Application of mulberry transcription factor MaMYB93 or its coding gene in improving drought resistance of plants

Abstract

This invention discloses the application of mulberry transcription factor MaMYB93 or its encoding gene in improving plant drought resistance, belonging to the field of plant stress resistance genetic engineering. This invention discloses the application of mulberry transcription factor MaMYB93 or its encoding gene in promoting plant suberin production and increasing the expression levels of drought-resistant genes, and provides methods for improving plant drought resistance and enhancing root suberization. This invention is the first to discover that mulberry transcription factor MaMYB93 and its encoding gene are related to plant drought resistance, and can enhance plant tolerance to drought stress by increasing the expression levels of drought-resistant genes and upregulating suberin synthesis.

Description

Technical Field

[0001] This invention relates to the field of plant stress resistance genetic engineering, specifically to the application of mulberry transcription factor MaMYB93 or its encoding gene in improving plant drought resistance. Background Technology

[0002] Drought is one of the major abiotic stresses that inhibit plant growth and development. The economic losses it causes often exceed the sum of all other abiotic stresses, making it a significant factor threatening the economic and ecological benefits of agriculture and forestry. As a key organ for plants to sense water stress and absorb mineral nutrients, the physiological characteristics of the root system directly affect the plant's drought tolerance. Studies have shown that root suberin is one of the key apoplastic barriers controlling short-distance radial water transport, and its deposition level directly affects the root system's water absorption capacity and the plant's drought tolerance.

[0003] In recent years, the role of transcription factors in plant stress response and root development regulation has attracted much attention. Among them, MYB family transcription factors have been shown to be widely involved in regulating suberin deposition in plants. In Arabidopsis thaliana, suberin deposition in the root endodermis is mainly regulated by redundancy of AtMYB41, AtMYB53, AtMYB92, and AtMYB93; in rice, OsMYB39a, OsMYB41, OsMYB92a, and OsMYB92b synergistically regulate suberin deposition in the root endodermis. In addition, MdMYB93 and MdMYB68 in apple participate in regulating the synthesis of suberin in pericarp scars, and AcMYB41 and AcMYB107 in kiwifruit have also been shown to have similar functions.

[0004] Wubu mulberry is a superior mulberry variety cultivated and selected over hundreds of years, mainly distributed in Yan'an City in northern Shaanxi Province, as well as Wubu County and Suide County in Yulin City, my country. This variety has a well-developed root system and exhibits strong adaptability and resistance to the arid, cold, and barren growing conditions of the Loess Plateau, making it an important mulberry germplasm resource in the region. However, its root corking response and regulation under drought stress have not yet been reported. This gap limits a deeper understanding of the molecular mechanisms of drought resistance in mulberry and also restricts the breeding of drought-resistant varieties of mulberry and other perennial trees. Summary of the Invention

[0005] The purpose of this invention is to compensate for the deficiencies in the regulation of root suberization response and drought resistance by mulberry MYB transcription factor, and to provide an application of mulberry transcription factor MaMYB93 or its encoding gene in improving plant drought resistance.

[0006] In a first aspect, the present invention provides an application of overexpression of mulberry transcription factor MaMYB93 or its encoding gene in improving plant drought resistance, wherein the amino acid sequence of mulberry transcription factor MaMYB93 is shown in SEQ ID NO:1, and the encoding gene of mulberry transcription factor MaMYB93 is... MaMYB93 The nucleotide sequence is shown in SEQ ID NO:2.

[0007] Secondly, the present invention provides an application of overexpression of mulberry transcription factor MaMYB93 or its encoding gene in improving the expression level of genes related to drought resistance in plants.

[0008] Preferably, the plant drought resistance-related genes include MaCYP86B1 (XM_010095782.2) MaKCS1 (XM_010102367.2) MaABCG1 (XM_010103205.2) MaCASP1 (XM_010095469.2) MaCOMT (XM_010113910.2) One or more of the genes.

[0009] Thirdly, the present invention provides an application of overexpression of mulberry transcription factor MaMYB93 or its encoding gene in promoting the formation of cork in plants.

[0010] Fourthly, this invention provides an application of overexpressing the mulberry transcription factor MaMYB93 or its encoding gene in the creation of plant germplasm or the breeding of drought-resistant plant varieties.

[0011] Preferably, the above-mentioned plants include Arabidopsis thaliana, cash crops (such as rice, wheat, corn, etc.) and economic forest trees (such as mulberry, poplar, apricot, fig, apple, etc.).

[0012] Preferably, the above-mentioned plants exhibit better drought resistance compared to wild-type plants under drought stress induced by cessation of watering and drought stress simulated by the use of 100-300 mM mannitol.

[0013] Fifthly, the present invention provides a nucleic acid molecule of the above-mentioned mulberry transcription factor MaMYB93.

[0014] In a sixth aspect, the present invention provides an expression vector comprising the above-described nucleic acid molecule.

[0015] In a seventh aspect, the present invention provides a host cell comprising the above-described nucleic acid molecule or the above-described expression vector.

[0016] Eighthly, the present invention provides a method for improving the drought resistance of plants, comprising the following steps: overexpressing the mulberry transcription factor MaMYB93 or its encoding gene in a target plant.

[0017] In a ninth aspect, the present invention provides a method for enhancing the corking of plant roots, comprising the following steps: overexpressing mulberry transcription factor MaMYB93 or its encoding gene in a target plant.

[0018] Compared with the prior art, the present invention has the following advantages: This invention is the first to discover that the mulberry transcription factor MaMYB93 and its encoding gene are related to plant drought tolerance. It can enhance plant tolerance to drought stress by increasing the expression levels of drought-related genes and upregulating the synthesis of suberin. The drought resistance of plants can be directionally modified using the mulberry transcription factor MaMYB93 and its encoding gene, providing a reliable gene resource for molecular breeding of plants to withstand stress and a theoretical basis for cultivating new drought-resistant varieties. Attached Figure Description

