Application of mulberry NAC transcription factor gene in environmental stress regulation

Abstract

The invention provides a mulberry NAC transcription factor gene MnNAC72. A gene sequence of a mulberry NAC transcription factor is as shown in SEQ ID NO: 1. The cDNA sequence is as shown in SEQ ID NO: 2; the amino acid sequence coded by the cDNA is as shown in SEQ ID NO: 3. The MnNAC72 gene disclosed by the invention can be used for regulating and controlling saline-alkali stress of plants, especially salt stress caused by sulfate and chloride.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to the application of the mulberry NAC transcription factor gene in the regulation of environmental stress. Background Technology

[0002] The results of the Third National Land Survey show that my country has 115 million mu (approximately 70 million hectares) of saline-alkali land, mainly distributed in three major regions: First, the arid and semi-arid regions of central and western China, primarily composed of sulfate-chloride compounds, including Xinjiang, Qinghai, Inner Mongolia, and Ningxia, accounting for 96.1% of the country's total saline-alkali land; second, the soda saline-alkali land of northeastern China, primarily composed of carbonate compounds, including Jilin, Inner Mongolia, and Heilongjiang, accounting for 3.2% of the country's total; and third, the coastal saline-alkali land of eastern China, primarily composed of chloride compounds, including Shandong, Jiangsu, and Hebei, accounting for less than 1% of the country's total. Based on current resource conditions and technological levels, and considering factors such as ecology, climate, soil, and location, the saline-alkali land suitable for development into arable land is currently mainly distributed in Jilin, Inner Mongolia, Xinjiang, and Heilongjiang provinces (regions).

[0003] Mulberry is an important economic forest tree in my country, and my country is one of the countries with the most extensive mulberry planting area. In ancient China, mulberry planting and silkworm raising created the Silk Road. Today, mulberry trees can not only be used to raise silkworms, but also have edible value such as mulberry leaf tea, fruit and juice. They also have medicinal value, such as mulberry leaves, branches and fruits can be used as medicines. In addition, mulberry trees have ecological value, such as drought resistance, tolerance to poor soil, salt and alkali resistance and the ability to resist wind and sand (Yu et al., 2025).

[0004] Saline-alkali land accounts for 69.03% of the national total in Northwest China, and it is an important reserve of arable land (Feng Qi et al., 2024). Biological improvement is the most environmentally friendly, long-term, and effective method for improving saline-alkali land. Planting salt-tolerant plants such as mulberry can not only improve soil structure, enhance soil permeability, and reduce soil moisture evaporation through their root systems, but also increase soil enzyme activity and the accumulation of soil organic matter. The huge biomass of the aboveground parts can also absorb and accumulate salt to reduce the salt content in the soil. Therefore, discovering salt-tolerant genes is the foundation for breeding salt-tolerant mulberry trees (Zeng Yuli et al., 2024). Therefore, functional analysis of salt-tolerant genes in mulberry trees is of great significance for screening and using transgenic technology to breed salt-tolerant mulberry varieties, improving the quality of saline-alkali land, and increasing the utilization rate of saline-alkali land. Currently, there are few salt-tolerant genes studied in mulberry trees, and existing studies mostly use NaCl as the experimental treatment, corresponding to saline-alkali land with chloride as the main component. However, there is limited research on complex saline-alkali land, which is dominated by sulfate and chloride. Therefore, it is urgent to explore salt-alkali tolerant genes in mulberry trees that are mainly composed of sulfate and chloride, so as to provide a theoretical basis for screening and breeding salt-alkali tolerant mulberry varieties.

[0005] NAC transcription factors are among the most numerous and unique transcription factors in plants, playing crucial roles in plant growth and development, stress response, and hormone signal transduction. Based on phylogenetic analysis, protein domain composition, and functional studies, the NAC gene family is mainly divided into two major lineages (Groups): Group I and Group II, each further subdivided into multiple subfamilies. Group II members are involved in initiating secondary cell wall biosynthesis and determining xylem vessel and fiber cell differentiation. Group I family members typically include NAC gene members related to developmental processes and stress responses; NAM and NAC1 subfamily members play a role in shoot tip formation and development, and cell morphogenesis; the SENU5 subfamily is closely linked to stress signals and senescence; NAP, AtNAC3, and ATAF subfamily members are widely involved in stress responses; and NAC2 and OsNAC8 subfamily genes regulate plant responses to various nutrient stresses. In summary, NAC transcription factors exhibit diverse biological activities.

[0006] There are 96 genes in mulberry trees that belong to the NAC transcription factor family. Due to the polyploidy and frequent hybridization events in mulberry trees, the genetic background of mulberry trees is very complex, so research on how the NAC transcription factor gene family plays an environmental regulatory role is very limited. Summary of the Invention

[0007] Based on this, the purpose of this invention is to provide a mulberry NAC transcription factor NAC72, its encoding gene, and its application. This mulberry NAC transcription factor NAC72 gene can be used to regulate saline-alkali stress, and is particularly suitable for saline-alkali lands in arid and semi-arid regions of central and western China where sulfate-chloride is the main alkali.

[0008] In a first aspect, the present invention provides an application of the mulberry NAC transcription factor gene in regulating plant salt and alkali stress, wherein the amino acid sequence of the protein encoded by the mulberry NAC transcription factor gene is shown in SEQ ID NO: 3.

[0009] In some embodiments, the salt stress is salt stress caused by sulfates and chlorides.

