MdSAUR15 gene for promoting root development under salt stress, protein and application of MdSAUR15 gene

Abstract

The invention belongs to the field of plant genetic engineering, and discloses an apple MdSAUR15 gene for promoting root development under salt stress, a protein and application of the apple MdSAUR15 gene and the protein. The invention provides the MdSAUR15 gene for promoting plant adventitious root development under the salt stress condition, the number and fresh weight of the adventitious roots can be increased through overexpression of the MdSAUR15 gene in apples, it is indicated that the MdSAUR15 gene can promote plant adventitious root development, can be used for cultivating plant varieties with strong adventitious root development capacity, reduces the plant asexual breeding cost, and has good application prospects. The plant breeding efficiency is improved and the seedling production period is shortened. Meanwhile, the overexpressed apple MdSAUR15 gene can also increase the number and fresh weight of adventitious roots of apple transgenic plants under the salt stress condition, which indicates that the MdSAUR15 gene can promote the development of the adventitious roots of the plants under the salt stress condition, can be used for cultivating salt-alkali tolerant plant varieties with strong root development ability, improves the salt tolerance of the plants, and improves the salt tolerance of the plants. The method can be widely used for saline-alkaline tolerance genetic improvement of plants.

Description

Technical Field

[0001] The present invention belongs to, but is not limited to, the technical field of plant genetic engineering, and particularly relates to an apple MdSAUR15 gene, protein for promoting root development under salt stress and their applications. Background Art

[0002] Apple (Malus×domestica Borkh.) is one of the important economic fruit trees in China. Currently, both the planting area and yield account for more than 50% of the world (FAO. 2022). Making full use of saline-alkali land has become one of the important ways for fruit tree development in China. The northwest is an important characteristic and advantageous apple production area in China, with rich light and heat resources, flat terrain, and extensive distribution of saline-alkali land. The selection of suitable rootstocks is the key to the efficient utilization of saline-alkali land (Zhang Yiman et al., 2018). Saline-alkali stress can significantly inhibit the root development of apple rootstock seedlings, while salt-tolerant rootstocks can improve the salt tolerance of apple seedlings by inducing the root development of rootstocks to adapt to the saline-alkali environment (Zhang et al., 2022).

[0003] Auxin up-regulated small RNA gene (SAUR) is a class in the large family of genes responsive to auxin. SAUR genes can participate in regulating the development of plant lateral roots and hypocotyls. Arabidopsis AtSAUR15 can directly bind to the promoter of the gene AHA encoding H + ATPase, and promote the development of Arabidopsis lateral roots by regulating the activity of plasma membrane H + ATPase and auxin biosynthesis (Yin et al., 2020). Transgenic plants overexpressing AtSAUR63 have longer hypocotyls, and the accumulation level of auxin transported in the hypocotyl is also higher than that of wild-type seedlings, indicating that Arabidopsis AtSAUR63 promotes auxin-stimulated organ elongation (Chae et al., 2012). In addition, SAUR also plays a role in stress responses. Under salt stress, the expression of TaSAUR75 in wheat roots is down-regulated. Overexpression of TaSAUR75 can reduce the accumulation of H2O2 in transgenic lines by increasing the root length and survival rate of transgenic lines and up-regulating the expression of some stress-responsive genes, and plays a positive regulatory role in the responses to drought and salt stress (Guo et al., 2018b). Wheat TaSAUR78 improves the tolerance of plants to abiotic stresses (salt, drought and low temperature) by regulating TaVDAC1 (voltage-dependent anion channel) (Guo et al., 2019).

[0004] At present, the SAUR gene has been proven to play a role in root development and stress response in model plants such as Arabidopsis thaliana, providing an idea for analyzing the regulation of SAUR on root development under salt stress in apples in this study. As a perennial woody plant, the adaptive development state of the roots of apples in saline-alkali land is particularly crucial for tree growth, yield formation, and fruit quality. Moreover, the function of SAUR in regulating the adaptive development of roots under saline-alkali stress in apples has not been analyzed yet.

[0005] In view of the above analysis, the technical problems urgently needed to be solved in the existing technology are as follows:

[0006] Studying the function of MdSAUR15 in regulating root development under saline-alkali stress in apples is expected to further enrich the gene resource library of apples that regulate root development under saline-alkali stress, which is of great significance for the genetic improvement of apple salt tolerance and resistance breeding research. Summary of the Invention

[0007] In view of the problems existing in the prior art, the present invention provides an apple MdSAUR15 gene, protein and their applications for promoting root development under salt stress.