[0019] Figure 1 In Example 1 MaMYB93 Agarose gel electrophoresis image of the gene coding region amplification products; Figure 2 This is a laser confocal microscope image of MaMYB93 subcellular localization in Example 2; Figure 3 This is a graph showing the results of MaMYB93 transcriptional activity detection in Example 2; Figure 4 This is a diagram showing the results of a yeast one-hybrid experiment involving MaMYB93 and related genes in Example 2. Figure 5 This is a diagram showing the results of the dual-luciferase assay of MaMYB93 and related genes in Example 2; Figure 6 The figure shows the EMSA experimental results in Example 2; Figure 7 This is an agarose gel electrophoresis image of the transgenic Arabidopsis identified in Example 2; Figure 8 This is a diagram showing the real-time quantitative PCR results of transgenic Arabidopsis thaliana in Example 2; Figure 9 The results of cork staining in transgenic Arabidopsis thaliana in Example 2; Figure 10 This is a gas chromatography-mass spectrometry (GC-MS) analysis of the transgenic Arabidopsis thaliana in Example 2. Figure 11 This is a diagram showing the results of treating Arabidopsis thaliana seedlings with 200mM mannitol in Example 3. Figure 12The image shows the results of soil drought stress treatment on Arabidopsis seedlings in Example 3. Detailed Implementation

[0020] To better understand the present invention, the following embodiments are further illustrations of the present invention, but the content of the present invention is not limited to the following embodiments.

[0021] Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional methods. The reagents and materials used in the embodiments are all commercially available, and the quantitative experiments involved in the embodiments are all performed in at least three replicates.

[0022] The white mulberry used in this invention ( Morus alba The variety “Wubao Mulberry” is preserved by the National Mulberry Germplasm Resource Center (Zhenjiang).

[0023] The Arabidopsis thaliana ecotype used was Columbia-0, preserved in the Mulberry Physiology and Cultivation Laboratory of Jiangsu University of Science and Technology. Unless otherwise specified, Arabidopsis thaliana seeds were cultured on 1 / 2 MS medium, vernalized at 4℃ in the dark for 2-3 days, and then transferred to the culture room. Seven days later, seedlings were transplanted into plastic pots (7cm×7cm×9cm), with 4 seedlings per pot. After transplanting, a 1:1 mixture of heat-treated potting soil and vermiculite was used for cultivation. Harvested seeds were dried at 28℃ for 2 days before storage or further germination on a substrate. The culture room was maintained at 22℃ during the day and 18℃ at night, with a 16-hour light-8-hour dark environment.

[0024] The subcellular localization vectors used in this invention are the overexpression vector pCambia1300-GFP, yeast expression vectors pGBKT7, pGADT7, pHIS2, dual-luciferase reporter vectors pGreenII-0800-luc, pGreenII-62-SK, and protein expression vector pMAL-c5x. DH5α Escherichia coli, Rosetta Escherichia coli, yeast strain AH109 and Y187 Agrobacterium tumefaciens GV3101 All of these can be obtained through commercial channels.

[0025] Example 1 Mulberry tree MaMYB93 Gene cloning, recombinant plasmid construction and transformation 1. Mulberry tree MaMYB93 Cloning of genes Total RNA was extracted from the roots of *Morus alba* wuburgensis* using the Omega Plant RNA Extraction Kit (R6827, Omega Bio-Tek, USA). The total RNA was reverse transcribed (HiScriptoIll 1st Strand cDNASynthesis Kit (+gDNA wiper), R312, Novizan, China) to obtain cDNA. Using the cDNA as a template, PCR amplification was performed using primer pairs MaMYB93-F and MaMYB93-R (2×Phanta Flash Master Mix, P520, Novizan, China). The amplified products were subjected to agarose gel electrophoresis. The gel containing the target band of approximately 1000 bp, consistent with the expected size, was excised, and the amplified fragment was recovered using a gel (SanPrep Column DNA Gel Extraction Kit, B518131, Sangon Biotech, China) to obtain the purified amplified product. The amplified product was ligated to the pCE2 TA / Blunt-Zero vector (5 min TA / Blunt-ZeroCloning Kit, C601, Novizan, China) and then sequenced. The sequence of primer MaMYB93-F is shown in SEQ ID NO:3, and the sequence of primer MaMYB93-R is shown in SEQ ID NO:4.

[0026] Agarose gel electrophoresis results are as follows Figure 1 As shown, lanes 2 and 3 are the target fragments obtained by PCR amplification. It can be seen that a single specific band appears at around 1000bp, which is about the same size as the expected amplified fragment, indicating that the target fragment has been amplified.

[0027] Sequencing results MaMYB93 The coding region of the gene is 1029 bp in length, and its sequence is shown in SEQ ID NO:2. It encodes 342 amino acids, and its sequence is shown in SEQ ID NO:1. Its molecular weight is 38.1 kDa.

[0028] 2. Construction and transformation of recombinant plasmids 2.1 Overexpression of recombinant plasmids The overexpression recombinant plasmid pCambia1300- was constructed using seamless cloning technology. MaMYB93 -GFP: Use B amHThe linearized pCambia1300-GFP vector was digested with enzymes I and SalI. Using the purified amplification product obtained in step 1 as a template, PCR amplification was performed using primers MaMYB93-Loc-F and MaMYB93-Loc-R to obtain the target DNA fragment. The obtained linearized pCambia1300-GFP vector and the target DNA fragment were then subjected to a recombination ligation reaction (Ready-to-Use Seamless Cloning Kit, B632219, Sangon Biotech, China) to obtain the recombination ligation product.

[0029] Add 10 μl of the recombinant ligation product to 100 μl of DH5α E. coli competent cells (B528413, Sangon Biotech, China) thawed on ice. Incubate on ice for 30 min, then incubate at 42°C for 90 s. After standing on ice for 2 min, add 700 μl of antibiotic-free LB broth and incubate at 37°C with shaking for 1 h. Collect the cells at 6000 rpm and resuspend them in 100 μl of ddH2O. Spread the bacterial culture evenly on LB agar plates containing kanamycin, and select healthy single colonies for sequencing. Extract plasmids from correctly sequenced single colonies (SanPrep Column Plasmid Mini-Preps Kit, B518191, Sangon Biotech, China) to obtain the recombinant plasmid pCambia1300- MaMYB93 -GFP. The sequence of primer MaMYB93-Loc-F is shown in SEQ ID NO:5, and the sequence of primer MaMYB93-Loc-R is shown in SEQ ID NO:6.

[0030] The recombinant plasmid was transformed into Agrobacterium GV3101 to obtain pCambia1300- MaMYB93 -GFP recombinant Agrobacterium.

