MdDEWAX protein and application thereof in regulating drought resistance by influencing wax biosynthesis

Abstract

The invention discloses an MdDEWAX protein and application of the MdDEWAX protein in regulation and control of drought resistance by influencing wax biosynthesis. The invention provides a protein which is any one of the following proteins: a protein consisting of an amino acid sequence as shown in a sequence 2; the N end or / and C end of the protein as shown in the sequence 2 is / are connected with a label to obtain a fusion protein; the amino acid sequence shown in the sequence 2 is subjected to substitution and / or deletion and / or addition of one or more amino acid residues, and the protein has the same function. The invention provides the MdDEWAX protein and the gene thereof, overexpression of the MdDEWAX protein finds that the MdDEWAX protein is a negative regulatory factor for wax accumulation, and the MdDEWAX protein and the gene thereof can inhibit the wax content and the main component content in plants, so that the drought resistance of the plants is reduced. Silencing shows that the wax content and the main component content in the plant can be improved.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to MdDEWAX and its application in regulating drought resistance by affecting wax biosynthesis. Background Art

[0002] The plant cuticle is a hydrophobic barrier covering the surfaces of above-ground organs such as leaves, flowers, and stems of terrestrial plants, and is composed of cutin and epicuticular waxes (Riederer and Schreiber, 2001; Kunst and Samuels, 2003; Pollard et al., 2008; Samuels et al., 2008; Bernard and Joubès, 2013; Yeats and Rose, 2013). Plant epicuticular waxes are a powerful barrier against biotic and abiotic stresses in the external environment and play important roles in regulating epidermal permeability, reducing non-stomatal water loss, adapting to external temperature changes, and protecting plants from ultraviolet rays, insects, and pathogens (Samuels et al., 2008; Buschhaus and Jetter, 2012; Li-Beisson et al., 2013; Olascoaga et al., 2014; Park et al., 2016). Drought can cause changes in the osmotic potential of plants, affect the photosynthetic system, inhibit growth, reduce yields, and even lead to plant death, severely limiting the normal growth and development of plants. To cope with this stress, plants maintain water in various ways to survive. Drought can cause an increase in the content of wax components in the cuticle on the surface of plant leaves, which can improve the drought resistance of plants to a certain extent (Ksoma et al., 2009; Islam et al., 2009; Bourdenx et al., 2011).

[0003] The synthesis and transport processes of plant waxes involve several main steps. First, C16 and C18 saturated fatty acids are synthesized in the plastids of epidermal cells and then esterified to the corresponding acyl-CoA by long-chain acyl-CoA synthetase (LACS). Each FAE cycle adds two carbon atoms to the long-chain fatty acid and finally elongates it into a very-long-chain fatty acid. The very-long-chain fatty acid can be modified to synthesize the main components of waxes, including alkanes, alcohols, aldehydes, fatty acids, ketones, and esters (Moggia et al., 2016; Yang et al., 2017). The synthesized wax is transported from the endoplasmic reticulum to the plasma membrane and crosses the cell wall through transporters to reach the epidermal cuticle that wraps the outer surface of the plant (Lee et al., 2022).

[0004] The biosynthesis of epidermal wax is regulated at the transcriptional level, and it has been confirmed that multiple transcription factors can induce or inhibit wax biosynthesis (Lee and Suh, 2015). For example, among the MYB transcription factors, MYB96 can directly bind to the promoters of wax synthesis-related genes KCS1, KCS2, KCR1, CER3, and CER6, thereby promoting wax accumulation in Arabidopsis. Similarly, MYB30 and MYB94 have also been shown to positively regulate wax synthesis (Seo et al., 2011; Oshima et al., 2013). Notably, the E3 ubiquitin ligase MIEL1 can negatively regulate epidermal wax biosynthesis by regulating the protein stability of MYB96 and MYB30 (Gil et al., 2017). A large number of studies have shown that AP2 transcription factors affect the accumulation of plant epidermal waxes by regulating wax biosynthesis-related genes (Zhang et al., 2005; Borisjuk et al., 2014; Park et al., 2016). WRI4 is an AP2 / ERF transcription factor that is highly expressed in stem epidermal tissues and can affect the production of waxes in the cuticle by activating the expression of downstream genes LACS1, KCR1, PAS2, and ECR, thereby mediating the synthesis of epidermal waxes in Arabidopsis stems (Park et al., 2016). The AP2 / ERF transcription factor DEWAX directly interacts with the promoters of wax biosynthesis genes CER1, LACS2, ACLA2, ECR, and FAR6, and transcriptional inhibition of their expression results in a decrease in the total wax content in Arabidopsis leaves and stems and changes in the ultrastructure of the cuticle (Go et al., 2014). In addition, DEWAX can also form a heterodimer with SPL9, interfering with the ability of SPL9 DNA to bind to CER1, thereby affecting epidermal wax accumulation (Li et al., 2019). However, whether DEWAX mediates the biosynthesis of cuticular waxes in apples has not been studied.

[0005] In apples, most studies on the regulatory mechanism of epidermal wax synthesis have focused on genes that promote epidermal wax accumulation and improve plant resistance to biotic and abiotic stresses, such as MdWRI4 (Zhang et al., 2020b), MdMYB30 (Zhang et al., 2019b), MdSHINE2 (Zhang et al., 2019a), and MdLACS1 (Li et al., 2022). There are few reports on genes related to the negative regulation of apple wax. Summary of the Invention

[0006] The object of the present invention is to provide MdDEWAX and its application in regulating drought resistance by affecting wax biosynthesis.

[0007] In the first aspect, the present invention provides a protein named MdDEWAX, which is any one of the following A1)-A4):

[0008] A1) A protein consisting of the amino acid sequence shown in SEQ ID NO: 2;

[0009] A2) A fusion protein obtained by linking a tag to the N-terminus and / or C-terminus of the protein shown in SEQ ID NO: 2;

[0010] A3) A protein obtained by substituting and / or deleting and / or adding one or several amino acid residues to the amino acid sequence shown in SEQ ID NO: 2 and having the same function;

[0011] A4) A protein having a homology of more than 99%, more than 95%, more than 90%, more than 85% or more than 80% with the amino acid sequence defined in any of A1)-A3) and having the same function.

[0012] In a second aspect, the present invention provides a nucleic acid molecule encoding the protein described in the first aspect.

[0013] The nucleic acid molecule described above is the MdDEWAX gene, which is any of the following:

[0014] B1) The DNA molecule shown in SEQ ID NO: 1;

[0015] B2) A DNA molecule having a homology of more than 98% with the DNA sequence defined in B1) and encoding a protein with the same function;

[0016] B3) A DNA molecule that hybridizes with the DNA sequence defined in B1) under stringent conditions and encodes a protein with the same function;

[0017] B4) A DNA molecule having a homology of more than 90% with the DNA sequence defined in B1) and encoding a protein with the same function.