[0008] The present invention is realized as follows. An MdSAUR15 gene for promoting root development under salt stress, characterized in that the nucleotide sequence of the apple MdSAUR15 gene is specifically as follows:

[0009] 5’ATGGGAATCAAGTTGATGGGGATAGCTCATGCCAAGCAGAAACTTCAGAGAACTCTTTCAGCAAGATATGGATCAGCTGCAGACAATACTAGTACTGAGGTTCCGAAAGGCCACTTTGCGGTTTATGTTGGAGAAGATGAAAAGAAGAGATTTGTGATTCCGATATCATATTTGAACCACCCTTTGTTCCAAGACTTGTTAAACAAGGCTGAGGAAGAGTTTGGATATGATCATCCTACAGGGGGACTCACAATTCCATGCAGTGAAGACTACTTCGTCAGTCTATCTTCATGTCTAAATTGTTCGTAA3’(SEQ ID NO.1).

[0010] The primer pair for cloning the cDNA sequence of the above gene MdSAUR15, the primer pair includes a forward primer and a reverse primer, and their nucleotide sequences are as follows:

[0011] Forward primer: 5’ATGGGAATCAAGTTGATGGGGA3’(SEQ ID NO.2)

[0012] Reverse primer: 5’CGAACAATTTAGACATGAAGATAGACTG3’ (SEQ ID NO.3)

[0013] Another object of the present invention is to provide an MdSAUR15 protein encoded by the apple MdSAUR15 gene, and the amino acid sequence of the MdSAUR15 protein is shown as SEQ ID NO.4:

[0014] MGIKLMGIAHAKQKLQRTLSARYGSAADNTSTEVPKGHFAVYVGEDEKKRFVIPISYLNHPLFQDLLNKAEEEFGYDHPTGGLTIPCSEDYFVSLSSCLNCS (SEQ ID NO.4).

[0015] The present invention also provides a recombinant expression vector containing the MdSAUR15 gene described in the above technical solution.

[0016] Furthermore, the recombinant expression vector containing the gene MdSAUR15 of the present invention is preferably obtained by ligating the apple MdSAUR15 gene with the vector pC2300 using pC2300 as the initial vector.

[0017] The present invention also provides an engineered bacterium comprising the recombinant expression vector of the MdSAUR15 gene described in the above technical solution.

[0018] The present invention also provides a host cell transformed with the expression vector described in the above technical solution.

[0019] Another object of the present invention is to provide the application of the MdSAUR15 gene, recombinant expression vector, engineered bacterium, or host cell in any one or more of the following I - IX:

[0020] I: Promoting the occurrence of apple adventitious roots;

[0021] II: Increasing the number of apple adventitious roots;

[0022] III: Increasing the fresh weight of apple adventitious roots;

[0023] IV: Promoting the occurrence of apple adventitious roots under salt stress

[0024] V: Increasing the number of apple adventitious roots under salt stress

[0025] VI: Increasing the fresh weight of apple adventitious roots under salt stress

[0026] VII: Regulating adventitious root development

[0027] Ⅷ: Regulation of adventitious root development under salt stress

[0028] Ⅸ: Vegetative propagation of plants

[0029] Ⅹ: Assisting molecular breeding for plant salt tolerance

[0030] Another object of the present invention is to provide a method for cultivating a plant with strong adventitious root development ability under salt stress conditions, by increasing the expression of MdSAUR15 in the target plant or increasing the content of MdSAUR15 protein in the target plant, to obtain the plant with strong adventitious root development ability under the salt stress conditions; the increase in the expression of the MdSAUR15 gene in the target plant or the increase in the content of MdSAUR15 protein in the target plant is achieved by introducing the MdSAUR15 gene into the plant.

[0031] Combined with the above technical solutions and the solved technical problems, the advantages and positive effects of the technical solutions to be protected by the present invention are as follows:

[0032] First, the present invention provides an MdSAUR15 gene that promotes adventitious root development of plants under salt stress conditions. By overexpressing the above MdSAUR15 gene in apples, the present invention can increase the number and fresh weight of adventitious roots, indicating that the MdSAUR15 gene can promote the development of adventitious roots of plants, and can be used to cultivate plant varieties with strong adventitious root development ability, reduce the cost of vegetative propagation of plants, improve the efficiency of plant breeding, and shorten the production period of seedling cultivation. At the same time, the overexpressed apple MdSAUR15 gene in the present invention can also increase the number and fresh weight of adventitious roots of apple transgenic plants under salt stress conditions, indicating that the MdSAUR15 gene can promote the development of adventitious roots of plants under salt stress conditions, and can be used to cultivate salt-tolerant plant varieties with strong root development ability, improve the salt tolerance of plants, and can be widely used for genetic improvement of plant salt tolerance.

