St. augustinegrass salt-tolerant gene zjchi1, plant expression vector and application thereof

Abstract

The application discloses a zoysia japonica salt-tolerant gene ZjCHI1, a plant expression vector and application thereof. The application discloses a new salt-tolerant gene ZjCHI1 in zoysia japonica, and the nucleotide sequence of the gene is shown as SEQ ID NO. 1. The plant expression vector is obtained by carrying out a homologous recombination reaction on a PCR product in which an upstream and a downstream of the ZjCHI1 gene are introduced into homologous recombination arms respectively and a linearized plant expression vector pCAMBIA1305.1 plasmid subjected to single enzyme digestion by using BamHI. The ZjCHI1 of the zoysia japonica provided by the application is a new salt-tolerant gene, the gene can improve the salt tolerance of plants, and can be applied in the creation of new salt-tolerant germplasm and the improvement of plant varieties.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering and transgenic breeding, and relates to the salt tolerance gene ZjCHI1 of Zoysia japonica, its plant expression vector, and its application. Background Technology

[0002] Soil salinization is a global problem. High salt and alkali content in saline-alkali areas severely restricts plant growth. However, saline-alkali land is an important reserve land resource, and cultivating new salt-tolerant plant varieties is an effective measure and important approach for improving and utilizing saline-alkali land. Traditional hybridization breeding and radiation-induced mutation breeding have made significant contributions to the cultivation of new stress-resistant plant varieties; however, they suffer from drawbacks such as long cycles and low efficiency. In recent years, with the rapid development of molecular biology, the discovery of superior stress-resistant genes and the use of rapid and efficient genetic engineering techniques to overexpress these genes in plants to enhance their stress resistance have become important methods in modern breeding.

[0003] Chitinase is a class of glycoside hydrolases. Studies have shown that plant chitinase is an important protein in response to biotic and abiotic stresses, and plays an important role in responding to salt stress and regulating salt tolerance. Studies have found that chitinase activity in Arabidopsis thaliana is induced by ultraviolet radiation, heat shock, salt stress, and mechanical damage (Lima et al., 2002; Gerhardt et al., 2004; Kwon et al., 2007); the expression of the chitinase (Class I, Chitinase) gene (ChitiWb1) in winged bean is upregulated under NaCl stress (Tateishi et al., 2001); the expression of the chitinase gene (BjCHI) in mustard (Brassica juncea) is highly induced by NaCl stress (Wu et al., 2009); and genetic transformation of tomatoes with osmotic regulatory protein (OLP) and chitinase gene (Chil1) revealed that transgenic plants had higher proline content and potassium content. + The content, relative water content, chlorophyll fluorescence, biomass and fruit yield were all higher than those of the wild type (Kumar et al., 2016).

[0004] This invention obtained a salt-tolerant candidate chitinase gene, ZjCHI1, from Zoysia japonica using GWAS association technology. This gene showed the highest homology (59.72%) with a homology gene from millet; its homology with other monocotyledonous plants (sorghum, rice, maize, and foxtail millet) was 56.18%, 53.71%, 52.30%, and 54.77%, respectively; and its homology with dicotyledonous plants (Arabidopsis thaliana and tobacco) was 44.17%. Therefore, ZjCHI1 is a novel salt-tolerant candidate gene. The motivation for this invention is to construct the Zoysia japonica ZjCHI1 gene into a plant expression vector and perform genetic transformation using Agrobacterium-mediated transformation to obtain new germplasm with salt-tolerant characteristics. This patent is of great significance for the breeding of superior new plant varieties and their widespread application in production.

[0005] References:

[0006] Lima VM, Magioli C, Gerhardt LBDA, Tarre E, Menezes RMG (2002) Bean class IV chitinase promoter is modulated during plant development and under ahioticstress. Physiol Plantamm, 116(4):512-521.

[0007] Gerhardt LBDA, Magioli C, Perez ABUCM, Margis R, Sachetto-Martins G (2004) AtchitlV gene expression is stimulated under abiotic stresses and isspatially and temporally regulated during embryo development. Genet Mol Biol27:118-123.

