Salt-tolerant gene ZjCNGC15 and application thereof in improving plant salt tolerance

Abstract

This invention relates to the field of genetic engineering technology, and in particular to a salt tolerance gene. ZjCNGC15 And its application in improving plant salt tolerance. This invention provides a salt tolerance gene. ZjCNGC15 The salt tolerance gene ZjCNGC15 The amino acid sequence of the encoded protein is shown in SEQ ID NO.1. This invention provides... ZjCNGC15 The gene is a salt tolerance gene, which is overexpressed in plants. ZjCNGC15 Genes can enhance a plant's salt tolerance and photosynthetic pigment content (chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids) through overexpression in plants, particularly Zoysia japonica or Arabidopsis thaliana. ZjCNGC15 It is possible to obtain highly salt-tolerant plants.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to a salt tolerance gene. ZjCNGC15 And its application in improving plant salt tolerance. Background Technology

[0002] plant CNGC There have been reports on the relationship between genes and plant salt tolerance, but these studies have mainly focused on sweet soil plants, while studies on halophytes have been less extensive. CNGC Research on this topic is still relatively scarce. Currently, plants... CNGC Genes exhibit varying degrees of salt tolerance, such as those expressed heterologously in yeast. AtCNGC3 The gene increases yeast salt sensitivity and exacerbates sodium ion accumulation. Knockout AtCNGC10 Genes can enhance salt tolerance in Arabidopsis thaliana, while overexpression of these genes can improve salt tolerance. AtCNGC10 Genes negatively regulate salt tolerance in Arabidopsis thaliana. AtCNGC5 and AtCNGC17 Genes can confer salt tolerance in Arabidopsis thaliana. A literature review revealed limited research on the salt tolerance function of the CNGC gene, primarily focusing on the RNA level. Reported... CNGC Genes, mainly concentrated in negative regulation CNGC Salt tolerance, such as AtCNGC10 And so on. CNGC There are few reports on the positive regulation of plant salt tolerance.

[0003] Sodium-potassium ion balance is an important strategy for salt tolerance in plants, and this process is closely related to cyclic nucleotide-gated channels (CNGCs) that mediate the transport of cations such as sodium, potassium, and calcium ions. Previous studies have shown that Arabidopsis thaliana has six genes (including...) CNGC3, CNGC5, CNGC10, CNGC17, CNGC19 and CNGC20 It is associated with salt stress. CNGC3 Expression in yeast may act on Na + and K + The absorption mechanism, knockout CNGC3 Seed germination rate is affected by NaCl. Knockout CNGC10 It will affect the germination and seedling growth of Arabidopsis thaliana, and knocking out CNGC10 The mutant body will reduce Na + and K + Accumulation in Arabidopsis thaliana. According to literature reports, CNGC5 and CNGC17 may also be associated with salt tolerance. CNGC19 and CNGC20 may be involved in various cellular responses to salt stress in shoots and may be related to sodium redistribution in Arabidopsis thaliana. Therefore, there are significant differences among CNGC family members in terms of the sites of function and the specificity of stress responses. Understanding the differences in salt tolerance among CNGC family members is crucial for identifying genes involved in salt stress responses. Summary of the Invention

[0004] To address the above problems, this invention provides a salt-tolerant gene. ZjCNGC15 And its application in improving plant salt tolerance. The present invention provides... ZjCNGC15 The gene is a salt tolerance gene, which is overexpressed in plants. ZjCNGC15 Genes can improve a plant's salt tolerance.

[0005] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a salt tolerance gene ZjCNGC15 The salt tolerance gene ZjCNGC15 The amino acid sequence of the encoded protein is shown in SEQ ID NO.1.

[0006] Preferably, the salt tolerance gene ZjCNGC15 The nucleotide sequence is shown in SEQ ID NO.2.

[0007] This invention provides the salt tolerance gene described in the above technical solution. ZjCNGC15 Or improve the salt tolerance gene described in the above technical solution. ZjCNGC15 Application of biological materials expressing certain levels in one or more of 1)-4): 1) Improve plant salt tolerance; 2) Increase plant photosynthetic pigment content; 3) Improve yeast cell salt tolerance; 4) Improve yeast cell potassium absorption capacity.

[0008] Preferably, the plant includes Arabidopsis thaliana or Zoysia japonica.

[0009] Preferably, the photosynthetic pigments include one or more of chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids.

[0010] Preferably, the biomaterial includes the salt tolerance gene described in the above-described technical solution. ZjCNGC15 Recombinant carriers.

