig. 15), indicating that the interaction mechanism of BBX19 and ABF3 in chrysanthemum is not conserved in arabidopsis thaliana.

To understand how CmBBX19 alters expression of ABA signaling genes, we isolated a genomic 455bp DNA sequence upstream from the CmRAB18 start codon. Sequence analysis showed that the CmRAB18 promoter contains three ABRE motifs (fig. 6a), and in yeast single-hybrid experiments, it was CmABF3, rather than CmBBX19, that binds directly to the CmRAB18 promoter. We used a 50bp fragment from-388 to-339 of the CmRAB18 promoter containing two ABRE motifs as a probe for Electrophoretic Mobility Shift Analysis (EMSA) and observed that the ABRE cis-element in the CmRAB18 promoter binds directly to CmABF3 but not to CmBBX19 (fig. 6c,7 a). We also tested with EMSA whether CmBBX19 interfered with the affinity of CmABF3 for its target gene promoter binding. We found that even if the amount of CmBBX19 protein was increased in the reaction, CmBBX19 had no effect on the affinity of CmABF3 for binding to the CmRAB18 promoter fragment.

CmBBX19 inhibits activation of CmABF3 on CmRAB18

To investigate whether CmBBX19 regulates ABA-responsive genes by inhibiting the transactivating activity of ABF3, we performed a dual luciferase reporter experiment. We fused the CmRAB18 promoter to firefly luciferase to construct a reporter with 35S: CmABF3,35S: CmBBX19 and 35S: CmBBX19-mut (with Cys-25 replacing Ser in the B-box1 region) as effectors (FIG. 7B). Co-transformation of 35S: CmABF3 and proCmRAB18: LUC into tobacco lamina we observed a LUC activity significantly higher than when the empty vector and proCmRAB18: LUC were co-transformed. When we co-transformed 35S: CmABF3,35S: CmBBX19, proCmRAB18: LUC, the LUC activity was significantly reduced. However, when CmBBX19 was replaced with the mutated CmBBX19, the LUC activity was restored again (fig. 7 c). It is shown that CmBBX19 inhibits the activation of CmRAB18 by CmABF 3.

To further validate at the genetic level the mechanism of action of CmBBX19 with CmABF3, we silenced CmABF3 with a modified brassica oleracea leaf bisvirus vector (CaLCuV) containing artificial microRNA-ABF3(CaLCuV-amiR-ABF3) in the context of WT and CmBBX19-RNAi strains (fig. 8 a). In the context of WT and CmBBX19-RNAi, both silencing lines of CmABF3 showed more severe wilting symptoms after 20 days of drought stress treatment, and the plants had lower transpiration rate, stomatal conductance and net photosynthetic rate (fig. 8 b-e). Silencing CmABF3 significantly inhibited the up-regulation of CmBBX 19-RNAi-induced abiotic stress response genes, in particular the LEA protein genes CmRAB18, CmRD29B, CmERD7 and CmLTI65 (fig. 16). These results indicate that CmABF3 mediates the effect of CmBBX19 on drought tolerance in chrysanthemum. Figure 17 shows a schematic representation of CmBBX19 involvement in drought stress responses, from which it can be seen that CmBBX19 is predominantly via the abscisic acid (ABA) dependent pathway.

Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.

SEQUENCE LISTING

<110> university of agriculture in China

<120> application of chrysanthemum zinc finger protein BBX19 and related factors thereof in adjusting drought stress tolerance

<130> GY20100190

<160> 50

<170> PatentIn version 3.5

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gttctagaat gcgaacatta tgtgatgttt gtg 33

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tccccgggtt tacttgtcag cttctctttt aaagg 35

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ttgctcgagt tgagcaacaa gatggtcttg gag 33

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ttatcgatag aatcactcgt aacgacatag aac 33

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tgtctagatt gagcaacaag atggtcttgg ag 32

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tttggtacca gaatcactcg taacgacata gaac 34

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ttgacgagta cggtgggacc ggtatgcgaa cattatgtga 40

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aatgaaacca gagttaaagg cctttacttg tcaccttctc 40

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cttctcaata gaaatgcccc cccgaccgat 30

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cccaccgtac tcgtcaattc 20

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gacgaattca tgcgaacatt atgtgatgtt tgtg 34

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gtcgacgctt gtcaccttct cttttaaagg 30

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gacgaattca tgagttcgtt tatcaacttc a 31

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gtcgacccaa ggtccagtca atgttcttc 29

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gtcgacatct attgcaaaat ttgaggttg 29

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gacgaattcg gaagtgtgtc gaatgatgg 29

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gtcgactaaa ccaccttcac caaatgtat 29

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gacgaattcc gagggaggaa aagcagtgg 29

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gtcgacctga ttgttatgca tatccatc 28

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ccaaatcgac tctagtctag aatgcgaaca ttatgtgatg t 41

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agcggtaccc tcgaggtcga cttacttgtc accttctctt tt 42

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gagaacacgg gggactctag aatgagttcg tttatcaact tcaagaac 48

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agcggtaccc tcgaggtcga cccaaggtcc agtcaatgtt cttc 44