[0008] Kwon YR, Kim SH, Jung MS, Kim MS, Oh JE (2007) Arabidopsis hot2 encodes anendochitinase-like protein that is essential for tolerance to heat, salt and drought stresses. The Plant Journal 49:184-193.

[0009] Tateishi Y,Umemura Y,Esaka M(2001)A basic class I chitinaseexpressing in winged bean is up-regulated by osmotic stress.Biosci BiotechnolBiochem 65:1663-1668.

[0010] Wu XF, Wang CL, Xie EB, Gao Y, Fan YL et al. (2009) Molecular cloning and characterization of the promoter for the multiple stress-inducible gene BjCHI1 from Brassica juncea. Planta229:1231-1242.

[0011] Kumar SA, Kumari PH, Jawahar G, Prashanth S, Suravajhala P, et al. (2016) Beyond just being foot soldiers-osmotin like protein(OLP) and chitinase(Chi11) genes act as sentinels to confront salt, drought, and fungal stress tolerance in tomato. Environmental and Experimental Botany132:53-65. Summary of the Invention

[0012] In response to the problem of cultivating new salt-tolerant plant germplasm mentioned in the background art, the purpose of this invention is to provide a new salt-tolerant gene ZjCHI1 of Zoysia japonica and its application, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0013] Another objective of this invention is to provide a biomaterial containing the above-mentioned Zoysia japonica salt tolerance gene ZjCHI1 and its application.

[0014] Another objective of this invention is to provide a method for improving plant salt tolerance or cultivating new salt-tolerant plant germplasm. The plant expression vector constructed by this invention can be directly used for Agrobacterium-mediated plant genetic transformation to create new salt-tolerant germplasm, and can be used for plant variety improvement.

[0015] The objective of this invention can be achieved through the following technical solutions:

[0016] In a first aspect, the present invention provides a Zoysia japonica salt-tolerant gene ZjCHI1, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0017] Secondly, the present invention provides biological materials containing the above-mentioned Zoysia japonica salt tolerance gene ZjCHI1; further, the biological materials are recombinant vectors, expression cassettes, transgenic cell lines or recombinant bacteria.

[0018] More preferably, the recombinant vector is a recombinant cloning vector or a recombinant expression vector.

[0019] More preferably, the recombinant expression vector is a Zoysia japonica salt tolerance gene ZjCHI1 plant expression vector constructed by inserting the above-mentioned Zoysia japonica salt tolerance gene ZjCHI1 into a plant expression vector.

[0020] More preferably, the recombinant expression vector is a ZjCHI1 salt-tolerant gene expression vector for Zoysia japonica obtained by homologous recombination of the ZjCHI1 gene fragment with a homologous recombination arm and the linearized pCAMBIA1305.1 expression vector plasmid. Specifically, the vector is constructed using the ClonExpress II One Step Cloning Kit (Vazyme, Nanjing) homologous recombination cloning method, with BamHI as a single restriction enzyme site, and is obtained by recombination reaction with the pCAMBIA-1305 expression vector plasmid.

[0021] In a specific embodiment of the present invention, the method for constructing the Zoysia japonica salt tolerance gene ZjCHI1 plant expression vector includes the following steps:

[0022] (1) Using Zoysia japonica cDNA as a template, primers ZjCHI1-F and ZjCHI1-R were designed for PCR reaction. The PCR product was ligated into pMD19-T vector, transformed into DH5α competent cells, and positive plasmids were extracted.

[0023] Upstream primer ZjCHI1-F: 5′-ATGGCGAATTCACCATGGCCGACGAAC-3′ (SEQ ID NO.2),

[0024] Downstream primer ZjCHI1-R: 5′-CTAGCAGGTGAGGTTGCCCCCCGGGTCG-3′ (SEQ ID NO.3),

[0025] (2) Using the extracted positive plasmid as a template, primers 1305-ZjCHI1-F and 1305-ZjCHI1-R were designed to construct the plant expression vector pCAMBIA1305.1. PCR reaction was carried out, and the PCR product was homologous recombination reaction was performed with the plant expression vector pCAMBIA1305.1 plasmid linearized by BamHI single enzyme digestion. The vector was transformed, and the positive plasmid was extracted. The plant expression vector pCAMBIA1305.1-ZjCHI1 was successfully constructed.