[0011] This invention provides a recombinant vector to improve the salt tolerance of plant or yeast cells, comprising a backbone vector and the salt tolerance gene described in the above-mentioned technical solution inserted into the backbone vector. ZjCNGC15 .

[0012] Preferably, the skeleton carrier includes: pYES2 carrier or PCAMBIA1305 carrier.

[0013] This invention provides a method for improving the salt tolerance of plants, comprising: applying the salt tolerance gene described in the above-mentioned technical solution... ZjCNGC15 Alternatively, the recombinant vector described in the above technical solution may be introduced into plants; the plants include Zoysia japonica or Arabidopsis thaliana.

[0014] This invention provides a salt-tolerant plant material, wherein the salt-tolerant plant material is formed by exogenously introducing the salt-tolerant gene described in the above-mentioned technical solution. ZjCNGC15 The genetically modified plant material; the plant includes Zoysia japonica or Arabidopsis thaliana.

[0015] Beneficial effects: This invention provides a salt tolerance gene ZjCNGC15 The salt tolerance gene ZjCNGC15 The amino acid sequence of the encoded protein is shown in SEQ ID NO.1. This invention provides... ZjCNGC15 The gene is a salt tolerance gene, which is overexpressed in plants. ZjCNGC15 Genes can enhance a plant's salt tolerance and photosynthetic pigment content (chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids) through overexpression in plants, particularly Zoysia japonica or Arabidopsis thaliana. ZjCNGC15 It is possible to obtain highly salt-tolerant plants. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0017] Figure 1 Zoysia japonica ZjCNGC14, ZjCNGC15 Results of salt tolerance testing for genes; among them, 14 and 15 respectively contain... ZjCNGC14 and ZjCNGC15 Gene recombination vector, where p is an empty vector.

[0018] Figure 2 The results are from the growth curve determination in liquid culture medium.

[0019] Figure 3 These are the enzyme digestion identification results of transgenic Arabidopsis mutants.

[0020] Figure 4 The results of hygromycin screening for transgenic Arabidopsis mutants.

[0021] Figure 5 The results of a vertical plate experiment were used to identify the salt tolerance of transgenic Arabidopsis thaliana.

[0022] Figure 6 The results of root length measurement in a vertical plate experiment for salt tolerance identification of transgenic Arabidopsis thaliana.

[0023] Figure 7 The results show the chlorophyll content of transgenic Arabidopsis thaliana under salt stress.

[0024] Figure 8 The results show the salt tolerance assessment of transgenic Arabidopsis thaliana. Detailed Implementation

[0025] This invention provides a salt tolerance gene ZjCNGC15 The salt tolerance gene ZjCNGC15 The amino acid sequence of the encoded protein is shown in SEQ ID NO.1, as follows: .

[0026] As one implementation method, the salt tolerance gene ZjCNGC15 The nucleotide sequence is shown in SEQ ID NO.2, as follows:

[0027] This invention focuses on the ZjCNGC gene family of the halophyte *Zoysia japonica*, evaluating its salt tolerance by expressing it in the salt-sensitive yeast G19 and detecting its growth under salt stress. The results showed that *Zoysia japonica*... ZjCNGC15 The gene has salt tolerance, and Zoysia japonica... ZjCNGC14 The gene does not possess salt tolerance. Based on the results of yeast salt tolerance identification, salt-sensitive genes were selected. ZjCNGC14 and salt tolerance genes ZjCNGC15 Heterologous transformation experiments were conducted in Arabidopsis thaliana, and the results showed that... ZjCNGC14 It weakened the salt tolerance of Arabidopsis thaliana, while ZjCNGC15 It enhanced the salt tolerance of Arabidopsis thaliana, consistent with the above-mentioned results of the salt tolerance identification of yeast. ZjCNGC14 and ZjCNGC15 Genes can serve as an important genetic resource for the genetic improvement of salt tolerance in Zoysia japonica. This invention discovers a positive regulatory mechanism for salt tolerance. ZjCNGC15 Genes can be used for subsequent genetic modification or hybridization breeding to improve the salt tolerance of plants (such as Zoysia japonica). Knocking out salt-sensitive genes... ZjCNGC14 It is also expected to improve the salt tolerance of Zoysia japonica.

