ThNAC87 gene for regulating and controlling salt gland development of tamarix hispida and application of ThNAC87 gene

Abstract

The invention provides a ThNAC87 gene for regulating and controlling salt gland development of tamarix hispida and application of the ThNAC87 gene, and belongs to the technical field of gene engineering. The nucleotide sequence of the ThNAC87 gene disclosed by the invention is as shown in SEQ ID NO. 3. The ThNAC87 gene is constructed into a plant expression vector, tamarix hispida is transformed, the ThNAC87 gene is over-expressed in tamarix hispida, development of salt glands of tamarix hispida can be promoted, salt efflux is further promoted, phenotype and physiological index observation shows that compared with a non-transgenic plant, the over-expressed transgenic plant is high in resistance enzyme activity, and the resistance enzyme activity of the non-transgenic plant is high. The damage index is obviously lower than that of a non-transgenic plant, so that the salt stress resistance of the plant can be improved by overexpressing the ThNAC87 gene in the plant. The ThNAC87 gene disclosed by the invention has a relatively good application prospect in the field of improving the salt resistance of plants.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of genetic engineering, and in particular to a ThNAC87 gene for regulating development of salt glands of Tamarix hispida and application thereof. BACKGROUND

[0002] Soil salinization is a major environmental factor that limits plant growth and development, reduces crop yield, and even endangers the ecological environment, greatly hindering agricultural activities and sustainable use of resources. Due to global climate change and overdevelopment of land by humans and unreasonable use of agricultural fertilizers, the area of saline soil is still expanding year by year. Therefore, the development and utilization of saline-alkali land is an important problem faced by agricultural and forestry production in China and even the whole world.

[0003] Tamaricaceae Tamarix plants are mainly distributed in northwest and north China and are important afforestation tree species in arid, semi-arid or saline-alkali regions. Tamarix has high tolerance to salt stress, and has perfected a salt excretion system in the long-term evolution process, which can effectively excrete excess salt carried by the aboveground part of the plant through salt gland bodies distributed on the scale leaves and green nutrient branches of the current year (Li C and Lan H, 2021), thereby maintaining the balance of salt in the body. This mechanism not only can alleviate the damage of saline-alkali to the plant body (Wei et al., 2020; Chen et al., 2022; Yin DD et al., 2024). Therefore, studying the formation mechanism of salt glands of Tamarix provides a new way for analyzing salt-secreting plants and stress resistance mechanisms. In the present application, Tamarix hispida is used as a material to clarify the role of ThNAC87 gene of Tamarix in salt gland development and salt tolerance, which provides an important basis for improving plant stress resistance by using molecular biology methods in the later stage, and provides theoretical guidance for subsequent molecular design breeding of forest trees.

[0004] REFERENCES

[0005] Li C, Lan H. Research progress on stress resistance mechanisms of desert plant Tamarix. Biotechnol Bull, 2021, 37(5): 128-140.

[0006] Yin DD, Wang LQ, Cao Y, et al. Comparison of sample preparation methods for scanning electron microscopy of salt glands in Tamarix plants. Chinese Journal of Electron Microscopy, 2024, 43(3): 354-361.

[0007] Chen YH, Zhang SY, Du SF, et al. Effects of exogenous potassium(K +application on the antioxidant enzymes activities in leaves of Tamarix ramosissima under NaCl stress. Genes, 2022, 13: 1507.

[0008] Wei XC, Yan X, Yang Z, et al. Salt glands of recretohalophyte Tamarix under salinity: Their evolution and adaptation. Ecology and Evolution, 2020, 10: 9384-9395. SUMMARY

[0009] Therefore, the application provides a ThNAC87 gene for regulating salt gland development of Tamarix hispid, and application thereof.

[0010] In order to achieve the above-mentioned application purposes, the application provides the following technical solutions.

[0011] The application provides a ThNAC87 gene for regulating salt gland development of Tamarix hispid, wherein the nucleotide sequence of the ThNAC87 gene is shown as SEQ ID NO. 3.

[0012] The application also provides a Tamarix ThNAC87 protein, wherein the amino acid sequence of the Tamarix hispid ThNAC87 protein is shown as SEQ ID NO. 4.

[0013] The application also provides a protein derived from the amino acid sequence shown as SEQ ID NO. 4, wherein the derived protein is obtained by substituting, deleting or adding one or more amino acids on the basis of the amino acid sequence shown as SEQ ID NO. 4.

[0014] The application also provides application of overexpression of the ThNAC87 gene in promoting salt gland development of plants.

[0015] The application also provides application of overexpression of the ThNAC87 gene in improving salt resistance of plants.