[0026] Upstream primer 1305-ZjCHI1-F: 5′-aggaccggtcccgggggatccATGGCGAATTCACCATGGC-3′ (SEQ ID NO.4),

[0027] Downstream primer 1305-ZjCHI1-R: 5′-gcccttgctcaccatggatccGCAGGTGAGGTTGCCCCC-3′ (SEQ ID NO.5).

[0028] Thirdly, the primer pair used to clone the Zoysia japonica salt tolerance gene ZjCHI1 also falls within the scope of protection of this invention. The upstream and downstream primers of this primer pair are shown below:

[0029] Upstream primer ZjCHI1-F: 5′-ATGGCGAATTCACCATGGCCGACGAAC-3′,

[0030] Downstream primer ZjCHI1-R: 5′-CTAGCAGGTGAGGTTGCCCCCCGGGTCG-3′.

[0031] Fourthly, the present invention provides a method for improving the salt tolerance of plants or cultivating new salt-tolerant plant germplasm, the method being to overexpress the above-mentioned Zoysia japonica salt tolerance gene ZjCHI1 in plants to improve the salt tolerance of plants or cultivate new salt-tolerant plant germplasm.

[0032] Fifthly, the application of the aforementioned Zoysia japonica salt-tolerant gene ZjCHI1, the aforementioned biological materials, or the aforementioned primer pairs in improving plant salt tolerance or cultivating new salt-tolerant plant germplasm also falls within the scope of protection of this invention.

[0033] Furthermore, the plant or plant is a dicotyledonous plant or a monocotyledonous plant; specifically, the monocotyledonous plant is selected from Zoysia japonica, millet, sorghum, rice, corn and foxtail millet; the dicotyledonous plant is selected from cotton, tobacco or Arabidopsis thaliana.

[0034] Beneficial effects of the present invention

[0035] 1. The Zoysia japonica ZjCHI1 gene provided by this invention is a novel salt tolerance gene that can improve the salt tolerance of plants;

[0036] 2. The Zoysia japonica ZjCHI1 plant expression vector constructed in this invention is reported for the first time. It can be directly used for Agrobacterium-mediated genetic transformation to create new salt-tolerant germplasm, improve the salt tolerance of plants, and improve plant varieties. Attached Figure Description

[0037] Figure 1 Construction of pCAMBIA1305.1-ZjCHI1 plant vector.

[0038] Figure 2 Agarose gel electrophoresis image of the construction process of the plant expression vector pCAMBIA1305.1-ZjCHI1;

[0039] Wherein, M1: Marker 5000; 1: ZjCHI1; M2: Marker 5000; 2: pCAMBIA1305.1 plasmid digested with BamHI; 3: pCAMBIA1305.1-ZjCHI1 expression vector plasmid electrophoresis image.

[0040] Figure 3 Transgenic Arabidopsis thaliana (ZjCHI1) and wild-type (WT) were identified by PCR.

[0041] Figure 4 Evaluation of salt tolerance in wild-type and transgenic Arabidopsis seedlings (phenotypic observation);

[0042] Among them, WT: wild type; 1-4, 1-5 and 1-6 are transgenic progeny overexpressing ZjCHI1.