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cttgaattcg agctcggtac cctgaaatta gacacgtact tttca 45

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agcacatgcc tcgaggtcga caaccgtaca cgtgcaagtt tgta 44

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gacgaattca tgcgaacatt atgtgatgtt tgtg 34

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gtcgacgctt gtcaccttct cttttaaagg 30

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gaaattagac acgtactttt cagtgataac ataaacatac ttacgtgttc 50

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gaacacgtaa gtatgtttat gttatcactg aaaagtacgt gtctaatttc 50

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gaaattagac acgtactttt cagtgataac ataaacatac ttacgtgttc 50

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gaacacgtaa gtatgtttat gttatcactg aaaagtacgt gtctaatttc 50

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gatctggttc cgcgtggatc catgagttcg tttatcaact tc 42

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ctcgagtcga cccgggaatt ctcaccaagg tccagtcaat g 41

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gatctggttc cgcgtggatc catgcgaaca ttatgtgatg t 41

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ctcgagtcga cccgggaatt cttacttgtc accttctctt tt 42

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gacgaattca tgagttcgtt tatcaacttc 30

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gtcgactcac caaggtccag tcaatg 26

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cgtctagaat gcgaacatta tgtgatgttt gtg 33

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caggtacctt acttgtcacc ttctctttta aagg 34

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gtcgacggta tcgataagct tcaagagaat gaacaagatg cg 42

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tgtttttggc gtcttccatg gtcttaagtg aaaaagattt atttgag 47

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atgcgaacat tatgtgatgt ttgtg 25

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ttagcttgtc accttctctt ttaaagg 27

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<211> 639

<212> DNA

<213> Chrysanthemum morifolium, cv. Fall Color

<400> 44

atgcgaacat tatgtgatgt ttgtgaaagt gctgctgcga tcttgttttg tgctgctgat 60

gaagctgctc tttgtcgtgc ttgtgatgaa aaggtgcata tgtgtaataa gcttgccagt 120

aggcatgtac gagtaggact tgctgacccc agtgatgtac aacgttgtga catttgtgaa 180

aatgcaccag ctttcttcta ctgtggaatt gatggaagtt ctctatgttt acaatgtgac 240

atgaacgttc atgttggtgg taaacgaaca catggaagat atctgctatt aaggcaaaga 300

gtcgagtttc caggggatag aagtggtcgt gacgatgagt tagggttgca acccggtgaa 360

ccaggtggtg aagtaaggag ggaacaaaac aatcaaccga aggctacaac aagagataac 420

caacaccacc ggttatctgc cataggaatg ctggaaaata ataatgatgg tgctggcagg 480

atggaaaata agttgtttga tcttaatgcc agacctcaac ggatgcatgg tcaaacttca 540

aataaccagg aacaaggaat ggatattagc ggcagcggca ataacgattc tgctagtgtg 600

gttcctgttg gatcctttaa aagagaaggt gacaagtaa 639

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atgagttcgt ttatcaactt ca 22

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ctaccaaggt ccagtcaatg tt 22

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<213> Chrysanthemum morifolium, cv. Fall Color