[0031] 2.2 Yeast expression vector pGBKT7- MaMYB93 Build The construction method in this step is the same as in 2.1, except that the pGBKT7 vector is digested with SmaI, and the primers are MaMYB93-BK-F and MaMYB93-BK-R to obtain the recombinant plasmid pGBKT7- MaMYB93 The sequence of primer MaMYB93-BK-F is shown in SEQ ID NO:7, and the sequence of primer MaMYB93-BK-R is shown in SEQ ID NO:8.

[0032] 2.3 Yeast expression vector pGADT7- MaMYB93 Build The construction method in this step is the same as in 2.1, except that the pGADT7 vector is digested with SfiI enzyme, and the primers are MaMYB93-AD-F and MaMYB93-AD-R to obtain the recombinant plasmid pGADT7- MaMYB93 The sequence of primer MaMYB93-AD-F is shown in SEQ ID NO:9, and the sequence of primer MaMYB93-AD-R is shown in SEQ ID NO:10.

[0033] 2.4 Construction of yeast expression vectors for related genes DNA was extracted from the roots of *Morus alba* wuburgensis (FastPure Plant DNA Isolation Mini Ki, tDC104, Novizan, China).

[0034] The construction method in this step is the same as in 2.1, except that the pHIS2 vector is digested with SacI, using Wubu mulberry root DNA as a template, and primers MaCYP86B1pro-HIS-F and MaCYP86B1pro-HIS-R to obtain the recombinant plasmid pHIS2-MaCYP86B1pro. The sequence of primer MaCYP86B1pro-HIS-F is shown in SEQ ID NO:11, and the sequence of primer MaCYP86B1pro-HIS-R is shown in SEQ ID NO:12.

[0035] The construction method in this step is the same as in 2.1, except that the pHIS2 vector is digested with SacI, using Wubu mulberry root DNA as a template, and primers MaKCS1pro-HIS-F and MaKCS1pro-HIS-R to obtain the recombinant plasmid pHIS2-MaKCS1pro. The sequence of primer MaKCS1pro-HIS-F is shown in SEQ ID NO:13, and the sequence of primer MaKCS1pro-HIS-R is shown in SEQ ID NO:14.

[0036] The construction method in this step is the same as in 2.1, except that the pHIS2 vector is digested with SacI, using Wubu mulberry root DNA as a template, and primers MaABCG1pro-HIS-F and MaABCG1pro-HIS-R to obtain the recombinant plasmid pHIS2-MaABCG1pro. The sequence of primer MaABCG1pro-HIS-F is shown in SEQ ID NO:15, and the sequence of primer MaABCG1pro-HIS-R is shown in SEQ ID NO:16.

[0037] The construction method in this step is the same as in 2.1, except that the pHIS2 vector is digested with SacI, using Wubu mulberry root DNA as a template, and primers MaCASP1pro-HIS-F and MaCASP1pro-HIS-R to obtain the recombinant plasmid pHIS2-MaCASP1pro. The sequence of primer MaCASP1pro-HIS-F is shown in SEQ ID NO:17, and the sequence of primer MaCASP1pro-HIS-R is shown in SEQ ID NO:18.

[0038] The construction method in this step is the same as in 2.1, except that the pHIS2 vector is digested with SacI, using Wubu mulberry root DNA as a template, and primers MaCOMTpro-HIS-F and MaCOMTpro-HIS-R to obtain the recombinant plasmid pHIS2-MaCOMTpro. The sequence of primer MaCOMTpro-HIS-F is shown in SEQ ID NO:19, and the sequence of primer MaCOMTpro-HIS-R is shown in SEQ ID NO:20.

[0039] 2.5 Construction of Dual-Luciferase Reporter Vector The construction method in this step is the same as in 2.1, except that the pGreenII-0800-luc vector is digested with SmaI, using Wubu mulberry root DNA as a template, and primers MaCYP86B1pro-LUC-F and MaCYP86B1pro-LUC-R to obtain the recombinant plasmid pGreenII-0800-luc-MaCYP86B1pro. This recombinant plasmid is then transformed into Agrobacterium GV3101 to obtain the pGreenII-0800-luc-MaCYP86B1pro recombinant Agrobacterium. The sequence of primer MaCYP86B1pro-LUC-F is shown in SEQ ID NO:21, and the sequence of primer MaCYP86B1pro-LUC-R is shown in SEQ ID NO:22.

[0040] The construction method in this step is the same as in 2.1, except that the pGreenII-0800-luc vector is digested with SmaI, using Wubu mulberry root DNA as a template, and primers MaKCS1pro-LUC-F and MaKCS1pro-LUC-R to obtain the recombinant plasmid pGreenII-0800-luc-MaKCS1pro. This recombinant plasmid is then transformed into Agrobacterium GV3101 to obtain the pGreenII-0800-luc-MaKCS1pro recombinant Agrobacterium. The sequence of primer MaKCS1pro-LUC-F is shown in SEQ ID NO:23, and the sequence of primer MaKCS1pro-LUC-R is shown in SEQ ID NO:24.

[0041] The construction method in this step is the same as in 2.1, except that the pGreenII-0800-luc vector is digested with SmaI, using Wubu mulberry root DNA as a template, and primers MaABCG1pro-LUC-F and MaABCG1pro-LUC-R to obtain the recombinant plasmid pGreenII-0800-luc-MaABCG1pro. This recombinant plasmid is then transformed into Agrobacterium GV3101 to obtain the pGreenII-0800-luc-MaABCG1pro recombinant Agrobacterium. The sequence of primer MaABCG1pro-LUC-F is shown in SEQ ID NO:25, and the sequence of primer MaABCG1pro-LUC-R is shown in SEQ ID NO:26.

[0042] The construction method in this step is the same as in 2.1, except that the pGreenII-62-SK vector is digested with SmaI, and the primers are MaMYB93-0800-F and MaMYB93-0800-R to obtain the recombinant plasmid pGreenII-62-MaMYB93. The recombinant plasmid is then transformed into Agrobacterium GV3101 to obtain the pGreenII-62-MaMYB93 recombinant Agrobacterium. The sequence of primer MaMYB93-0800-F is shown in SEQ ID NO:27, and the sequence of primer MaMYB93-0800-R is shown in SEQ ID NO:28.

