Salix psammophila SpsRLCK1 upstream regulatory factor SpsZFP6 encoding protein and application thereof

Abstract

The invention discloses a salix psammophila SpsRLCK1 upstream regulatory factor SpsZFP6 encoding protein and application thereof, through a yeast one-hybrid library screening experiment, the upstream regulatory factor SpsZFP6 of a SpsRLCK1 promoter is successfully identified, and under the drought stress condition, transgenic arabidopsis thaliana over-expressing the SpsZFP6 shows significantly enhanced stress resistance. Specifically, the root length, the fresh weight and the plant height of a transgenic plant under drought treatment are all superior to those of a wild plant, the cell membrane integrity is good, the antioxidant enzyme activity is remarkably improved, and ROS accumulation is reduced. The result shows that the SpsZFP6 can specifically improve the stress resistance of the plant under the drought condition, and important theoretical basis and practical basis are provided for forest molecular breeding. According to the invention, not only is a new transgenic variety with better drought tolerance facilitated to be cultivated, but also a new direction is provided for further researching a plant stress resistance mechanism.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a Salix psammophila SpsRLCK1 upstream regulatory factor SpsZFP6 and an application thereof. Background Art

[0002] Salix psammophila, a shrub of the Salicaceae family, is native to western China. It has a well-developed root system, rapid growth, and is resistant to drought, cold, and wind. It is a key tree species in China's "Three Northern Shelterbelts" and one of the largest tree species planted in sandy areas. It holds significant value for ecological and socioeconomic development in western my country. Research on the stress-resistance mechanisms of Salix psammophila is crucial for ecological restoration, land reclamation, and economic improvement in arid and semi-arid regions.

[0003] Zinc finger proteins are a family of transcription factors that have been widely studied. These proteins contain highly conserved structural domains and can be divided into ten categories based on the position and number of histidine (His) and cysteine (Cys) residues: C2H2, C2C2, C3H, C3HC4, C2HC5, C4HC3, C2HC, C4, C6, and C8. Among them, C2H2-type zinc finger proteins (C2H2-ZFPS) constitute one of the largest families of transcriptional regulatory factors in plants. Current functional studies of C2H2-type zinc finger proteins mainly include: (1) regulating plant growth and development, morphological construction, and biochemical metabolism, such as flower development, floral organogenesis, and leaf initiation; (2) participating in multiple signal transduction pathways, acting as positive or negative regulatory factors, and possibly interacting with plant hormones to effectively enhance plant resistance to adversity. C2H2-type zinc finger proteins have been discovered and identified in multiple species, including Arabidopsis, rice, and potato, demonstrating their potential to regulate plant growth and development and enhance stress tolerance. Arabidopsis plants overexpressing ZAT18 exhibit enhanced drought tolerance, while ZAT18 mutants are more sensitive to drought, suggesting that this gene is involved in the drought stress response. ZFP245 can enhance rice drought resistance by scavenging peroxidases and superoxide dismutases through its ability to scavenge ROS. Similarly, overexpression of the OsDRZ1 gene in rice improves drought tolerance in seedlings. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a new zinc finger protein separated from Salix psammophila and its application.

[0005] The technical solution of the present invention is: Salix psammophila SpsZFP6 protein, the amino acid sequence of which is shown in SEQ ID No.2.

[0006] The SpsZFP6 gene encoding the above-mentioned Salix psammophila SpsZFP6 protein.

[0007] Furthermore, the nucleotide sequence of the SpsZFP6 gene is shown in SEQ ID No. 1.

[0008] An expression vector containing the above-mentioned SpsZFP6 gene.

[0009] The use of the above-mentioned SpsZFP6 gene or expression vector in improving plant drought tolerance.

[0010] Furthermore, the drought tolerance of plants is improved by overexpressing the Salix psammophila SpsZFP6 gene in plants.

[0011] Furthermore, the plant is a Willow plant or Arabidopsis thaliana.