<400> 47

atgagttcgt ttatcaactt caagaactat ggtgacacat cgcaacaaga cctgaatgga 60

agtaaacaaa tggtgtcaaa ttcgccacta aaccgacaat cttctattta ctcattaact 120

ttcgatgagt tacaaaacac atttggtgga ggtggtaagg attttggatc aatgaatatg 180

gatgaacttt taaagaacat ttggactgtg gaagagactc aaacggtcgc atcaacctca 240

aattttgcaa tagatggaag tgtgtcgaat gatggaaata ataatattaa taatatacaa 300

agacaaggat ctttgacatt gccacgaaca cttagccaga aaacagtcga ggaagtgtgg 360

cgagagttgc tcaagagtag tactaatggt gtagttaaag aaggggattt gctgagtgaa 420

actaacttac gaccggagca aagggaacca actttaggcg agatgacact cgaggatttc 480

ttgtccaaag ctggtgtagt aaccgaaaac aatcaagttc agaagggttc atattttggt 540

gatatttcac aacagaatgg tgaaaacagc agtatcatat tcgggtttca gaaccctaat 600

cagaatcaag cgtttcaaca accaccacct cggaacaatg caccaatggt taaccaaatt 660

acgcagactc aaaatggact aaaccttcag ccaaaaccaa aaccactacc aatttttcca 720

aagcaagctg ccttggactt caccgcacca ttgactggtc agagggctag ccccgccact 780

gggattcgga aatctgagca accgataagt agtagcgtgg ttcaatctaa tggtatggaa 840

agtggtgcaa tgaatgttaa gacacttggg ggaattatgg ccgggtctcc acggaatatt 900

attccaaaaa ctaactttga ttcaactcca tcacctccgt attatacatt tggtgaaggt 960

ggtttacgag ggaggaaaag cagtgggact ttagagaaag tagtggaaag aaggcggagg 1020

agaatgatta agaatagaga atctgctgca cgatcacggg cccgaaagca ggcctatact 1080

ttggagttgg aagccgaagt tgcaaagctt aaagaaatga ataacgagtt gcttaagaaa 1140

caggaagaga tgatggatat gcataacaat caggtagtgg agaagatgaa actgccatgg 1200

ggaaataaaa gactatgctt acgaagaaca ttgactggac cttggtag 1248

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atggcacaat acggagga 18

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ttactggtga cctcctcctg g 21

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<211> 477

<212> DNA

<213> Chrysanthemum morifolium, cv. Fall Color

<400> 50

atggcacaat acggaggaga acaatacaag cagcaagagg gtcaccacac tgatgagcat 60

gctcaaaacc aacttcactc cactgctggt catggtatcg gaggtactgg aaccaacgtc 120

cactcgacga tgactggtca aaatattgga ggtactggaa ctaatatggg gatgggaggt 180

ggtcacggct atgaagaagg gaaacaaggt ggtggaatac tccaccgttc tgggagtggc 240

agttctagct cttcggagag cgatggagaa ggtggaagaa ggaagaagaa aggtgtggtg 300

gagaagatca aggagaagct gcctggcggt gatcatggta gcgaacagca cacagctccg 360

gccagtgcca ccgtaggtgg tactggtggc tatggaaatg taggagagga aggacatgag 420

aagaagggac tcatggacaa gattaaggac aagttaccag gaggaggtca ccagtaa 477

Claims

1. A method for modulating drought stress tolerance in a plant using a CmBBX19 and/or CmBBX 19-related factor, wherein the method is performed by modulating expression of a CmBBX19 and/or CmBBX 19-related factor, wherein the CmBBX 19-related factor is a factor that is modulated by CmBBX19 in an ABA-dependent pathway.

2. The method of claim 1, wherein:

the modulating the plant drought stress tolerance is decreasing the plant drought stress tolerance and is performed by overexpressing the CmBBX19 and/or decreasing the expression of the CmBBX 19-related factor.

3. The method of claim 1, wherein:

the modulating the plant drought stress tolerance is increasing the plant drought stress tolerance and is performed by decreasing the expression of CmBBX19 and/or increasing the expression of the CmBBX 19-related factor.

4. The method according to any one of claims 1 to 3, wherein the modulation is a transcription level modulation and/or a translation level modulation.

5. The method according to any one of claims 1 to 4, characterized in that:

the CmBBX 19-related factor comprises a CmBBX19 interaction factor or a factor downstream of the interaction factor;

preferably, the CmBBX19 interaction factor is ABF3, especially CmABF 3; and/or the downstream factor is selected from the group consisting of RAB18, RD29B, ERD7 and LTI65, in particular from the group consisting of CmRAB18, CmRD29B, CmERD7 and CmLTI 65.

6. The method of claim 3, wherein the method is performed by at least one of:

(1) knocking out the CmBBX19 gene or overexpressing the CmBBX 19-related factor gene;

(3) blocking protein interaction of CmBBX19 and CmABF 3;

wherein the CmBBX19 related gene comprises a CmBBX19 interacting factor gene or a downstream factor gene of the interacting factor;

7. Method according to any one of claims 1 to 6, wherein the plant is a Compositae plant, preferably a Chrysanthemum morifolium, more preferably a Chrysanthemum morifolium, even more preferably a Chrysanthemum morifolium variety (cv. Fall Color).

Use of the CmBBX19 gene and/or CmBBX19 related factor in:

(1) restoring continued growth of top shoots of the plant after drought stress; (2) increasing the survival rate of plants under drought stress;

(3) simultaneously, the transpiration rate, the stomatal conductance, the water loss and the photosynthetic rate of the plants are reduced;

wherein the CmBBX 19-related factor comprises a CmBBX19 interaction factor or a factor downstream of the interaction factor;

9. A method for drought tolerant breeding of a plant, said method comprising at least one of:

(1) (ii) reduces expression of CmBBX 19;

(2) increasing expression of a CmBBX 19-related factor in an ABA-dependent pathway, wherein the CmBBX 19-related factor comprises a CmBBX19 interacting factor or a factor downstream of the interacting factor; preferably, the CmBBX19 interaction factor is ABF3, especially CmABF 3; and/or the downstream factor is selected from the group consisting of RAB18, RD29B, ERD7 and LTI65, in particular from the group consisting of CmRAB18, CmRD29B, CmERD7 and CmLTI 65;

(3) blocking the protein interaction of CmBBX19 with CmABF 3.

10. A method of modulating expression of a plant ABA-dependent pathway-associated factor, comprising:

the method is achieved by modulating expression of the CmBBX19 factor; the ABA dependent pathway related factor is an ABA signal pathway related factor;

preferably, the ABA signal path correlation factor is a CmBBX19 correlation factor;

more preferably, the CmBBX 19-related factor comprises a CmBBX19 interaction factor or a factor downstream of the interaction factor;

it is further preferred that the CmBBX19 interaction factor is ABF3, in particular CmABF 3; and/or the downstream factor is selected from the group consisting of RAB18, RD29B, ERD7 and LTI65, in particular from the group consisting of CmRAB18, CmRD29B, CmERD7 and CmLTI 65.