[0018] In a third aspect, the present invention provides an expression cassette, a recombinant vector or a recombinant microorganism containing the nucleic acid molecule described in the second aspect.

[0019] In a fourth aspect, the present invention provides the use of the protein described in the first aspect, the nucleic acid molecule described in the second aspect, or the expression cassette, recombinant vector or recombinant microorganism described in the third aspect in any of the following:

[0020] C1) Reducing the accumulation of wax crystals in plant tissues;

[0021] C2) Reducing the transcriptional level of plant wax-related genes;

[0022] C3) Reducing the wax content in plant tissues;

[0023] C4) Improving the permeability of the plant leaf cuticle;

[0024] C5) Increasing the water loss rate of plant leaves;

[0025] C6) Enhance the sensitivity of plants to ABA;

[0026] C7) Regulate plant drought resistance;

[0027] C8) Cultivate plants with low accumulation of wax crystals in tissues;

[0028] C9) Cultivate plants with low wax content in tissues;

[0029] C10) Cultivate plants with high permeability of leaf cuticle;

[0030] C11) Cultivate plants with high leaf water loss rate;

[0031] C12) Cultivate plants with high ABA sensitivity;

[0032] C13) Cultivate plants with low drought resistance;

[0033] C14) Serve as or prepare a regulatory factor that negatively regulates wax biosynthesis in plant tissues.

[0034] The above regulation of plant drought resistance specifically refers to reducing plant drought resistance.

[0035] The above-mentioned tissue is the above-ground tissue of plants. Further, in the present invention, the above-ground tissue of plants is the stem, leaf epidermis or pericarp.

[0036] The above-mentioned wax-related genes in plants are specifically, in Arabidopsis thaliana, the wax-related genes are AtCER1, AtECH, AtKCS1, AtMYB96, AtCER3 and AtSHINE3, and in apples, the wax-related genes are MdKCS1, MdLACS2 and MdSHINE2.

[0037] In the above, the wax is specifically as follows:

[0038] In Arabidopsis thaliana, the main components of the wax include alkanes, alcohols, fatty acids, aldehydes, ketones and / or esters; further, the alkanes with significantly reduced content are specifically C29 alkanes; the alcohols with significantly reduced content are specifically C28, C29 and C30 alcohols; the fatty acid with significantly reduced content is C18; the aldehyde with significantly reduced content is C30; the ketone with significantly reduced content is C29; the ester with significantly reduced content is C42 or C44;

[0039] In apples, the wax is alkanes, alcohols, fatty acids, aldehydes and / or triterpenoids. Further, the alkanes with significantly reduced content are specifically C29 alkanes; the alcohols with significantly reduced content are specifically C29 alcohols; the fatty acid with significantly reduced content is C30; the aldehyde is C30.

[0040] The above reduction of plant drought resistance is further manifested as the accumulation of malondialdehyde, reactive oxygen species and / or anthocyanins in plants under drought conditions.

[0041] In a fifth aspect, the present invention provides the use of a substance for reducing the activity or content of the protein described in the first aspect, or for inhibiting the expression of the nucleic acid molecule described in the second aspect, in any of the following:

[0042] D1) enhancing the accumulation of wax crystals in plant tissues;

[0043] D2) enhancing the transcriptional level of wax-related genes in plants;

[0044] D3) enhancing the wax content in plant tissues;

[0045] D4) enhancing the drought resistance of plants.

[0046] In the above, the plant is specifically an apple in the examples of the present invention, and the wax-related genes in the apple are MdKCS1, MdLACS2, and MdSHINE2.

[0047] The tissue described above is the above-ground tissue of the plant. Further, in the present invention, the above-ground tissue of the plant is the apple peel.

[0048] In the above, the plant wax is specifically apple wax, and the main components of the wax include alkanes, alcohols, fatty acids, aldehydes, and / or triterpenoids; further, the alkane with increased content is specifically C29 alkane; the alcohol with increased content is specifically C29 alcohol; the fatty acid with increased content is C30; the aldehyde with increased content is C30.

[0049] In a sixth aspect, the present invention provides a method for cultivating a plant with low wax crystal accumulation in tissues, low wax content in tissues, high cuticular permeability of leaves, high leaf water loss rate, high ABA sensitivity, and / or low drought resistance, comprising the following steps: enhancing the activity or content of the protein described in the first aspect in a target plant, or enhancing the expression level of the nucleic acid molecule described in the second aspect in the target plant, to obtain a transgenic plant;

[0050] The transgenic plant has at least one of the following characteristics:

[0051] E1) the amount of wax crystal accumulation in the tissues of the transgenic plant is less than that of the target plant;

[0052] E2) the wax content in the tissues of the transgenic plant is less than that of the target plant;

[0053] E3) the cuticular permeability of the leaves of the transgenic plant is greater than that of the target plant;

[0054] E4) the leaf water loss rate of the transgenic plant is higher than that of the target plant;

[0055] E5) the sensitivity of the transgenic plant to ABA is higher than that of the target plant;

[0056] E6) The drought resistance of the transgenic plant is lower than that of the target plant. Seventh aspect, the present invention provides a method for cultivating a plant with high wax crystal accumulation and / or high wax content in tissues, comprising the following steps: reducing the activity or content of the protein described in the first aspect in the target plant, or reducing the expression level of the nucleic acid molecule described in the second aspect in the target plant, to obtain a transgenic plant;

[0057] The transgenic plant has at least one of the following characteristics:

[0058] F1) The amount of wax crystal accumulation in the transgenic plant tissues is greater than that of the target plant;

[0059] F2) The wax content in the transgenic plant tissues is greater than that of the target plant.

[0060] The plants described above are monocotyledonous plants or dicotyledonous plants, specifically taking Arabidopsis thaliana or apple as examples.

[0061] In this study, a key transcription factor MdDEWAX that inhibits wax synthesis was isolated from apples. It was found that this gene negatively regulates the biosynthesis of apple wax and thus reduces drought resistance, and this gene was proven to be a negative regulator of wax accumulation. By measuring the expression levels of MdDEWAX under PEG and ABA treatment conditions, it was found that MdDEWAX could respond to both of the above stresses. As a transcription factor that negatively regulates wax biosynthesis, the total wax content in the stems of transgenic Arabidopsis thaliana of MdDEWAX was significantly lower than that of the wild type, and the main components of wax were all reduced. Among them, C29 alkane, C28 alcohol, C29 alcohol, C30 alcohol, C30 aldehyde and C29 ketone were the components with the most significant reduction. It has been reported that alkanes are the most important components affecting epidermal water loss, and their content increases significantly under drought stress, and aldehydes also play an important role in drought resistance (Leide et al., 2011). Here, it is speculated that MdDEWAX may accelerate the water loss of the Arabidopsis thaliana epidermis by reducing the content of alkanes and aldehydes, thereby reducing its drought resistance.