[0043] 2.6 EMSA Experiment: Construction of MaMYB93 Protein Expression Vector The construction method in this step is the same as in 2.1, except that the pMAL-c5x vector is double-digested with XmnI and HindIII, and the primers are MaMYB93-Mal-F and MaMYB93-Mal-R to obtain the recombinant plasmid pMAL-c5x-MaMYB93. The positive plasmid is then transformed into the expression strain Rosetta to obtain the pMAL-c5x-MaMYB93 recombinant E. coli. The sequence of primer MaMYB93-Mal-F is shown in SEQ ID NO:29, and the sequence of primer MaMYB93-Mal-R is shown in SEQ ID NO:30.

[0044] Example 2: Functional Analysis of Mulberry MaMYB93 1. Subcellular localization of MaMYB93 The pCambia1300-MaMYB93-GFP recombinant Agrobacterium obtained in Example 1 was activated, and single clones were selected and cultured until OD600 = 0.2-0.8. 10 μM acetylsyringone (As) was added and the culture was continued for 30 min on a shaker at 28℃. Tobacco leaves were transiently infected by injection, and the leaves were collected after 72 hours for observation using a laser confocal microscope.

[0045] The results of laser confocal microscopy observation are as follows Figure 2 As shown, GFP is the control group expressing GFP protein alone via the nucleoprotein marker, while GFP-MaMYB93 is the experimental group infected with recombinant Agrobacterium. It can be seen that the fluorescence in the control group is diffusely distributed throughout the cell, present in both the nucleus and cytoplasm; the green fluorescence signal in the experimental group is concentrated in the nucleus, and shows that MaMYB93 co-localizes with the nucleoprotein marker, indicating that MaMYB93 is mainly located in the nucleus.

[0046] 2. Analysis of MaMYB93 transcriptional activity and its interaction with downstream target genes 2.1 MaMYB93 transcriptional activity analysis Carrier DNA was heated in a 95°C metal bath for 5 min, then quickly inserted into ice. 100 µl of AH109 competent cells (YC1010, Weidi Biotechnology, China) thawed on ice was taken, and 2–5 µg of pre-chilled recombinant plasmid pGBKT7- was added sequentially. MaMYB93 10 µl of pretreated carrier DNA was mixed with 500 µl of PEG / LiAc and incubated at 30 °C for 30 min. The mixture was then incubated at 42 °C for 15 min, centrifuged at 5000 rpm for 40 s, the supernatant was discarded, and the culture was resuspended in 400 µl of ddH2O. After centrifugation for 30 s, the supernatant was discarded again, and the culture was resuspended in 50 µl of ddH2O. The resulting culture was plated and incubated at 29 °C for 48–96 h. Single colonies were picked and cultured in yeast tryptophan synthesis auxotroph (SD / -Trp) liquid medium to obtain cells carrying pGBKT7-. MaMYB93 The AH109 yeast strain was used as a vector. The bacterial culture was spotted onto yeast tryptophan-histidine synthesis auxotrophic (SD / -His / -Trp) solid medium and cultured at 30°C in the dark for 3 days.

[0047] Culture results as follows Figure 3 As shown, pGBKT7 is the control group transformed only with the pGBKT7 blank vector. It can be seen that the pGBKT7- MaMYB93The AH109 yeast strain containing the vector could grow normally on SD / -Trp / -His medium, while the control group could not grow normally on SD / -Trp / -His medium, indicating that MaMYB93 has transcriptional activation activity.

[0048] 2.2 Yeast one-hybrid Using the same method as in step 2.1 of this embodiment, the recombinant plasmid pGBKT7- obtained in Example 1 was processed. MaMYB93 The recombinant plasmids pHIS2-MaCYP86B1pro, pHIS2-MaABCG1pro, pHIS2-MaKCS1pro, pHIS2-MaCASP1pro, and pHIS2-MaCOMTpro obtained in Example 1 were co-transformed into yeast strain AH109. The strain was cultured in yeast tryptophan-leucine synthesis auxotroph (SD / -Trp / -Leu) liquid medium to obtain a yeast strain carrying the recombinant plasmids. The bacterial culture was spotted onto yeast tryptophan-leucine-histidine synthesis auxotroph (SD / -Trp / -Leu / -His) solid medium supplemented with 80 mM 3AT (3-amino-1,2,4-triazole) and cultured in the dark at 30°C for 3 days.

[0049] Culture results as follows Figure 4 As shown, these are the transfers to pGADT7- MaMYB93 Y187 yeast strains transfected with the pHIS2 vector containing 5 gene promoters; and a control group of Y187 yeast strains transfected only with the blank pGADT7 vector and the pHIS2 vector containing 5 gene promoters. It can be seen that transfecting with pGADT7- MaMYB93 Y187 yeast strains containing the pHIS2 vector with five gene promoters can grow normally on SD / -Trp / -Leu / -His + 80mM 3AT medium, while the control group cannot grow normally, indicating that MaMYB93 promotes the growth of yeast cells containing five gene promoters. MaCYP86B1 , MaKCS1 , MaABCG1 , MaCASP1 , MaCOMT It has transcriptional activation function.

[0050] 2.3 Dual-luciferase reporter assay The recombinant Agrobacterium tumefaciens pGreenII-0800-luc-MaCYP86B1pro, pGreenII-0800-luc-MaKCS1pro, pGreenII-0800-luc-MaABCG1pro, and pGreenII-62-MaMYB93 obtained in Example 1 were activated, and single clones were selected and cultured to OD. 600=0.2~0.8, then acetylsyleugenol (As) was added to the bacterial culture to a final concentration of 10 μM, and the culture was continued for 30 min on a shaker at 28℃. Recombinant Agrobacterium pGreenII-62-MaMYB93 was mixed with recombinant Agrobacterium pGreenII-0800-luc-MaCYP86B1pro, pGreenII-0800-luc-MaKCS1pro, and pGreenII-0800-luc-MaABCG1pro at a ratio of 10:1, and injected transiently into tobacco leaves. Leaves were collected 72 hours after infection. Recombinant Agrobacterium transformed with the blank plasmid pGreenII-62-SK was mixed with recombinant Agrobacterium pGreenII-0800-luc-MaCYP86B1pro, pGreenII-0800-luc-MaKCS1pro, and pGreenII-0800-luc-MaABCG1pro in the same proportion as a control group. After infection, LUC / REN was determined using the Dual Luciferase Reporter Assay Kit from Novizan and a luminescence detector, and fluorescence images of tobacco leaves were analyzed using the Tanon Chemi Dog Ultra.