[0012] Furthermore, the Salix plant is Salix psammophila.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] The present invention conducted a yeast one-hybrid library screening experiment on the promoter SpsRLCK1 and successfully identified its upstream regulatory factor SpsZFP6. By observing and analyzing the growth status and physiological indicators of SpsZFP6 transgenic and wild-type Arabidopsis under drought stress, it was found that the transgenic Arabidopsis under drought stress had a stronger growth status than the wild-type Arabidopsis. At the same time, physiological indicators also confirmed that Arabidopsis overexpressing SpsZFP6 had significantly better drought resistance than wild-type Arabidopsis. This discovery illustrates that overexpressing SpsZFP6 can specifically enhance a plant's drought resistance. Therefore, the isolation and identification of the C2H2-type zinc finger protein transcription factor SpsZFP6 from Salix psammophila and the analysis of its stress resistance function provide important theoretical and practical significance for the development of new drought-resistant transgenic varieties in the field of forest molecular breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The results of the yeast one-hybrid library screening of the SpsRLCK1 upstream protein provided in Example 1 of the present invention are as follows;

[0016] Figure 2 The results of the luciferase assay for detecting the interaction between the SpsRLCK1 promoter and the upstream regulatory factor SpsZFP6 provided in Example 1 of the present invention, A is the luciferase imaging assay of tobacco leaves, and B is the luciferase activity (LUC) analysis;

[0017] Figure 3 The expression level of the Salix psammophila SpsZFP6 gene under drought stress was analyzed by real-time fluorescence quantitative PCR provided in Example 3 of the present invention;

[0018] Figure 4 This is a schematic diagram of the quantitative data of transgenic Arabidopsis expression provided in Example 5 of the present invention;

[0019] Figure 5 Schematic diagram of fresh weight and root length of transgenic Arabidopsis thaliana and wild-type Arabidopsis thaliana after drought treatment provided in Example 6 of the present invention, A shows the growth of WT and SpsZFP6 transgenic Arabidopsis seedlings in 1 / 2 MS medium, B shows the growth of WT and SpsZFP6 transgenic Arabidopsis seedlings in 1 / 2 MS medium containing 150 mM mannitol, C shows the effect of drought stress on root length of WT and SpsZFP6 transgenic Arabidopsis thaliana, and D shows the effect of drought stress on fresh weight of WT and SpsZFP6 transgenic Arabidopsis thaliana;

[0020] Figure 6 Schematic diagram of the phenotypes of transgenic Arabidopsis thaliana and wild-type Arabidopsis thaliana in soil provided in Example 6 of the present invention;

[0021] Figure 7 Schematic diagram of the relative electrical conductivity of transgenic Arabidopsis thaliana and wild-type Arabidopsis thaliana after drought treatment provided in Example 6 of the present invention;

[0022] Figure 8 A schematic diagram of the relative water content of transgenic Arabidopsis thaliana and wild-type Arabidopsis thaliana after drought treatment provided in Example 6 of the present invention;

[0023] Figure 9 Schematic diagram of NBT and DAB staining of leaves of transgenic Arabidopsis thaliana and wild-type Arabidopsis thaliana after drought treatment provided in Example 6 of the present invention, A is NBT staining, and B is DAB staining;

[0024] Figure 10 A schematic diagram of the MDA content in leaves of transgenic Arabidopsis thaliana and wild-type Arabidopsis thaliana after drought treatment provided in Example 6 of the invention;

[0025] Figure 11 Schematic diagram of POD content in leaves of transgenic Arabidopsis thaliana and wild-type Arabidopsis thaliana after drought treatment, provided in Example 6 of the invention;

[0026] Figure 12 A schematic diagram of CAT content in leaves of transgenic Arabidopsis thaliana and wild-type Arabidopsis thaliana after drought treatment provided in Example 6 of the invention;

[0027] Figure 13 Schematic diagram of the SOD content in leaves of transgenic Arabidopsis thaliana and wild-type Arabidopsis thaliana after drought treatment provided in Example 6 of the invention;

[0028] Figure 14 Schematic diagram of the AsA content in leaves of transgenic Arabidopsis thaliana and wild-type Arabidopsis thaliana after drought treatment provided in Example 6 of the invention. DETAILED DESCRIPTION

[0029] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from commercial channels unless otherwise specified.

[0030] Example 1 Screening and Verification of the Upstream Regulatory Gene SpsZFP6 of Salix psammophila SpsRLCK1

[0031] 1.1 Screening of upstream regulatory genes of Salix psammophila SpsRLCK1 by yeast one-hybridization

[0032] The SpsRLCK1 promoter was amplified by PCR and incorporated into the phis2 vector between the EcoRI and SacI restriction sites via homologous recombination. Y187 yeast cells were co-transformed with a bait vector containing the SpsRLCK1 promoter attached to the pHis2 reporter gene and a prey vector containing a Salix psammophila cDNA library fused to the GAL4 activation domain. Monoclonal colonies were grown in DDO (DO / -Leu / -Trp) and TDO (DO / -His / -Leu / -Trp) medium, respectively. pHis2-p53 and pGADT7-Rec2-p53 were used as positive controls, while pHis2-p53 and pGADT7-Rec2-SpsZFP6 were used as negative controls. Figure 1 The results showed that pHis2-SpsRLCK1 pro The +pGADT7-Rec2-SpsZFP6 cells could grow in both DDO and 60 mM 3AT TDO medium, while the negative control cells could only be cultured in DDO, indicating that SpsZFP6 interacts with the SpsRLCK1 promoter.