[0028] This invention selects salt-sensitive genes screened from yeast. ZjCNGC14 and salt tolerance genes ZjCNGC15 Heterologous transformation of Arabidopsis thaliana was performed. Salt tolerance assessment results for the transgenic Arabidopsis thaliana showed that, compared to the wild type, the salt tolerance of the transgenic Arabidopsis thaliana was significantly higher. ZjCNGC14 Genetically modified Arabidopsis thaliana is more salt-sensitive, while transgenic Arabidopsis thaliana... ZjCNGC15 The gene is more salt-tolerant. ZjCNGC14 The photosynthetic pigment content of gene-modified Arabidopsis thaliana was significantly lower than that of WT, while the content of transgenic Arabidopsis thaliana was significantly lower. ZjCNGC15 The photosynthetic pigment content in *Arabidopsis thaliana* was significantly increased. The results in *Arabidopsis thaliana* were consistent with the results of yeast salt tolerance identification, indicating that using a yeast system for initial screening of CNGC salt tolerance has significant reference value. ZjCNGC14 and ZjCNGC15 Genes can serve as an important genetic resource for the genetic improvement of salt tolerance in Zoysia japonica.

[0029] Based on the above advantages, the present invention provides the salt tolerance gene described in the above technical solution. ZjCNGC15 Or improve the salt tolerance gene described in the above technical solution. ZjCNGC15 Application of biological materials expressing certain levels in one or more of 1)-4): 1) Improve plant salt tolerance; 2) Increase plant photosynthetic pigment content; 3) Improve yeast cell salt tolerance; 4) Improve yeast cell potassium absorption capacity.

[0030] In one embodiment, the plant includes Arabidopsis thaliana or Zoysia japonica.

[0031] In one embodiment, the photosynthetic pigments include one or more of chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids.

[0032] In one embodiment, the biomaterial includes the salt tolerance gene described in the above-described technical solution. ZjCNGC15 Recombinant carriers.

[0033] Based on the above advantages, the present invention provides a recombinant vector for improving the salt tolerance of plant or yeast cells, comprising a backbone vector and the salt tolerance gene described in the above-mentioned technical solution inserted into the backbone vector. ZjCNGC15 .

[0034] In one embodiment, the skeleton carrier includes: pYES2 carrier or PCAMBIA1305 carrier.

[0035] Based on the above advantages, the present invention provides a method for improving the salt tolerance of plants, including the salt tolerance gene described in the above technical solution. ZjCNGC15 Alternatively, the recombinant vector described in the above technical solution may be introduced into plants; the plants include Zoysia japonica or Arabidopsis thaliana.

[0036] Based on the above advantages, the present invention provides a salt-tolerant plant material, wherein the salt-tolerant plant material is prepared by exogenously introducing the salt-tolerant gene described in the above-mentioned technical solution. ZjCNGC15 The transgenic plant material includes Zoysia japonica or Arabidopsis thaliana. In this invention, the salt-tolerant plant material is an exogenously introduced salt-tolerant gene. ZjCNGC15 Afterwards, salt tolerance genes ZjCNGC15 Non-stable genetic materials with significantly upregulated expression levels are different from new plant varieties. They lack uniformity and stability, meaning that there is phenotypic segregation in other phenotypic traits.

[0037] To further illustrate the present invention, the following description, in conjunction with embodiments and accompanying drawings, describes a salt-tolerant gene provided by the present invention. ZjCNGC15 The invention describes in detail its application in improving plant salt tolerance, but this should not be construed as limiting the scope of protection of the invention.

[0038] Preparation Example The plant materials, main reagents, and culture media used in the examples are as follows: YNB (Weidi, Shanghai), DOSupplement-His / -Ura (Weidi, Shanghai), glucose (Shoude, Nanjing), agar (Shoude, Nanjing), sucrose (Shoude, Nanjing), NaCl (Shoude, Nanjing), KCl (Shoude, Nanjing), Ap medium (Zhuangmeng, Beijing), MS medium (Shoude, Nanjing), NaCl (Shoude, Nanjing), antibiotics (Kanamycin, Rifampicin, etc.), 70% ethanol solution, 5% sodium hypochlorite solution, Silwet L-77 (surfactant, to promote infection); SD-Ura medium: 6.7 g / L YNB, 20 g / L glucose and 0.76 g / L Do-Ura, pH 5.8. Solid medium requires the addition of 20 g / L Agar and the corresponding concentration of NaCl.

[0039] Ap medium: 20 mL Ap medium and 80 mL water, pH 5.8. For solid medium, add 20 g / L Agar and the corresponding concentration of KCl.

[0040] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, pH 7.0. Solid medium requires the addition of 15 g / L agar.

[0041] 1 / 2MS medium: 2.215 g / L MS basal medium, 15 g / L sucrose, pH 5.8. Solid medium requires the addition of 8 g / L agar.