[0051] Fluorescence images such as Figure 5 As shown, the LUC / REN values ​​of the tobacco regions co-transformed with pGreenII-62-MaMYB93, pGreenII-0800-luc-MaCYP86B1pro, pGreenII-0800-luc-MaKCS1pro, and pGreenII-0800-luc-MaABCG1pro were significantly higher than those of the control group, and the fluorescence intensity was significantly higher than that of the other injection regions. This indicates that MaMYB93 has a transcriptional activating effect on MaCYP86B1, MaKCS1, and MaABCG1.

[0052] 2.4 EMSA Experiment (1) Predicted Synthetic Probes: The MYB binding elements of the promoter regions of MaCYP86B1, MaKCS1, MaABCG1, and MaCOMT were predicted using the PlantCARE online software (https: / / bioinformatics.psb.ugent.be / webtools / plantcare / html / ), and the corresponding biotin-labeled wild-type probes, unlabeled competitive cold probes, and non-competitive probes with sequence mutations were synthesized. The probe types and sequences are shown in Table 1. The biotin-labeled site is at the 5′ end; the mutation sites in the mutant probes are indicated by the underlined sites; all probes were synthesized by Boyuan Biotechnology (Wuhan, China).

[0053] Table 1 Probe Types and Sequences

[0054] (2) Recombinant protein purification: The pMAL-c5x-MaMYB93 recombinant Escherichia coli obtained in Example 1 was activated and then inoculated into 400 mL of LB medium and cultured until OD. 600 Value 0.6. Add 0.5 mM IPTG and induce expression at 16℃ for 16 h, then centrifuge to collect the bacterial cells. Add Lysis buffer (10 mL Lysis buffer per 1 g of bacterial cells) to resuspend the bacterial cells, then sonicate to disrupt the cells, collect the cell lysate and filter. Incubate the filtered cell lysate with a highly crosslinked purification resin for MBP-tagged proteins (MBPSep Dextrin Agarose Resin 6FF, 20515ES25, Yeasen, China) at 4℃ for 3 h. After incubation, collect the affinity resin and wash thoroughly with washing buffer (20 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA, pH 7.4) to remove non-specifically bound contaminating proteins. Finally, perform gradient elution with Elution buffer (maltose) and collect the eluent to obtain the purified MBP-MaMYB93 protein. Take 30 μL of elution buffer, add 10 μL of 5×SDS Sample Buffer (final concentration composition: 250 mM Tris-HCl (pH 6.8), 10% (w / v) SDS, 0.5% (w / v) bromophenol blue, 50% (v / v) glycerol, 5% (v / v) β-mercaptoethanol), mix well, incubate at 100 °C for 10 min, centrifuge, and then perform SDS-PAGE analysis to verify the correct purification of the protein.

[0055] (3) Gel migration assay: The probe, MBP-MaMYB93 protein, and binding buffer (GS005, Beyotime, China) were added sequentially and mixed gently. The mixture was incubated at 25°C for 30 min in a PCR instrument to obtain the binding reaction product. A 6% non-denaturing polyacrylamide gel was prepared (1 ml 10×TBE, 4 ml 30% Acrylamide / Bis, 625 μl glycerol, 14.2 ml deionized water, 150 μl 10% APS). Pre-electrophoresis was performed at 80V for 10 min in pre-cooled 0.5×TBE electrophoresis buffer. The binding reaction product was mixed with 6× loading buffer and loaded onto the gel. Electrophoresis was performed at 80V at 4°C until the bromophenol blue indicator migrated to a position approximately 2-3 cm from the bottom of the gel. After electrophoresis, the nucleic acids and complexes in the gel were transferred to a positively charged nylon membrane using a semi-dry transfer method. The membrane transfer was performed at 4°C in 0.5×TBE buffer for 1 hour at a constant current of 200 mA. Immediately after transfer, the membrane was placed in a UV crosslinker and crosslinked for 60 seconds using 254 nm UV light at an energy of 120 mJ / cm² to immobilize the biotin-labeled probe on the membrane. The membrane was then placed in blocking buffer and blocked at room temperature for 45 minutes. Subsequently, the membrane was incubated with streptavidin-HRP conjugate (SA00001-0, Proteintech, USA) diluted 1:3000 with blocking buffer at room temperature for 25 minutes. The membrane was washed three times with PBST buffer for 10 minutes each time. Finally, equal volumes of ECL chemiluminescence solution A and solution B (K-12045-D50, Advansta, USA) were mixed and uniformly applied to the membrane. Exposure and image acquisition were performed using a chemiluminescence imaging system.

[0056] Chemiluminescence detection results as follows Figure 6As shown, probe is a biotin-labeled wild-type probe, MBP is the MBP-tagged protein control group, competitor probe is an unlabeled competitive cold probe, and mutant probe is a non-competitive probe with a sequence mutation. + indicates the addition of this component to the reaction system, - indicates its absence, and 10× and 50× represent the addition of 10-fold and 50-fold of the corresponding component, respectively. It can be seen that in the wells containing only the biotin-labeled wild-type probe, the signal band is located at the bottom of the gel, indicating a free probe. When MBP-MaMYB93 protein is added to the reaction system, a significant hysteresis band appears above the free probe band, indicating the formation of a protein-probe complex; however, this hysteresis band does not appear in the control wells containing the MBP-tagged protein. When an unlabeled competitive cold probe is added to the reaction of MBP-MaMYB93 protein with the labeled probe, the hysteresis band signal is significantly weakened or disappears; when a non-competitive probe with a sequence mutation is added, the hysteresis band signal is unaffected. This indicates that MaMYB93 protein can react with... MaCYP86B1 CAACCA element in the starter sub-region, CAACAG element in the MaKCS1 starter sub-region, MaABCG1 CAACCA components in the starter sub-region and MaCOMT The specific binding of the TAACAG element in the promoter region demonstrates that MaMYB93 has a transcriptional activating effect on the above genes.

