Medicago sativa polygalacturonase MsPG2, coding gene thereof and application of MsPG2 in salt stress resistance of plants

Abstract

The invention belongs to the technical field of plant genetic engineering, and relates to medicago sativa polygalacturonase MsPG2, a coding gene thereof and application of the medicago sativa polygalacturonase MsPG2 in plant salt stress resistance, and an amino acid sequence of the polygalacturonase MsPG2 is shown as SEQ ID NO.1 or a derivative sequence or a homologous sequence of the amino acid sequence shown as SEQ ID NO.1. The invention further discloses a preparation method of the medicago sativa polygalacturonase MsPG2. The invention also provides a coding gene of the polygalacturonase MsPG2, wherein the nucleotide sequence of the coding gene is shown as SEQ ID NO. 2; the coding gene can be expressed by salt induction. According to the invention, the cultivation of the MsPG2 transgenic plant is realized by utilizing a genetic engineering technology, the salt tolerance of the plant is obviously improved, an important theoretical basis is provided for the cultivation and breeding work of a new salt-tolerant variety of medicago sativa, and the MsPG2 transgenic plant has a great application value.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and specifically relates to an alfalfa polygalacturonase MsPG2 and a coding gene thereof and an application thereof in plant salt stress tolerance. Background Art

[0002] Alfalfa (Medicago sativa L.) is an important leguminous forage grass. It is known as the "king of forage grasses" for its high yield, excellent grass quality, and good palatability. Alfalfa is a perennial, cross-pollinated autotetraploid with a complex genetic background. Traditional breeding methods face great challenges in the selection of stress-resistant varieties. Therefore, it is very necessary to explore resistance genes through reverse genetics and analyze their molecular and physiological mechanisms for alfalfa breeding.

[0003] Soil salinization accounts for about 20% of the world's arable land, which seriously inhibits plant growth and causes yield reduction. Therefore, salt tolerance is one of the most important agronomic traits for crop variety breeding. The salt tolerance of plants depends on the body's ability to absorb and transport Na ions, reactive oxygen ROS metabolism, cell plasticity and other aspects. Among them, the cell wall is extremely important for plants to adapt to salt stress, and plays a role in plants sensing salt stress signals and avoiding salt growth. When the integrity of the cell wall and the cell wall remodeling pathway are damaged, plants show a phenotype that is more sensitive to salt stress. Among the components of the cell wall, cellulose, hemicellulose and pectin are cross-linked to form a complex and dynamically changing network structure to maintain the mechanical properties and ion adsorption properties of the cell wall. Pectin and hemicellulose are negatively charged and reversibly bind cations (Ca 2+ 、Na + etc.), for example, pectin and Ca 2+ Combined to form an "egg box structure" to enhance the rigidity of the cell wall. However, in a high salt environment, Ca 2+ Will be partially + Replacement destroys the "egg box structure" of pectin and inhibits cell elongation growth.

[0004] At present, polygalacturonase has been widely reported to be involved in the softening and shedding of fruit trees (Southern China Fruit Trees, 2017, 46 (3): 14-19), and to promote plant disease resistance by affecting cell wall composition (Microbiology Bulletin, 2023, 50 (8): 3440-3453), but no research has been reported on its involvement in plant response to salt stress. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and provide an alfalfa polygalacturonase (MsPG2) and its encoding gene and its application in plant salt stress tolerance. The alfalfa polygalacturonase MsPG2 has the function of improving the salt tolerance of alfalfa and can provide theoretical reference and practical guidance for the breeding of salt-tolerant alfalfa varieties.

[0006] In order to achieve the above object, the present invention provides an alfalfa polygalacturonase MsPG2, the amino acid sequence of the alfalfa polygalacturonase MsPG2 is shown in (a) or (b) or (c):

[0007] (a) having the amino acid sequence shown in SEQ ID NO.1;

[0008] Furthermore, the amino acid sequence contains four conserved domains (NTD, DD, GHG, and RIK) typical of plant MSPG2 proteins and belongs to the F subfamily of the MSPG2 family;

[0009] (b) an amino acid sequence derived from (a) which is a substitution, deletion or addition of one or more amino acids to the amino acid sequence shown in SEQ ID NO.1 and has the characteristics of polygalacturonase MsPG2;

[0010] (c) an amino acid sequence having at least 92.44% homology to the amino acid sequence shown in SEQ ID NO.1.