[0010] In some embodiments, the cDNA sequence of the mulberry NAC transcription factor gene is shown in SEQ ID NO: 2. It should be understood that, considering codon degeneracy, modifications to the nucleotide sequence of the above-mentioned encoding gene without altering the amino acid sequence are also within the scope of protection of this invention.

[0011] In some embodiments, the mulberry NAC transcription factor gene is amplified using primers shown in SEQ ID NO: 6 and SEQ ID NO: 7, or SEQ ID NO: 8 and SEQ ID NO: 9.

[0012] Secondly, the present invention provides an application of a recombinant expression vector in regulating salt and alkali stress in plants, wherein the recombinant expression vector is inserted with the above-mentioned mulberry NAC transcription factor gene.

[0013] In some embodiments, the expression vectors are PBI121 and pCAMBIAsuper1300.

[0014] Thirdly, the present invention provides an application of engineered bacteria in regulating salt and alkali stress in plants, wherein the engineered bacteria are loaded with the above-mentioned recombinant expression vector.

[0015] In some embodiments, the engineered bacteria is Agrobacterium rhizogenes GV3101.

[0016] Fourthly, the present invention provides a method for improving the salt and alkali stress tolerance of plants, the method comprising increasing the expression of the above-mentioned mulberry NAC transcription factor gene in host plant cells, tissues or individual plants.

[0017] In some embodiments, the plant is Arabidopsis thaliana (Brassica family), Nicotiana spp. (Solanaceae family), or Morus spp. (Moraceae family).

[0018] In some embodiments, the plant is a mulberry tree, tobacco, or Arabidopsis thaliana.

[0019] In some embodiments, the Arabidopsis thaliana is Col-0 wild type, the tobacco is Yunyan 87 and Benshiyan, and the mulberry variety is mainly Guisangyou 12.

[0020] The present invention has the following beneficial effects:

[0021] This invention reveals that sulfate and chloride can stimulate the high expression of the mulberry NAC transcription factor MnNAC72, and reports the function of this mulberry NAC transcription factor for the first time. The application of this mulberry NAC transcription factor gene NAC72 in regulating salt stress, particularly sulfate and chloride-induced salt stress, enriches the gene function of this type of NAC transcriptome factor.

[0022] This invention reveals that MnNAC72 responds to sulfate-chloride salt stress in mulberry trees, and its expression level is significantly higher in salt-tolerant mulberry varieties than in salt-sensitive varieties. Expression of MnNAC72 in Arabidopsis and tobacco endows transgenic Arabidopsis and tobacco with enhanced salt tolerance not found in non-transgenic plants. Comparative analysis demonstrates that transgenic mulberry NAC transcription factor plants exhibit improved salt tolerance compared to non-transgenic plants. The mulberry NAC transcription factor gene NAC72 described in this invention can be widely used for screening and breeding salt-tolerant plant varieties, and is suitable for saline-alkali lands in arid and semi-arid regions dominated by sulfate-chloride salts. Attached Figure Description

[0023] Figure 1 Evolutionary tree of NAC family genes in mulberry, poplar, and Arabidopsis was constructed.

[0024] Figure 2 Phylogenetic trees were constructed by comparing the NAC72 sequences of mulberry with those of Arabidopsis thaliana, maize, crabapple, salt mustard, and poplar.

[0025] Figure 3 The NAC72 sequence of mulberry was compared with those of several other species, including Arabidopsis thaliana, maize, crabapple, salt mustard, and poplar.

[0026] Figure 4 Subcellular localization of MnNAC72.

[0027] Figure 5 The expression pattern of the MnNAC72 gene under salt stress was analyzed. Among them, (A) is the expression pattern of the MnNAC72 gene under sulfate-chloride salt stress; (B) is the expression pattern of the MnNAC72 gene in multiple mulberry species.

[0028] Figure 6 The pBI121 spectrum is used as an introductory vector.

[0029] Figure 7 The pCAMBIAsuper1300 spectrum is used as an introductory vector.

[0030] Figure 8 The results of RNA extraction and reverse transcription from wild-type and transgenic Arabidopsis thaliana are presented. (A) shows the molecular identification of the transgenic Arabidopsis thaliana, specifically the overexpression lines OE1, OE4, and OE5 of *Arabidopsis thaliana* overexpressing *Mulberry NAC72*. (B) shows the growth status of the overexpressing Arabidopsis thaliana lines in the selection medium. (C) and (D) show the germination rate and survival rate of the overexpressing Arabidopsis thaliana lines in the selection medium. (E) shows the phenotypic diagrams of the overexpressing Arabidopsis thaliana lines and the control after 3 days of treatment with 200 mM sulfate-chloride saline.

[0031] Figure 9The results of RNA extraction and reverse transcription in wild-type and transgenic tobacco were validated. (A) shows the phenotypes of tobacco lines overexpressing the NAC72 gene and wild-type after treatment with 200 mM sulfate-chloride salt; (B) shows the molecular identification of transgenic tobacco; (C) shows the change in malondialdehyde (MDA) content in transgenic tobacco lines after salt-alkali treatment; (D) shows the change in proline (Pro) content in transgenic tobacco lines after salt-alkali treatment; (E) shows the change in superoxide dismutase (SOD) activity in transgenic tobacco lines after salt-alkali treatment; (F) shows the change in peroxidase (POD) activity in transgenic tobacco lines after salt-alkali treatment; (G) shows the change in catalase (CAT) activity in transgenic tobacco lines after salt-alkali treatment; and (H) shows the change in NtSOD gene expression in transgenic tobacco lines after salt-alkali treatment. Detailed Implementation

[0032] To facilitate understanding of the present invention, a more complete description will be provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0033] Unless otherwise specified, experimental methods in the following examples were performed under standard conditions, such as those described in the fourth edition of *Molecular Cloning: A Laboratory Manual*, edited by Green and Sambrook, published in 2013, or according to the manufacturer's recommendations. All commonly used chemical reagents used in the examples are commercially available products.