[0043] Figure 5 Comparison of biomass determination between wild-type and transgenic Arabidopsis thaliana under different salt concentration treatments;

[0044] Among them, WT: wild type; 1-4, 1-5 and 1-6 are transgenic progeny overexpressing ZjCHI1. Detailed Implementation

[0045] The specific embodiments of the invention are described in detail below: This embodiment is implemented based on the technical solution of the present invention, and provides detailed implementation methods and specific operation processes, as follows:

[0046] Example 1. Cloning of the ZjCHI1 gene from Zoysia japonica

[0047] Using Zoysia japonica seed material 'Z011' as material, healthy stolons were selected and cut into small segments with 2-3 nodes. After hydroponics in 1 / 2 Hongland nutrient solution for 30 days, the segments were transferred to 1 / 2 Hongland nutrient solution containing 340mM NaCl for 24 hours. 0.1g of young leaves were taken, and total RNA was extracted from the leaves according to the instructions of the Trizol RNA Extraction Kit (TaKaRa). 1μg of total RNA was reverse transcribed into cDNA according to the M-MLV Reverse Transcription Kit (TaKaRa). The cDNA product was digested with RNase, and primers were designed using Primer 5 software to amplify ZjCHI1.

[0048] Upstream primer ZjCHI1-F: 5′-ATGGCGAATTCACCATGGCCGACGAAC-3′ (SEQ ID NO.2);

[0049] Downstream primer ZjCHI1-R: 5′-CTAGCAGGTGAGGTTGCCCCCCGGGTCG-3′ (SEQ ID NO.3).

[0050] Using extracted leaf cDNA as a template, PCR was performed. The 50 μL reaction system consisted of: 5.0 μL of 10×PCR Buffer, and 2.5 μL each of ZjCHI1-F and ZjCHI1-R primers (10 μmol·L⁻¹). -1 ), dNTP mix 1.0μL (10.0mmol·L -1 The reaction mixture consisted of 0.5 μL of TaqDNA Polymerase, 1 μL of cDNA template, and 37.5 μL of ddH2O. The reaction program was as follows: pre-denaturation at 95 °C for 5 min, followed by denaturation at 94 °C for 45 sec, annealing at 55 °C for 45 sec, extension at 72 °C for 1 min, for 30 cycles, and a final extension at 72 °C for 10 min. The product was recovered using a gel extraction kit (AXYGEN, USA), ligated into the pMD19-T vector (TaKaRa) using T4 DNA ligase (TaKaRa), transformed into DH5α competent cells, and sequenced as SEQ ID NO.1.

[0051] Example 2. Construction of plant expression vector pCAMBIA1305.1-ZjCHI1

[0052] Primers were designed based on the full-length ZjCHI1 gene sequence for PCR reaction. Using the positive plasmid from Example 1 above as a template, homologous recombination arms were introduced upstream and downstream of the ZjCHI1 gene, respectively.

[0053] Upstream primer 1305-ZjCHI1-F: 5′-aggaccggtcccgggggatccATGGCGAATTCACCATGGC-3′ (SEQ ID NO.4);

[0054] Downstream primer 1305-ZjCHI1-R: 5′-gcccttgctcaccatggatccGCAGGTGAGGTTGCCCCC-3′ (SEQ ID NO.5).

[0055] Use high-fidelity enzyme (PrimeSTAR) TM PCR was performed using HS DNA Polymerase (TaKaRa). The 50 μL reaction system consisted of: 5.0 μL of 10×HS PCR Buffer, and 2.5 μL each of primers 1305-ZjCHI1-F and 1305-ZjCHI1-R (10 μmol·L⁻¹). -1 ), dNTP mix 1.0μL (10.0mmol·L -1 PrimeSTAR TM HSDNA Polymerase 0.4 μL, cDNA template 1 μL, ddH2O 37.6 μL; reaction program: 95℃ pre-denaturation for 5 min, then 94℃ denaturation for 45 sec, 55℃ annealing for 30 sec, 72℃ extension for 1 min, 30 cycles, 72℃ extension for 10 min; PCR products were recovered using a gel extraction kit (AXYGEN, USA).