[0016] Preferably, the plant is Tamarix hispid.

[0017] The application also provides an expression vector for improving salt resistance of plants, wherein the expression vector comprises the ThNAC87 gene and a basic vector.

[0018] Preferably, the basic vector comprises pMDC32.

[0019] The application further provides a recombinant bacteria for improving the salt tolerance of plants, wherein the recombinant bacteria is transformed with the expression vector.

[0020] The application further provides a method for improving the salt tolerance of plants, wherein the expression vector carrying the ThNAC87 gene is transformed into plants to overexpress the ThNAC87 gene in the plants, and the nucleotide sequence of the ThNAC87 gene is shown as SEQ ID NO. 3.

[0021] Preferably, the plants are Tamarix hispida.

[0022] By adopting the technical scheme, the application has the following beneficial effects: the nucleotide sequence of the ThNAC87 gene is shown as SEQ ID NO. 3. The ThNAC87 gene is constructed into a plant expression vector and transformed into Tamarix hispida, so that the ThNAC87 gene is overexpressed in the Tamarix hispida, which can promote the development of salt glands of the Tamarix hispida and further promote the excretion of salt. It is found through phenotype and physiological index observation that the resistance enzyme activity of the overexpression transgenic plants is higher than that of non-transgenic plants, and the damage index of the overexpression transgenic plants is significantly lower than that of the non-transgenic plants, which indicates that overexpression of the ThNAC87 gene in plants can improve the salt stress tolerance of the plants. The ThNAC87 gene has a good application prospect in the field of improving the salt tolerance of plants. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 It is a prediction diagram of the three-dimensional structure of Tamarix ThNAC87 protein.

[0024] Figure 2 It is a hydrophilic-hydrophobic analysis diagram of Tamarix ThNAC87 protein.

[0025] Figure 3 It is a transmembrane structure prediction diagram of Tamarix ThNAC87 protein.

[0026] Figure 4 It is a signal peptide analysis diagram of Tamarix ThNAC87.

[0027] Figure 5 It is a subcellular localization diagram of Tamarix ThNAC87.

[0028] Figure 6 It is a diagram of the plant overexpression vector pMDC32-ThNAC87.

[0029] Figure 7The image shows the RNA level detection of *Tamarix pubescens* plants overexpressing the ThNAC87 gene and non-transgenic *Tamarix pubescens* plants; where WT represents non-transgenic *Tamarix pubescens*, and OE1 and OE2 represent transgenic lines overexpressing the ThNAC87 gene in *Tamarix pubescens*.

[0030] Figure 8 The image shows the salt glands of *Tamarix pubescens* plants overexpressing the ThNAC87 gene and non-transgenic *Tamarix pubescens* plants; where WT represents non-transgenic *Tamarix pubescens*, and OE1 and OE2 represent transgenic lines overexpressing the ThNAC87 gene.

[0031] Figure 9 Figure 1 shows the salt stress resistance of *Tamarix fusiforme* plants overexpressing the ThNAC87 gene and non-transgenic *Tamarix fusiforme* plants; where A is H2O2 content, B is electrical conductivity, C is ABA content, D is superoxide dismutase activity, E is superoxide dismutase activity, and F is proline content. Detailed Implementation

[0032] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0033] Example 1. Cloning of the ThNAC87 gene in Tamarix chinensis

[0034] (1) Using *Tamarix fusiforme* as material, total RNA was extracted from *Tamarix fusiforme* leaves using a plant total RNA extraction kit (Tiangen Biotech Co., Ltd.). After removing genomic DNA contamination, PrimeScript was used to extract the RNA. TM The RT reagent Kit (TaKaRa, catalog number: RR037A) was used to synthesize the first strand of cDNA. For specific reaction systems, please refer to the kit instructions.

[0035] (2) Based on the *Tamarix chinensis* ThNAC87 gene sequence in the *Tamarix chinensis* genome data, primer sequences (the amplified fragments contain start and stop codons) were designed. The forward primer ThNAC87-F sequence is 5'-TGTCGCCGTTACAGGACATGTTCAT-3' (SEQ ID NO.1), and the reverse primer ThNAC87-R sequence is 5'-TTAGAAAGGCTTGGGCAGGGGTGTG-3' (SEQ ID NO.2). Using cDNA as a template, [the primers were then applied]. HSDNA Polymerase (TaKaRa, catalog number: R010A) amplifies the full-length coding region of the ThNAC87 gene. The specific reaction mixture is: 1 μL cDNA template, 1 μL each of forward and reverse primers (10 μM), and 10 μL...