[0057] 3. Overexpression MaMYB93 Components and content of suberin in Arabidopsis roots and leaves 3.1 Agrobacterium tumefaciens-mediated transformation in Arabidopsis thaliana The carrier pCambia1300- obtained in Example 1 MaMYB93 -GFP was transformed into Agrobacterium GV3101 and cultured for 2-3 days. Subsequently, the culture was inoculated into liquid LB medium containing 50 mg / L rifamycin and 50 mg / L kanamycin, and cultured with shaking at 28°C and 200 rpm. The bacterial culture was cultured until the OD value reached approximately 0.8, and the bacterial pellet was collected by centrifugation at 5000 rpm for 5 min at room temperature. The collected bacterial pellet was resuspended in a suspension (10 μmol L⁻¹ MgCl₂, 10 μmol L⁻¹ MES, 200 μmol L⁻¹ Acetosyringone) to obtain the Agrobacterium solution.

[0058] Immerse the main inflorescence of Arabidopsis thaliana in an Agrobacterium solution for 2-3 seconds, gently stirring to cover the plant with a thin film of liquid. After immersion, wrap the Arabidopsis plant with absorbent paper to gently blot out excess Agrobacterium solution. Then, gently wrap the plant with plastic wrap and cover the entire pot with a black plastic bag. Place it in a growth chamber in the dark for 16-24 hours. Remove the black plastic bag and culture under long-day conditions until the siliques turn yellow. Collect the seeds. Culture the seeds to obtain transgenic Arabidopsis thaliana.

[0059] 3.2 Identification of positive transgenic Arabidopsis thaliana DNA and RNA were extracted from transgenic Arabidopsis leaves using the same method as in Example 1. Using leaf DNA as a template, PCR amplification was performed using primers 35s-MaMYB93-F and 35s-MaMYB93-R, followed by agarose gel electrophoresis of the amplified products. The sequence of primer 35s-MaMYB93-F is shown in SEQ ID NO:31, and the sequence of primer 35s-MaMYB93-R is shown in SEQ ID NO:32.

[0060] Using RNA from transgenic Arabidopsis leaves as a template, cDNA was obtained by reverse transcription using the HiScriptoIll 1st Strand cDNA Synthesis Kit (+gDNA wiper) (R312, Novizan, China). Using the cDNA as a template, real-time quantitative PCR (ChamQ SYBR qPCR Master Mix, Q341-02, Novizan, China) was performed using primers MaMYB93-RT-F and MaMYB93-RT-R to identify the different transgenic Arabidopsis lines. MaMYB93 Expression level.

[0061] Agarose gel electrophoresis results are as follows Figure 7 As shown, lanes 1-14 represent different transgenic Arabidopsis plants, WT is the wild-type Arabidopsis control group, H2O is the blank control, and PC is the positive control group for the recombinant plasmid vector. It can be seen that a specific band was detected in the transgenic plants at approximately 1500 bp, and the band size was consistent with that of the positive control group, indicating successful Agrobacterium transformation.

[0062] Real-time quantitative PCR results are as follows Figure 8 As shown, AtActin7 is the internal reference gene, WT is the wild-type plant control group, and OE-2 to OE-14 are the numbers of different positive transgenic Arabidopsis plants. It can be seen that AtActin7 can be detected in the positive transgenic plants. MaMYB93 The expression level was significantly higher than that of the wild-type control group.

[0063] 3.3 Staining and content determination of suberin in roots and leaves of Arabidopsis thaliana overexpressing MaMYB93 according to Figure 8 The positive transgenic plants shown MaMYB93 Expression levels were selected from the gradient overexpression in step 3.2. MaMYB93 The three transgenic Arabidopsis thaliana lines, numbered OE-8, OE-10, and OE-11, were used to obtain homozygous transgenic Arabidopsis thaliana by culturing these three lines to the T3 generation. Seeds of the homozygous transgenic Arabidopsis thaliana were cultured to 7 days of age, and the roots were stained with cork using dye FY088 (Fluorol Yellow 088).

[0064] Homozygous transgenic Arabidopsis thaliana was cultured to 5 weeks of age, and its various monomers of cork were analyzed using gas chromatography-mass spectrometry. Fresh 5-week-old homozygous transgenic Arabidopsis thaliana was ground into a powder, and 1 g of plant tissue was weighed and added to a chloroform-methanol mixture (v / v 1:2). The mixture was extracted at room temperature with shaking for 3 h. After centrifugation at 10000×g for 15 min, the supernatant was discarded, and the internal standard tetracosane (C24:0) was added to the precipitate, followed by 4 mL of sulfuric acid-methanol mixture (v / v 5:95). The mixture was reacted at 85℃ for 2 h. Subsequently, 3 mL of 0.9% sodium chloride solution and 8 mL of dichloromethane were added, and the mixture was thoroughly vortexed and centrifuged at 800×g for 10 min. The lower organic phase was carefully aspirated, washed repeatedly with 0.5 mol / L sodium chloride solution, dehydrated with anhydrous sodium sulfate, and then dried under nitrogen. Add 0.1 mL of bis(trimethylsilyl)trifluoroacetamide (BSTFA) and 0.1 mL of pyridine to the dried residue, and react at 70 °C for 40 min. After cooling to room temperature, dry the reagent with nitrogen gas, and dissolve it in 0.5 mL of dichloromethane for analysis.

[0065] The analysis was performed using an Agilent 7890A-5975C gas chromatograph-mass spectrometer (Agilent, Germany) equipped with a DB-5MS capillary column (30 m long, 0.25 mm inner diameter, 0.25 μm film thickness). The column temperature program was as follows: initial temperature 80 °C, held for 2 min; increased to 200 °C at 15 °C / min, then to 280 °C at 5 °C / min, and finally held at 300 °C for 10 min. Helium was used as the carrier gas at a flow rate of 1.0 mL / min. Mass spectrometry was performed in 70 eV electron impact ionization mode, with the ion source temperature set to 230 °C. The identification of cork monomers was performed by comparing the retention times and mass spectra of standards, and by referring to the NIST 2011 standard mass spectrum library. Quantitative analysis was conducted using tetracosane (C24:0) as an internal standard, and the final results were converted to the dry weight content of the sample.

[0066] The results of cork staining are as follows: Figure 9As shown, WT is the wild-type plant control group. It can be seen that overexpression... MaMYB93 The suberization level of roots in transgenic Arabidopsis thaliana OE-8, OE-10, and OE-11 was significantly higher than that in the control group.