[0011] Furthermore, the alfalfa polygalacturonase MsPG2 is an amino acid sequence obtained by deleting, inserting and / or replacing 1 to 33 amino acids of the amino acid sequence shown in SEQ ID NO.1.

[0012] Furthermore, the alfalfa polygalacturonase MsPG2 is an amino acid sequence obtained by adding 1 to 20 amino acids to the C-terminus and / or N-terminus of the amino acid sequence shown in SEQ ID NO.1.

[0013] Furthermore, the alfalfa polygalacturonase MsPG2 is artificially modified and has an amino acid sequence with an identity of ≥92.44% compared with the amino acid sequence of SEQ ID NO.1.

[0014] The present invention also provides a gene encoding alfalfa polygalacturonase MsPG2, the nucleic acid sequence of which is as follows (a) or (b) or (c) or (d):

[0015] (a) having a base sequence as shown in SEQ ID NO.2;

[0016] (b) a sequence having at least 95.73% homology to the nucleic acid sequence shown in SEQ ID NO.2;

[0017] (c) a nucleic acid sequence complementary to the base sequence of SEQ ID NO.2;

[0018] (d) A deletion, insertion and / or substitution of 1 to 49 nucleotides in the nucleic acid sequence shown in SEQ ID NO. 2, or a sequence formed by adding up to 60 nucleotides at the 5′ and / or 3′ end.

[0019] Furthermore, the nucleic acid sequence of the encoding gene is obtained by alfalfa cloning and / or artificial synthesis methods.

[0020] Furthermore, the nucleic acid sequence of the alfalfa polygalacturonase MsPG2 encoding gene is derived from the alfalfa variety 'WL525'.

[0021] The present invention also provides application of the alfalfa polygalacturonase MsPG2 or the encoding gene in improving the salt stress resistance of transgenic alfalfa.

[0022] The present invention also provides a plant expression vector containing the coding gene. When constructing the plant expression vector, any enhancing promoter or inducible promoter can be added before the transcription start nucleotide.

[0023] The present invention also provides a host cell transformed by genetic engineering, which contains the coding gene of the polygalacturonase MsPG2, or contains a recombinant cloning vector and an expression vector constructed by the coding gene.

[0024] Furthermore, the host cell is an Escherichia coli cell, a Saccharomyces cerevisiae cell or an Agrobacterium cell.

[0025] The present invention also provides the use of the polygalacturonase MsPG2 or the encoding gene or the expression vector or the host cell in genetic engineering.

[0026] The present invention also provides application of the polygalacturonase MsPG2 or the encoding gene or the expression vector or the host cell in genetic engineering for improving plant salt tolerance.

[0027] Furthermore, the expression vector carrying the MsPG2 of the present invention can be used to transform plant cells or tissues by conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation, Agrobacterium-mediated transformation, and the transformed plant tissues can be cultivated into plants.

[0028] Furthermore, the transformed host can be either a monocot or a dicot.

[0029] The present invention also provides application of the polygalacturonase MsPG2 or the encoding gene or the expression vector or the host cell in improving the salt stress resistance of alfalfa.

[0030] Furthermore, an expression vector containing the coding gene of the alfalfa polygalacturonase MsPG2 is constructed, and a plant host is transformed, and transgenic plants are obtained by cultivation and screening.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] (1) The present invention cloned a polygalacturonase encoding gene MsPG2 from alfalfa that responds to salt stress. The gene can be induced to express by salt and has a high expression level in the stem. MsPG2 mainly alleviates the damage of salt stress and enhances the salt tolerance of plants by changing the cell wall structure of plant cells and reducing the accumulation of Na in the body.

[0033] (2) The present invention provides a basis for its effective application and has important theoretical value and application significance for breeding salt-tolerant alfalfa varieties.