[0034] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this invention includes any and all combinations of one or more of the associated listed items.

[0035] This invention, based on transcriptome data of mulberry trees under sulfate-chloride complex salt stress, has for the first time screened and identified a key member of the AtNAC3 subfamily (NAC72). The traditional functional localization of this family of genes is stress response. While AtNAC3 has been reported in relation to salt stress, these have all been related to chloride salt stress, and no reports have been made regarding sulfate-chloride complex salt stress, which accounts for the largest proportion of salt stress in China. This study reveals for the first time its significant transcriptional response to sulfate-chloride complex saline-alkali land, expanding our understanding of the functional role of the AtNAC3 subfamily of genes in abiotic stress adaptation, and disclosing the application value of this gene in salt-alkali tolerant breeding of mulberry trees against sulfate-chloride complex salt stress and in the ecological restoration of saline-alkali land.

[0036] This invention transfers the mulberry NAC72 gene into Arabidopsis thaliana and tobacco to obtain transgenic Arabidopsis thaliana and tobacco with the MnNAC72 gene. Physiological and biochemical tests confirmed that the sulfate-chloride salt tolerance of the transgenic Arabidopsis thaliana and tobacco was significantly improved.

[0037] This invention provides a mulberry NAC transcription factor and its corresponding gene, comprising the following:

[0038] (a) The nucleotide sequence shown in SEQ ID No: 1;

[0039] (b) The cDNA sequence shown in SEQ ID No: 2;

[0040] (c) The amino acid sequence shown in SEQ ID No: 3;

[0041] Specifically, (a) the nucleotide sequence of the mulberry NAC transcription factor gene MnNAC72 is (SEQ ID No: 1):

[0042]

[0043]

[0044] (c) Amino acid sequence of mulberry NAC transcription factor MnNAC72 (SEQ ID No: 3):

[0045] MGVPETDLLSQLSLPPGFRFFPTDEELLVQYLCRKVAGQQFSLQIIGEIDLYKFDPWVLPSKAIFGEKEWYFFSPRDRKYPNGSRPNRVAGSGYWKATGTDKVITTEGRKVGIKKALVFYVGKAPKGTKTNWIMHEYRLIESSRKHGSSKLDEWVLCRIYKKNSSSQKPMISSS LTSKEQYSNGSSSSSSSHLDDVLDSLPTIDHRFFSLPRVSSLKALQDDDKLGLYNLGSGNFDWASLVGLNSVPELVPSNQAQTQYQGTVGFNNGDVYAPSIPPLCHVESPAKRLGNSVEEEVQSGLRTQRVENSGLFQSNSNVLTQNFSNSLDPYGFRYPTQSAGFGFGR (344 aa).

[0046] In some embodiments of the present invention, the application of expressing the MnNAC72 gene in the mulberry NAC transcription factor gene MnNAC72 and improving salt tolerance is involved.

[0047] The plant in question is a mulberry tree, tobacco, or Arabidopsis thaliana.

[0048] In some embodiments, the Arabidopsis thaliana is Col-0 wild type, the tobacco is Yunyan 87 and Benshiyan, and the mulberry variety is mainly Guisangyou 12.

[0049] In some embodiments, the expression pattern of the mulberry NAC transcription factor gene MnNAC72 described in this invention was examined at five time points (0, 12 h, 1 d, 3 d, and 5 d) during treatment with 200 mM sulfate-chloride salt on Gui Sang You 12 mulberry trees to verify the response of MnNAC72 to salt stress. Furthermore, the expression pattern of the mulberry NAC transcription factor gene MnNAC72 was detected in nine different mulberry varieties to verify the relationship between the expression pattern of the MnNAC72 gene and the salt tolerance of mulberry trees.

[0050] In some embodiments, the application of the mulberry NAC transcription factor gene MnNAC72 described in this invention in expressing MnNAC72 in Arabidopsis thaliana or tobacco plants.

[0051] This invention first cloned the NAC transcription factor gene MnNAC72 from the Guisangyou 12 plant. Using homologous recombination, the MnNAC72 gene was ligated into the PBI121 plasmid. Then, a large number of clones were obtained in *E. coli* DH5α via heat shock transformation. The MnNAC72 gene was ligated into the expression vector pCAMBIAsuper1300 and expressed in *E. coli* DH5α. Positive transgenic *E. coli* strains were then screened, and transformation plasmids were extracted and finally transformed into *Agrobacterium* strain GV3101. The transformed *Agrobacterium* strain was then transformed into *Arabidopsis thaliana* and tobacco to verify the function of MnNAC72.

[0052] In this embodiment of the invention, salt stress refers to a 1:1 molar ratio of 200 mM NaCl: Na2SO4.

[0053] The present invention will be further described in detail below with reference to specific embodiments.

[0054] Example 1: The role of the mulberry NAC transcription factor gene NAC72 in the salt stress response of mulberry.

