Mulberry MaBEH1 / 2 gene for improving salt tolerance of plants and application of mulberry MaBEH1 / 2 gene

Abstract

The invention relates to a mulberry MaBEH1 / 2 gene for improving plant salt tolerance and application thereof, and belongs to the technical field of plant genetic engineering and biology. Wherein the coding nucleotide sequence of the mulberry MaBEH1 / 2 gene for regulating and controlling the salt tolerance of the woody plant is shown as a sequence 3; and the amino acid sequence of the MaBEH1 / 2 expression protein is shown as a sequence 4. According to the invention, MaBEH1 / 2 is transferred into 84K poplars to obtain MaBEH1 / 2-OE transgenic poplars; compared with a non-transgenic poplar, the salt stress tolerance of the MaBEH1 / 2-OE transgenic poplar can be remarkably enhanced. The invention provides key gene resources and technical support for genetic improvement of salt-tolerant forest varieties.

Description

TECHNICAL FIELD

[0001] The application relates to a mulberry MaBEH1 / 2 gene for improving plant salt tolerance and application thereof, and belongs to the technical field of plant genetic engineering and biotechnology. BACKGROUND

[0002] Soil salinization often damages agricultural and forestry ecosystems by inhibiting plant growth and reducing crop productivity, and further has a series of negative effects on food security, ecological balance and long-term social and economic stability. Therefore, improving plant salt tolerance is crucial for sustainable agricultural development. In recent years, with the promotion of the second-generation sequencing technology, the genome sequences of a large number of plants have been resolved. How to use these genetic resources to breed new crop varieties with better stress resistance has become a top priority. At present, traditional genetic breeding methods need to go through multiple generations such as hybridization and selfing, which is time-consuming and often accompanied by unclear genetic background. Therefore, using mature biotechnological means (such as genetic engineering) to improve plant salt tolerance has become an important way for directional and efficient breeding.

[0003] Mulberry (Morus alba) is an excellent fruit tree, which can not only breed silkworms, but also process mulberry leaves to make tea, and can also be used for landscaping. However, due to the difficulty of asexual reproduction, slow development of adventitious roots, and difficulty in establishing a mulberry tissue culture regeneration system, the breeding, popularization and demonstration application of mulberry varieties are hindered. In recent years, as an important model species of woody plants, Populus has a stable genetic transformation system and plays an important role in studying the functions of genes in woody plants. Brassinosteroids (BRs) are an important class of plant sterol hormones that play a core role in plant growth and development, morphological development and stress resistance regulation. In the BR signal transduction pathway, BZR (Brassinazole-Resistant) transcription factors are a new class of plant-specific transcription factors that positively mediate the regulation of brassinosteroids (BR) through phosphorylation and dephosphorylation processes. At the same time, BZR belongs to the helix-loop-helix (bHLH) transcription regulator superfamily, which regulates plant growth and development by participating in the transcription process of downstream genes. Members of the BZR family in some species have been identified, and the role of BZR in plant stress response has been confirmed, indicating that BZR transcription factors are closely related to plant stress resistance.

[0004] This invention uses the coding genome sequence of the superior mulberry variety Lusang No. 1 as a template to clone the MaBEH1 / 2 gene. Simultaneously, the nucleic acid fragment of MaBEH1 / 2 is constructed into the modified overexpression vector pK2GW7. Driven by a strong 35S promoter, MaBEH1 / 2 is overexpressed in poplar trees, significantly enhancing their tolerance to salt stress. Therefore, this invention provides a mulberry MaBEH1 / 2 gene for improving plant salt tolerance and its application, providing key gene resources and technical support for the cultivation of superior salt-tolerant forest tree varieties. Summary of the Invention

[0005] In view of the fact that most plants are restricted by salt stress, resulting in stunted plant growth, the main purpose of this invention is to provide a mulberry MaBEH1 / 2 gene to improve the salt tolerance of plants.

[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0007] A mulberry MaBEH1 / 2 gene that enhances salt tolerance has the following nucleotide sequence: as shown in Sequence 3; and the following amino acid sequence: as shown in Sequence 4.