[0062] In this study, the heterologous expression of MdDEWAX in Arabidopsis thaliana enhanced the sensitivity of the plant to ABA, but reduced the drought resistance of the plant. The research shows that the reduction of drought resistance by MdDEWAX is not dependent on ABA. Transgenic Arabidopsis thaliana of MdDEWAX is more vulnerable to stress damage when suffering from drought. Based on this, it is speculated that there may be no direct relationship between drought resistance and ABA sensitivity. It is worth noting that compared with the wild type, the accumulation of anthocyanins in transgenic Arabidopsis thaliana after drought treatment increased significantly. It is speculated that this may be due to the reduction of wax biosynthesis in transgenic Arabidopsis thaliana, resulting in more carbon sources flowing to the biosynthesis of anthocyanins. The conversion relationship between wax and anthocyanins will be an interesting point for further research in the future.

[0063] Through scanning electron microscopy and gas chromatography-mass spectrometry analysis, overexpression or silencing of MdDEWAX in apple peel significantly altered the wax accumulation in apple peel. In this study, the differences in apple peel wax components were caused by the contents of C29 alkane, C29 alcohol, C30 aldehyde, C30 fatty acid, and triterpenoids. Overexpression of MdDEWAX in apple peel and heterologous expression of MdDEWAX in Arabidopsis both led to a decrease in alkane content. Currently, it is widely believed that alkanes are important components of waxes that can prevent water loss. Therefore, MdDEWAX may accelerate water loss in apple fruits. By analyzing the stem waxes of Arabidopsis and apple peel waxes, it was found that triterpenoids were the components with the greatest differences between the two materials.

[0064] Therefore, the present invention provides the MdDEWAX protein and its gene. Overexpression of it was found to be a negative regulator of wax accumulation, capable of inhibiting the wax content and the contents of main components in plants, thereby reducing the drought resistance of plants. Knocking it out was found to be able to increase the wax content and the contents of main components in plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 is the genomic structure of the MdDEWAX sequence.

[0066] Figure 2 is the subcellular localization and tissue expression pattern of MdDEWAX. (A) Subcellular localization of MdDEWAX. Scale bar = 10 μm. (B) Relative expression levels of MdDEWAX in apple roots, stems, leaves, flowers, and fruits. (C) Relative expression levels of MdDEWAX in apple peel at different stages after flowering.

[0067] Figure 3 is the response of the expression level of MdDEWAX to PEG (A), NaCl (B), and ABA (C). Error bars represent standard deviation (SD; n = 3). Data are the mean ± standard deviation of three independent replicates. Different lowercase letters indicate significant differences (P < 0.05).

[0068] Figure 4 is the identification of MdDEWAX transgenic Arabidopsis.

[0069] Figure 5 is the observation of the cuticular wax crystal morphology of MdDEWAX transgenic Arabidopsis and Col Arabidopsis by scanning electron microscopy. A - B, ultrastructure of the epidermis of the stems (A) and leaves (B) of MdDEWAX transgenic lines and Col. A, observation results of Arabidopsis stem wax crystals at magnification of 500 times and 1000 times, with scale bars of 50 μm and 10 μm respectively. B, observation results of Arabidopsis leaf wax crystals at magnification of 500 times and 1000 times, with scale bars of 50 μm and 10 μm respectively.

[0070] Figure 6 Effect of MdDEWAX on wax biosynthesis in Arabidopsis thaliana. (A) Expression levels of genes related to wax biosynthesis in Col-0 and MdDEWAX transgenic lines. (B) Total wax content in the stems of Col-0 and MdDEWAX transgenic lines. (C) Total amount of wax components in the stems of Col-0 and MdDEWAX transgenic lines. (D-I) Wax components in the stems of MdDEWAX transgenic lines and Col determined by GC-MS: alkanes (D), alcohols (E), fatty acids (F), aldehydes (G), C29 ketones (H), and esters (I). Data are the mean ± standard deviation of three independent replicates. Different lowercase letters indicate significant differences (P<0.05).

[0071] Figure 7 Change in cuticle permeability of MdDEWAX transgenic Arabidopsis thaliana. A-C, Toluidine blue-stained inflorescences (A), stems (B), and leaves (C) of MdDEWAX transgenic lines and Col. Scale bar is 1 cm. D, Water loss rate of rosette leaves of MdDEWAX transgenic lines and Col. E, Chlorophyll content of rosette leaves of MdDEWAX transgenic lines and Col. FW is fresh weight.

[0072] Figure 8 MdDEWAX transgenic Arabidopsis thaliana can improve plant sensitivity to ABA and PEG. (A) Phenotypes of MdDEWAX transgenic Arabidopsis thaliana and Col Arabidopsis thaliana on 1 / 2MS, 1 / 2MS + 10 μmol·L -1 ABA and 1 / 2MS + 6% PEG media, scale bar = 1 cm. (B) Primary root lengths of Col and transgenic Arabidopsis thaliana. Error bars represent standard deviation (SD; n = 3). Data are the mean ± standard deviation of three independent replicates. Different lowercase letters indicate significant differences (P<0.05).

[0073] Figure 9 MdDEWAX reduces the drought resistance of Arabidopsis thaliana. (A) Col and MdDEWAX transgenic Arabidopsis thaliana grown under normal and drought treatment conditions. (B-E) Physiological indices of MdDEWAX Arabidopsis thaliana and Col Arabidopsis thaliana under drought and non-drought conditions: MDA (B), H2O2 (C), O 2- (D), anthocyanin (E). Values are the mean ± standard deviation of at least three biological replicates. Different lowercase letters indicate significant differences (P<0.05).

[0074] Figure 10 Expression level of MdDEWAX around the injection site detected by qRT-PCR.

[0075] Figure 11 To observe the wax crystal morphology of apple peel overexpressing or silencing MdDEWAX by scanning electron microscopy. (A) MdDEWAX was overexpressed in apple peel, and the cuticular wax crystal morphology in the peel was observed at 500× and 1000×. The scale bars are 50 μm and 10 μm, respectively. (B) MdDEWAX was silenced in apple peel, and the morphology of cuticular wax crystals in apple peel was observed at 500× and 1000×. The scale bars are 50 μm and 10 μm, respectively.

[0076] Figure 12 For the expression of wax synthesis-related genes MdKCS1, MdLACS2, and MdSHINE2 around the injection site.

[0077] Figure 13 For the effect of MdDEWAX on apple peel wax biosynthesis. (A) Total wax content in apple peel of MdDEWAX overexpression, silencing, and control groups. (B) Total amount of apple peel wax components in MdDEWAX overexpression, silencing, and control groups. (C-F) Apple peel wax components of MdDEWAX overexpression, silencing, and control groups were determined by GC-MS, including alkanes (C), alcohols (D), aldehydes (E), and fatty acids (F). Data are the mean ± standard deviation of three independent replicates. Different lowercase letters indicate significant differences (P < 0.05). Specific implementation manner

[0078] The experimental methods used in the following examples are all conventional methods unless otherwise specified.