[0034] (3) The present invention uses genetic engineering technology to cultivate MsPG2 transgenic plants, significantly improving the salt tolerance of the plants, providing an important theoretical basis for the cultivation and breeding of new salt-tolerant alfalfa varieties, and has great application value. At the same time, as an excellent salt-tolerant gene resource, it also has important application potential and value in the molecular breeding of salt-tolerant plants in other plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Schematic diagram of the PCR amplification results of the MsMG2 gene involved in the present invention; wherein M: Trans2K ® PlusDNA marker; Lanes 1-3: MsPG2 PCR amplification results.

[0036] Figure 2 This is a schematic diagram of the expression pattern of the MsPG2 gene involved in the present invention under different salt treatment time.

[0037] Figure 3 The figure is a schematic diagram of the expression pattern of the MsPG2 gene involved in the present invention in different tissues.

[0038] Figure 4 This is a growth phenotype diagram of the MsPG2 transgenic alfalfa involved in the present invention under salt treatment.

[0039] Figure 5The height and biomass of the MsPG2 transgenic alfalfa involved in the present invention under control and salt treatment, wherein A is plant height, B is aboveground biomass; GN3 is wild type Gannong No. 3; OE1 and OE19 are MsPG2 overexpression transgenic plants; Ri5B and Ri6B are MsPG2 interference expression transgenic plants.

[0040] Figure 6 The sodium ion content of different parts of the MsPG2 transgenic alfalfa involved in the present invention under control and salt treatment; A is leaf; B is stem; C is root; GN3 is wild type Gannong No. 3; OE1 and OE19 are MsPG2 overexpression transgenic plants; Ri5B and Ri6B are MsPG2 interference expression transgenic plants; different letters indicate significant differences (P<0.05, ANOVA analysis and LSD test).

[0041] Figure 7 This is a diagram of the cell wall structure of the MsPG2 transgenic alfalfa involved in the present invention under control and salt treatment. DETAILED DESCRIPTION

[0042] The technical solution of the present invention is further described in detail below through specific embodiments and in conjunction with the accompanying drawings.

[0043] Example 1: Cloning and sequence analysis of MsPG2 gene

[0044] Extraction of total RNA and synthesis of cDNA from alfalfa: Total RNA from leaves of alfalfa 'WL525' was extracted using EasyPure Plant RNA Kit (purchased from Quanshijin), and reverse transcribed using TransScript One-Step gDNA Removal and cDNASynthesis SuperMix (purchased from Quanshijin) to synthesize cDNA.

[0045] Primer design and synthesis: Based on the transcriptome data of alfalfa (Medicago sativa L.) 'WL525' in salt treatment and control group, the differentially expressed MsPG2 gene fragment was selected and compared in NCBI, and the MtPG2 gene with high homology to the gene in Medicago truncatula was found. Primers were designed based on the gene sequence, and the upstream primer sequence and downstream primer sequence are as follows: MsPG2-F1: 5'-ATGCAGGACTTGTTTATATC-3' (SEQID NO.3); MsPG2-R1: 5'-TTACTGGTGTAGACACAGTG -3' (SEQ ID NO.4). The above-mentioned alfalfa cDNA was used as a template for homologous cloning, and PCR amplification was performed according to the following reaction system and conditions: 20 μL system, containing 10 μL of 2×EXTaq super PCR Mix (purchased from TaKaRa), 0.8 μL of 10 μM MsPG2-F1 and MsPG2-R1 primers, 1 μL of cDNA, and deionized water to 20 μL. Reaction conditions: pre-denaturation at 94℃ for 5min; 94℃ for 30 s, 57℃ for 30 s, 72℃ for 1 min, 30 cycles; 72℃ extension for 7 min. The above-mentioned amplified fragment was recovered and linked to the cloning vector pMD18-T (purchased from TaKaRa), and the bacterial solution was sent to Sangon Biotech for sequencing after PCR identification. The sequence result is shown in SEQ ID NO.2.

[0046] The 1194 bp MsPG2 gene sequence was obtained by PCR amplification. The amino acid sequence of the encoded protein is shown in SEQ ID NO.1, which consists of 397 amino acid residues and contains the four typical conserved domains (NTD, DD, GHG and RIK) of plant MsPG2 protein. Figure 1 shown.