[0055] To investigate the role of the mulberry NAC transcription factor gene NAC72 in the salt stress response of mulberry, transcriptome sequencing was performed to screen for the gene MnNAC72, which was simultaneously regulated by five time periods of salt stress. Then, RT-PCR was used to determine its response to salt stress.

[0056] 1. Cultivation and growth of Mulberry Tree Sand 2 × Lun 109, Gui Sang You 5, Gui Sang You 6, Sang Te You 2, Gui Sang You 12, Gui Sang You 62, Kang Qing 283 × Kang Qing 10, Yue Sang 51 and Yue Sang 120:

[0057] Several varieties, including Gui Sang You 12, germinated in an incubator and were cultivated in an indoor environment at a temperature of 23-25℃, with a light cycle of 16 hours of light and 8 hours of darkness, and a humidity of 70%. Treatment was carried out when the plants reached 5-6 leaves.

[0058] In this embodiment, seedlings of Gui Sang You 12 were treated with a 1:1 NaCl:Na2SO4 salt concentration of 200 mM. Morphological changes were observed after 0 h, 12 h, 1 d, 3 d, and 5 d of salt treatment. Mulberry leaves were then sampled and cryopreserved in liquid nitrogen. Mulberry leaves from Sha 2×Lun 109, Gui Sang You 5, Gui Sang You 6, Sang Te You 2, Gui Sang You 12, Gui Sang You 62, Kang Qing 283×Kang Qing 10, Yue Sang 51, and Yue Sang 120 were collected as verification materials and cryopreserved in liquid nitrogen.

[0059] 2. RNA extraction from mulberry leaves:

[0060] 0.1 g of mulberry leaf material was placed in a mortar, liquid nitrogen was added, and the mixture was rapidly ground into a fine powder. Extraction was performed using the TIANGEN RNAprep Pure Polysaccharide-Polyphenol Plant Total RNA Extraction Kit, following the instructions in the manufacturer's manual. The concentration and quality of the extracted RNA were determined using a Nanodrop 2000 spectrophotometer.

[0061] 3. Reverse transcription:

[0062] Step (1), cDNA synthesis: RNA reverse transcription was performed using a reverse transcription kit from TAKARA. The procedure is as follows (all steps were performed on ice to prevent RNA degradation):

[0063] Synthesis of the first strand of cDNA:

[0064] DNA removal

[0065] Add the following ingredients to a 200 μL centrifuge tube:

[0066] 5× gDNA Eraser Buffer, 2 μL

[0067] gDNA Eraser, 1 μL

[0068] Total RNA, 1000 ng

[0069] Rnase Free H2O, add to 10 μL

[0070] Place the RNAase Fress centrifuge tube containing the prepared solution into a PCR instrument and incubate at 42°C for 2 minutes.

[0071] Step (2), reverse transcription reaction:

[0072] The reaction solution in step (1), 10 μL

[0073] primerScript RT Enzyme Mix Ⅰ, 1 μL

[0074] RT Primer Mix, 4 μL

[0075] 5× PrimerScript Buffer 2, 4 μL

[0076] dd H2O, 1 μL

[0077] Total, 20 μL

[0078] Place the centrifuge tube containing the prepared solution into a PCR instrument, set the temperature to 37℃ for 15 min, then to 85℃ for 5 s. After the reaction is complete, dilute with 80 μL of DEPC water and store at -20℃.

[0079] Quantitative primers were designed based on the known NAC72 gene sequence published in GenBank:

[0080] NAC072F: TTGTGCCGAAAAGTTGCTGG (SEQ ID No: 4).

[0081] NAC072R: TTCGGGTACTTCCGATCCCT (SEQ ID No: 5).

[0082] Design PCR conditions according to the instructions for the Vazyme Real-Time Fluorescence Kit.

[0083] A total of 96 NAC gene family members were identified from the mulberry genome and named MnNAC1-MnNAC96 based on their alignment with Arabidopsis thaliana. The phylogenetic tree results (e.g.) are presented below. Figure 1 The results show that the NAC gene family is divided into 14 subfamilies. MnNAC72 is grouped into one subfamily with Arabidopsis NAC019, NAC055 and NAC72. Arabidopsis NAC019, NAC055 and NAC72 play a key role in resisting drought stress.

[0084] Furthermore, sequence alignment of the mulberry NAC72 gene with those of Arabidopsis thaliana, Haloxylon ammodendron, Malus baccata, Populus tomentosa, and Maize was performed. Phylogenetic analysis showed that MnNAC72 (mulberry) is closely related to MdNAC72 (Malus baccata), while it is more distantly related to ZmNAC (Maize). Figure 2 Multiple sequence alignment results showed that the NAC protein sequences of all species contained a total of 5 conserved AE domains, demonstrating sequence conservation. Figure 3 Subcellular localization also confirmed that the mulberry NAC72 gene is located in the cell nucleus (e.g., Figure 4 ).

[0085] To investigate the changes in MnNAC72 expression induced by salt stress, mulberry seedlings were treated with a 1:1 molar ratio of NaCl:Na2SO4 at 200 mM for five time points: 0, 12 h, 1 d, 3 d, and 5 d. Figure 5 As shown in Figure A, MnNAC72 expression was induced by salt stress and increased by 110-fold, 77-fold, 101-fold, and 165-fold at four different time points compared to the control. The expression level reached its highest value after 5 days of salt treatment, indicating that the salt stress effect significantly promoted the expression of this gene.