[0036] HSDNA Polymerase, supplemented with Nuclease-free H2O to 20 μL; the reaction procedure was 94 °C pre-denaturation for 3 min; 94 °C denaturation for 30 s, 58 °C annealing for 30 s, 72 °C extension for 90 s, 30 cycles; 72 °C extension for 7 min.

[0037] (3) The amplified target fragment was purified by using a PCR purification kit (OMEGA, item number: D6492), and the purified product was connected with a cloning vector pMD TM 19-T (TaKaRa, item number: D102A), and the specific reaction system was referred to the instruction manual of the kit. 5 μL of the ligation product was transformed into E. coli DH5α competent cells (Shenzhen Kangti Life Science and Technology Co., Ltd.) by heat shock, blue-white spot screening was performed, white clones were selected for expansion culture, 2 μL of bacterial liquid was taken as a template for colony PCR detection, 5 μL of the amplified product was subjected to agarose gel electrophoresis detection, positive transformants were sent to Shengong Bioengineering Co., Ltd. for Sanger sequencing, and finally the nucleotide sequence of the ThNAC87 gene coding region was obtained, with a length of 927 bp, which could encode 308 amino acid residues, the nucleotide sequence and the amino acid sequence were shown as SEQ ID NO. 3 and SEQ ID NO. 4, respectively.

[0038] >SEQ ID NO. 3

[0039] ATGGCCGGAGAATTGCAGTTACCGCCGGGATTCAGATTCCATCCAACCGATGAGGAGCTAGTAAAGCACTACCTTATCCGTAGGTGTGCGTCCCAGCCTATTGCTGTTCCAATTATCGCCGAAATCGATCTCTACAGATACGATCCTTGGGACCTTCCAGAATTGGCACGGTATGGCGAGAAAGAATGGTATTTCTTTTCACCGAGAGACAGGAAGTATCCTAATGGCTCGAGACCGAATCGAGCTGCCGGAAGCGGCTACTGGAAAGCTACCGGAGCCGACAAACCTATCGGTCAGCCGAAAGCCTTAGCTATTAAGAAGGCCTTGGTGTTCTACGCTGGAAAGGCGCCTAAAGGCGAGAAGACTAATTGGATTATGCACGAGTACCGTTTAGCTGATGTCGATCGCTCTGCTCGTAAGAAAGGCAATGGTCTTCGGCTCGATGATTGGGTGCTATGCCGCATTTACAACAAAAAAGGCACAATCGAGAAACAACCCAGCGCCGTCAAAAAGCAACAGCCAGTCAGCCAACCCTTCTCCACTCCAAACCACCCGACCATCAAAACCGAAGATATAAGGCCGGACGTCATGGCTCATACACCACCATCCATCTCTAACACGACTACATTCAACGACTGCATCCATTTCGACACGTCTGAATCGATCCCTAGGCTTCACACCGACTCGAGCTCTTCCGATCATGTCGTTTCCCCAGAGTTCCACCCTGAGGTTCAGAGCCAGCCTAAAATGTTAGAACTCGATGGGGTCTTCGATTATTCGTTTAATTACGACATGGGCGCCCCCACTGCCATGGACAGTGCATTTGTTTCTCCCTTGCAGCAGCTGCAGGGTGGTTGTATTGGTAATCCTCTGTCGCCGTTACAGGACATGTTCATGTACAACACACCCCTGCCCAAGCCTTTCTAA.

[0040] >SEQ ID NO. 4

[0041] MAGELQLPPGFRFHPTDEELVKHYLIRRCASQPIAVPIIAEIDLYRYDPWDLPELARYGEKEWYFFSPRDRKYPNGSRPNRAAGSGYWKATGADKPIGQPKALAIKKALVFYAGKAPKGEKTNWIMHEYRLADVDRSARKKGNGLRLDDWVLCRIYNKKGTIEKQPSAVKKQQPVSQPFSTPNHPTIKTEDIRPDVMAHTPPSISNTTTFNDCIHFDTSESIPRLHTDSSSSDHVVSPEFHPEVQSQPKMLELDGVFDYSFNYDMGAPTAMDSAFVSPLQQLQGGCIGNPLSPLQDMFMYNTPLPKPF.

[0042] The ThNAC87 protein of Tamarix was subjected to three-dimensional structure prediction, hydrophobicity analysis, transmembrane structure prediction, signal peptide analysis and subcellular localization, and the results are shown in Figure 1 、 Figure 2 、 Figure 3 、 Figure 4 and Figure 5 .