[0008] This invention also provides a method for cloning the mulberry MaBEH1 / 2 gene, which enhances plant salt tolerance, comprising the following steps:

[0009] Step 1: Using Lusang No. 1 improved variety (provided by the Sericulture Research Institute of Shandong Academy of Agricultural Sciences) as experimental material, RNA was extracted using the FastPure Universal Plant Total RNA Isolation Kit (Vazyme, NanJing, China); cDNA of Lusang No. 1 was obtained by reverse transcription of RNA using the SuperScript III first-strand synthesis system (Life Technologies, Carlsbad, CA, USA).

[0010] Step 2: Obtain the cDNA sequence of the target gene MaBEH1 / 2 from the mulberry genome website (https: / / morus.biodb.org / ), and design primers using Oligo7 software; the MaBEH1 / 2-F forward primer is shown in Sequence 1; the MaBEH1 / 2-R reverse primer is shown in Sequence 2.

[0011] Step 3: Amplification of the target gene cDNA sequence

[0012] Using cDNA from Lusang 1 as a template, and amplification was performed using PrimeSTAR HS (TaKaRa, BeiJing, China) R040A high-fidelity enzyme, in a 25 μL reaction system, the full-length 1038 bp cDNA sequence was finally obtained and named MaBEH1 / 2 gene (its nucleotide sequence is shown in Sequence 3; its amino acid sequence is shown in Sequence 4).

[0013] Preferably, in step 2, the primer length is 15-25 bp, and the Tm value of the designed primer is suitable at 65℃, and the pK2GW7 adapter is added to the primer.

[0014] Another object of the present invention is to provide a plant expression vector carrying the mulberry MaBEH1 / 2 gene.

[0015] The above-mentioned objective of this invention is achieved through the following technical solution:

[0016] The construction of a plant expression vector for the mulberry MaBEH1 / 2 gene is as follows:

[0017] Step 1: Using the mulberry coding genome sequence as a template, specific PCR primers (MaBEH1 / 2-F and MaBEH1 / 2-R) are used to amplify the coding sequence of the MaBEH1 / 2 gene by PCR.

[0018] Step 2: The obtained coding sequence of the MaBEH1 / 2 gene is inserted into the multiple cloning site of the overexpression vector pK2GW7 using DNA homologous recombination seamless cloning technology to obtain the constructed MaBEH1 / 2 overexpression vector. The sequence of the overexpression vector pK2GW7 is shown in Sequence 5.

[0019] Step 3: Expression vector construction: According to the reaction system, fresh PCR products, pK2GW7 overexpression vector and Basic Mix enzyme (Basic Seamless Cloning and Assembly Kit, Beijing TransGen Biotech Co., Ltd.) were added respectively, and placed in a PCR instrument. The reaction was carried out for 1.5 h to obtain the constructed overexpression vector. The mixture after the above reaction was transformed into E. coli and plated on LB solid selection medium (LB solid medium: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, agar powder 8 g / L, spectinomycin 50 mg / L). Single clones were picked from the selection medium for PCR detection and sequencing verification to confirm that the MaBEH1 / 2 sequence was successfully inserted into the pK2GW7 overexpression vector and named MaBEH1 / 2-OE.

[0020] Step 4: In the constructed MaBEH1 / 2-OE vector, the 5' end of the MaBEH1 / 2 gene is equipped with a strong 35S promoter, which enables efficient expression of the MaBEH1 / 2 protein in poplar. The expression vector pK2GW7 contains LB and RB sequences as well as kanamycin (Kan). The LB and RB sequences promote the integration of the 35S::MaBEH1 / 2 expression framework and the selection marker gene Kan assembled therein into the poplar chromosome. Kan is used as a selection marker for transgenic poplar to select transgenic poplar, thereby obtaining MaBEH1 / 2-OE transgenic poplar.

[0021] Another object of the present invention is to provide the application of the above-mentioned MaBEH1 / 2 gene in improving the salt tolerance of mulberry trees.

[0022] The above-mentioned objective of this invention is achieved through the following technical solution:

[0023] Application of MaBEH1 / 2 gene in improving salt tolerance of mulberry trees.