[0086] The expression levels of MnNAC72 in several mulberry varieties, including Sha 2×Lun 109, Gui Sang You 5, Gui Sang You 6, Sang Te You 2, Gui Sang You 12, Gui Sang You 62, Kang Qing 283×Kang Qing 10, Yue Sang 51, and Yue Sang 120, were detected. The results confirmed that the MnNAC72 gene expression level was highest in Gui Sang You 6, Gui Sang You 62, and Gui Sang You 5. Previous studies have shown that Gui Sang You 62, Sang Te You 6, and Sang Te You 5 are salt-tolerant mulberry varieties, and the MnNAC72 expression level in these three varieties was significantly higher than in other varieties. Figure 5 B).

[0087] Our study found that MnNAC72 expression was low in Gui Sang You 12 mulberry, and some mulberry varieties did not express it at high levels. Furthermore, we discovered significant differences in NAC72 expression among different mulberry varieties. Whether the NAC72 gene can resist salt stress, especially sulfate and chloride-induced salt stress, and whether it can be used to screen for salt-tolerant varieties, requires further research.

[0088] Example 2 Construction of MnNAC72 binary expression vector

[0089] Using cDNA and genomic DNA as templates, high-fidelity enzyme amplification was performed, and the obtained gene was named MnNAC72 (mulberry NAC72 gene).

[0090] PBIBamhI NAC72F: cgggggactctagagATGGGTGTGCCGGAAACAGA (SEQ ID No: 6).

[0091] PBISacⅠNAC72R: ggggaaattcgagctTCACCGCCCAAACCCGAA (SEQ ID No: 7).

[0092] 1300GFPF: ATACACCAAATCGACTCTAGAATGGGTGTGCCGGAAA (SEQ ID No: 8).

[0093] 1300GFPR: CATGGTACCGGATCCACTAGTCCGCCCAAACCCGAACCCT (SEQ ID No: 9).

[0094] PBI121 vector (such as Figure 6 The vector pCAMBIAsuper1300 was transdigested with restriction endonucleases Bamh I and Sac I, and then expressed transiently. Figure 7The MnNAC72 gene was double-digested with restriction endonucleases Xba I and Spe I. The MnNAC72 gene amplified by PCR was ligated to PBI121 and pCAMBIAsuper1300 using homologous recombination ligase to obtain plasmids PBI121-NAC72 and pCAMBIAsuper1300-NAC72.

[0095] Example 3: Arabidopsis thaliana transgenic with the MnNAC72 gene was obtained using Agrobacterium-mediated transformation.

[0096] 1. Transformation of Agrobacterium tumefaciens GV3101

[0097] 1) Add 1 μL of plant expression vector plasmid DNA to Agrobacterium competent cells, mix gently, freeze in liquid nitrogen for 5 min, incubate at 37°C for 5 min, and then incubate on ice for 5 min.

[0098] 2) Add 600 μL of LB liquid medium, gently shake at 28°C for 2 h, centrifuge at 6000 rpm for 3 min at room temperature to enrich the bacterial cells;

[0099] 3) Retain 50-200 μL of bacterial culture, mix well, and spread evenly on LB selective plates containing an appropriate amount of antibiotics. Incubate upside down at 28°C for two days.

[0100] 4) Pick fresh colonies, perform PCR identification, and screen for positive clones.

[0101] 2. Genetic transformation and selection of positive lines in Arabidopsis thaliana

[0102] 1) Activate the Agrobacterium for transformation on a plate, then pick single clones and culture them in LB liquid medium with the corresponding antibiotic. On the second day, expand the culture at a ratio of 1:100 for 12–16 h.

[0103] 2) At room temperature, centrifuge at 5000 rpm for 10 min to collect the precipitate, discard the supernatant, resuspend the precipitate in a solution containing 0.01% Silwet-77 and 5% sucrose, and adjust the OD600 concentration to 0.8 as the staining solution;

[0104] 3) When Arabidopsis thaliana (wild type Col-0) grows to 1 cm in length, the top is cut off to induce the formation of lateral inflorescences. Select healthy plants, cut off the fruit pods, and soak the inflorescences in the prepared infusion solution for 5 min.

[0105] 4) Remove the inflorescence from the infusion solution and place it in a dark incubator at 24°C for 1 day.

[0106] 5) After dark culture, transfer the plants to a normal growth environment. After one week of growth, repeat the above steps to complete the second transformation to improve the transformation success rate.

[0107] 6) After the seeds mature, harvest and dry them, and place them in a 4℃ refrigerator for vernalization treatment for 2 days to break dormancy;

[0108] 7) Seeds were sown in 1 / 2 MS medium containing kanamycin resistance for positive screening;

[0109] 8) Directly screen out T1 generation positive seedlings, transplant them into plug trays, and harvest seeds from each individual plant; these are T2 generation seeds. Continue screening the seeds collected from each plant. Transplant positive plants with a segregation ratio of 3:1 (positive:negative) and grow them to harvest T3 generation transgenic seeds. Harvest seeds from each individual plant; all T3 generation seeds collected from each plant constitute one line. Sow the T3 generation seeds on a culture medium containing antibiotics. If all seedlings grown on the medium are positive, the seeds collected from this plant are the desired purebred transgenic line. All T3 generation purebred transgenic Arabidopsis seeds collected from a single plant constitute one line. Name the collected lines OE1 (overexpression 1), OE2, OE3, OE4, and OE5. Select OE1, OE5, and OE4 according to high, medium, and low MnNAC72 gene expression levels to verify the function of the MnNAC72 gene.