[0079] The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

[0080] Plant materials and growth conditions in the following examples:

[0081] The materials used in this experiment mainly include tissue-cultured 'GL3' apple seedlings (wild-type apple seedlings), Arabidopsis thaliana, tobacco, and apple fruits. Apple tissue-cultured seedlings were grown in MS medium supplemented with 0.5 mmol·L -1 6-BA, 0.1 mmol·L -1 IAA and 0.8% agar. The cultivation temperature was 25°C, the relative humidity was 60%, and the photoperiod was 16 hours of light / 8 hours of darkness. Treated with 100 μmol·L -1 ABA, 10% polyethylene glycol (PEG 6000), or 100 mmol·L -1NaCl-treated tissue culture seedlings for 0, 1, 3, 6, 12, 24, and 48 hours. Arabidopsis thaliana was grown under conditions of 21 °C, 60% relative humidity, and a photoperiod of 16 hours of light / 8 hours of darkness. Tobacco was grown in a growth chamber under natural light at 25 °C and was infected after growing six leaves. The roots, stems, leaves, flowers, and fruits of apples were taken from "Royal Gala" apple trees planted at the experimental station of Shandong Agricultural University.

[0082] For the quantitative experiments in the following examples, there were 10 plants for each strain, and the results were averaged.

[0083] Each result in the following examples was based on parallel experiments with at least three technical replicates. All data were analyzed using data processing system software and compared using the one-way factor Tukey method. Treatment means with different lowercase letters were significantly different statistically (P < 0.05).

[0084] Table 1 shows the primer sequences related to the experiment

[0085]

[0086]

[0087] Example 1. Cloning of the MdDEWAX gene

[0088] I. Cloning of the MdDEWAX gene

[0089] To identify the MdDEWAX gene in the apple genome, the Arabidopsis thaliana DEWAX gene (AT5G61590.1) was used as a bait, and similar sequences in apples were screened using the NCBI database (https: / / www.ncbi.nlm.nih.gov / ).

[0090] Using the cDNA of "Royal Gala" fruits as a template, PCR amplification was performed using the specific primers MdDEWAX-F and MdDEWAX-R for MdDEWAX (Table 1). An open reading frame of 702 bp was amplified and named the MdDEWAX gene (MD07G1248700). The MdDEWAX gene encodes 233 amino acids and contains one exon ( Figure 1 ), named the MdDEWAX protein.

[0091] The nucleotide sequence of the MdDEWAX gene is shown in Sequence 1, and the amino acid sequence of the protein it encodes is shown in Sequence 2.

[0092] II. Subcellular localization

[0093] Construct the MdDEWAX-TOPO vector, and then ligate it with the GFP vector through LR replacement reaction to form the fusion expression vector 35S::MdDEWAX-GFP.

[0094] Transfer the obtained vector into tobacco leaf epidermal cells by Agrobacterium-mediated infection method. Detect the fluorescence signal using a laser confocal microscope (Zeiss LSM 510META, Jena, Germany).

[0095] The results are as Figure 2 shown in A. The fluorescence signal of the control group 35S::GFP is distributed in the nucleus and cytoplasm, while the fluorescence signal of 35S::MdDEWAX-GFP is distributed in the nucleus of tobacco epidermal cells ( Figure 2 A). This result indicates that MdDEWAX is a nuclear localization protein.

[0096] III. Detect the tissue expression of MdDEWAX

[0097] To detect the tissue expression pattern of MdDEWAX, quantitative RT-qPCR method was used to analyze the transcriptional levels of MdDEWAX in apple roots, stems, leaves, flowers and fruits.

[0098] Total RNA was extracted from apple peels, apple seedlings and different apple organs using an RNA extraction kit (Tiangen Biotech, Beijing), and cDNA was reverse transcribed using a reverse transcription kit (Perfect Real Time, TaKaRa, Dalian). Real-time fluorescence quantitative PCR was performed for analysis, and real-time fluorescence quantitative PCR was carried out according to the method of An et al. (2018). MdActin was used as the internal reference gene for apples, and 18S was used as the internal reference gene for Arabidopsis thaliana (Zhang et al., 2020b).

[0099] RNA was extracted from apple roots, stems, leaves, flowers and fruits, reverse transcribed to obtain cDNA, and RT-qPCR amplification was performed. The primers were MdDEWAX-qPCR-F and MdDEWAX-qPCR-R.

[0100] The results are as Figure 2 shown in B, indicating that the relative expression level of MdDEWAX is the highest in apple fruits, followed by flowers, the expression levels in stems and leaves are lower, and the expression level in roots is the lowest. This shows that this gene may mainly play a role in apple fruits.

[0101] To further verify the function of MdDEWAX in fruit development, the relative expression levels of MdDEWAX in apple peels at different stages were measured. The method was the same as above, except for the period after fruit flowering.

[0102] The results are asFigure 2 As shown in C, it was found that its expression level decreased rapidly at 60 days after flowering.

[0103] IV. MdDEWAX Responds to Multiple Abiotic Stresses

[0104] The transcriptional changes of the MdDEWAX gene in apples under drought stress (PEG), salt (NaCl), and ABA treatments were studied.

[0105] The results are as Figure 3 shown in A-3C, indicating that under PEG treatment, the expression level of MdDEWAX changed little in the initial stage of treatment. As the treatment time extended, the expression of MdDEWAX was rapidly up-regulated at 24 h and remained at a relatively high level at 48 h ( Figure 3 A). Under NaCl treatment, the expression of MdDEWAX was immediately up-regulated and then rapidly down-regulated, slightly up-regulated at 24 h and then down-regulated again ( Figure 3 B). Under ABA treatment, the expression of MdDEWAX showed an obvious trend of first rising and then falling, and reached the highest expression level at 3 h ( Figure 3 C).

[0106] Example 2. Application of MdDEWAX in Regulating the Function of Arabidopsis

[0107] I. MdDEWAX Negatively Regulates Wax Accumulation in Arabidopsis

[0108] To explore whether MdDEWAX is involved in wax biosynthesis, three MdDEWAX transgenic Arabidopsis lines (MdDEWAX#1, #2, and #3) were obtained.

[0109] 1. Construction of MdDEWAX-Transgenic Arabidopsis

[0110] 1) Construction of Overexpression Vector

[0111] The full-length cDNA of the MdDEWAX gene from 'Royal Gala' apples was cloned into the pRI-101 plant expression vector using amplification primers as shown by MdDEWAX-PRI-F and MdDEWAX-PRI-R in Table 1.