[0056] Plasmid pCAMBIA1305.1 was digested with BamHI. The digestion system (40 μL) consisted of: 10×Buffer 4.0 μL, plasmid 2.0 μg, BamHI 2.5 μL, and ddH2O to a total volume of 40 μL. The reaction was carried out at 37℃ for 2 h, and plasmid pCAMBIA1305.1 was recovered using a gel extraction kit (AXYGEN). The PCR product was then subjected to LR recombination with the linearized plant expression vector pCAMBIA1305.1 plasmid after BamHI digestion. The reaction system (20 μL) consisted of: pCAMBIA1305.1 linearized vector 0.03 pmol, ZjCHI1 insert 0.06 pmol, 5×Buffer 4.0 μL, Exnase II 2.0 μL, and ddH2O to a total volume of 20 μL. The reaction was carried out at 37℃ for 30 min, and then cooled to 4℃. 10 μL of the recombinant product was transformed into DH5α competent cells and cultured overnight at 37°C. Positive clones were selected for amplification, and the plasmid pCAMBIA1305.1-ZjCHI1 was extracted and verified by electrophoresis and sequencing. The plant expression vector pCAMBIA1305.1-ZjCHI1 was successfully constructed. Figure 1 , Figure 2 ).

[0057] Example 3. Genetic transformation of Arabidopsis thaliana using plant expression vector pCAMBIA-1305-ZjCHI1 and identification of its salt tolerance

[0058] ① The ZjCHI1 gene overexpression vector pCAMBIA-1305-ZjCHI1 was transformed into Agrobacterium strain EHA105 using the freeze-thaw method: 5 μg of the constructed expression vector plasmid was added to 100 μl of Agrobacterium competent cells and mixed well; the competent cells were then incubated sequentially at 4°C for 5 min on ice, 5 min in liquid nitrogen, 5 min in a 37°C water bath, and 5 min on ice at 4°C, followed by the addition of 600 μl of LB liquid medium and incubation at 28°C in a shaker for 3 h; the cultured competent cells were centrifuged at 6000 rpm, and 100 μl of supernatant was resuspended and plated on LB / c media. + / Rif + Incubate on solid culture medium at 28°C for 2 days, then select single clones for screening. Store the obtained positive clones at -80°C for subsequent Arabidopsis transformation.

[0059] ② Arabidopsis thaliana transformation was performed using the flower-dipping method: Columbia (Col-0) wild-type Arabidopsis thaliana was cultivated in culture containers containing a 1:1 (w:w) ratio of vermiculite and nutrient soil. It was cultured at 23℃ under a 10h / 14h light / dark cycle for approximately 3 weeks. Infection was then carried out during its peak flowering period; preserved Agrobacterium was then used in LB / Kan... + / Rif + Stir-fry on solid medium until single colonies emerge, then pick 5-8 single colonies and inoculate into 150 mL LB / Kan. + / Rif + In liquid culture medium, the culture was carried out at 28°C for approximately 16 hours. The cultured bacterial suspension was centrifuged at 5000 rpm for 10 minutes, and the precipitate was resuspended completely in 1 / 2 MS transformation buffer containing 50 g / L sucrose and 200 μL / L Silwet L-77. The suspension containing Agrobacterium was then used to soak wild-type Arabidopsis inflorescences for 1 minute. After slight drying, the inflorescences were wrapped in plastic wrap and cultured in the dark for 12 hours. The plastic wrap was then removed, and the inflorescences were cultured under light. After approximately 2-3 weeks, mature seeds of the infected T0 generation were harvested, dried, and stored at 4°C for further screening.

[0060] ③ Sowing after seed disinfection: Place the received T0 generation seeds into a 1.5mL centrifuge tube, add 1mL of 75% alcohol, shake to sterilize for 15min, and in a clean bench, use a sterilized pipette tip to draw out the 75% alcohol, add 1mL of anhydrous ethanol, shake well, and then draw out the seeds and place them on sterile filter paper to dry. Then fold the filter paper in half, gently tap the filter paper, and evenly sow the seeds onto the selection medium (1 / 2MS + 25mg / L hygromycin + 25mg / L ampicillin) for selection culture for 10 days. Then transplant the resistant seedlings into the soil, cover them with plastic wrap to keep them moist for about 1 week; when the resistant seedlings have 7-8 leaves, extract the DNA from the Arabidopsis leaves, and identify them by PCR (primers 1305-ZjCHI1-F, 1305-ZjCHI1-R). Figure 3 ).