[0043] Example 2. Construction of Tamarix ThNAC87 gene plant overexpression vector

[0044] After the correctly sequenced clones were expanded, the pMD19-T vector containing the ThNAC87 gene was extracted using a plasmid extraction kit (OMEGA, item number: D6943), and the specific steps are described in the kit instruction manual; using GATEWAY technology, the primers were designed as follows: TM 19-T vector plasmid, and the specific steps are described in the kit instruction manual; using GATEWAY technology, the primers were designed as follows:

[0045] The sequence of the forward primer is shown in SEQ ID NO. 5:

[0046] 5'-GGGGACAACTTTGTACAAAAAAGTTGGA TGTCGCCGTTACAGG-3' (SEQ ID NO. 5);

[0047] The sequence of the reverse primer is shown in SEQ ID NO. 6:

[0048] 5'-GGCGGCCGCACAACTTTGTACAAGAAAGTTGGGTATTAGAAAG GCTTGGGC-3' (SEQ ID NO. 6).

[0049] The primers were used to amplify the full-length sequence of the ThNAC87 gene coding region using the extracted plasmid as a template. After purification of the amplified target fragment, the purified product was ligated to the intermediate vector pDNOR207 by BP reaction. The reaction system was as follows: 150 ng of PCR product, 75 ng of pDNOR207 vector, 0.8 μL of BP enzyme, and 20 μL of nucleic acid-free water; the reaction program was 25°C for 6 h. The ligation solution was transformed into E. coli DH5α competent cells by heat shock, and the positive single clones with resistance were selected for expansion culture. Then, 2 μL of bacterial liquid was used as a template for colony PCR detection, and the reaction system and program were the same as those in step (2) of Example 1. The positive transformants were sent to Shengong Bioengineering Co., Ltd. for Sanger sequencing verification.

[0050] The positive clone strain with correct sequencing was expanded and cultured, and the plasmid was extracted. Then, the LR reaction was performed between the plasmid and the plant expression vector pMDC32. The reaction system and program were the same as those in the BP reaction. After the reaction, the full-length sequence of the ThNAC87 gene coding region was introduced into the plant expression vector pMDC32 (the pMDC32 vector contains a strong promoter CaMV 35S, which can drive the expression of the ThNAC87 gene). The positive single clones with resistance were selected for expansion culture, and then 2 μL of bacterial liquid was used as a template for colony PCR detection. The reaction system and program were the same as those in step (2) of Example 1. The positive transformants were sent to Shengong Bioengineering Co., Ltd. for Sanger sequencing verification, and the plant overexpression vector pMDC32-ThNAC87 containing the full-length sequence of the ThNAC87 gene coding region was obtained. Figure 6

[0051] Example 3. Genetic transformation and detection of the Tamarix ThNAC87 overexpression vector

[0052] 1. Genetic transformation of the Tamarix ThNAC87 overexpression vector

[0053] The overexpression vector pMDC32-ThNAC87 obtained in Example 2 was transformed into Agrobacterium competent cells GV3101 (Shanghai Weidi Biotechnology Co., Ltd.) by electroporation, and the recombinant bacteria GV3101-pMDC32-ThNAC87 were obtained.

[0054] ​The recombinant bacteria GV3101-pMDC32-ThNAC87 were used to transform Tamarix austromongolica by the genetic transformation method of agrobacterium infection of Tamarix stem tips. The specific method refers to the invention patent with the application number "202210951880.5" and the invention name "a construction method of an agrobacterium-mediated Tamarix austromongolica genetic transformation system". Tamarix austromongolica was transformed, and after induction culture, the resistant adventitious buds were transferred to a rooting medium containing hygromycin and timentin until rooting was induced to form complete plants.

[0055] 2. Tamarix ThNAC87 transgenic plant detection

[0056] After antibiotic screening, a total of 2 resistant transgenic plants ThNAC87-OE1 and ThNAC87-OE2 were obtained. Leaf samples were extracted for total RNA and reverse transcribed with non-transgenic plants (WT, as a control), and the relative expression of ThNAC87 gene in each transgenic line was detected at the RNA level with quantitative primers.

[0057] ThNAC87-qPCR-F: 5'-TAACACGACTACATTCAACGA-3' (SEQ ID NO. 7)

[0058] ThNAC87-qPCR-R: 5'-GAGGATTACCAATACAACCAC-3' (SEQ ID NO. 8) as quantitative primers, the relative expression of ThNAC87 gene in each transgenic line was detected at the RNA level, and the results are shown in Figure 7 The results show that the expression amount of ThNAC87 gene in overexpression transgenic lines OE1 and OE2 is 20 and 25 times that of non-transgenic plants, respectively.