[0042] Infection medium: 1 / 2 MS, 5% sucrose and 0.02% Silwet L-77 (surfactant).

[0043] Zoysia japonica Z011 material was cultured in a greenhouse at 28℃, 70% relative humidity, and a photoperiod of 12 h / 12 h (day / night). Zoysia japonica leaf material was obtained from mature Zoysia japonica Z011 material grown hydroponically in the greenhouse. The Zoysia japonica Z011 material is preserved at the Institute of Botany, Chinese Academy of Sciences, Jiangsu Province, and is published in the literature [Li Shan, Chen Jingbo, Guo Hailin, Zong Junqin, Zhang Fang, Xiao Qing, Jiang Qiaofeng, Ding Wanwen, Liu Jianxiu. Salt tolerance evaluation of Zoysia japonica turfgrass germplasm resources. Acta Prataculturae Sinica, 2012, 21(4): 41-53.].

[0044] Arabidopsis thaliana columbia: wild type, published in the literature [Yu Chen, Lanlan Li, Junqin Zong, Jinbo Chen, Hailin Guo, Aigui Guo, Jianxiu Liu]. .Heterologous expression of the halophyte Zoysia matrella H + - pyrophosphatase geneimproved salt tolerance in Arabidopsis thaliana . Plant Physiol. Bioch. 2015,91: 49-55]; Transgenic Arabidopsis thaliana: ZjCNGC14-1 / 2 and ZjCNGC15-1 / 2. Grown in a temperature-controlled chamber at 20 ± 1 °C with 16 h of light (75 μmol·m⁻¹). -2 ·s -1 ) and 8 hours of dark cycle growth.

[0045] Nutrient solution formula: Ca(NO3)2·4H2O 2mM, KNO3 2mM, MgSO4·7H2O 1mM, K2H2PO4 1mM, EDTA-Fe 25μM, H3BO3 46μM, ZnSO4·7H2O 0.8μM, CuSO4·5H2O 0.3μM, MnCl2·4H2O 9μM and (NH4)2MoO4 0.1μM.

[0046] Example 1 I. Experimental Design 1. Gene cloning and vector construction RNA extraction: Mature leaf material of Zoysia japonica Z011 was immediately frozen in liquid nitrogen and ground into powder. RNA was extracted from Zoysia japonica using an RNA extraction kit (#RC201, vazyme, Nanjing, China).

[0047] cDNA synthesis: The extracted RNA was reverse transcribed into cDNA using a reverse transcription kit (#R211, vazyme, Nanjing, China).

[0048] ZjCNGC Gene cloning: Genes were synthesized using designed primers containing multiple cloning site arms, and the products were enriched by PCR. The primer sequences are as follows: ZjCNGC14-pYES2-F: 5'-CCCAAGCTTATGGACGACGCTATGGCGAGG-3' (SEQ ID NO. 3); ZjCNGC14-pYES2-R: 5'-CCGGAATTCTCTAGTTTATGCCGTCATCC-3' (SEQ ID NO. 4); ZjCNGC15-pYES2-F: 5'-CCGGAATTCATGTTTTGGGTCCAGGGCTCAG-3' (SEQ ID NO. 5); ZjCNGC15-pYES2-R: 5'-ATAAGAATGCGGCCGCTCAGTCAAAATGGTCTG-3' (SEQ ID NO. 6); Among them, the bolded sequences CCCAAGCTT, CCGGAATTCT (SEQ ID NO.15), CCGGAATTC and ATAAGAATGCGGCCGC (SEQ ID NO.16) are multiple cloning site arms.

[0049] The PCR amplification reaction program is as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 15 s, 65℃ for 15 s, 72℃ extension for 2 min, 30 cycles; 72℃ final extension for 5 min, and storage at 4℃.

[0050] Vector construction: The Zoysia japonica cloned above was used as a vector. ZjCNGC The gene sequences were inserted into the two restriction enzyme sites of the pYES2 vector via homologous recombination, resulting in two recombinant vectors. Sequence information was verified by sequencing (General Biology company, Chuzhou, China). The insertion positions of the different genes are as follows: ZjCNGC14 HindIII-EcoRI; ZjCNGC15 EcoRI- NotI.