[0033] Second, after the technical solution of the present invention is transformed, it can expand the apple planting area, provide salt-tolerant germplasm for planting fruit trees in the saline-alkali land in the northwest, and adjust the proportion of apples in the fruit tree industry; at the same time, it can save the soil improvement cost of planting apple fruit trees in saline-alkali land, as well as other agronomic operation management costs such as water and fertilizer. Description of the drawings

[0034] Figure 1 It is the analysis of the expression pattern of the apple MdSAUR15 gene in the salt stress process provided by the embodiment of the present invention: a is the expression level of MdSAUR15 after 24 hours of treatment with 30 mM NaCl; b is the GUS staining and activity determination of pMdSAUR15:GUS transgenic apple callus after treatment with 75 mM NaCl.

[0035] Figure 2It is the subcellular localization result of the MdSAUR15 gene in Example 2 provided by the embodiments of the present invention. Among them, 35S:GFP serves as a positive control, GFP is green fluorescence, mCherry contains the nuclear localization signal of transgenic tobacco with NLSmCherry, BF is the bright field, and Merge is the combination of the three fields of view of GFP, DAPI, and BF;

[0036] Figure 3 It is the detection result of the overexpressed MdSAUR15 overexpressed transgenic apple in Example 3 provided by the embodiments of the present invention. a is the DNA detection result of the MdSAUR15 overexpressed apple transgenic line. WT is the wild type 'GL3', P is the MdSAUR15 recombinant plasmid as a positive control, and bands of MdSAUR15 were detected in the MdSAUR15 overexpressed apple transgenic lines #1, #2, #3, #5, #6, #8, #9, and #10; b is the RNA detection result of the apple 'GL3' overexpressed MdSAUR15 transgenic apple line;

[0037] Figure 4 It is the morphological phenotype observation and statistical result of the transgenic apple line overexpressing MdSAUR15 and the wild type 'GL3' provided in Example 3 of the embodiments of the present invention. a is the phenotype observation of the MdSAUR15 transgenic apple line and the wild type apple 'GL3'; b is the statistical result of the number of adventitious roots of the MdSAUR15 transgenic apple line and the wild type apple 'GL3'; c is the statistical result of the length of adventitious roots of the MdSAUR15 transgenic apple line and the wild type apple 'GL3'; d is the statistical result of the fresh weight of adventitious roots of the MdSAUR15 transgenic apple line and the wild type apple 'GL3'; e is the statistical result of the rooting rate of adventitious roots of the MdSAUR15 transgenic apple line and the wild type apple 'GL3'.

[0038] Figure 5 It is the evidence related to the technical effects obtained in the embodiments of the present invention. Heterologous overexpression of apple MdSAUR15 promotes the development of adventitious roots of Populus tomentosa under salt stress. a is the genomic PCR identification result of the MdSAUR15 overexpressed poplar line; b is the RT-qPCR identification result of the MdSAUR15 overexpressed poplar line; c is the phenotypic analysis of the adventitious root development of wild type Populus tomentosa and the MdSAUR-15 overexpressed poplar line grown on the rooting medium under control (0 mM) and salt (75 mM NaCl) stress conditions after 11 days of subculture; d is the number of adventitious roots, the length of adventitious roots, the fresh weight of adventitious roots, and the incidence rate of adventitious roots of wild type Populus tomentosa and the MdSAUR15 overexpressed line under control and salt stress conditions. Detailed implementation manners

[0039] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below in conjunction with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0040] An embodiment of the present invention provides an MdSAUR15 gene that promotes root development under salt stress. The nucleotide sequence of the apple MdSAUR15 gene is shown in SEQ ID NO.1 in the sequence listing. The nucleotide sequence shown in SEQ ID NO.1 of the present invention is specifically:

[0041] 5’ATGGGAATCAAGTTGATGGGGATAGCTCATGCCAAGCAGAAACTTCAGAGAACTCTTTCAGCAAGATATGGATCAGCTGCAGACAATACTAGTACTGAGGTTCCGAAAGGCCACTTTGCGGTTTATGTTGGAGAAGATGAAAAGAAGAGATTTGTGATTCCGATATCATATTTGAACCACCCTTTGTTCCAAGACTTGTTAAACAAGGCTGAGGAAGAGTTTGGATATGATCATCCTACAGGGGGACTCACAATTCCATGCAGTGAAGACTACTTCGTCAGTCTATCTTCATGTCTAAATTGTTCGTAA3’.

[0042] An embodiment of the present invention provides a primer pair for cloning the cDNA sequence of the above gene MdSAUR15. The primer pair includes a forward primer and a reverse primer, and their nucleotide sequences are as follows:

[0043] Forward primer MdSAUR15-F1: 5’ATGGGAATCAAGTTGATGGGGA3’ (SEQ ID NO.2); Reverse primer MdSAUR15-R1: 5’CGAACAATTTAGACATGAAGATAGACTG3’ (SEQ ID NO.3)

[0044] The MdSAUR15 gene described in the embodiment of the present invention is isolated from the roots of ‘Qingzhen No.1’ and has the function of regulating the adventitious root development of plants under salt stress. Experiments show that overexpressing the MdSAUR15 gene in apples and poplars can promote the adventitious root development of transgenic lines under salt stress conditions.