[0112] The overexpression vector MdDEWAX-PRI is a vector obtained by replacing the fragment between the SalI and BamHI restriction sites of the pRI-101 vector (Reference: 1. Sugio, T., Satoh, J., Matsuura, H., Shinmyo, A., & Kato, K. (2008). The 5′-untranslated region of the Oryza sativa alcohol dehydrogenase gene functions as a translational enhancer in monocotyledonous plant cells. Journal of bioscience and bioengineering, 105(3), 300-302) with the MdDEWAX gene shown in Sequence 1, and this vector overexpresses the MdDEWAX gene.

[0113] 2) Construction of MdDEWAX-overexpressing Arabidopsis

[0114] According to the method of Zhou et al. (2019), the MdDEWAX-PRI vector was introduced into the Agrobacterium strain GV3101. Arabidopsis Col-0 (hereinafter referred to as wild-type Arabidopsis) was transformed by the Agrobacterium-mediated floral dip method to obtain T0-generation MdDEWAX-overexpressing Arabidopsis.

[0115] RNA was extracted from the leaves of T0-generation MdDEWAX-overexpressing Arabidopsis, reverse-transcribed into cDNA, and RT-PCR amplification was performed. The primers were MdDEWAX-PRI-F and MdDEWAX-PRI-R in Table 1.

[0116] The results are as Figure 4 shown. The left figure is the electrophoresis result, and the right figure is the expression level. It can be seen that the expression levels of the three T0-generation MdDEWAX-overexpressing Arabidopsis lines MdDEWAX#1, MdDEWAX#2, and MdDEWAX#3 are higher than those of wild-type Arabidopsis, proving that the construction of overexpressing plants was successful.

[0117] The T0-generation MdDEWAX-overexpressing Arabidopsis lines MdDEWAX#1, MdDEWAX#2, and MdDEWAX#3 were sown to obtain purified T3-generation MdDEWAX-overexpressing Arabidopsis lines MdDEWAX#1, MdDEWAX#2, and MdDEWAX#3.

[0118] 2. Accumulation of epidermal wax crystals in Arabidopsis stems and leaves

[0119] The accumulation of epidermal wax crystals in the stems and leaves of Col-0 (denoted as Col in the figure) and the T3 generation of Arabidopsis thaliana transgenic lines overexpressing MdDEWAX, namely MdDEWAX#1, MdDEWAX#2, and MdDEWAX#3, was detected as follows:

[0120] Leaves and stems of the above-mentioned Arabidopsis thaliana lines at 5 weeks old were frozen in liquid nitrogen and then transferred to a freeze dryer, where they were vacuum-dried for 24 h at -80 °C and 110 Pa (FDU1110, 50 / 60 Hz, 1.7 kVA). Samples were observed using a scanning electron microscope (JSM-6610LV, JEOL) after ion sputtering.

[0121] Observation by scanning electron microscope revealed that, compared with Col, the number of wax crystals in the stem epidermis of the T3 generation of Arabidopsis thaliana transgenic lines overexpressing MdDEWAX, namely MdDEWAX#1, MdDEWAX#2, and MdDEWAX#3, decreased significantly ( Figure 5 A). Compared with Col, the wax crystals in the leaves of the T3 generation of Arabidopsis thaliana transgenic lines overexpressing MdDEWAX, namely MdDEWAX#1, MdDEWAX#2, and MdDEWAX#3, decreased slightly, but not as significantly as those in the stems ( Figure 5 B).

[0122] The above results indicate that MdDEWAX reduces the wax crystals in the stem epidermis and leaves of Arabidopsis thaliana.

[0123] 3. Detection of the expression levels of wax-related genes

[0124] RNA was extracted from the stems of Col-0 and the T3 generation of Arabidopsis thaliana transgenic lines overexpressing MdDEWAX, namely MdDEWAX#1, MdDEWAX#2, and MdDEWAX#3, and reverse-transcribed into cDNA. RT-PCR was performed to amplify wax-related genes AtCER1, AtECH, AtKCS1, AtMYB96, AtCER3, and AtSHINE3, with the primers shown in Table 1.

[0125] The results are shown in Figure 6 A. It can be seen that, compared with Col-0, the transcriptional levels of the above genes in the transgenic Arabidopsis thaliana were significantly down-regulated, indicating that MdDEWAX plays a negative regulatory role in wax accumulation.

[0126] 4. Detection of the total wax in Arabidopsis thaliana stems

[0127] To further determine the role of MdDEWAX in wax accumulation, the total wax in Arabidopsis thaliana stems was extracted and its components were analyzed.

[0128] Select the stems of Col-0 and the T3 generation of transgenic Arabidopsis thaliana lines MdDEWAX#1, MdDEWAX#2, and MdDEWAX#3 that have been transformed with MdDEWAX, measure their surface areas, immerse the plant materials in chloroform, perform rotary evaporation, and dissolve them again using a solvent. After the derivatization reaction, add the internal standard for injection, filter, and transfer to the injection vial. Analyze the composition and content of wax using triple quadrupole gas chromatography.

[0129] The gas chromatography uses temperature-programmed injection. The injection temperature is 50 °C, held for 2 minutes at 50 °C, then heated to 200 °C at a rate of 40 °C / min and held for 2 minutes at 200 °C, and then heated to 320 °C at a rate of 3 °C / min and held for 30 minutes at 320 °C. Use a capillary gas chromatograph (Agilent; 30 m HP-1, 0.32-mm i.d., df = 1 μm) and flame ionization detection technology to study the quantitative components of the mixture. The conditions of the gas chromatograph are the same as above, but when injecting, set the H2 carrier gas inlet pressure to 50 kPa, hold for 5 minutes, then increase to 150 kPa at a rate of 3 kPa / min, and hold at 150 kPa for 40 minutes. Manually integrate the peak areas and quantify the individual compounds against the internal standard. (Reference: Aharoni A, Dixit S, Jetter R, Thoenes E, van Arkel G, Pereira A. The SHINE clade of AP2 domain transcription factors activates wax biosynthesis, alters cuticle properties, and confers drought tolerance when overexpressed in Arabidopsis. Plant Cell. 2004 Sep;16(9):2463-80.)

[0130] The results are as Figure 6 shown in B. The total wax content of Col-0 is 1.36 times, 1.33 times, and 1.41 times that of the transgenic Arabidopsis thaliana lines MdDEWAX#1 / 2 / 3, respectively. This indicates that MdDEWAX reduces the total amount of wax in the plant stems.

[0131] Use gas chromatography-mass spectrometry to analyze the composition of wax.