[0047] SEQ ID NO.1:

[0048] MQDLFISGLLIFYIVSCNSFVGFGQNTFNVLQYGAKGDGTSDDTQAFLEAWKALCAANQGTPTLVVPAEHTFFVRQATFKGPCKSQNFHIQIMGNIIAPHRDAWGTCSKRWLYFLDVHGMTVDGSGVINGQGEAWWGNLNGTKGCAANPPPTALLFERCNELQLSGLTHINGMSMHVYVVHSQDVTISHINVSSPKDSHNTDGIDLSNSVRVNIHDSIIQSGDDCIAIKGGSQFINVTQVTCGMSTHGISVGSLGGGGAEEFADHVNVRNCTFNGADSAARIKTWMSGKGYAKAIIFDNIMVNQIRNPIYIQQHYMGAPEQKDAVKVSDVTFSNIYGTCSGDDAIFLDCANIGCDNITLEQIDITSVGPKKPNSAICNNVQGKANNISSPPFHCLHQ*, * indicates the termination of protein translation.

[0049] SEQ ID NO.2

[0050]

[0051] Example 2: Response pattern of alfalfa MsPG2 gene to salt treatment

[0052] Cultivation and treatment of alfalfa plants: Remove the coating of alfalfa 'WL525' seeds (purchased from Beijing Zhengdao Ecological Technology Co., Ltd.), wash them and evenly distribute them in a growth tray lined with filter paper, keep them moist, and when they germinate and grow the first true leaf, select seedlings with consistent growth and transplant them into 1 / 2 Hoagland nutrient solution for hydroponics. The formula of Hoagland culture medium is as follows: Ca(NO3)2·4H2O 0.62 g / L, KNO3 0.34 g / L, KH2PO4 0.06 g / L, NH4NO3 0.053g / L, MgSO4 0.24 g / L, MgCl2 0.67 mg / L, H3BO3 0.38 mg / L, MnSO4 0.2 mg / L, ZnSO4·7H2O0.29 mg / L, CuSO4 0.01 mg / L, FeSO4·7H2O 0.02785 g / L, EDTA-Na2 0.0373 g / L, (pH 5.8), cultured at 25℃ with 16 h light / 8 h dark conditions.

[0053] One week later, two treatments of 0 and 200 mM NaCl were set up, and underground root and aboveground leaf samples of alfalfa were collected at 0, 1, 3, 6, 9, 12, and 24 h after treatment, and quickly placed in liquid nitrogen and then stored in a -80°C ultra-low temperature freezer.

[0054] Total RNA was extracted from the root and leaf samples, and reverse transcribed to synthesize cDNA, the method was the same as in Example 1. Real-time fluorescence quantitative PCR primers were designed according to the cDNA sequence of MsPG2, and the upstream and downstream primer sequences were as follows: MsPG2-qRTF1: 5′-GGTGGCTTTATTTCCTTGACG -3′ (SEQ ID NO.5); MsPG2-qRTR1: 5′- GTTCCATTAAGATTGCCCCAC-3′ (SEQ ID NO.6). The constitutively expressed gene EF-α of alfalfa was used as the internal reference gene, and the upstream and downstream primer sequences were as follows: MsEF-α-qRTF1: 5'-GCACCAGTGCTCGATTGC-3' (SEQ ID NO.7); MsEF-α-qRTR1: 5'-TCGCCTGTCAATCTTGGTAACAA-3' (SEQ ID NO.8). qRT-PCR was performed using a Bio-rad real-time quantitative PCR instrument with the cDNA of the above-mentioned samples as templates. The reaction system contained 10 μL of 2×SYBR qPCR SuperMix (purchased from Quanshijin), 0.4 μL of primers F / R, 2 μL of cDNA, and water to a total volume of 20 μL. The reaction program was 94°C for 30 s; 95°C for 5 s, 57°C for 15 s, and 72°C for 15 s, for 40 cycles. Each treatment was repeated 3 times for biological replication and 3 times for technical replication. The data were analyzed by SPSS14.0 and the graphs were drawn by EXCEL.

[0055] The response patterns of MsPG2 in roots and aboveground leaves to salt treatment are as follows Figure 2 As shown. Figure 2 It can be seen that under salt stress, the expression level of MsPG2 is upregulated, the expression level is the highest after 3-6 hours of treatment, and then the expression level decreases. The above results indicate that MsPG2 is induced to express under salt stress.