[0059] Example 4. Determination of salt resistance of ThNAC87 transgenic Tamarix plants

[0060] 1. Observation of salt glands

[0061] The transgenic Tamarix and non-transgenic Tamarix twigs were observed by scanning electron microscopy. The Tamarix twigs were fixed in 4% glutaraldehyde fixative overnight, washed, and then the samples were dried and observed by scanning electron microscopy, and the results are shown in Figure 8 It was found that the number of salt glands in transgenic plants increased Figure 8 ).

[0062] 2. Determination of physiological indicators

[0063] The 30-day-old ThNAC87 overexpression transgenic plants (OE-1 and OE-2) and non-transgenic Tamarix austriaca (WT) tissue culture seedlings in good growth state and uniform size were transplanted into plastic pots filled with artificial soil, and then placed in a greenhouse for 2 months, and then placed in a 300 mM sodium chloride salt solution stress (ST) for 2 days, and then the mature twigs were taken for physiological index determination, and 3 biological replicates were set in each test.

[0064] The H2O2 content and conductivity of the Tamarix austriaca plants were determined to evaluate the degree of cell membrane damage and the stress response of the antioxidant system. The H2O2 content and conductivity determination results show that, under normal treatment, the H2O2 content and conductivity of the overexpression transgenic Tamarix austriaca and the non-transgenic Tamarix austriaca have no significant difference compared with the WT. However, under salt stress, the H2O2 content and conductivity of the overexpression transgenic plants are obviously lower than those of the non-transgenic plants, which indicates that the overexpression of ThNAC87 enhances the salt resistance of the transgenic plants Figure 9 A and B).

[0065] The plant hormone abscisic acid (ABA) is an important signal molecule for plant response to stress, and under salt stress, the ABA content of the overexpression transgenic plants is higher than that of the wild type Figure 9 C).

[0066] Malondialdehyde (MDA) is an effective cell membrane oxidative damage index. Under normal growth conditions, the MDA content of the overexpression transgenic Tamarix austriaca and the non-transgenic Tamarix austriaca leaves is basically the same. However, under salt stress, the MDA level of the overexpression transgenic Tamarix austriaca leaves is significantly lower than that of the non-transgenic Tamarix austriaca Figure 9 D).

[0067] Antioxidant enzymes, such as peroxidase (POD) and superoxide dismutase (SOD), are main ROS scavengers and play a key role in ROS homeostasis. Under normal growth conditions, the POD and SOD activity levels and proline content of the overexpression ThNAC87 transgenic Tamarix austriaca are similar to those of the non-transgenic plants. However, under salt stress, the POD and SOD activity levels of the overexpression ThNAC87 transgenic Tamarix austriaca are obviously higher than those of the non-transgenic Tamarix austriaca. At the same time, the proline content of the overexpression ThNAC87 transgenic Tamarix austriaca is significantly higher than that of the non-transgenic plants Figure 9 E and F).

[0068] The above results show that the overexpression of ThNAC87 enhances the salt stress tolerance of the transgenic plants.

[0069] It can be known from the above examples that the application provides a ThNAC87 gene for regulating the development of salt glands of Tamarix hispidus and application thereof. The overexpression of the ThNAC87 gene can promote the number of salt glands of Tamarix austriaca and improve the salt resistance of Tamarix austriaca.

[0070] The above merely describes the preferred embodiments of the present application, and it should be pointed out that those skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered as falling within the protection scope of the present application.

Claims

1. A ThNAC87 gene for regulating the development of salt glands of A. strictus, characterized in that, The nucleotide sequence of the ThNAC87 gene is shown as SEQ ID NO.

3.

2. A Tamarix ThNAC87 protein characterized in that, The amino acid sequence of the ThNAC87 protein of the Adenium obesum is shown as SEQ ID NO.

4.

3. Application of overexpression of the ThNAC87 gene in promoting development of salt glands of plants.

4. Application of overexpression of the ThNAC87 gene in improving salt resistance of plants.

5. Use according to claim 3 or 4, characterized in that, The plant is Adenium obesum.

6. An expression vector for improving salt tolerance of a plant, characterized by comprising the polynucleotide of claim 1. The expression vector comprises the ThNAC87 gene and a basic vector.

7. The expression vector of claim 6, wherein, The basic vector comprises pMDC32.

8. A recombinant microorganism for improving salt tolerance in plants, characterized in that, The recombinant bacteria are transformed with the expression vector of claim 6 or 7.

9. A method for improving salt tolerance in plants, characterized by, The expression vector carrying the ThNAC87 gene is transformed into plants to overexpress the ThNAC87 gene in the plants.

10. The method of claim 9, wherein, The plant is Adenium obesum.

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