[0110] 3. Nine wild-type and nine transgenic Arabidopsis thaliana lines OE1, OE4 and OE5 were used to extract RNA and reverse transcribe it, and then verified by PCR.

[0111] (1) RNA extraction steps:

[0112] 0.1 g of wild-type and transgenic Arabidopsis leaf material were placed in a mortar, liquid nitrogen was added, and the mixture was rapidly ground into a fine powder. RNA was extracted using the TIANGEN RNA Easy Fast Plant Tissue RNA Rapid Extraction Kit (centrifuge column type), following the instructions in the manufacturer's manual. The concentration and quality of the extracted RNA were determined using a Nanodrop 2000 spectrophotometer.

[0113] (2) Reverse transcription step:

[0114] Step 1: cDNA synthesis was performed using a reverse transcription kit from TAKARA, with the following steps (all steps were performed on ice to prevent RNA degradation):

[0115] Synthesis of the first strand of cDNA:

[0116] DNA removal

[0117] Add the following ingredients to a 200 μL centrifuge tube:

[0118] 5× gDNA Eraser Buffer, 2 μL

[0119] gDNA Eraser, 1 μL

[0120] Total RNA, 1000 ng

[0121] Rnase Free H2O, add to 10 μL

[0122] Place the RNAase Fress centrifuge tube containing the prepared solution into a PCR instrument and incubate at 42°C for 2 minutes.

[0123] Step 2, Reverse Transcription Reaction:

[0124] The reaction solution in step 1, 10 μL

[0125] primerScript RT Enzyme Mix Ⅰ, 1 μL

[0126] RT Primer Mix, 4 μL

[0127] 5× PrimerScript Buffer 2, 4 μL

[0128] dd H2O, 1 μL

[0129] Total volume, 20 μL.

[0130] Place the centrifuge tube containing the prepared solution into a PCR instrument, set the temperature to 37℃ for 15 min, then to 85℃ for 5 s. After the reaction is complete, dilute with 80 μL of DEPC water and store at -20℃.

[0131] (3) Design quantitative primers based on the NAC72 gene sequence:

[0132] NAC072F: TTGTGCCGAAAAGTTGCTGG (SEQ ID No: 4).

[0133] NAC072R: TTCGGGTACTTCCGATCCCT (SEQ ID No: 5).

[0134] Design PCR conditions according to the instructions for the Vazyme Real-Time Fluorescence Kit.

[0135] See results Figure 8A. Expression levels of the MnNAC72 gene in wild-type Arabidopsis thaliana WT and transgenic Arabidopsis thaliana lines OE1, OE4, and OE5. The wild-type Arabidopsis thaliana showed almost no expression, while the transgenic Arabidopsis thaliana lines showed an expression level of approximately 1000 times that in the wild-type Arabidopsis thaliana, indicating that the overexpression lines were successfully constructed and can be used for subsequent verification experiments.

[0136] 3. Salt stress treatment of Arabidopsis thaliana (1:1 molar ratio of NaCl: Na2SO4)

[0137] 1) Germination rate: Transgenic Arabidopsis seeds were treated at 4℃ for 48 h and sown separately from wild-type seeds on opposite sides of a 1 / 2 MS medium containing 100 mM NaCl (e.g., Figure 8 (B) 61 and 144 seeds were sown from OE1 and wild-type, respectively; 125 and 132 seeds were sown from OE4 and wild-type, respectively; and 90 and 138 seeds were sown from OE5 and wild-type, respectively. These seeds were cultured in a long-day artificial climate chamber, and the seed germination rate was recorded. Subsequent culture was conducted, with 10 wild-type and 10 overexpression lines transferred to 1 / 2 MS medium containing 100 mM NaCl to examine seed survival in the saline medium. Results are shown below. Figure 8 B-8D, the number of germinating seeds was counted. The germination rate of seeds from overexpression lines OE1, OE4, and OE5 was higher than that of wild type. After 3-5 days of culture, the survival rate of OE1 and OE5 was lower than that of wild type. The reason for the low survival rate of overexpression lines may be that the NAC72 gene is mainly involved in the abscisic acid pathway. Overexpression of NAC72 will lead to increased sensitivity of plants to ABA, resulting in a lower green embryo rate in overexpression plants.

[0138] 2) Seedling tolerance test: Transgenic Arabidopsis seeds were treated at 4℃ for 48 h and then sown on 1 / 2 MS medium. When the seeds had 4 true leaves, they were transplanted into pots containing a substrate of vermiculite and nutrient soil and placed in a long-day artificial climate chamber for cultivation. After 50 days, when the seedlings were about to bolt and flower, salt stress treatment was initiated: the seedlings were first thoroughly watered, and then irrigated with 200 mM NaCl and Na2SO4 solutions. The salt tolerance phenotypes of wild-type and transgenic lines were observed and statistically analyzed. The results are shown in […]. Figure 8 E. Transgenic plants and control plants were treated with 200 mM salt and pure water for 3 days respectively. After pure water treatment, there was no difference in growth and biomass between wild-type and transgenic lines. After salt treatment, the yellowing and wilting of leaves of wild-type Arabidopsis thaliana was significantly greater than that of transgenic lines. The phenotype showed that transgenic Arabidopsis thaliana plants significantly improved the salt tolerance of Arabidopsis thaliana.