[0056] Example 3: Expression pattern of MsPG2 gene in different tissues

[0057] Plant seedlings were cultured according to the method shown in Example 2 without any treatment. After about two weeks, the root tip, root base, stem, leaves (young leaves, mature leaves and old leaves), petiole, stipules and terminal buds were sampled, RNA was extracted, reverse transcribed and then subjected to real-time fluorescence quantitative PCR. The implementation method was the same as in Example 2 above, and the results were as follows: Figure 3 As shown. Figure 3 It can be seen that the expression level of MsPG2 is highest in the stem, followed by the expression levels in the petiole, node and root base, and lower in the leaf blade, stipule, root tip and terminal bud.

[0058] Example 4 Growth phenotype of alfalfa plants overexpressing or interfering with MsPG2 under salt treatment

[0059] Construction of plant overexpression vector: According to the plant overexpression vector pHB sequence and MsPG2 gene sequence, the upstream primer MsPG2-F2: 5′-CTTGATATCGAATTCCTGCAG ATGCAGGACTTGTTTATATC-3′ (SEQ ID NO.9) and the downstream primer MsPG2-R2: 5′-TTATCGATACCGTCACTAGT CTGGTGTAGACAGTGGAAGG-3′ (SEQ ID NO.10) were designed. The pMD18-T-MsPG2 plasmid in Example 1 was used as a template and PCR amplification was performed using the following reaction system and conditions: 50 μL system, containing 25 μL of 2×Phanta Max Master Mix (purchased from Vazyme), 2 μL of 10 μM MsPG2-F2 and MsPG2-R2 primers each, 2 μL of plasmid template, and deionized water to 50 μL. Reaction conditions: pre-denaturation at 95℃ for 3min; 95℃ for 15 s, 57℃ for 15 s, 72℃ for 1 min 30 s, 30 cycles; extension at 72℃ for 5 min. The above PCR products were detected by electrophoresis and recovered by gel excision. The plant overexpression vector pHB was double-digested with restriction endonucleases Pst1 and Spe1, and the digestion products were detected by electrophoresis and recovered by gel excision. The above two gel-recovered products were linked by seamless cloning according to the following system and conditions to obtain pHB-MsPG2 plasmid: MsPG2 fragment product 4 μL, pHB vector digestion product 3 μL, 5× CE II Buffer 4 μL, Exnase II 2 μL, deionized water to 20 μL, 37°C reaction for 30 min (purchased from Vazyme). The above linkage products were transformed into Escherichia coli competent cells and sequenced.

[0060] Plant interference expression vector construction: Primer MsPG2-Ri-F: 5′-GAAGGAGCCCTTCACC was designed based on the non-conserved domain segment of the MsPG2 nucleotide sequence GGATCC ACAATATCATGGTCAATCAA-3′ (SEQ ID NO. 11); MsPG2-Ri-R: 5′-CGGGTTTTGAGCTCAAA GAATTCCTGGTGTAGACAGTGGAAGGGA-3′ (SEQ ID NO.12) was amplified by PCR with high fidelity using pMD18-T-MsPG2 plasmid as template (same PCR reaction system and conditions in the construction of plant overexpression vectors above). The amplified fragment was constructed into the pTOPO vector by seamless cloning to obtain the pTOPO-MsPG2 plasmid (same seamless cloning reaction system and conditions in the construction of plant overexpression vectors above); the reaction system of 3 µL of linearized pHellsgate12 plasmid, 1 µL of pTOPO-MsPG2, and 1 µL of LR clone Mix (purchased from Invitrogen) was connected in a PCR instrument at 25°C for 1h. The above products were transformed into E. coli competent cells and sequenced.

[0061] Agrobacterium transformation: pHB-MsPG2 and pHellsgate12-MsPG2 plasmids were transformed into Agrobacterium GV3101 competent cells, and the bacterial liquid PCR was performed for verification. Positive clones were selected and transferred to YEB liquid medium containing 50 mg / L Kan and 20 mg / L Rif, 100 mg / L Spe and 20 mg / L Rif, respectively, and cultured on a shaker at 28°C at 200 rpm until OD600 = 0.8 for infection.