[0051] 2. Yeast Transformation Experiment Yeast conversion is the process of converting the yeast into a yeast-containing compound constructed in step 1. ZjCNGCThe pYES2 recombinant vector and empty pYES2 vector were transformed into salt-sensitive yeast G19 and potassium-deficient yeast R5421 (Shanghai Weidi Biotechnology Co., Ltd.) using a yeast transformation kit (#sk2400, Coolaber, Beijing, China). Transformants were screened using SD-Ura (100mM KCl) liquid medium to obtain positive yeast single colonies. The salt-sensitive yeast G19 is disclosed in the literature [Chen Yu(#), Zong Junqin(#), Tan. Zhiqun, Li Lanlan, Hu Baoyun, Chen Chuanmin, Chen Jingbo, Liu Jianxiu(#). ). Systematic mining of salt-tolerantgenes in halophyte-Zoysia matrella through cDNA expression library screening. Plant Physiology and Biochemistry. 2015, 89: 44-52.].

[0052] The specific steps for yeast conversion are as follows: (1) Add 310 μL of premix to one yeast competent cell and repeatedly blow and aspirate to precipitate the yeast cells so that they are completely suspended in the premix.

[0053] (2) Incubate in a water bath at 30°C for 30 min, mixing once every 10 min.

[0054] (3) Heat the mixture in a water bath at 42°C for 15 minutes, mixing it every 5 minutes.

[0055] (4) Centrifuge at 12000 rpm for 15 s and discard the supernatant.

[0056] (5) Add 0.1-1 mL of sterile deionized water or 0.9% sodium chloride solution to resuspend the precipitate (resuspend with 200 μL of liquid, take 100 μL to spread), spread on screening medium plates, and incubate at 30℃ for 2-4 days.

[0057] 3. Arabidopsis thaliana genetic transformation inflorescence infection method 1) Material preparation: Agrobacterium tumefaciens EHA105 (Weidi Biotechnology, Nanjing), Arabidopsis thaliana seeds (Columbia ecotype).

[0058] 2) The vector construction method is similar to step 1, the main difference being that the inserted vector is the PCAMBIA1305 vector (General Biotechnology, Anhui). ZjCNGC The primer sequences for gene cloning are as follows: ZjCNGC14-1305-F: 5'-CATGCCATGGACGACGCTATGGCGAG-3' (SEQ ID NO. 7); ZjCNGC14-1305-R: 5'-GAAGATCTCTAGTTTATGCCGTCATCCCT-3' (SEQ ID NO. 8); ZjCNGC15-1305-F: 5'-GAAGATCTATGTTTGGGTCCAGGGCTCAG-3' (SEQ ID NO. 9); ZjCNGC15-1305-R: 5'-GAAGATCTCAGTCAAAATGGTCTGCTGA-3' (SEQ ID NO. 10).

[0059] The positions of different genes inserted into the 1305 vector are as follows: ZjCNGC14: NcoI-BglII; ZjCNGC15: BglII.

[0060] 3) Agrobacterium-mediated transformation: ① Take the competent Agrobacterium cells stored at -80℃ and leave them at room temperature or in your palm for a moment until they partially thaw. When they are in an ice-water mixture, insert them into ice.

[0061] ② Add 0.01-1 μg of plasmid DNA to every 100 μl of competent cells, gently tap the bottom of the tube to mix, and incubate on ice for 5 minutes, in liquid nitrogen for 5 minutes, in a 37°C water bath for 5 minutes, and in an ice bath for 5 minutes.

[0062] ③ Add 700 μl of antibiotic-free LB liquid medium and incubate at 28°C with shaking for 2-3 h.

[0063] ④ Centrifuge at 6000 rpm for one minute to collect the bacteria. Take about 100 μl of supernatant, gently pipette and resuspend the bacterial block, spread it on an LB agar plate containing the appropriate antibiotic, and incubate upside down in a 28℃ incubator for 2-3 days.

[0064] ⑤ Liquid incubation: Add 1 ml of LB containing antibiotics to a 12-15 ml round-bottomed aerated test tube, inoculate 1-2 single colonies from a fresh Agrobacterium plate, and incubate at 30 ℃ and 200 rpm for 24-48 h.

[0065] ⑥ Expanded culture: Select single clones and inoculate them into 5 mL of LB liquid medium (containing antibiotics), and culture at 28°C with shaking at 200 rpm until OD.600 ≈0.8.

[0066] 4) Agrobacterium infection in Arabidopsis thaliana: ① Cultivate to OD 600 Centrifuge the Agrobacterium tumefaciens culture at approximately 0.8 μL (5000 rpm, 5 min).

[0067] ② Discard the supernatant, resuspend the bacterial culture in infection medium (1 / 2 MS + 5% sucrose + 0.02% Silwet L-77), and adjust the OD. 600 Up to 0.8-1.0.