[0024] The genetic transformation of MaBEH1 / 2 transgenic mulberry trees was carried out as follows: The constructed MaBEH1 / 2-OE expression vector was transferred into Agrobacterium tumefaciena GV3101 by electroporation. Then, using Agrobacterium-mediated transformation, the 35S::MaBEH1 / 2 expression framework and the selection marker gene NPTII were integrated into the chromosome of 84K poplar (Silver gland poplar, Populusalba x Populus glandulosa) to create MaBEH1 / 2 overexpression transgenic poplar trees (MaBEH1 / 2-OE).

[0025] Preferably, the specific transformation steps are as follows: One-month-old 84K poplar tissue culture seedlings are cultured at a temperature of 23-25℃ and a light / dark cycle of 16 / 8h (day / night). Agrobacterium containing the MaBEH1 / 2-OE expression vector is cultured in LB liquid medium (tryptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L, spectinomycin 50mg / L, rifampin 20mg / L) until the OD600 reaches 0.6-0.8. The infected leaves are then transferred to co-culture medium (2.2g / L Murashige-Skoog (MS) + 0.5g / L 4-morpholineethanesulfonic acid). Leaf samples were co-cultured for 2-3 days in the dark on a medium containing 50 mg / L naphthaleneacetic acid (MA) and 200 mg / L termetin (Tim) (MS + 0.5 g / L MES + 0.5 mg / L 6-benzylaminopurine (6-BA) + 0.05 mg / L naphthaleneacetic acid (NAA) + 20 g / L sucrose + 4 g / L termetin (MA) + 20 g / L sucrose + 4 g / L termetin (MA) + 20 g / L acetosyringone (AS). After co-culture, the leaves were transferred to a rooting medium containing 200 mg / L termetin (MA) (MS + 0.5 g / L termetin (MA) + 20 g / L sucrose + 4 g / L termetin (MA) + 20 g / L acetosyringone (AS ...termetin (MA) + 20 g / L acetosyringone (AS) + 20 g / L termetin (MA) + 20 g / L acetosyringone (AS) + 20 g / L termetin (MA) + 20 g / L acetosyringone (AS) + 20 g / L termetin (MA) + 20 g / L acetosyringone (AS) + 20 g / L termetin (MA) MES + 0.05 mg / L indole-3-butyric acid (IBA) + 0.02 mg / L NAA + 20 g / L sucrose + 8 g / L agar powder were added until adventitious roots were induced. DNA was extracted from the leaves of rooted plants that had developed Kan resistance, and PCR verification was performed using the extracted DNA as a template with MaBEH1 / 2 cloning primers. Finally, transgenic 84K poplar containing the MaBEH1 / 2-OE vector sequence was obtained.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0027] This invention patent describes the creation of a transgenic poplar tree, MaBEH1 / 2-OE, which overexpresses the homologous gene MaBEH1 / 2 of Arabidopsis thaliana BEH1 and BEH2 in mulberry. This resulted in transgenic poplar lines (OE2, OE3, and OE5) with significantly increased salt tolerance. Phenotypic analysis in asexual reproduction revealed that the salt tolerance of the MaBEH1 / 2-OE transgenic poplar was significantly higher than that of the non-transgenic poplar (WT), indicating that overexpression of the mulberry MaBEH1 / 2 gene can improve the plant's tolerance to salt stress.

[0028] The present invention will be further described below with reference to specific embodiments and accompanying drawings, but this does not imply any limitation on the scope of protection of the present invention. Attached Figure Description

[0029] Figure 1 This is a phylogenetic tree of the BZR gene family in Arabidopsis thaliana and mulberry trees, as presented in this invention; At represents Arabidopsis thaliana, and Ma represents Morus alba.

[0030] Figure 2 This is a schematic diagram of the structure of the MaBEH1 / 2 overexpression vector pK2GW7 constructed in this invention;

[0031] Figure 3 Semi-quantitative electrophoresis was used to identify the MaBEH1 / 2-OE transgenic poplar in this invention; the amplified bands of the internal reference gene Actin showed basically consistent brightness, while the brightness of the target gene MaBEH1 / 2 showed OE2>OE5>OE3.