[0067] Gas chromatography-mass spectrometry analysis results are as follows Figure 10 As shown, (B) is a graph showing the content of suberin monomers in roots, (C) is a graph showing the content of suberin monomers in leaves, (D) is a graph showing the content of suberin in roots, and (E) is a graph showing the content of suberin in leaves; FA represents fatty acids, including C16:0, C18:0, C18:1, C18:2, C20:0, C22:0, and C24:0; ω-OH represents ω-hydroxy fatty acids, including C20:0; DCA represents dicarboxylic acids, including C16:0; -OH represents intermediate hydroxy fatty acids, including C22:0; Ferulate represents ferulic acid; Coumarate represents coumaric acid; aliphatic suberin represents aliphatic suberin, and aromatic suberin represents aromatic suberin; a, b, c, and d in the graph are markers used to indicate statistically significant differences. Different letters represent statistically significant differences, while the same letters indicate no statistically significant differences.

[0068] It can be seen that, with the development of Arabidopsis thaliana... MaMYB93 Gradual overexpression resulted in a gradual increase in suberin content in roots, while no significant difference was observed in leaves. In roots, the content of most suberin monomers increased with increasing overexpression, exhibiting a dose-dependent change: low expression (OE-8) only altered monomer composition (e.g., a significant increase in C16:0 DCA); while medium-to-high expression (OE-10 / 11) simultaneously increased the content and total deposition of key monomers such as ω-hydroxy acids (e.g., C18:1 ω-OH) and dicarboxylic acids (e.g., C16:0 DCA). In leaves, medium-to-high expression lines (OE-10 / 11) induced a 3-6 fold accumulation of ω-OH and DCA monomers.

[0069] Example 3 Overexpression MaMYB93 Improve plant drought resistance The homozygous transgenic Arabidopsis thaliana OE-8, OE-10, and OE-11 from Example 2 were cultured to 7 days old. Some seedlings were transferred to 1 / 2 MS medium containing 200 mM mannitol and cultured for 24 h. Mannitol was used to simulate the effect of drought stress, and then phenotypic observation and FY088 cork staining were performed.

[0070] Two seedlings were cultured until they reached two weeks of age, then transplanted into plastic flowerpots (7cm×7cm×9cm), with four seedlings per pot. They were cultured in a 1:1 mixture of heat-treated potting soil and vermiculite, maintaining a daytime temperature of 22℃ and a nighttime temperature of 18℃, with 16 hours of light and 8 hours of darkness. The relative soil moisture content was standardized for each seedling line, and watering was then stopped for 14 days to induce drought stress.

[0071] Results of treatment with 200mM mannitol are as follows Figure 11 As shown, it can be seen that under 200mM mannitol treatment, the wild-type control group and the overexpressing group... MaMYB93 Root corkation in Arabidopsis thaliana was enhanced compared to the control group, and overexpression was observed. MaMYB93 The level of corkage in Arabidopsis thaliana was still higher than that in the wild type, and the inhibitory effect of drought stress on its root elongation was significantly weaker than that in the wild type.

[0072] Results under soil drought stress are as follows Figure 12 As shown, under drought stress, the WT wild-type control group exhibited severe wilting, stunted growth, and significant leaf curling, wrinkling, and yellowing; while the overexpressing... MaMYB93 The leaves of Arabidopsis thaliana did not show significant curling, wrinkling, or yellowing, demonstrating stronger drought resistance.