[0045] The embodiment of the present invention also provides the MdSAUR15 protein encoded by the MdSAUR15 gene described in the above technical solution, and the sequence of the MdSAUR15 protein is shown in SEQ ID NO.4. The specific amino acid sequence shown in SEQ ID NO.4 of the present invention is as follows:

[0046] MGIKLMGIAHAKQKLQRTLSARYGSAADNTSTEVPKGHFAVYVGEDEKKRFVIPISYLNHPLFQDLLNKAEEEFGYDHPTGGLTIPCSEDYFVSLSSCLNCS(SEQ ID NO.4).

[0047] The embodiment of the present invention also provides a recombinant expression vector containing the MdSAUR15 gene described in the above technical solution.

[0048] The initial vector for preparing the recombinant expression vector in the embodiment of the present invention preferably includes a plasmid vector, more preferably includes the pCAMBIA2300 vector, and even more preferably is 35S:pCAMBIA2300GFP. The pCAMBIA2300 of the present invention is preferably used for overexpression, and the 35S:pCAMBIA2300GFP containing the 35s promoter can improve the gene expression level.

[0049] The recombinant vector in the embodiment of the present invention preferably includes the MdSAUR15GFP overexpression vector. The MdSAUR15GFP overexpression vector in the present invention is preferably a recombinant plasmid obtained by introducing the nucleotide sequence shown in SEQ ID NO.1 into the vector pCAMBIA2300; the MdSAUR15GFP can express the MdSAUR15 protein shown in SEQ ID NO.4. After overexpression using the recombinant vector of the present invention, it can promote the development of adventitious roots of plants under salt stress conditions, promote the number and fresh weight of adventitious roots, assist in the asexual reproduction of plants and cultivate plants with strong adventitious root development ability under salt conditions, optimize the adventitious root development system of plants, improve the salt tolerance of plants, and can be widely used in the genetic improvement of plant salt tolerance.

[0050] The embodiment of the present invention also provides an engineered bacterium, including the MdSAUR15 gene or the recombinant expression vector described in the above technical solution. The original strain for preparing the engineered bacterium in the present invention is preferably Agrobacterium, more preferably Agrobacterium tumefaciens, and even more preferably Agrobacterium EHA105. The engineered bacterium of the present invention is preferably obtained by transferring the recombinant expression vector into the original strain. The present invention has no special limitation on the transfer method, and conventional methods in the art are adopted.

[0051] The embodiment of the present invention also provides a host cell transformed by the expression vector described in the above solution. The host cell of the present invention is preferably a horticultural crop cell, and further optimized to be an apple cell. The present invention has no special limitation on the method for transforming the expression vector into the host cell, and conventional genetic transformation methods in the art can be used.

[0052] The embodiment of the present invention also provides the application of the MdSAUR15 gene, recombinant expression vector, engineering bacterium or host cell described in the above technical solution in any one or more of the following I - IX: I: Promote the occurrence of adventitious roots; II: Increase the number of adventitious roots; III: Increase the fresh weight of adventitious roots; IV: Promote the occurrence of adventitious roots under salt stress conditions; V: Increase the number of adventitious roots under salt stress conditions; VI: Increase the fresh weight of adventitious roots under salt stress conditions; VII: Regulate adventitious root development; VIII: Regulate adventitious root development under salt stress conditions; IX: Plant asexual reproduction; X: Assist in plant salt tolerance molecular breeding. The plants of the present invention include horticultural crops, and further preferably include apples.

[0053] The embodiment of the present invention also provides a method for cultivating a plant with strong adventitious root development ability under salt stress conditions, by increasing the expression of MdSAUR15 in the target plant or increasing the content of MdSAUR15 protein in the target plant to obtain the plant with strong adventitious root development ability under salt stress conditions. The increase in the expression of the MdSAUR15 gene in the target plant or the increase in the content of MdSAUR15 protein in the target plant is achieved by introducing the MdSAUR15 gene into the plant. The plants of the present invention include horticultural crops, and further preferably include apples.

[0054] Example 1 Analysis of the expression pattern of the MdSAUR15 gene during salt stress

[0055] (1) Test materials:

[0056] Apple samples, namely 'Qingzhen No. 1', Wanglin callus (kindly provided by Professor Haoyujin of Shandong Agricultural University) and 'GL3' (kindly provided by Professor Zhangzhihong of Shenyang Agricultural University). 'Qingzhen No. 1' and 'GL3' are both preserved under tissue culture conditions and transferred to the rooting medium for 2 days. The medium formula is 2.215 g of MS powder, 30 g of sucrose, 7.8 g of agar and 1 mg of IBA per liter. Culture conditions: 16 h of light and 8 h of darkness, temperature is 24 °C. Apple callus is cultured in MS liquid medium (MS519 + 2, 4D 1.5 mg / L + 6BA 0.4 mg / L, pH = 5.8) and cultured in suspension in the dark at 25 °C.