[0032] Figure 4 The salt tolerance phenotypes of WT and MaBEH1 / 2-OE transgenic poplars in tissue culture bottles are shown in this invention; Control represents the growth status of non-transgenic poplar WT and MaBEH1 / 2-OE transgenic poplars (OE2, OE3 and OE5) in normal rooting medium; 125mM NaCl represents the growth status of WT and MaBEH1 / 2-OE transgenic poplars in rooting medium containing 125mM NaCl.

[0033] Figure 5 This study analyzed the salt tolerance of WT and MaBEH1 / 2-OE transgenic poplar seedlings grown in soil for one month in this invention. Among them, (AB) are the plant status of the control group (Control) and the salt-treated group (200mM NaCl) before salt stress treatment, respectively; (CF) are the phenotypes of the control group and the treated group after 7 days and 10 days of salt stress treatment, respectively. Detailed Implementation

[0034] The present invention will be further described below with reference to specific embodiments. It should be understood that the following text is only used to describe one or more specific implementations of the present application and does not strictly limit the scope of protection specifically claimed in the present application. In the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other.

[0035] Unless otherwise specified, the reagents involved in the following examples are all commercially available conventional reagents, and the methods used are all methods commonly used in this technical field.

[0036] Example 1

[0037] 1. Cloning of the MaBEH1 / 2 gene

[0038] In response to the current situation where the growth of most plants is inhibited due to salt stress, this invention provides a gene that improves the salt tolerance of plants and its application in transgenic poplar trees overexpressed with it.

[0039] First, the homologous gene MaBEH1 / 2 of Arabidopsis thaliana AtBEH1 and AtBEH2 was selected for cloning. The specific steps are as follows:

[0040] (1) Using the superior variety of Lu Sang No. 1 (Morus alba L.) (provided by the Sericulture Research Institute of Shandong Academy of Agricultural Sciences) as the experimental material, RNA was extracted using the FastPure Universal Plant Total RNA Isolation Kit (Vazyme, NanJing, China); cDNA was synthesized by reverse transcription of RNA using the SuperScript III first-strand synthesis system (Life Technologies, Carlsbad, CA, USA);

[0041] (2) The cDNA sequence of the target gene MaBEH1 / 2 was obtained through the mulberry genome website (https: / / morus.biodb.org / ). Primers were designed using Oligo7 software. The primer length was 15-25 bp. At the same time, the Tm value of the designed primers was suitable at 65℃ (vector adapters were added to the primers).

[0042] The forward primer MaBEH1 / 2-F is shown in sequence 1; the reverse primer MaBEH12 / -R is shown in sequence 2.

[0043] Table 1

[0044]

[0045] Table 2

[0046]

[0047] (3) Using the cDNA of Lusang No. 1 as a template, and simultaneously using TaKaRa Prime STAR HS (premix) R040A high-fidelity enzyme for amplification, the 25μL reaction system is shown in Table 3 below; (3) Amplification of the target gene cDNA sequence

[0048] Using mulberry cDNA as a template, and amplification was performed using TaKaRa PrimeSTAR HS(premix)R040A high-fidelity enzyme. The 25μL reaction system is shown in Table 3 below.

[0049] Table 3

[0050]

[0051]

[0052] The PCR reaction procedure is shown in Table 4 below:

[0053] Table 4

[0054]

[0055] The final cDNA sequence of the gene, 1038 bp in length, was obtained and named MaBEH1 / 2 gene; its nucleotide sequence is shown in Sequence 3; the amino acid sequence of MaBEH1 / 2 gene is shown in Sequence 4.

[0056] Table 5

[0057]

[0058] The amino acid sequence of the MaBEH1 / 2 gene is shown in sequence 4;

[0059] Table 6

[0060]

[0061] 2. Construction of plant overexpression vector for MaBEH1 / 2 gene

[0062] The method for constructing the overexpression vector of the MaBEH1 / 2 gene is as follows:

[0063] (1) The overexpression vector of the MaBEH1 / 2 gene was constructed using cloning technology. Using specific PCR primers (MaBEH1 / 2-F and MaBEH1 / 2-R primers), the coding sequence fragment of the obtained PCR amplification product MaBEH1 / 2 gene was constructed into the pK2GW7 overexpression vector by homologous recombination. The sequence is shown in Sequence 5.