[0061] ④ Salt tolerance evaluation: T1 generation transgenic seedlings identified by PCR continued to grow, and T2 generation seeds were harvested. Wild-type and T2 generation resistant transgenic Arabidopsis were subjected to salt treatments (0 mM NaCl + MS medium, 125 mM NaCl + MS medium, 150 mM NaCl + MS medium, 175 mM NaCl + MS medium) for 14 days. It was found that the growth of the three ZjCHI1 gene-transgenic Arabidopsis was significantly better than that of wild-type Arabidopsis. Figure 4 , Figure 5 Salt tolerance is significantly improved.

[0062] In summary, this invention constructed a plant expression vector pCAMBIA-1305-ZjCHI1 containing a salt tolerance gene, of which ZjCHI1 is reported for the first time. The constructed vector can be introduced into plants to enhance their salt tolerance.

[0063] The Zoysia japonica 'Z011'ZjCHI1 gene (SEQ ID NO.1)

[0064]

Claims

1. A method for improving plant salt tolerance or cultivating new salt-tolerant plant germplasm, characterized in that, Overexpression of the Zoysia japonica salt tolerance gene with the nucleotide sequence shown in SEQ ID NO.1 in plants. ZjCHI1 To improve the salt tolerance of plants or to cultivate new salt-tolerant plant germplasm; the plants mentioned are Zoysia japonica or Arabidopsis thaliana.

2. The salt tolerance gene of Zoysia japonica with a nucleotide sequence as shown in SEQ ID NO.1 ZjCHI1 Or contains the salt tolerance gene of Zoysia japonica. ZjCHI1 The application of biomaterials in improving plant salt tolerance or cultivating new salt-tolerant plant germplasm is characterized by, The biological material is a recombinant vector, expression cassette, or recombinant bacteria; the plant is Zoysia japonica or Arabidopsis thaliana.

3. The application according to claim 2, characterized in that, The recombinant vector is a recombinant cloning vector or a recombinant expression vector.

4. The application according to claim 3, characterized in that, The recombinant expression vector is used to express the salt tolerance gene of Zoysia japonica. ZjCHI1 Salt tolerance gene of Zoysia japonica obtained by inserting plant expression vector ZjCHI1 Plant expression vectors.

5. The application according to claim 4, characterized in that, The recombinant expression vector is a vector containing homologous recombination arms. ZjCHI1 The salt tolerance gene of Zoysia japonica was obtained by homologous recombination of the gene fragment with the linearized pCAMBIA1305.1 expression vector plasmid. ZjCHI1 Plant expression vectors.

6. The application according to claim 5, characterized in that, The salt tolerance gene of Zoysia japonica ZjCHI1 The construction method of plant expression vectors includes the following steps: (1) Using Zoysia japonica cDNA as a template, primers ZjCHI1-F and ZjCHI1-R were designed for PCR reaction. The PCR product was ligated into the pMD19-T vector and transformed. DH5α Competent cells, positive plasmids extracted; Upstream primer ZjCHI1-F: 5′-ATGGCGAATTCACCATGGCCGACGAAC-3′ Downstream primer ZjCHI1-R: 5′-CTAGGCAGGTGAGGTTGCCCCCCGGGTCG-3′ (2) Using the extracted positive plasmid as a template, primers 1305-ZjCHI1-F and 1305-ZjCHI1-R were designed to construct the plant expression vector pCAMBIA1305.

1. PCR reactions were performed, and the PCR products were then mixed with the positive plasmids extracted from the positive plasmid. Bam The plant expression vector pCAMBIA1305.1 plasmid, linearized by HI single enzyme digestion, was subjected to homologous recombination reaction, transformed, and positive plasmid was extracted. The plant expression vector pCAMBIA1305.1-ZjCHI1 was successfully constructed. Upstream primer 1305-ZjCHI1-F: 5′- aggaccggtcccgggggatccATGGCGAATTCACCATGGC -3′ Downstream primer 1305-ZjCHI1-R: 5′- gcccttgctcaccatggatccGCAGGTGAGGTTGCCCCC -3′.

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