[0033] 1.2 Dual luciferase assay

[0034] The SpsZFP6 sequence was cloned into the pCAMBIA1302 vector using homologous recombination. The SpsRLCK1 promoter was inserted into the pGreenII0800-LUC vector to generate a reporter vector. The constructed effector vector containing the SpsZFP6 sequence and the reporter vector containing the promoter sequence, as well as the empty vectors pCAMBIA1302 and pGreenII0800-LUC, were transformed into Agrobacterium GV3101 competent cells using the heat shock method. The cells were then plated onto LB solid medium containing three antibiotics (kanamycin 0.05 g / L, rifampicin 0.34 g / L, and gentamicin 0.01 g / L) and cultured in a 28°C constant temperature incubator until plaques formed. After verification, positive bacterial cultures were stored for future use. The stored positive colonies were inoculated into LB liquid medium containing antibiotics and cultured in a shaking incubator at 28°C for 6 hours. Then, 20 mL of LB culture medium containing the same antibiotics was added to a 50 mL centrifuge tube, and 200 μl of the microbial suspension was added. After 6 hours, the cells were centrifuged at 6000 rpm for 10 minutes to collect the cells. The cells were resuspended twice with 1 / 2 MS liquid medium and then diluted with infiltration buffer to an OD of 0. 600 =1.0. The diluted bacterial solution was mixed at a ratio of 1:1 and then incubated in the dark for 2 hours. The bacterial solution composition was: I: 35S::SpsZFP6+SpsRLCK1 pro -Luc, Ⅱ: 35S::SpsZFP6+pGreen II 0800-Luc, Ⅲ: pCAMBIA1302+SpsRLCK1 pro -Luc, IV: pCAMBIA1302+pGreen II 0800-Luc. Select tobacco leaves with good growth status and inject the mixed bacterial solution into different parts of the same tobacco leaf from the back of the tobacco using a 1ml syringe. Two days after infection, use a small spray bottle to evenly spray 1mm D-luciferin potassium salt on the back of the tobacco leaves and then treat them at 37℃ in the dark for 7 minutes. After treatment, the fluorescence signal intensity of the tobacco leaves was observed by scanning and imaging using PlantView 100 (BLT, China). The results are shown in the figure. Figure 2 , experimental group 35S::SpsZFP6+SpsRLCK1 pro The -Luc fluorescence signal intensity was stronger and significantly higher than that of the control group, indicating that SpsZFP6 bound to the SpsRLCK1 promoter, thereby stimulating the expression of SpsRLCK1.

[0035] Example 2 Cloning of Salix psammophila SpsZFP6 gene

[0036] 2.1 Obtaining the target gene sequence

[0037] Based on the sequence numbers in the Phytozome database for Populus trichocarpa, cDNA sequences homologous to Salix psammophila SpsZFP6 were screened. Primers were then designed using Primer5 software based on the screened sequences. Finally, the complete gene sequence was amplified using PCR. The primers are listed in Table 1.

[0038] Table 1 Cloning primers

[0039]

[0040] Gene cloning was performed using a high-fidelity PCR reaction system using total RNA extracted from Salix psammophila leaves and reverse-transcribed cDNA as a template. The specific reaction system (20 μl) was as follows: PrimeSTAR, 10 μl; Clone F, 1 μl; Clone R, 1 μl; Salix psammophila cDNA, 2 μl; ddH2O, 6 μl.

[0041] The PCR cloning reaction program was as follows: 95°C for 5 minutes, 95°C for 10 seconds, 57°C for 30 seconds, and 72°C for 50 seconds, 40 cycles, and 72°C for 2 minutes. DNA fragments were recovered using a DNA product purification kit (Novozymes, Nanjing).

[0042] 2.2 Target gene connected to T vector

[0043] The recovered target fragment was ligated to a T-vector. The reaction system consisted of: 1 μl of T-vector; 30 ng of the aforementioned DNA product; and 5 μl of Solution I. The total volume was adjusted to 10 μl with ddH₂O, and the ligation was incubated overnight at 16°C. After the ligation reaction, the product was transformed into E. coli, and individual colonies were selected for PCR verification. Positive colonies were confirmed by sequencing. Sequencing confirmed the nucleotide sequence shown in SEQ ID No. 1. The gene fragment was named SpsZFP6, consisting of 753 base pairs (bp) encoding 250 amino acids (SEQ ID No. 2).