[0062] Explant preparation and infection: The explants were made from leaves of alfalfa 'Gannong No. 3'. A 0.1 v / v% Tween20 solution and a 30 wt% sodium hypochlorite aqueous solution were added to the culture bottle containing the leaves, and the surface was sterilized on a shaker at 100 rpm for 8-10 min. Then, the leaves were rinsed three times with sterile distilled water. The leaves were sandwiched into the bacterial solution containing the overexpression plasmid or the interference expression plasmid prepared for infection, vacuumed for 10 min, ultrasonicated for 40 s, and vacuumed again for 10 min. The bacterial solution was discarded, and the leaves were sandwiched between two layers of sterile filter paper. When the residual bacterial solution on the surface of the leaves was dried, they were transferred to the co-culture solid culture medium for dark culture for 4 days. After co-culture, the leaves were transferred to the SM4 screening medium, and after about 4 weeks of light culture, they were transferred to the MSBK differentiation induction medium. After the buds are differentiated, the callus is transferred to MSS medium. After about 1-2 months, complete leaves grow out and are transferred to MSR rooting medium. The medium formula is based on the literature of Fu Chunxiang et al. (Fu, C., Hernandez, T., Zhou, C., et al., Alfalfa (Medicago sativaL.). In Agrobacterium protocols[M]. Springer, New York, 2015: 213-221.). After about 1-2 months, complete alfalfa overexpression transgenic plants or interference expression transgenic plants can be grown. Wild-type, overexpression transgenic plants and interference expression transgenic plants are identified by PCR and qRT-PCR, and positive plants are selected for cutting propagation and subsequent physiological phenotype analysis.

[0063] Plant salt treatment: When the wild type, overexpression plants and interference expression plants grew to a height of 10 cm, they were treated with 1 / 2 Hoagland nutrient solution containing 0 mM NaCl (control group) and 400 mM NaCl (treatment group). After about 2-3 weeks of treatment, the growth phenotype was observed and photographed, and the plant height and aboveground fresh weight were counted. The results are as follows: Figure 4 and Figure 5 shown.

[0064] from Figure 4 It can be seen that under salt treatment, the growth of wild-type and interference expression plants was significantly inhibited, and the leaves turned yellow significantly. The inhibition degree of interference expression plants was significantly higher than that of wild type, but overexpression plants still maintained normal growth, and the leaves did not turn yellow significantly. In the control group, wild-type, overexpression transgenic plants and interference expression transgenic plants all grew normally, and the growth of overexpression plants was slightly better than that of wild-type and interference expression plants. Figure 5It can be seen that compared with the control group, the plant height and aboveground fresh weight of the wild type, overexpression plants (OE1 and OE19) and interference expression plants (Ri5B and Ri6B) in the salt treatment group showed the same change trend.

[0065] Determination of ion content: Samples were taken from the wild type, overexpression transgenic plants and interference expression transgenic plants in the control group and salt treatment group, and placed in a 60℃ oven for 3 days. Weigh the same amount of dry weight of roots, stems and leaves into 50mL centrifuge tubes, add 1mL HNO3 and 1mL H2O2, and digest at 120℃ for 2 hours until the sample is completely digested. Use deionized water to make the same volume, and use ICP-MS to determine the Na ion content. The results are as follows: Figure 6 As shown. Figure 6 It can be seen that under salt treatment, the Na content in the roots, stems and leaves of the overexpression plants was significantly lower than that of the wild type, while the Na content in the interference plants was higher than that of the wild type or had no significant difference from the wild type.