[0064] Table 7

[0065]

[0066]

[0067]

[0068]

[0069]

[0070] Construction of overexpression vector: (1) According to the reaction system shown in Table 8 below, add fresh PCR product, pK2GW7 overexpression vector and Basic Mix enzyme respectively, and then place in a 50℃ metal bath for 0.5h.

[0071] Table 8

[0072] PCR product Overexpression vector Basic Mix enzyme 100 ng 20 ng 2.5 μL

[0073] (2) The mixture after the above reaction was transformed into Escherichia coli and plated on LB solid selection medium (tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, agar powder 8 g / L, spectinomycin 50 mg / L). Single clones were picked from the selection medium for PCR detection and sequencing verification to confirm that the MaBEH1 / 2 overexpression vector was successfully constructed and named MaBEH1 / 2-OE.

[0074] In the constructed MaBEH1 / 2-OE vector, the 5' end of the MaBEH1 / 2 gene is equipped with a strong 35S promoter, which enables efficient expression of the MaBEH1 / 2 protein in poplar. The expression vector pK2GW7 contains LB and RB sequences as well as kanamycin (Kan). The LB and RB sequences promote the integration of the 35S::MaBEH1 / 2 expression framework and the selection marker gene Kan assembled therein into the poplar chromosome. Kan serves as a selection marker for transgenic poplar and is used to select transgenic poplar, thereby obtaining MaBEH1 / 2-OE transgenic poplar.

[0075] 3. Genetic transformation of MaBEH1 / 2 transgenic mulberry trees

[0076] The genetic transformation steps of the MaBEH1 / 2 transgenic mulberry tree are as follows:

[0077] The constructed MaBEH1 / 2-OE expression vector was transformed into Agrobacterium GV3101 by electroporation; subsequently, the MaBEH1 / 2-OE expression vector was integrated into the 84K poplar chromosome using Agrobacterium-mediated transformation, thereby obtaining MaBEH1 / 2-OE transgenic poplar.

[0078] The specific transformation steps are as follows: One-month-old 84K poplar tissue culture seedlings were cultured at a temperature of 23-25℃ and a light intensity of 16 / 8h (day / night). Agrobacterium containing the MaBEH1 / 2-OE expression vector was cultured in LB liquid medium (10g / L tryptone, 5g / L yeast extract, 10g / L sodium chloride, 50mg / L spectinomycin, 20mg / L rifampin) until the OD600 reached 0.6-0.8. The infected leaves were then transferred to co-culture medium (2.2g / L Murashige-Skoog, MS) + 0.5g / L 4-morpholineethanesulfonic acid. The leaves were co-cultured for 2-3 days in a medium containing 50 mg / L Kan and 200 mg / L Timentin (Tim) and MS (2.2 g / L MS + 0.5 g / L MES + 0.5 mg / L 6-Benzyl Aminopurine (6-BA) + 0.05 mg / L Naphthaleneacetic acid (NAA) + 20 g / L sucrose + 4 g / L plant gel in the dark. After co-culture, the leaves were transferred to an adventitious shoot induction medium (2.2 g / L MS + 0.5 g / L MES + 0.5 mg / L 6-Benzyl Aminopurine (6-BA) + 0.05 mg / L Naphthaleneacetic acid (NAA) + 20 g / L sucrose + 4 g / L plant gel) to induce and screen for resistant adventitious shoots. After 20 days of induction culture, the resistant adventitious shoots were transferred to a medium containing 200 mg / L acetosyringone (AS) and MES. Tim was used to induce adventitious roots in the rooting medium (2.2 g / L MS + 0.5 g / L MES + 0.05 mg / L indole-3-butyric acid (IBA) + 0.02 mg / L NAA + 20 g / L sucrose + 8 g / L agar powder); DNA was extracted from the leaves of the rooted plants that had developed Kan resistance, and PCR verification was performed using the MaBEH1 / 2 cloning primers, ultimately obtaining transgenic 84K poplar containing the MaBEH1 / 2-OE vector sequence;