[0068] ③ Select vigorous Arabidopsis thaliana plants, completely immerse the inflorescences in the bacterial solution, and gently shake for 5-10 minutes.

[0069] ④ Remove the plant and cover it with plastic wrap to maintain humidity.

[0070] ⑤ Place the infected plants in the dark and high humidity conditions for 24 hours.

[0071] ⑥ After 24 hours, transfer to normal culture conditions (22℃, 16 hours light / 8 hours dark).

[0072] ⑦ Harvest seed (T1 generation).

[0073] The primers in Table 1 were used to determine whether Arabidopsis thaliana plants were successfully infected.

[0074] Table 1 Transgenic Arabidopsis thaliana ZjCNGCs Identification of primer sequences

[0075] 4. Salt tolerance identification test of salt-sensitive G19 yeast 1) Solid plate salt tolerance evaluation test: The obtained G19 yeast transformants were cultured in SD-Ura / -His liquid medium for 2 days (30 ℃). When OD 600 Once the OD value reaches approximately 0.8, the yeast is collected and its OD value is adjusted using sterile water. 600 =1, and then successively diluted 10, 100, and 1000 times to obtain diluted bacterial solutions of different concentrations. All yeast suspensions were spotted at 3 μL volumes onto solid culture media containing 0 mM, 50 mM, 100 mM, 150 mM, and 300 mM NaCl. The basic components of the solid culture medium were: AP-Ura / -His containing 2 mM KCl, pH 6.0. The spotted yeast was incubated upside down in a 30℃ incubator, and photographs were taken and observed daily.

[0076] 2) Liquid culture salt tolerance evaluation test: The same method as the solid plate test was used to obtain the OD.600 =1 G19 yeast suspensions transformed with each gene. Take 10 μL and add it to 5 ml of AP liquid medium containing 150 mM NaCl. Incubate at 30℃ and 200 rpm for 3 days, and measure the OD every 24 h. 600 Numerical values. Significance analysis was performed using a t-test using SPSS 26.0 software. p <0.05), the image was generated using Prism 9.5 software.

[0077] 5. Aseptic sowing of Arabidopsis thaliana seeds Arabidopsis seeds were treated with 5% sodium hypochlorite for 10 min, repeated once. They were then surface-sterilized with 70% ethanol for 30 s, rinsed three times with sterile water, and sown on 1 / 2 MS solid medium. The seeds were cultured in an incubator under the above conditions. T1 and T2 generation transgenic Arabidopsis lines were obtained by repeatedly harvesting and sowing transgenic Arabidopsis seeds on 1 / 2 MS medium (containing 25 mg / L hygromycin). Homozygous lines were verified using 1 / 2 MS medium (containing 25 mg / L hygromycin). A T2 generation homozygous line was obtained.

[0078] 6. Salt tolerance assessment of transgenic Arabidopsis thaliana 1) Vertical plate salt tolerance identification: Arabidopsis seedlings were obtained using the above method. Lines with similar growth were transferred to CK (1 / 2 MS solid medium) and 50 mM / 75 mM NaCl + 1 / 2 MS solid medium, with 5 seedlings per line. The culture dishes were placed vertically in the growth chamber (25 / 20 ℃, 14 / 10 h day / night cycle, 120 μmol·m³ / h). -2 ·s -1 After 7 days of salt stress culture, their phenotypes were observed, and root length and photosynthetic pigment content were measured.

[0079] 2) Root length measurement: Seeds of homozygous Arabidopsis thaliana lines were sown on 1 / 2 MS medium using the aseptic sowing method described above. When the seedlings reached the "two-leaf, two-heart stage" (approximately 5-7 days), five seedlings from each line were transferred to 1 / 2 MS medium and 50 mM / 75 mM NaCl + 1 / 2 MS medium for vertical salt stress treatment. After 7 days of salt stress culture, the root length was measured.

[0080] 3) Determination of photosynthetic pigment content: 0.01 g of Arabidopsis leaves treated with vertical plate salt for 7 days were placed in a 2 mL centrifuge tube, submerged in 95% ethanol, and placed in the dark overnight. The next day, the photosynthetic pigment content was measured at 665 nm using a spectrophotometer. 665 ), 649 nm (A 649 ) and 470 nm (A 470The content of each photosynthetic pigment was calculated by colorimetric analysis at a specific wavelength. The calculation formula is as follows: Chlorophyll a (mg / L) = 13.95 × A 665 - 6.88 × A 649 ; Chlorophyll b (mg / L) = 24.96 × A 649 - 7.32 × A 665 ; Total chlorophyll (mg / L) = chlorophyll a + chlorophyll b; Carotenoids (mg / L) = (1000 × A) 470 - 2.05 × chlorophyll a - 114.8 × chlorophyll b) / 245.