[0139] Example 4: Obtaining tobacco transgenic with the MnNAC72 gene using Agrobacterium-mediated transformation.

[0140] 1. After transformation and identification by Agrobacterium tumefaciens GV3101, the Agrobacterium tumefaciens bacterial solution that tested positive was expanded and cultured. 100 µL of the positive stock solution was added to 5 mL of LB liquid medium containing kanamycin and rifampin. The culture was carried out at 28℃ and 200 rpm for 16 h. Then, the culture was centrifuged at 4000 rpm for 5 min, the supernatant was discarded, and the precipitate was resuspended with freshly prepared infiltration buffer (10 mM MgCl2, 10 mM MMEs, 200 μM acetylsyl syringone). After washing twice, the OD600 concentration was measured using a microplate reader and adjusted to 0.8.

[0141] 2. Explant preparation

[0142] Aseptic seedling culture: Select healthy Yunyan 87 tobacco seeds, disinfect them in a clean bench for 2 minutes with 75% ethanol and 10% sodium hypochlorite for 15 minutes, rinse them with sterile water 5-10 times to prevent sodium hypochlorite residue, place the rinsed seeds on sterile paper, absorb the surface moisture, and then sow them in 1 / 2 MS medium. Place them in a tissue culture room for constant temperature and light culture at 28℃. After two weeks of culture, obtain tobacco seedlings under sterile conditions.

[0143] Leaf cutting: Sterile tobacco leaves are cut into leaf discs of 1 cm × 1 cm or 4-6 mm to serve as recipient materials for transformation.

[0144] 3. Agrobacterium infection and co-culture

[0145] Infection: Place the cut leaves into the Agrobacterium bacillus solution and gently shake or stir. The infection time is usually 5-10 minutes to ensure that the Agrobacterium bacillus comes into full contact with the leaves.

[0146] Co-culture: After infection, the leaves were blotted with sterile filter paper to remove excess bacterial solution, laid flat on sterilized filter paper to dry, and then transferred to co-culture medium (MS medium + 6BA 1 mg / L + NAA 0.1 mg / L). They were then incubated in the dark at 23-25℃ for 3 days to promote the interaction between Agrobacterium and tobacco cells.

[0147] 4. Screening and Cultivation

[0148] Transfer medium: The co-cultured leaves were transferred to a selection medium containing selective antibiotics (such as kanamycin) and cephalosporins (MS medium + 6BA 1 mg / L + NAA 0.1 mg / L + Kan 100 mg / L + Cb 400 mg / L) and cultured at 25°C under light.

[0149] Callus induction: Callus tissue gradually grows from leaf wounds on the selection medium. The culture continues until the callus tissue grows well and has resistance.

[0150] 5. Bud strengthening and rooting culture

[0151] Bud growth culture: Bud clusters were cut from the callus tissue and inoculated onto bud growth culture medium (MS medium + 6BA 0.1 mg / L + NAA 0.01 mg / L + Kan 100 mg / L + Cb 400 mg / L) and cultured for about 1 month to allow the bud clusters to grow vigorously.

[0152] Rooting culture: Select vigorous resistant shoots, inoculate them onto rooting medium (MS medium), and culture for about half a month to induce rooting and form complete transgenic plants.

[0153] 6. Identification of positive plants and hardening off seedlings

[0154] Molecular identification: Transgenic plants are identified using PCR molecular biology methods, and positive plants that have successfully integrated the target gene are screened out.

[0155] Hardening off and transplanting: Transgenic plants identified as positive were removed from the culture medium and hardened off. After adapting to the natural environment, they were transplanted into the soil for further cultivation and seed collection. Quantitative verification was performed using NAC072F / R primers. The control plants showed almost no expression of the mulberry NAC72 gene, while the transgenic plants showed high expression. All purebred transgenic tobacco seeds collected from one selected plant were considered as one line, and the collected lines were named OEnt1 (overexpression Nicotiana tabacum1), OEnt2, OEnt3, OEnt4, OEnt5, OEnt6, OEnt7, OEnt8, etc. OEnt1, OEnt8, and OEnt4 were selected as examples based on high, medium, and low MnNAC72 gene expression levels to verify the function of the MnNAC72 gene.

[0156] 7. Leaves from 5 wild-type and 5 transgenic tobacco plants after culture were used as materials. RNA was extracted and reverse transcribed, and then verified by PCR.

[0157] (1) RNA extraction steps:

[0158] 0.1 g of wild-type and transgenic tobacco leaf material were placed in a mortar, liquid nitrogen was added, and the mixture was rapidly ground into a fine powder. RNA was extracted using the TIANGEN RNA Easy Fast Plant Tissue RNA Rapid Extraction Kit (centrifuge column type), following the instructions in the manufacturer's manual. The concentration and quality of the extracted RNA were determined using a Nanodrop 2000 spectrophotometer.

[0159] (2) Reverse transcription step:

[0160] cDNA synthesis was performed using a reverse transcription kit from TAKARA, with RNA reverse transcription performed according to standard procedures (all steps were performed on ice to prevent RNA degradation). After the reaction was completed, 80 μL of DEPC water was added for dilution, and the mixture was stored at -20°C.

[0161] (3) Design quantitative primers based on the NAC72 gene sequence:

[0162] NAC072F: TTGTGCCGAAAAGTTGCTGG (SEQ ID No: 4).