[0079] 4. Phenotypic analysis of MaBEH1 / 2-OE transgenic mulberry trees

[0080] This invention patent mainly clones the mulberry MaBEH1 / 2 gene and constructs it into the pK2GW7 expression vector. Figure 2Subsequently, transgenic poplar lines overexpressing the MaBEH1 / 2 gene (named MaBEH1 / 2-OE) were obtained through Agrobacterium-mediated genetic transformation. Semi-quantitative analysis of the expression levels of five transgenic lines (OE1-OE5) revealed that the gene expression level in line OE4 was not significant; the expression levels in lines OE1 and OE5 were comparable. Based on this, three positive lines, MaBEH1 / 2-OE (OE2, OE3, and OE5), were finally selected. The expression levels of the MaBEH1 / 2 gene in the three lines were: OE2 > OE5 > OE3. Figure 3 The shoot tips of WT and MaBEH1 / 2-OE transgenic poplars, which had been cultured for one month, were transferred to Control (normal rooting medium) and 125 mM NaCl (rooting medium with 125 mM NaCl added), respectively. After 20 days of growth, the salt tolerance of the seedlings was observed and analyzed. It was found that the leaves of the control group plants were all bright green, indicating that the different strains were growing well under normal conditions. However, the leaves of WT plants in the salt stress group showed obvious yellowing and browning (marked by red arrows), showing salt sensitivity. But the leaves of the MaBEH1 / 2 overexpressing plants OE2, OE3, and OE5 remained green, indicating that MaBEH1 / 2 gene overexpression enhanced the salt tolerance of the plants. Figure 4 In summary, the preliminary results indicate that the overexpression lines (OE2, OE3, OE5) are more salt-tolerant than WT.

[0081] Further salt stress treatment experiments were conducted using soil-grown seedlings, verifying that the salt tolerance of the MaBEH1 / 2 overexpressing transgenic poplar was significantly stronger than that of the non-transgenic WT poplar. Before salt treatment, the WT and MaBEH1 / 2 overexpressing poplar (OE2, OE3, OE5) in both the control group (watered) and the salt-treated group (200Mm NaCl treatment) showed good growth and consistent growth status. Figure 5 AB); After 7 days of salt treatment, the control group plants maintained good growth, while the WT plants in the salt-treated group showed obvious wilting and drooping leaves, indicating a significant impact from salt damage; although the leaves of the MaBEH1 / 2 overexpressing plants (OE2, OE3, and OE5) also experienced some water loss, their overall growth was better than that of the WT plants, with less wilting. Figure 5 CD); After 10 days of salt treatment, the plant height growth of WT plants in the salt-treated group completely stopped, and the leaves drooped and curled; while the MaBEH1 / 2 overexpressing plants maintained a normal growth rate, with only a few leaves drooping slightly, and the overall growth was good, with biomass accumulation significantly better than the control group. Figure 5 EF); meanwhile, the OE3 line, with the lowest MaBEH1 / 2 overexpression level, showed significantly greater leaf drooping than OE2 and OE5 (EF); Figure 5F); Based on the above phenotypic differences, transgenic poplar trees with overexpression of the mulberry MaBEH1 / 2 gene can significantly enhance their tolerance to salt stress, and the strength of their tolerance to salt stress depends on the fold increase in the expression of the MaBEH1 / 2 gene.