[0057] (2) Extraction of total RNA from apple samples and cDNA synthesis:

[0058] Total RNA of adventitious root samples at 0, 1, 3, 5, 7, 9, 12, and 24 h after treatment with NaCl (30 mmol L 1 ) was extracted using a polysaccharide polyphenol RNA extraction kit (FOREGENE, Chengdu, China). The quality of the extracted total RNA was detected using 1% agarose gel and a NanoDrop 2000c micro-ultraviolet spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). RNA reverse transcription was performed using a PrimeScriptTM RT Reagent Kit with gDNA Eraser reverse transcription kit (Takara Bio, Japan).

[0059] (3) Relative expression level of MdSAUR15 gene in the roots of 'GL3' under salt stress

[0060] Specific primers for qRT-PCR were designed within the open reading frame of the MdSAUR15 gene using Primer Premier 6.0. The nucleotide sequences of the primers are as follows:

[0061] Forward primer MdSAUR15-F2: 5'-GCCACTTTGCGGTTTATGTTG-3' (SEQ ID NO.5); Reverse primer MdSAUR15-R2: 5'-CAAACTCTTCCTCAGCCTTGTT-3' (SEQ ID NO.6).

[0062] The apple MdACTIN gene was used as an internal reference control. The nucleotide sequences of the primers are as follows:

[0063] Forward primer MdACTIN-F: 5'-TGACCGAATGAGCAAGGAAATTACT-3 (SEQ ID NO.7); Reverse primer MdACTIN-R: 5'-TACTCAGCTTTGGCAATCCACATC-3' (SEQ ID NO.8).

[0064] qRT-PCR was performed using a SYRB kit (Takara, Japan) according to the kit instructions. The 20 μl reaction system included: 10 μl 2×SYRB Premix ExTaq, 1 μl cDNA, 8.2 μl sterile double-distilled water, 0.4 μl forward primer, and 0.4 μl reverse primer. PCR was carried out using a qRT-PCR instrument (LightCycler480, Roche). The PCR reaction program was: 95°C, pre-denaturation for 3 minutes; 95°C, denaturation for 15 seconds, 60°C, annealing for 15 seconds, 72°C, extension for 30 seconds, for 40 cycles. Each sample was repeated 3 times, and the average Ct value of each sample was calculated. By calculating 2-△△Ct Obtain the relative expression level of the MdSAUR15 gene.

[0065] (3) Analysis of the response pattern of the MdSAUR15 gene promoter to salt stress

[0066] Furthermore, the promoter sequence of MdSAUR15 (2000 bp upstream of ATG) was ligated to the pCAMBIA1381GUS expression vector by homologous recombination to form the pMdSAUR15GUS fusion vector. After transforming Escherichia coli DH5α and detecting the correct band position by colony PCR, the positive bacterial solution was sent to the company for sequencing. The plasmid was extracted from the positive bacterial solution with correct sequencing alignment and then transformed into Agrobacterium tumefaciens GV3101. The promoter sequence of MdSAUR15 is shown as follows:

[0067]

[0068] The Agrobacterium liquid containing pMdSAUR15GUS was activated. After shaking the bacteria, it was suspended with MMA infection solution and injected into tobacco leaves. The infected tobacco was statically cultured in a light incubator for 2 - 3 days. Some of the injected leaves were treated with NaCl (75 mmol L 1 ), and the other part was treated with water as a control. After 24 hours, some leaves were collected for GUS histochemical staining. The formula of the GUS staining solution (200 mL) is as follows:

[0069] Reagent dosage: EDTANa2, 0.744 g; NaH2PO4·H2O, 2.760 g; K4Fe(CN)6·3H2O, 0.042 g; Triton X100, 200 μL; XGluc (100 mM), 1 mL; ddH2O was added to make up to 200 mL.

[0070] The transiently transformed apple callus pMdSAUR15:GUS was immersed in the GUS staining buffer composed of 50 mM NaPO4 (pH 7.2), 0.5 mM K3Fe(CN)6, 0.5 mM K4Fe(CN)6 and 2 mM XGLUC, and soaked at 37°C in the dark for 12 hours. According to the staining effect, observation and photography were carried out. The total GUS protein was quantified using the Bradford protein detection kit (BioRad, USA).

[0071] It can be seen from Figure 1 that when treated with NaCl, the expression of MdSAUR15 in the adventitious roots of 'GL3' apples was significantly up-regulated nearly 9-fold after 24 hours; the GUS staining analysis in the apple callus transiently expressing the pMdSAUR15:GUS vector further confirmed that the expression level of MdSAUR15 was significantly increased under salt stress conditions. The above results indicate that the MdSAUR15 gene is significantly induced by salt stress, suggesting that it may play a role in the response of apple roots to salt stress.