[0132] The results show that the main components of wax are: alkanes, alcohols, fatty acids, aldehydes, ketones, and esters ( Figure 6C). Compared with Col, the contents of C29 alkane in the T3 generation of MdDEWAX transgenic Arabidopsis lines MdDEWAX#1, MdDEWAX#2, and MdDEWAX#3 were reduced by 30.03%, 24.70%, and 33.56% respectively ( Figure 6 D). The contents of C28, C29, and C30 alcohols in the T3 generation of MdDEWAX transgenic Arabidopsis lines MdDEWAX#1, MdDEWAX#2, and MdDEWAX#3 were significantly reduced, and the total alcohol contents were reduced by 27.43%, 32.50%, and 29.89% respectively ( Figure 6 E). Among the fatty acids, the content of C16 increased slightly, the content of C18 decreased significantly, but the total amount of fatty acids decreased ( Figure 6 F). The reduction of aldehyde content was mainly concentrated in C30, which decreased by 35.84%, 35.39%, and 31.43% respectively ( Figure 6 G). The contents of C29 ketone decreased by 26.13%, 24.11%, and 26.10% respectively ( Figure 6 H). Among the esters, the contents of C42 and C44 decreased more significantly. The content of C42 decreased by 33.11%, 26.98%, and 26.80% respectively, and the content of C44 decreased by 23.41%, 22.22%, and 32.72% respectively ( Figure 6 I).

[0133] The above data were summarized in Table 2.

[0134] Table 2 shows the cutin wax components (μg / dm 2 )

[0135]

[0136]

[0137] II. The cutin permeability of MdDEWAX transgenic Arabidopsis is improved

[0138] Wax directly determines the permeability of the cutin. Since MdDEWAX negatively regulates wax accumulation, the cutin permeability of Col-0 (denoted as Col in the figure) and the T3 generation of MdDEWAX transgenic Arabidopsis lines MdDEWAX#1, MdDEWAX#2, and MdDEWAX#3 was studied.

[0139] 1. Toluidine blue staining

[0140] The rosette leaves, stems, and inflorescences of Arabidopsis of the above-mentioned lines were cut off, soaked in 0.05% toluidine blue staining solution at room temperature for 4 hours, then rinsed three times with deionized water and photographed.

[0141] The results showed that, compared with Col-0, the inflorescences, stems and leaves of MdDEWAX transgenic Arabidopsis thaliana MdDEWAX#1, MdDEWAX#2, and MdDEWAX#3 were more easily stained with toluidine blue ( Figure 7 A-C).

[0142] 2. Chlorophyll leaching experiment

[0143] To further investigate whether MdDEWAX altered the permeability of the cuticle, a chlorophyll leaching experiment was conducted. The rosette leaves of Arabidopsis thaliana of each of the above-mentioned lines grown for 5 weeks were weighed and immersed in 30 mL of 80% absolute ethanol. The absorbance was measured at wavelengths of 664 and 647 nm at 10, 30, 60, 120, and 180 minutes respectively. Chlorophyll content = 7.93 * A664 + 19.53 * A647.

[0144] The results showed that, compared with Col-0, chlorophyll was extracted from the leaves of MdDEWAX transgenic plants MdDEWAX#1, MdDEWAX#2, and MdDEWAX#3 at a faster rate ( Figure 7 E).

[0145] These results indicate that MdDEWAX significantly increased the permeability of the cuticle of Arabidopsis thaliana rosette leaves.

[0146] 3. Water loss rate

[0147] The water loss rates of MdDEWAX transgenic lines and Col-0 were measured: The rosette leaves of Arabidopsis thaliana of each of the above-mentioned lines at 5 weeks old were soaked in deionized water for 1 hour under dark conditions, and then the excess water on the leaf surface was blotted with filter paper. The leaves were placed in the dark, and the leaf weight was measured every 20 minutes within 120 minutes, and the water loss was calculated.

[0148] The results are as Figure 7 shown in D. Compared with the leaves of Col, the water loss rates of the leaves of MdDEWAX transgenic MdDEWAX#1, MdDEWAX#2, and MdDEWAX#3 were faster, indicating that MdDEWAX increased the leaf water loss rate through a strong effect on cuticle permeability and has a potential role in drought stress.

[0149] III. MdDEWAX transgenic Arabidopsis thaliana enhances the sensitivity of plants to ABA and reduces drought resistance

[0150] 1. Detection of ABA sensitivity

[0151] After growing T3-generation transgenic Arabidopsis thaliana lines overexpressing MdDEWAX, namely MdDEWAX#1 (which can also be denoted as 35S::MdDEWAX#1 in the figure), MdDEWAX#2 (which can also be denoted as 35S::MdDEWAX#2 in the figure), MdDEWAX#3 (which can also be denoted as 35S::MdDEWAX#3 in the figure), and Col-0 (denoted as Col in the figure) on normal 1 / 2 MS medium for 5 days, they were respectively transferred to 1 / 2 MS medium supplemented with 10 μM ABA or 6% (mass percentage) PEG and grown for 14 days, and then the root lengths of the seedlings were measured.

[0152] The results are shown in Figure 8 Figures 5A and 5B. It can be seen that under normal conditions, there was no significant difference in the root lengths between MdDEWAX transgenic Arabidopsis thaliana and Col-0 Arabidopsis thaliana. However, under ABA and PEG treatments, the root lengths of MdDEWAX transgenic plants were significantly shorter than those of Col, which confirmed that MdDEWAX increased the sensitivity to ABA and negatively regulated drought stress.

[0153] 2. Drought treatment

[0154] The T3-generation transgenic Arabidopsis thaliana lines overexpressing MdDEWAX, namely MdDEWAX#1, MdDEWAX#2, MdDEWAX#3, and Col-0 Arabidopsis thaliana, were grown under normal conditions for 15 days and then subjected to drought treatment. After 14 days of water deprivation until the leaves withered and turned yellow, their phenotypes were observed and relevant physiological indices were measured. The normal condition culture was used as a control. The drought treatment was as follows: Arabidopsis thaliana seedlings were grown in a 1:1 mixture of nutrient soil and vermiculite. After the plants grew normally for 15 days, the seedlings were subjected to a 14-day drought treatment until the leaves withered and turned yellow.

[0155] The physiological indices included malondialdehyde (MDA), hydrogen peroxide (H2O2), and superoxide anion (O 2- ), and were detected using kits provided by Kemin Bio. The anthocyanin content was measured according to the method of An et al. (2018).

[0156] The results are shown in Figure 9 Figure 6. Col-0 (also denoted as col in the figure) and the T3-generation transgenic Arabidopsis thaliana lines overexpressing MdDEWAX, namely MdDEWAX#1, MdDEWAX#2, MdDEWAX#3, had the same growth status under normal conditions. However, compared with Col-0, the leaves of MdDEWAX transgenic Arabidopsis thaliana lines MdDEWAX#1, MdDEWAX#2, MdDEWAX#3 showed severe leaf withering and anthocyanin accumulation after drought treatment ( Figure 9 Figure 6A).

[0157] Meanwhile, compared with Col, transgenic plants MdDEWAX#1, MdDEWAX#2, and MdDEWAX accumulated more malondialdehyde and reactive oxygen species under drought conditions ( Figure 9 B-D), indicating that their cell membranes were severely damaged. The increased accumulation of the stress-responsive substance anthocyanin indicates an increased degree of stress ( Figure 9 E).