[0082] This invention uses the coding genome sequence of mulberry tree 'Lu Sang 1' as a template to clone the MaBEH1 / 2 gene. Subsequently, the coding sequence of MaBEH1 / 2 is constructed into the plant overexpression vector pK2GW7 using a seamless DNA homologous recombination cloning system. Transgenic poplar trees overexpressing MaBEH1 / 2 (MaBEH1 / 2-OE, OE2, OE3, and OE5) are obtained through stable genetic transformation of poplar trees. Finally, by comparing and analyzing the salt stress tolerance of MaBEH1 / 2-OE seedlings with those of WT seedlings and soil-grown seedlings, this invention finds a positive correlation between plant salt stress tolerance and the expression level of the MaBEH1 / 2 gene. Ultimately, this invention clarifies the function of MaBEH1 / 2 in improving plant salt tolerance and simultaneously creates a new salt-tolerant poplar germplasm of MaBEH1 / 2-OE.

[0083] This invention provides the mulberry MaBEH1 / 2 gene for improving salt tolerance in plants, and also provides the application of salt-tolerant transgenic poplar with enhanced MaBEH1 / 2 expression, belonging to the fields of plant genetic engineering and biotechnology. The nucleotide sequence encoding the mulberry MaBEH1 / 2 gene, which regulates salt tolerance in woody plants, is shown in Sequence 3; the amino acid sequence of the MaBEH1 / 2 expressed protein is shown in Sequence 4. This invention obtains MaBEH1 / 2-OE transgenic poplar by transferring MaBEH1 / 2 into 84K poplar. Compared with non-transgenic poplar, MaBEH1 / 2-OE transgenic poplar significantly enhances its tolerance to salt stress. This invention provides key gene resources and technical support for the genetic improvement of salt-tolerant forest tree varieties.

[0084] Although the foregoing has described the inventive concept and embodiments in detail, those skilled in the art will recognize that various improvements and modifications can be made to the invention without departing from the scope of the claims, and such improvements and modifications should still fall within the protection scope of the invention.

Claims

1. A mulberry MaBEH1 / 2 gene for improving salt tolerance, the nucleotide sequence of which is shown in Sequence 3; the amino acid sequence of which is shown in Sequence 4.

2. The cloning method for the mulberry MaBEH1 / 2 gene that enhances plant salt tolerance as described in claim 1, comprising the following steps: Step 1: Using mulberry trees as experimental material, RNA was extracted using the FastPure Universal Plant Total RNA Isolation Kit; cDNA was synthesized by reverse transcription of RNA using the SuperScript III first-strand synthesis system. Step 2: Obtain the cDNA sequence of the target gene MaBEH1 / 2 from the mulberry genome website https: / / morus.biodb.org / , and design primers using Oligo7 software; the forward primer MaBEH1 / 2-F is shown in Sequence 1; the reverse primer MaBEH1 / 2-R is shown in Sequence 2. Step 3: Amplification of the target gene cDNA sequence. Using mulberry genomic cDNA as a template, amplification was performed using TaKaRaPrimeSTAR HS R040A high-fidelity enzyme. The reaction system was 25 μL, and the PCR reaction program was used to obtain the full-length 1038 bp cDNA sequence of the gene, which was named MaBEH1 / 2 gene.

3. The cloning method for the mulberry MaBEH1 / 2 gene to improve plant salt tolerance according to claim 2, characterized in that: In step 2, the primer length is 15-25 bp, and the Tm value of the designed primer is suitable at 65℃. The primer is combined with the adapter for seamless cloning of the pK2GW7 vector.

4. The construction of the plant expression vector for the mulberry MaBEH1 / 2 gene, which enhances salt tolerance, according to claim 1, comprises the following steps: Step 1: Using the mulberry coding genome sequence as a template, specific PCR primers: MaBEH1 / 2-F and MaBEH1 / 2-R are used to amplify the coding sequence of the MaBEH1 / 2 gene by PCR. Step 2: Construction of expression vector using DNA homologous recombination seamless cloning: The coding sequence of the obtained MaBEH1 / 2 gene is inserted into the multiple cloning site of vector pK2GW7 to obtain the constructed overexpression vector; Step 3: According to the reaction system, add fresh PCR product, pK2GW7 overexpression vector and Basic Mix enzyme respectively, and react in a PCR instrument for 1.5h to obtain the constructed overexpression vector; transform E. coli into the above reaction mixture and plate it on LB solid selection medium. Pick single clones from the selection medium for PCR detection and sequencing verification to confirm that the MaBEH1 / 2 overexpression vector was successfully constructed and named MaBEH1 / 2-OE; Step 4: In the constructed MaBEH1 / 2-OE vector, the 5' end of the MaBEH1 / 2 gene is assembled with a strong 35S promoter, which enables efficient expression of the MaBEH1 / 2 protein in poplar. The overexpression vector pK2GW7 contains kanamycin as a selection marker for transgenic poplar, and kanamycin is used to select transgenic poplar. At the same time, it contains LB and RB sequences, which promote the integration of the 35S::MaBEH1 / 2 expression framework and the selection marker gene Kan assembled therein into the poplar chromosome, thereby obtaining MaBEH1 / 2-OE transgenic poplar.