[0072] Example 2 Subcellular localization of MdSAUR15

[0073] Cloning of the coding region (CDS) of MdSAUR15

[0074] (1) Extraction of total RNA from apple samples and cDNA synthesis:

[0075] When cloning the coding region (CDS) of MdSAUR15, the root samples of 'Qingzhen No. 1' were used. The extraction of total RNA and cDNA synthesis of the samples were referred to the methods in Example 1.

[0076] (2) Amplification of the full-length CDS sequence of the MdSAUR15 gene.

[0077] According to the full-length CDS sequence of the MdSAUR15 gene predicted in the apple reference genome (GDDH13), gene quantitative primers were designed using the online tool PrimerBLAST of NCBI (https: / / www.ncbi.nlm.nih.gov / ). The forward primer sequences of the cloned primers are shown in SEQ ID NO.2 and SEQ ID NO.3.

[0078] PCR amplification system: 25 μL of purified cDNA product, 10 μL of 5×TdT buffer, 5 μL of 0.1% BSA, 2.5 μL of 10 mM dCTP, 15 U of TdT, 1 μL of each upstream and downstream primer (stock solution concentration 5 μM), and made up to 50 μL with double-distilled water. PCR amplification program: pre-denaturation at 94°C for 5 minutes; the cycling parameters were denaturation at 94°C for 30 seconds, annealing at 56°C for 30 seconds, extension at 72°C for 90 seconds, for 32 cycles; full extension at 72°C for 10 minutes.

[0079] After the PCR reaction, the PCR product was recovered and ligated to the pMD18T vector to obtain the pMD18T-MdSAUR15 plasmid. Sequencing was completed by Sangon Biotech (Shanghai) Co., Ltd. The results showed that the nucleotide sequence of the MdSAUR15 gene was as shown in SEQ.ID.NO.1; the amino acid sequence encoded by it was as shown in SEQ.ID.NO.2. For the correctly sequenced monoclonal, the plasmid DNA of Pmd18T-MdSAUR15 was extracted and stored at 20°C for subsequent functional verification experiments.

[0080] The above-obtained nucleotide sequence of SEQ.ID.NO.1 was introduced into the pCAMBIA2300-GFP expression vector by homologous recombination method, and the restriction enzyme sites were SacⅠ and XbalⅠ to form the MdSAUR15-GFP overexpression vector, and further transferred into the Agrobacterium tumefaciens strain EHA105 by the liquid nitrogen quick-freezing method. The specific steps are as follows: (1) Take 1 - 2 μg of the MdSAUR15-GFP recombinant plasmid and add it to 50 μL of Agrobacterium competent cells and mix gently; (2) Incubate on ice for 30 min, quick-freeze in liquid nitrogen for 5 min, water bath at 37°C for 5 min, and incubate on ice for 2 min; (3) Add 600 μL of liquid LB medium, culture at 28°C and 200 rpm on a shaker for 4 - 5 h; (4) Centrifuge at 12000 rpm for 1 min, discard the supernatant, retain 200 μL of the liquid, pipette and mix well, and spread it on solid LB medium containing antibiotics (containing 50 mg / L kanamycin sulfate and 50 mg / L rifampicin), culture at 28°C for 48 - 72 h; (5) Pick monoclonal colonies for PCR identification. After shaking the positive colonies, store them with an equal volume of 50% glycerol for subsequent genetic transformation.

[0081] The stored Agrobacterium was streaked and activated on LB solid medium containing 50 mg / L rifampicin and 50 mg / L kanamycin sulfate, and cultured in a constant temperature incubator at 28 °C for 48 h. A transgenic tobacco material containing a nuclear localization signal (NLSmCherry) was used to observe the subcellular localization of MdSAUR15. The specific method was as follows: (1) After activating the Agrobacterium carrying 35S:MdSAUR15GFP and GFP empty plasmid, it was inoculated into 5 mL of LB liquid culture medium (containing 50 mg / L rifampicin and 50 mg / L kanamycin sulfate), and cultured overnight at 28 °C and 200 rpm; (2) 100 - 200 μL of the bacterial liquid from the previous step was taken into 50 mL of LB culture medium and continued to be cultured until the OD600 value was about 0.8, centrifuged at 12000 rpm for 1 min, and the bacterial cells were collected; (3) Suspended with MMA infection solution (100 mL of MMA infection solution contains 0.048 g of MgCl·6H2O, 0.042 g of MES, 100 μL of 100 mM acetosyringone, pH = 5.5), and the final concentration was adjusted to OD600 of 0.4 - 0.6; (4) Tobacco leaf infection: The suspended bacterial liquid was left standing at room temperature for 2 h, and the bacterial liquid was injected into the back of the tobacco leaf with a 1 ml syringe; after injection, it was cultured in the dark for 2 - 3 d; (5) The GFP fluorescence signal was detected and photographed using a laser confocal microscope LSCM (IX83FV1200).