[0081] 4) Salt tolerance assessment of potted plants: The above Arabidopsis seedlings were transferred to flower pots for cultivation. During this period, they were watered with 1 / 2 MS nutrient solution. When they grew to about 40 days, the control group was watered with 1 / 2 MS and the salt treatment group was watered with 350 mM NaCl + 1 / 2 MS liquid solution, once every 3 days, for a total of 5 times.

[0082] II. Results and Analysis 1. Salt tolerance assessment of Zoysia japonica ZjCNGCs Will ZjCNGCs Salt tolerance was assessed by transferring the gene into G19 salt-sensitive yeast. High concentrations of sodium ions inhibited the growth of G19 yeast, similar to previous reports using empty vectors. Furthermore, the gene was transferred into Zoysia japonica... ZjCNGCs After gene selection, it was found that transgenic yeast could grow normally on culture media without NaCl. After one day of salt treatment, transgenic G19 yeast cultured in 0 mM and 50 mM NaCl began to grow. On the second day of culture, transgenic G19 yeast cultured in 150 mM NaCl began to grow, while transgenic G19 yeast cultured in 300 mM NaCl did not grow at all during five consecutive days of observation. This shows that different salt concentrations have different effects on transgenic G19 yeast. The higher the salt concentration, the greater the impact on yeast growth, and... ZjCNGC15 The gene's salt tolerance is superior ZjCNGC14 Gene( Figure 1 ).

[0083] To verify the above salt tolerance identification results, samples containing salt tolerance genes were selected. ZjCNGC15 Salt-sensitive gene ZjCNGC14 The recombinant vector and the negative control pYES2 empty vector ， The liquid growth curve of transgenic yeast was determined, and the results are shown in the figure. Figure 2 The results showed that on day 3, the salt-tolerant gene was converted... ZjCNGC15G19 yeast OD 600 The value was greater than the negative control pYES2, but the differences between them were not significant. The transsensitive salt gene... ZjCNGC14 G19 yeast OD 600 The value was smaller than the negative control pYES2. These results demonstrate that *Zoysia japonica*... ZjCNGC15 It has salt tolerance, and ZjCNGC14 It does not have salt tolerance.

[0084] 2. Overexpression ZjCNGC14 and ZjCNGC15 Salt tolerance identification of Arabidopsis thaliana Salt-tolerant genes selected from salt-sensitive yeast ZjCNGC15 Hemin Salt Gene ZjCNGC14 The seedlings were transferred to Arabidopsis thaliana for further salt tolerance assessment. The selected T0 generation seedlings were further cultured and seeded to obtain T1 generation seeds. The T1 generation seeds were then further screened, and gene identification was performed using PCR and gel electrophoresis. Figure 3 The T1 seedlings obtained from the identification were further cultured, and individual plants were harvested to obtain T2 generation seeds. The obtained T2 generation seeds were screened using hygromycin. Those T2 generation seeds that all germinated in 1 / 2 MS solid medium were identified as homozygous lines. Figure 4 The transgenic homozygous lines were used for subsequent experiments such as salt tolerance identification and determination of related indicators.

[0085] 3. Salt tolerance assessment of transgenic Arabidopsis vertical plates The identified transgenic Arabidopsis seeds were sown on 1 / 2 MS medium. When they reached the stage of two leaves and two buds, they were transferred to 1 / 2 MS medium (containing NaCl). Salt tolerance was assessed by comparing the effects of NaCl addition on Arabidopsis growth. The results are shown below. Figure 5 and Figure 6 The result was that the transfer ZjCNGC14 The genetically modified Arabidopsis thaliana exhibited weaker growth and reduced salt tolerance compared to the wild type. Root measurements after 7 days of treatment with 75 mM NaCl revealed that the salt treatment resulted in decreased growth and salt tolerance. ZjCNGC14 The root lengths of the *Arabidopsis thaliana* gene were 6.925 cm and 6.275 cm, respectively, significantly shorter than the WT root length of 8.575 cm. These results collectively indicate that... ZjCNGC14 After being transferred to Arabidopsis thaliana, the salt tolerance of Arabidopsis decreased, which is consistent with the results of yeast salt tolerance identification. The transfer... ZjCNGC15 The genetically modified Arabidopsis thaliana exhibits stronger salt tolerance than the wild type. Measurements of the root system of Arabidopsis thaliana treated with 50 mM NaCl revealed that after salt treatment, the modified species showed increased salt tolerance. ZjCNGC15The root lengths of the transformed Arabidopsis thaliana were 9.325 cm and 9.400 cm, respectively, significantly longer than the WT root length of 7.600 cm. These results collectively indicate that ZjCNGC15 enhances the salt tolerance of Arabidopsis thaliana after transformation, which is consistent with the results of yeast salt tolerance identification.