[0158] These results indicate that MdDEWAX reduces the drought resistance of plants.

[0159] Example 3. Application of MdDEWAX in regulating apple functions

[0160] I. MdDEWAX reduces the accumulation of wax in apple fruits

[0161] 1. Construction of MdDEWAX transgenic apples

[0162] 1) Construction of the overexpression vector MdDEWAX-IL60 and the silencing vector MdDEWAX-TRV

[0163] The overexpression vector MdDEWAX-IL60 is a vector obtained by replacing the fragment between the SalI and XbaI restriction enzyme sites of the IL60-2 vector (reference: Peretz, Y., Mozes-Koch, R., Akad, F., Tanne, E., Czosnek, H., & Sela, I. (2007). A universal expression / silencing vector in plants. Plant physiology, 145(4), 1251-1263., named IL60 in the text) with the MdDEWAX gene shown in Sequence 1. This vector overexpresses the MdDEWAX gene.

[0164] The silencing vector MdDEWAX-TRV is a vector obtained by replacing the fragment between the BamHI and SmaI restriction enzyme sites of the TRV-2 vector (reference: Bachan, S., & Dinesh-Kumar, S.P. (2012). Tobacco rattle virus(TRV)-based virus-induced gene silencing. Antiviral resistance in plants: methods and protocols, 83-92., named TRV in the text) with the base sequence shown at positions 510-690 of Sequence 1. This vector silences the MdDEWAX gene.

[0165] 2). Transient transformation into apple fruits

[0166] The above-mentioned silencing vector MdDEWAX-TRV and the empty vector TRV-2 were transformed into Agrobacterium tumefaciens LBA4404 to obtain the corresponding recombinant bacteria MdDEWAX-TRV and recombinant bacteria TRV-2.

[0167] Select bagged apples with uniform fruit size and no mechanical damage on the surface (“Fuji”, also referred to as wild-type apples below). After diluting the bacterial liquid or plasmid with the infection solution (formula: 100 μM Ace, 10 mM MgCl2, and 10 mM MES), inject it into the surface of the apple fruit with a disposable sterile syringe, and finally place it in the dark for subsequent experiments. For the specific method, refer to Zhang et al. (2023).

[0168] The above plasmids are the overexpression vector MdDEWAX-IL60 and the empty vector IL60-2, with a concentration of 10 μg / ml.

[0169] The above bacterial liquid is the recombinant bacteria MdDEWAX-TRV and recombinant bacteria TRV-2, with a concentration of OD 600 value of 0.6.

[0170] Extract the RNA from the tissue around the injection site of the apple three days after injection, reverse transcribe it to obtain cDNA, and perform RT-PCR amplification. The primers are MdDEWAX-PRI-F and MdDEWAX-PRI-R in Table 1.

[0171] The results are as Figure 10 shown. It can be seen that compared with the apples injected with the empty vector IL60-2 (denoted as IL60 in the figure), the expression level of the MdDEWAX gene in the apples injected with the overexpression vector MdDEWAX-IL60 (denoted as MdDEWAX-IL60 in the figure) is higher; compared with the apples injected with the empty vector TRV-2 (denoted as TRV in the figure), the expression level of the MdDEWAX gene in the apples injected with the silencing vector MdDEWAX-TRV (denoted as MdDEWAX-TRV in the figure) is lower.

[0172] A total of 3 apples with transient transformation of the overexpression vector MdDEWAX-IL60 were obtained, denoted as MdDEWAX-IL60#1, MdDEWAX-IL60#2, MdDEWAX-IL60#3; a total of 3 apples with transient transformation of the silencing vector MdDEWAX-TRV were obtained, denoted as MdDEWAX-TRV#1, MdDEWAX-TRV#2, MdDEWAX-TRV#3.

[0173] II. MdDEWAX reduces the wax accumulation in apple fruits

[0174] 1. Detection of wax accumulation in apple fruits

[0175] The peels of apples MdDEWAX-IL60#1, MdDEWAX-IL60#2, MdDEWAX-IL60#3 that had been injected with the overexpression vector MdDEWAX-IL60 for ten days, apples MdDEWAX-TRV#1, MdDEWAX-TRV#2, MdDEWAX-TRV#3 that had been injected with the silencing vector MdDEWAX-TRV for ten days, apples injected with the empty vector TRV-2 for ten days (denoted as IL60 in the figure), and apples injected with the empty vector IL60-2 for ten days (denoted as TRV in the figure) were frozen in liquid nitrogen and then transferred to a freeze dryer, where they were vacuum dried at -80 °C and 110 Pa (FDU1110, 50 / 60 Hz, 1.7 kVA) for 24 h. After ion sputtering, the samples were observed using a scanning electron microscope (JSM-6610LV, JEOL).

[0176] The results are as Figure 11 shown. Compared with the IL60 control group, the fruits of MdDEWAX-IL60 transiently transfected apples MdDEWAX-IL60#1, MdDEWAX-IL60#2, MdDEWAX-IL60#3 accumulated less wax crystals ( Figure 11 A). Compared with the TRV control, the fruits of MdDEWAX-TRV transiently transfected apples MdDEWAX-TRV#1, MdDEWAX-TRV#2, MdDEWAX-TRV#3 accumulated more wax crystals, which were connected into blocks ( Figure 11 B).

[0177] 2. Detect the expression changes of genes related to wax synthesis around the injection site

[0178] RNA was extracted from the peels of apples injected with the empty vector IL60-2 for ten days (denoted as IL60 in the figure), apples injected with the empty vector TRV-2 for ten days (denoted as TRV in the figure), MdDEWAX-IL60#1 (denoted as MdDEWAX-IL60 in the figure), and MdDEWAX-TRV#1 (denoted as MdDEWAX-TRV in the figure) around the injection site of the apples. cDNA was obtained by reverse transcription, and RT-PCR was performed to amplify the wax-related genes MdKCS1, MdLACS2, and MdSHINE2, and the primers are shown in Table 1.

[0179] The results are as Figure 12 shown. It can be seen that overexpression of MdDEWAX reduces the expression of MdKCS1, MdLACS2, and MdSHINE2, while inhibition of MdDEWAX significantly increases the expression of these genes.

[0180] These results indicate that MdDEWAX inhibits the accumulation of wax in apple fruits, and inhibiting the expression of MdDEWAX can increase the accumulation of wax in apple fruits.