5. The construction of a plant expression vector for the mulberry MaBEH1 / 2 gene, which enhances salt tolerance, according to claim 4, is characterized in that: In step 2, the sequence of the plant overexpression vector pK2GW7 is shown in sequence 5.

6. The construction of a plant expression vector for the mulberry MaBEH1 / 2 gene, which enhances salt tolerance, according to claim 4, is characterized in that: In step 3, the composition of the LB solid screening medium is as follows: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 8 g / L agar powder, and 50 mg / L spectinomycin.

7. The application of the mulberry MaBEH1 / 2 gene for improving plant salt tolerance as described in claim 1 in improving the salt tolerance of poplar.

8. The genetic transformation of the mulberry MaBEH1 / 2 gene for improving plant salt tolerance as described in claim 1 into transgenic poplar trees comprises the following steps: the constructed MaBEH1 / 2-OE expression vector is transformed into Agrobacterium GV3101 by electroporation, and the 35S::MaBEH1 / 2 expression framework and the selection marker gene Kan are integrated into the 84K poplar chromosome using Agrobacterium-mediated transformation, thereby creating MaBEH1 / 2 overexpression transgenic poplar MaBEH1 / 2-OE.

9. The genetic transformation of the mulberry MaBEH1 / 2 gene, which enhances salt tolerance in plants, according to claim 8, in transgenic mulberry trees, characterized in that: The specific transformation steps are as follows: One-month-old 84K poplar tissue culture seedlings were cultured at a temperature of 23-25℃ and a light intensity of 16 / 8h. Agrobacterium containing the MaBEH1 / 2-OE expression vector was cultured in LB liquid medium until the OD600 reached 0.6-0.8, and then infected the leaf discs of 84K poplar. The infected leaves were then transferred to a co-culture medium and co-cultured in the dark for 2-3 days. The co-cultured leaves were then transferred to an adventitious bud induction medium containing 50 mg / L kanamycin and 200 mg / L termethin to induce and screen for resistant adventitious buds. After 20 days of induction culture, the resistant adventitious buds were transferred to a rooting medium containing 200 mg / L termethin until adventitious roots were induced. DNA was extracted from the leaves of the rooted plants that had developed Kan resistance, and PCR verification was performed using the Kan resistance as a template with MaBEH1 / 2 cloning primers. Finally, transgenic 84K poplar containing the MaBEH1 / 2-OE vector sequence was obtained.

10. The genetic transformation of the mulberry MaBEH1 / 2 gene, which enhances salt tolerance in plants, according to claim 9, into transgenic poplar trees, characterized in that: The LB liquid medium consisted of: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 50 mg / L spectinomycin, and 20 mg / L rifampin; the co-culture medium consisted of: 2.2 g / L Murashige-Skoog + 0.5 g / L 4-morpholine ethanesulfonic acid + 20 g / L sucrose + 4 g / L plant gel + 100 μmol / L acetosyringone; the adventitious shoot induction medium consisted of: 2.2 g / L MS + 0.5 g / L MES + 0.5 mg / L 6-benzylaminopurine + 0.05 mg / L naphthaleneacetic acid + 20 g / L sucrose + 4 g / L plant gel; and the rooting medium consisted of: 2.2 g / L MS + 0.5 g / L MES + 0.05 mg / L indolebutyric acid + 0.02 mg / L NAA + 20 g / L sucrose + 8 g / L agar powder.

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