[0086] 4. Determination of photosynthetic pigment content in transgenic Arabidopsis thaliana Chlorophyll content was measured in salt-treated transgenic Arabidopsis thaliana, and it was found that... ZjCNGC14 The chlorophyll a content of the transgenic Arabidopsis thaliana was 3.998 mg / L and 3.743 mg / L, significantly lower than the 5.183 mg / L of the whole-wheat thaliana (WT); the chlorophyll b content was 1.321 mg / L and 1.242 mg / L, significantly lower than the 1.492 mg / L of the WT. Similarly, the total chlorophyll content was also lower than that of the WT. The carotenoid content was 1.068 mg / L and 0.977 mg / L, significantly lower than the 1.418 mg / L of the WT. The transgenic Arabidopsis thaliana... ZjCNGC15 The chlorophyll a, chlorophyll b, total chlorophyll content, and carotenoids in the gene-modified Arabidopsis thaliana were significantly higher than those in the WT group. Figure 7 ).

[0087] 5. Salt tolerance assessment of transgenic Arabidopsis thaliana in pot experiments Salt treatment was applied to transgenic Arabidopsis thaliana in small pots, and it was found that... ZjCNGC14 Compared to WT, the leaf condition and other phenotypes of the gene-modified Arabidopsis thaliana showed significant wilting and yellowing, exhibiting a salt-sensitive phenotype, consistent with the results of the vertical plate salt tolerance identification mentioned above. Meanwhile, the transgenic Arabidopsis thaliana... ZjCNGC15 It exhibits a salt-tolerant phenotype, which is consistent with the results of the salt tolerance assessment of the vertical plate. Figure 8 ).

[0088] In summary, ZjCNGC15 It is a salt-tolerant gene, compared to WT, it is a variant. ZjCNGC15 Genes in Arabidopsis thaliana are more salt-tolerant and have significantly increased levels of photosynthetic pigments (chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids), as observed by overexpression in Zoysia japonica. ZjCNGC15 It can produce salt-tolerant Zoysia japonica.

[0089] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A salt tolerance gene ZjCNGC15 Its characteristics are, The salt tolerance gene ZjCNGC15 The amino acid sequence of the encoded protein is shown in SEQ ID NO.

1.

2. The salt tolerance gene according to claim 1 ZjCNGC15 Its characteristics are, The salt tolerance gene ZjCNGC15 The nucleotide sequence is shown in SEQ ID NO.

2.

3. The salt tolerance gene according to claim 1 or 2 ZjCNGC15 Or improve the salt tolerance gene as described in claim 1 or 2 ZjCNGC15 Application of biological materials expressing certain levels in one or more of 1)-4): 1) Improve plant salt tolerance; 2) Increase plant photosynthetic pigment content; 3) Improve yeast cell salt tolerance; 4) Improve yeast cell potassium absorption capacity.

4. The application according to claim 3, characterized in that, The plants mentioned include Arabidopsis thaliana or Zoysia japonica.

5. The application according to claim 3, characterized in that, The photosynthetic pigments include one or more of chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids.

6. The application according to claim 3, characterized in that, The biomaterial includes the salt tolerance gene as described in claim 1 or 2. ZjCNGC15 Recombinant carriers.

7. A recombinant vector for improving the salt tolerance of plant or yeast cells, characterized in that, Includes a backbone vector and the salt tolerance gene of claim 1 or 2 inserted into the backbone vector. ZjCNGC15 .

8. The recombinant vector according to claim 7, characterized in that, The skeletal carrier includes either the pYES2 carrier or the PCAMBIA1305 carrier.

9. A method for improving the salt tolerance of plants, characterized in that, Including the salt tolerance gene as described in claim 1 or 2 ZjCNGC15 Or the recombinant vector of claim 7 or 8 is introduced into a plant; the plant includes Zoysia japonica or Arabidopsis thaliana.

10. A salt-tolerant plant material, characterized in that, The salt-tolerant plant material is prepared by exogenously introducing the salt-tolerant gene as described in claim 1 or 2. ZjCNGC15 The genetically modified plant material; the plant includes Zoysia japonica or Arabidopsis thaliana.

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