[0181] 3. Detection of Total Wax on Apple Skin

[0182] Select the apple peels of apples injected with empty vector IL60-2 for ten days (denoted as IL60 in the figure), apples injected with empty vector TRV-2 for ten days (denoted as TRV in the figure), MdDEWAX-IL60#1 (denoted as MdDEWAX-IL60 in the figure), and MdDEWAX-TRV#1 (denoted as MdDEWAX-TRV in the figure). Measure their surface areas. Immerse the plant materials in chloroform, perform rotary evaporation, and dissolve them again with a solvent. After the derivatization reaction, add the sample internal standard, filter, and transfer to a sample vial. Similar to item 4 in Example 2, use triple quadrupole gas chromatography to analyze the composition and content of wax. Use gas chromatography-mass spectrometry to analyze the composition of wax.

[0183] The results are as Figure 13 shown. Overexpression of MdDEWAX results in a 15.87% decrease in the total wax content of apples. On the contrary, inhibition of MdDEWAX results in a 19.26% increase in the total wax content ( Figure 13 A).

[0184] Gas chromatography-mass spectrometry analysis of the specific components of wax reveals that they are mainly composed of alkanes, alcohols, aldehydes, fatty acids, and triterpenoids. Overexpression or silencing of MdDEWAX will respectively lead to different degrees of decrease or increase in the content of each component ( Figure 13 B). For example, overexpression of MdDEWAX reduces the content of triterpenoids by 18.66%, while inhibition of this gene increases the content of triterpenoids by 21.51%. In addition, the main components with the largest differences include C29 alkane, C29 alcohol, C30 aldehyde, and C30 fatty acid, which are reduced by 18.33%, 15.96%, 11.50%, and 31.04% respectively in the fruits overexpressing MdDEWAX, while increased by 15.93%, 18.50%, 22.97%, and 30.99% respectively in the fruits with silenced MdDEWAX ( Figure 13 C-F).

[0185] Summarize each data as shown in Table 3.

[0186] Table 3 shows the wax components of apple peel (μg / cm 2 )

[0187] IL60 MdDEWAX - IL60 TRV MdDEWAX - TRV alkane C27 26.43 23.89 19.16 24.01 C28 26.46 26.96 25.37 28.83 C29 210.28 171.74 205.83 238.62 C30 25.01 22.84 25.40 32.81 C31 25.86 18.57 20.99 31.98 total amount 314.04 264.00 296.74 356.25 alcohol C26 21.20 20.73 20.89 22.79 C28 21.06 19.07 21.97 24.26 C29 84.32 70.86 89.07 105.55 C30 32.21 27.26 27.96 31.55 total amount 158.79 137.92 159.89 184.15 aldehyde C28 11.06 9.07 9.97 11.92 C29 3.65 3.86 3.73 4.55 C30 48.88 43.26 44.63 54.88 total amount 63.59 56.19 58.33 71.35 fatty acid C26 17.23 14.11 17.95 18.02 C28 14.90 14.39 13.72 16.50 C30 34.60 23.86 29.82 39.06 total amount 66.73 52.36 61.49 73.58 triterpenoid 209.28 170.23 200.25 243.32 others 6.56 8.31 7.21 6.22 total wax 818.99 689.02 783.92 934.87

Claims

1. A protein, which is any one of A1)-A4) below: A1) A protein consisting of the amino acid sequence shown in Sequence 2; A2) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein shown in Sequence 2; A3) A protein obtained by substituting and / or deleting and / or adding one or several amino acid residues to the amino acid sequence shown in Sequence 2 and having the same function; A4) A protein having a homology of more than 99%, more than 95%, more than 90%, more than 85% or more than 80% with the amino acid sequence defined in any one of A1)-A3) and having the same function.

2. A nucleic acid molecule encoding the protein according to claim 1.

3. The nucleic acid molecule according to claim 2, wherein: The nucleic acid molecule is any one of the following: B1) The DNA molecule shown in Sequence 1; B2) A DNA molecule having a homology of more than 98% with the DNA sequence defined in B1) and encoding the same functional protein; B3) A DNA molecule that hybridizes with the DNA sequence defined in B1) under stringent conditions and encodes the same functional protein; B4) A DNA molecule having a homology of more than 90% with the DNA sequence defined in B1) and encoding the same functional protein.

4. An expression cassette, recombinant vector or recombinant microorganism containing the nucleic acid molecule according to claim 2 or 3.

5. Use of the protein according to claim 1 or the nucleic acid molecule according to claim 2 or 3 or the expression cassette, recombinant vector or recombinant microorganism according to claim 4 in any of the following: C1) Reducing the accumulation of wax crystals in plant tissues; C2) Reducing the transcriptional level of wax-related genes in plants; C3) Reducing the wax content in plant tissues; C4) Increasing the permeability of the cuticle of plant leaves; C5) Increasing the water loss rate of plant leaves; C6) Enhancing the sensitivity of plants to ABA; C7) Regulating the drought resistance of plants; C8) Cultivating plants with low accumulation of wax crystals in tissues; C9) Cultivating plants with low wax content in tissues; C10) Cultivating plants with high permeability of the cuticle of leaves; C11) Cultivating plants with high water loss rate of leaves; C12) Cultivating plants with high sensitivity to ABA; C13) Cultivating plants with low drought resistance; C14) Acting as or preparing a regulatory factor for negatively regulating wax biosynthesis in plant tissues.

6. Use of a substance that reduces the activity or content of the protein according to claim 1, or inhibits the expression of the nucleic acid molecule according to claim 2 or 3, in any of the following: D1) Enhancing the accumulation of wax crystals in plant tissues; D2) Enhancing the transcriptional level of wax-related genes in plants; D3) Enhancing the wax content in plant tissues; D4) Enhancing the drought resistance of plants.

7. A method for cultivating plants with low wax crystal accumulation in tissues, low wax content in tissues, high cuticular permeability of leaves, high leaf water loss rate, high ABA sensitivity and / or low drought resistance, comprising the following steps: enhancing the activity or content of the protein according to claim 1 in a target plant, or enhancing the expression level of the nucleic acid molecule according to claim 2 or 3 in the target plant, to obtain a transgenic plant; The transgenic plant has at least one of the following characteristics: E1) The amount of wax crystal accumulation in the transgenic plant tissues is less than that of the target plant; E2) The wax content in the transgenic plant tissues is less than that of the target plant; E3) The cuticular permeability of the transgenic plant leaves is greater than that of the target plant; E4) The leaf water loss rate of the transgenic plant is higher than that of the target plant; E5) The transgenic plant has a higher sensitivity to ABA than the target plant; E6) The transgenic plant has lower drought resistance than the target plant.

8. A method for cultivating plants with high wax crystal accumulation and / or high wax content in tissues, comprising the following steps: reducing the activity or content of the protein according to claim 1 in a target plant, or reducing the expression level of the nucleic acid molecule according to claim 2 or 3 in the target plant, to obtain a transgenic plant; The transgenic plant has at least one of the following characteristics: F1) The amount of wax crystal accumulation in the transgenic plant tissues is greater than that of the target plant; F2) The wax content in the transgenic plant tissues is greater than that of the target plant.

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