[0082] The steps for observing GFP fluorescence were as follows: (1) Cut a small piece of the above-mentioned tobacco leaf cultured for 2 - 3 d (about 5 * 5 cm), place the epidermis upwards on a glass slide, and drop 1 - 2 drops of distilled water to make a temporary slide. (3) Invert the slide and detect the GFP fluorescence signal and photograph it using a laser scanning confocal microscope LSCM (IX83FV1200). The subcellular localization of MdSAUR15 was observed using a laser confocal microscope, and the results were shown as Figure 2 shown, where GFP is green fluorescence, mCherry is the red nuclear localization fluorescence of transgenic tobacco containing NLSmCherry, BF is the bright field, and Merge is the combination of the three fields of GFP, mCherry, and BF.

[0083] It can be seen from Figure 2 this that GFP signals can be observed in the cell membrane and nucleus in tobacco leaf cells of 35S:MdSAUR15GFP, and green GFP signals can be observed in the nucleus, cell membrane, and cytoplasm in control tobacco leaf cells, indicating that MdSAUR15 is localized in the nucleus and cell membrane, and further proving that the MdSAUR15 protein can function in the nucleus and cell membrane.

[0084] Example 3 Phenotypic Identification of MdSAUR15 Transgenic Apple Lines

[0085] The Agrobacterium tumefaciens EHA105 containing the MdSAUR15GFP overexpression vector obtained in Example 2 was streaked and activated on an LB solid medium containing 50 mg / L rifampicin and 50 mg / L kanamycin sulfate, and cultured in a constant temperature incubator at 28 °C for 48 h. The activated Agrobacterium tumefaciens was picked and cultured in liquid LB in a constant temperature incubator at 28 °C for 14 h until the OD value reached 0.8. The Agrobacterium tumefaciens solution was centrifuged at 5000 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in a resuspension solution (50 mL of sterile water containing 100 μM acetosyringone) for apple transgenic transformation.

[0086] Using the tissue culture seedlings of Gala seedling 'GL3' as transgenic materials, transgenic apple plants were obtained by an Agrobacterium-mediated leaf disc transformation system. The specific method was as follows: (1) For the 'GL3' apple tissue culture seedlings in good growth condition (grown in the medium for 25 d), select the top leaves with uniform and tender growth, and gently make wounds with a scalpel for later use; (2) Immerse the cut leaves in the infection solution and shake them clockwise for 8 - 10 min; (3) Place the infected leaves on a sterilized filter paper to absorb the excess bacterial solution, and then transfer them to a medium without antibiotics for co-culture, and culture them in the dark for 1 - 3 d. (4) Transfer the above-mentioned leaves to a differentiation medium containing cefotaxime (250 mg / L) and kanamycin (50 mg / L), culture them in the dark for one week and then transfer them to light for culture, and change the medium every 10 d during this period; (5) Transfer the new shoots differentiated on the medium to a new medium for growth and propagation; (6) Detect the plants induced after differentiation culture at the DNA and RNA levels to obtain stable transgenic apple tissue culture seedlings. For DNA level detection, the cloning primers of MdSAUR15 with sequences SEQ ID NO.2 and SEQ ID NO.3 were used, and the PCR procedure was the same as described in Example 2; for RNA level detection, the quantitative primers of MdSAUR15 with sequences SEQ ID NO.5 and SEQ ID NO.6 were used, and the qPCR procedure was the same as described in Example 1.

[0087] It can be concluded from Figure 3 a that no MdSAUR15 gene bands were detected in the wild type 'GL3' (WT) and the negative control (H2O is sterile water), while MdSAUR15 gene bands were detected in the positive plasmid and the overexpression lines MdSAUR15OE#1, #2, #3, #5, #6, #8, #9 and #10. Figure 3b shows the RNA detection results of the wild-type 'GL3' (WT) and MdSAUR15 overexpression lines. Among them, MdSAUR15OE has a high expression level in three of the transformed lines (#3, #8, and #10), so these three lines were used for subsequent analysis. The three MdSAUR15 overexpression lines and the wild-type 'GL3' obtained were cultured in the multiplication medium for 5 months until a certain number was obtained. The multiplication medium contained 4.43 g of MS powder, 30 g of sucrose, 7.8 g of agar, 0.2 mg of IBA, and 0.2 mg of 6BA per liter of medium. The tissue culture seedlings of MdSAUR15 overexpression and wild-type 'GL3' grown to 1.5 cm in height were transplanted into the rooting medium. The formula of the rooting medium was 2.215 g of MS powder, 30 g of sucrose, 7.8 g of agar, and 0.2 mg of IBA per liter of medium. The rooting medium for the tissue culture seedlings used for salt stress treatment was additionally supplemented with 30 mmol L 1 of NaCl. The adventitious root growth phenotypes were counted on the 26th day of growth in the rooting medium. The statistical indicators included the number of adventitious roots, the length of adventitious roots, the fresh weight of adventitious roots, and the incidence of adventitious roots. The formula for calculating the incidence of adventitious roots was the number of tissue culture seedlings with adventitious roots / the total number of tissue culture seedlings × 100%. The obtained adventitious roots were scanned using a root scanner (WinRHIZO2003, Quebec, Canada), and the 2D images were analyzed using the WinRHIZO Pro root structure analysis system supporting the scanner to obtain parameter values such as root length.