[0073] The amino acid sequence of transcription factor MaMYB93 (SEQ ID NO:1) MGRSPCCDESGLKKGPWTPEEDQKLVKYIQKHGHGSWRALPRLAGLNRCGKSCRLRWTNYLRPDIKRGKFSQEEEETILNLHSILGNKWSAIAGHLPGRTDNEIKNFWNTHLKKKLIQMGIDPMTHRPRTDIFSSLPHLIALANLKELMTMDHPWEELQAMRSLQADQALQ MAKLQYLQYLLQPPNPQNNNNQMINMPDQSLNSLQSVQYDSVPFDNLPDLLQISDQTTPLQLMNKQQVVDHIIQAQDNFTVGFSQAGDIDNDDHIMMNIFPSSSSWVNPNSSASSTPSPPTAAAAAVEATSLSNLQGGDACSANSSYGEVAAPSVWPDLLLDDTLFQEVA* SEQ ID NO:2 MaMYB93 nucleotide sequence of a gene SEQ ID NO:3 Primer MaMYB93-F ATGGGAAGATCTCCTTGTTG SEQ ID NO:4 Primer MaMYB93-R TTAAGCAACCTCCTGAAACA SEQ ID NO:5 Primer MaMYB93-Loc-F AGGACAGGTACCCGGGGATCATGGGAAGATCTCCTTGTTGT SEQ ID NO:6 Primer MaMYB93-Loc-R ATCGTCTTTGTAGTCGTCGATAGCAACCTCCTGAAACAAAG SEQ ID NO:7 Primer MaMYB93-BK-F CCATGGAGGCCGAATTCCCGATGGGAAGATCTCCTTGTTG SEQ ID NO:8 Primer MaMYB93-BK-R CTGCAGGTCGACGGATCCCCTTAAGCAACCTCCTGAAACA SEQ ID NO:9 Primer MaMYB93-AD-F AGTGGTCTCTGTCCAGTCCTATGGGAAGATCTCCTTGTTG SEQ ID NO:10 Primer MaMYB93-AD-R GGTCTCAGCAGACCACAAGTTTAAGCAACCTCCTGAAACA SEQ ID NO:11 Primer MaCYP86B1pro-HIS-F TATAGGGCGAATTCCCGGGGTGCTTGCTCTTTTCCACACA SEQ ID NO:12 Primer MaCYP86B1pro-HIS-R GATCGATTCGCGAACGCGTGGTTGGTGGTGGTGGTGATCA SEQ ID NO:13 Primer MaKCS1pro-HIS-F ATACGACTCACTATAGGGCGGTGTCCACACTTTTCCTGTT SEQ ID NO:14 also encapsulates MaKCS1pro-HIS-R CGCGAACGCGTGAGCTCCCCGAAATCTAAGCCCTACACGTC SEQ ID NO:15 Protein MaABCG1pro-HIS-F FATHERGGCGAATTCCCGGGGGTCATCTTGTGATGCAAAAG SEQ ID NO:16 also encodes MaABCG1pro-HIS-R GATCGATTCGCGAACGCGTGTTGATTAGTACAACCCTTTT SEQ ID NO:17 also contains MaCASP1pro-HIS-F CACTATAGGGCGAAATCATCTGGTCGTTGGAGGCTTTGCA SEQ ID NO:18 for MaCASP1pro-HIS-R CGCGAACGCGTGAGCTCCCCAGATGGTTTTGAAGGCTTGA SEQ ID NO:1919 MaCOMTpro-HIS-F ATACGACTCACTATAGGGCGGTGGCATGACGATTGTTTTT SEQ ID NO:20 FROM MaCOMTpro-HIS-R CGCGAACGCGTGAGCTCCCCTGTTTTGGGATGCTTAGGCT SEQ ID NO:21 Protein MaCYP86B1pro-LUC-F ATATCGAATTCCTGCAGCCCTGCTTGCTCCTTTTCACACA SEQ ID NO:22 Protein MaCYP86B1pro-LUC-R CTAGAACTAGTGGATCCCCCGTTGGTGGTGGTGGTGATCA SEQ ID NO:23 Protein MaKCS1pro-LUC-F GGGCCCCCCCTCGAGGTCGAGTGTCCACACTTTTCCTGTT SEQ ID NO:24 Protein MaKCS1pro-LUC-R CAGGAATTCGATATCAAGCTGAAATCTAAGCCCTACACGTC SEQ ID NO:25 Primer MaABCG1pro-LUC-F GGGCCCCCCCTCGAGGTCGAGTCATCTTGTGATGCAAAAG SEQ ID NO:26 Primer MaABCG1pro-LUC-R CAGGAATTCGATATCAAGCTTTGATTAGTACAACCTTTT SEQ ID NO:27 Primer MaMYB93-0800-F CTAGAACTAGTGGATCCCCCATGGGAAGATCTCCTTGTTG SEQ ID NO:28 Primer MaMYB93-0800-R ATATCGAATTCCTGCAGCCCAGCAACCTCCTGAAACAAAG SEQ ID NO:29 Primer MaMYB93-Mal-F AACAACCTCGGGATCGAGGGAAGGGGCTCCGGCTCCGGCTCCATGGGAAGATCTCCTTGTTGTG SEQ ID NO:30 Primer MaMYB93-Mal-R GAGCCTTTCGTTTTATTTGAAGCTTTAAGCAACCTCCTGAAACAAAGTG SEQ ID NO:31 Primer 35s-MaMYB93-F GACGCACAATCCCACTATCC SEQ ID NO:32 Primer 35s-MaMYB93-R CGTCGTCCTTGAAGAAGATGG SEQ ID NO:33 Primer MaMYB93-RT-F GGACCAGACAACACCACTACA SEQ ID NO:34 Primer MaMYB93-RT-R TGGCTGAACCCTACTGTGAAAT SEQ ID NO:35 MaCYP86B1 Biotin-labeled wild-type probe CACAAAACCCTTCAACCAAGAAAGTCCATT SEQ ID NO:36 MaCYP86B1 Unlabeled competitive cold probe CACAAAACCCTTCAACCAAGAAAGTCCATT SEQ ID NO:37 MaCYP86B1 mutant non-competitive probe CACAAAACCCTTTTTTTTAGAAAGTCCATT SEQ ID NO:38 MaKCS1 biotin-labeled wild-type probe TTCATTTAAAACAACAGTTATACAGAAAAA SEQ ID NO:39 MaKCS1 unlabeled competitive cold probe TTCATTTAAAACAACAGTTATACAGAAAAA SEQ ID NO:40 MaKCS1 mutant non-competitive probe TTCATTTAAAAAAAAAATTATACAGAAAAA SEQ ID NO:41 MaABCG1 Biotin-labeled wild-type probe TTTAATTGTTTACAACCATATTGTTCAATC SEQ ID NO:42 MaABCG1 Unlabeled competitive cold probe TTTAATTGTTTACAACCATATTGTTCAATC SEQ ID NO:43 MaABCG1 mutant non-competitive probe TTTAATTGTTTAAAAAAATATTGTTCAATC SEQ ID NO:44 MaCOMT biotin-labeled wild-type probe TCTATGTAATAACAGAAATAAAATTAACAGACATAAAAAATGAAATAACAGAAATAAAAA SEQ ID NO:45 MaCOMT Unlabeled Competitive Cold Probe TCTATGTAATAACAGAAATAAAATTAACAGACATAAAAAATGAAATAACAGAAATAAAAA SEQ ID NO:46 MaCOMT mutant non-competitive probe TCTATGTAATTTTTTTAAATAAAATTTTTTTACATAAAAAATGAAATTTTTTAAATAAAAA The above description is merely a preferred embodiment of the present invention, and should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications are also considered to be within the scope of protection of the present invention.

Claims

1. The application of overexpression of mulberry transcription factor MaMYB93 or its encoding gene in improving plant drought resistance, characterized in that, The amino acid sequence of the mulberry transcription factor MaMYB93 is shown in SEQ ID NO:1, and the gene encoding the mulberry transcription factor MaMYB93 is described. MaMYB93 The nucleotide sequence is shown in SEQ ID NO:

2.

2. Application of overexpression of mulberry transcription factor MaMYB93 or its encoding gene in improving the expression level of genes related to plant drought resistance.

3. The application according to claim 2, characterized in that, The plant drought resistance-related genes include MaCYP86B1, MaKCS1 , MaABCG1, MaCASP1, MaCOMT One or more genes.

4. Application of overexpression of mulberry transcription factor MaMYB93 or its encoding gene in promoting plant suberin production.

5. Application of overexpression of mulberry transcription factor MaMYB93 or its encoding gene in the creation of plant germplasm or the breeding of drought-resistant plant varieties.

6. A nucleic acid molecule encoding the mulberry transcription factor MaMYB93 as described in any one of claims 1-5.

7. An expression carrier, characterized in that, Includes the nucleic acid molecule as described in claim 6.

8. A host cell, characterized in that, The host cell contains either the nucleic acid molecule of claim 6 or the expression vector of claim 7.

9. A method for improving plant drought resistance, characterized in that, Includes the following steps: Overexpression of mulberry transcription factor MaMYB93 or its encoding gene in target plants.

10. A method for enhancing the corkyization of plant roots, characterized in that, Includes the following steps: Overexpression of mulberry transcription factor MaMYB93 or its encoding gene in target plants.

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