[0163] NAC072R: TTCGGGTACTTCCGATCCCT (SEQ ID No: 5).

[0164] Design PCR conditions according to the instructions for the Vazyme Real-Time Fluorescence Kit.

[0165] 7. Detection of salt tolerance indicators in genetically modified tobacco

[0166] To understand the salt tolerance of tobacco transformed with the mulberry transcription factor NAC72 gene, 21-day-old transgenic plants and wild-type tobacco seedlings were treated with sulfate-chloride salts. The transgenic plants were then identified using PCR molecular biology methods, and positive plants with successful integration of the target gene (e.g., mulberry NAC72 gene) were screened. Figure 9 (B) The expression levels of the MnNAC72 gene in the overexpression lines OEnt1, OEnt4, and OEnt8 were significantly higher than those in the wild-type line (WT), indicating that the transgenic lines were successfully constructed and can be used for subsequent gene function verification experiments. The phenotypes after salt treatment (e.g., Figure 9 A) Wild-type tobacco (WT) exhibited significantly higher levels of leaf wilting and chlorosis under salt stress treatment compared to the overexpression lines OEnt1, OEnt4, and OEnt8, while no significant difference was observed in the absence of salt stress. This indicates that overexpression of MnNAC72 in tobacco can enhance its salt stress tolerance. The malondialdehyde (MDA) content, proline content, superoxide dismutase (SOD) activity, catalase (CAT) activity, and peroxidase (POD) activity of the treated materials were measured. The corresponding substance contents and enzyme activities were determined using the appropriate kits from BoxBio, following the manufacturer's instructions. The results are as follows: Figure 9As shown in C-9G, under salt-free conditions, the malondialdehyde (MDA) and proline contents were almost identical. However, under salt stress, the MDA content decreased in the overexpression lines, while the proline content significantly increased in the OEnt4 lines. The POD and CAT activities of the overexpression and wild-type lines were almost identical under control conditions. Under control conditions, the SOD activity of the overexpression lines was lower than that of the wild-type lines. However, under salt stress, the POD and CAT activities of the overexpression lines were significantly higher than those of the wild-type lines, and the SOD activity was also higher, indicating that the overexpression lines may increase salt stress resistance through an antioxidant enzyme system. Furthermore, we quantitatively detected the reactive oxygen species (ROS) gene SOD using primers NtSOD1F: GACGGACCTTAGCAACAGG (SEQ ID No: 10) and NtSOD1R: CTGTAAGTAGTATGCATGTTC (SEQ ID No: 11). The results are shown below. Figure 9 H. Under the absence of salt stress, there was no difference in the expression of the tobacco SOD gene between wild-type and transgenic lines. However, under salt stress, the expression level of the tobacco SOD gene in transgenic lines was higher than that in wild-type lines, which verifies that transgenic lines may resist salt stress through an antioxidant system.

[0167] Salt stress experiments using transgenic MnNAC72 gene and control groups demonstrated that after salt stress induced by sulfate and chloride, the leaves of wild-type Arabidopsis and tobacco showed significantly higher levels of yellowing and wilting than those of transgenic MnNAC72 gene lines. The activity of antioxidant enzyme systems in transgenic tobacco lines was significantly higher than that in the control group. This indicates that the NAC72 gene we cloned, as a transcription factor responding to salt stress in plants, may play a role in sulfate and chloride-induced salt stress by regulating the expression of antioxidant genes.

[0168] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. The application of a mulberry NAC transcription factor gene in regulating plant salt-alkali stress, wherein the amino acid sequence of the protein encoded by the mulberry NAC transcription factor gene is shown in SEQ ID NO:

3.

2. The application according to claim 1, characterized in that, The salt stress mentioned is salt stress caused by sulfates and chlorides.

3. The application according to claim 1, characterized in that, The cDNA sequence of the mulberry NAC transcription factor gene is shown in SEQ ID NO:

2.

4. The application according to claim 1, characterized in that, The mulberry NAC transcription factor gene was amplified using primers shown in SEQ ID NO: 6 and SEQ ID NO: 7, or the mulberry NAC transcription factor gene was amplified using primers shown in SEQ ID NO: 8 and SEQ ID NO:

9.

5. The application of a recombinant expression vector in regulating salt and alkali stress in plants, wherein the recombinant expression vector is inserted with the mulberry NAC transcription factor gene as described in any one of claims 1-4.

6. The application of an engineered microorganism in regulating salt and alkali stress in plants, characterized in that, The engineered bacteria are loaded with the recombinant expression vector as described in claim 5.

7. A method for improving plant tolerance to salt and alkali stress, characterized in that, The method includes increasing the expression of the mulberry NAC transcription factor gene in host plant cells, tissues or individual plants, wherein the amino acid sequence of the protein encoded by the mulberry NAC transcription factor gene is shown in SEQ ID NO:

3.

8. The method according to claim 7, characterized in that, The plant in question is either Arabidopsis thaliana (Brassicaceae), Nicotiana spp. (Solanaceae), or Morus spp. (Moraceae).

9. The method according to claim 8, characterized in that, The plant in question is a mulberry tree, tobacco, or Arabidopsis thaliana.

10. The method according to claim 8, characterized in that, The Arabidopsis thaliana is Col-0 wild type, the tobacco is Yunyan 87 and Benshiyan, and the mulberry is Guisangyou 12.

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