[0088] The morphological phenotype observations and statistical results of the transgenic apple lines overexpressing MdSAUR15 and the wild-type 'GL3' are as Figure 4 shown. Among them, a shows the phenotype observations of the MdSAUR15 transgenic apple lines and the wild-type apple 'GL3', b shows the statistics of the number of adventitious roots of the MdSAUR15 transgenic apple lines and the wild-type apple 'GL3', c shows the statistics of the length of adventitious roots of the MdSAUR15 transgenic apple lines and the wild-type apple 'GL3', d shows the statistics of the fresh weight of adventitious roots of the MdSAUR15 transgenic apple lines and the wild-type apple 'GL3', and e shows the statistics of the rooting rate of adventitious roots of the MdSAUR15 transgenic apple lines and the wild-type apple 'GL3'.

[0089] It can be concluded from Figure 4 that under non-stress conditions and NaCl treatment, the number, length, fresh weight, and incidence of adventitious roots of MdSAUR15OE plants were significantly higher than those of the wild-type 'GL3' plants ( Figure 4 be). Compared with the control, although the root development of the wild-type 'GL3' and MdSAUR15OE plants was inhibited to varying degrees under salt stress conditions ( Figure 4ae), but the number, length, fresh weight, and incidence of adventitious roots of MdSAUR15OE plants were still significantly higher than those of wild-type 'GL3' plants ( Figure 4 be).

[0090] In addition, by heterologously overexpressing the MdSAUR15 gene in poplar, it was also observed that under non-stress conditions and NaCl treatment, the number, length, fresh weight, and incidence of adventitious roots of poplar MdSAUR15 OE plants were significantly higher than those of wild-type poplar plants ( Figure 5 be).

[0091] Based on the above example results, it can be concluded that MdSAUR15 can promote the development of adventitious roots in apples and poplars, especially promoting the number, length, fresh weight, and incidence of adventitious roots, and playing a promoting and regulatory role in the root development of apples and poplars inhibited by salt stress.

[0092] As described above, it is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention by those skilled in the art within the technical scope disclosed by the present invention shall be covered by the protection scope of the present invention.

Claims

1. An MdSAUR15 gene that promotes root development under salt stress, characterized in that, The nucleotide sequence of the MdSAUR15 gene is shown as SEQ ID NO.

1.

2. The MdSAUR15 gene according to claim 1, wherein The primer pair for amplifying the cDNA sequence of the MdSAUR15 gene includes: Forward primer: 5’ATGGGAATCAAGTTGATGGGGA3’ (SEQ ID NO.2); Reverse primer: 5’CGAACAATTTAGACATGAAGATAGACTG3’ (SEQ ID NO.3).

3. An MdSAUR15 protein encoded by the MdSAUR15 gene according to claim 1, characterized in that, The amino acid sequence of the MdSAUR15 protein is shown as SEQ ID NO.

4.

4. The MdSAUR15 gene according to claim 1, wherein The MdSAUR15 gene is cloned into a recombinant expression vector, and the recombinant expression vector is the pC2300 vector.

5. The recombinant expression vector according to claim 4, characterized in that, The vector is transformed into a host cell or an engineered bacterium for the expression of foreign genes.

6. Use of the MdSAUR15 gene in plant root development, characterized in that, The MdSAUR15 gene is used to promote the formation of adventitious roots in plants, increase the number of adventitious roots, increase the fresh weight of adventitious roots, regulate the development of adventitious roots, or for salt tolerance breeding of plants.

7. A method for cultivating salt-tolerant plants, characterized in that, The method includes: introducing the MdSAUR15 gene described in claim 1 into a target plant, or increasing the expression level of the MdSAUR15 gene in the target plant to promote the occurrence of adventitious roots under salt stress conditions.

8. The method according to claim 7, characterized in that, The target plants include apples, Arabidopsis thaliana, tomatoes, cotton, soybeans or other cash crops.

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