[0066] Observation of cell wall structure: Scanning electron microscopy was used to observe the root tip cell wall structure of wild-type, overexpression transgenic plants and interference expression transgenic plants in the control group and salt treatment group. The specific method is as follows: the root tip (0-1 cm) of the control group or salt treatment was placed in 1 mL of 2.5wt% glutaraldehyde solution, the sample was completely immersed in the solution, and it was left to stand at 4°C overnight; the glutaraldehyde solution was aspirated and washed with 20 mM HEPES (pH7.0) at room temperature for 4 times, each time for 15 min; then dehydrated with ethanol aqueous solutions of different volume concentrations (30%, 40%, 50%, 70%, 85%, 100%) in turn, and be careful not to touch the root tip during the operation to avoid deformation of the root tip structure. After dehydration, the root apex was wrapped in clean and soft lens-wiping paper and critical point dried in anhydrous ethanol (EM CPD300, Leica). The dried root apex was gold-coated on a vacuum coater (Q 150T ES plus, England) for 1 min, and the root apex and surface cell wall structure were observed using a Raman image-scanning electron microscope (RISE-MAGNA). The results are shown in Figure 2. Figure 7 shown.

[0067] from Figure 7It can be seen that in the control group, when there is no salt stress, the structure of the fiber-microfibril of the root tip cell wall of the MsPG2 overexpressing transgenic plants and the wild type is relatively stretched. Under salt treatment, the arrangement of the fiber-microfibril of the root tip cell wall of the wild type and the interference expression plants becomes densely cross-linked and compact, and the porosity decreases; the fiber-microfibril structure of the root tip cell wall of the overexpressing plants is looser and stretched than that of other plants, and the porosity is also significantly increased. This shows that under salt stress, overexpression of MsPG2 can maintain the stretch of the cell wall structure of alfalfa, alleviate the damage to the root tip, reduce the accumulation of Na in the body, and thus improve the salt tolerance of alfalfa.

Claims

1. An alfalfa polygalacturonase MsPG2, characterized in that The amino acid sequence of the alfalfa polygalacturonase MsPG2 is shown in (a), (b) or (c): (a) having the amino acid sequence shown in SEQ ID NO.1; (b) an amino acid sequence derived from (a) which is a substitution, deletion or addition of one or more amino acids to the amino acid sequence shown in SEQ ID NO.1 and has the characteristics of polygalacturonase MsPG2; (c) an amino acid sequence having at least 92.44% homology to the amino acid sequence shown in SEQ ID NO.

1.

2. The alfalfa polygalacturonase MsPG2 according to claim 1, characterized in that The amino acid sequence of the alfalfa polygalacturonase MsPG2 is obtained by adding 1 to 20 amino acids to the C-terminus and / or N-terminus of the amino acid sequence shown in SEQ ID NO.

1.

3. The alfalfa polygalacturonase MsPG2 according to claim 1, characterized in that: The amino acid sequence of MsPG2 is the amino acid sequence shown in SEQ ID NO.1 with 1 to 33 amino acids deleted, inserted and / or substituted.

4. A gene encoding alfalfa polygalacturonase MsPG2 according to claim 1, characterized in that: The nucleic acid sequence encoding the gene is (a) or (b) or (c) or (d): (a) having a base sequence as shown in SEQ ID NO.2; (b) a sequence having at least 95.73% homology to the nucleic acid sequence shown in SEQ ID NO.2; (c) a nucleic acid sequence complementary to the base sequence of SEQ ID NO.2; (d) A deletion, insertion and / or substitution of 1 to 49 nucleotides in the nucleic acid sequence shown in SEQ ID NO. 2, or a sequence formed by adding up to 60 nucleotides at the 5′ and / or 3′ end.

5. A plant expression vector containing the coding gene according to claim 4.

6. A genetically engineered host cell, characterized in that: The host cell has the coding gene of the polygalacturonase MsPG2 according to claim 5, or has a recombinant cloning vector and an expression vector constructed by the coding gene according to claim 5.

7. Use of the polygalacturonase MsPG2 according to claim 1, the encoding gene according to claim 4, the expression vector according to claim 5 or the host cell according to claim 6 in genetic engineering for improving plant salt tolerance.

8. Use of the polygalacturonase MsPG2 according to claim 1 or the encoding gene according to claim 4 in improving the salt stress tolerance of alfalfa.

9. Use of the polygalacturonase MsPG2 or the encoding gene according to claim 8 in improving the salt stress tolerance of alfalfa, characterized in that: An expression vector containing the coding gene of the alfalfa polygalacturonase MsPG2 is constructed, and a plant host is transformed, and transgenic plants are obtained by cultivation and screening.

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