[0044] Example 3 Analysis of the expression characteristics of the Salix psammophila SpsZFP6 gene

[0045] 3.1 Primer design

[0046] Based on the obtained complete gene sequence, quantitative primers were designed using Primer3 software and quantitative fluorescence PCR was performed. The primers are listed in Table 2.

[0047] Table 2 Quantitative primers

[0048]

[0049] 3.2 Drought stress treatment of Salix psammophila tissue culture seedlings

[0050] Normally grown Salix psammophila tissue culture seedlings were transferred to a medium containing 100 mM Mannitol. Total RNA was extracted from the roots and reverse-transcribed into cDNA. qRT-PCR reactions were then performed using the following reaction system (20 μl): SYBR qPCR Master Mix, 10 μl; Quantitation F, 0.4 μl; Quantitation R, 0.4 μl; Salix psammophila cDNA, 2 μl; and ddH2O, 7.2 μl.

[0051] The PCR reaction program was set as: 95°C for 30 seconds, 95°C for 10 seconds, and 60°C for 30 seconds, for 39 cycles, and the instrument's default melting curve acquisition program was used.

[0052] The obtained data were analyzed, such as Figure 3 The results showed that under drought stress, the expression level of SpsZFP6 gene in Salix psammophila increased, so it was speculated that this gene might be induced by drought stress.

[0053] Example 4 Construction of Salix psammophila SpsZFP6 gene expression vector

[0054] 4.1 Primer design

[0055] Plant overexpression vectors were constructed and primers with homology arms to pCAMBIA1302 were designed. The primers are shown in Table 3.

[0056] Table 3 Overexpression primers

[0057]

[0058] 4.2 Construction of pCAMBIA1302-SpsZFP6 overexpression vector

[0059] The SpsZFP6 gene was amplified using primers Overexpression F and Overexpression R. The product was recovered, and the pCAMBIA1302 vector was double-digested and linearized. The target gene was then constructed into the vector using Norwegian's native recombinase. The reaction system (10 μl) was as follows: target gene, 15 ng; vector, 105 ng; 5× CE II Buffer, 2 μl; Exnase II, 1 μl; and finally, ddH2O was added to bring the volume to 10 μl.

[0060] The reaction procedure was: 37°C for 30 min and then cooled to 4°C or immediately placed on ice for cooling.

[0061] The ligated vector was transformed into E. coli, and single clones were selected for PCR verification and positive colony sequencing. The pCAMBIA1302-SpsZFP6 overexpression vector was successfully constructed.

[0062] Example 5 Genetic transformation of Arabidopsis thaliana SpsZFP6 gene

[0063] The constructed pCAMBIA1302-SpsZFP6 overexpression vector was transformed into Agrobacterium tumefaciens GV3101 via heat shock. The SpsZFP6 gene was then transferred into Arabidopsis thaliana via Agrobacterium-mediated transformation. The transformation steps were as follows: One-month-old robust Arabidopsis plants, 4-6 cm tall, with numerous partially opened flower buds and very few fruit pods, were selected. The culture temperature was 23-25°C under a 16 / 8 hour light cycle (day / night). The flower buds were inoculated with a bacterial solution containing the pCAMBIA1302-SpsZFP6 expression vector for 45-60 seconds. Water was then applied using a watering can. After incubation under shade for 12 hours, the plants were incubated under normal conditions. Seven days later, a second infection was performed using the same method. When the transformed pods matured, the seeds were collected and evenly plated on solid 1 / 2 MS medium containing antibiotics to screen for positive strains. Approximately 10 days later, seedlings that grew normally on the selective medium were considered positive strains. After the seedlings grew two true leaves, they were transplanted into the soil for cultivation. cDNA was extracted from the leaves of the rooted plants using the CTAB method and verified by PCR. RNA was extracted from the 20 positive seedlings screened and the transgenic expression was verified by qRT-PCR. The results are as follows Figure 4 As shown, OE-1 and OE-2 plants with appropriate expression levels were selected for subsequent phenotypic identification and analysis of drought resistance.

[0064] Example 6 Analysis of Drought Resistance of Arabidopsis SpsZFP6 Transgenic Plants

[0065] 6.1 Root Length and Fresh Weight of Transgenic Arabidopsis

[0066] The wild-type Arabidopsis WT and transgenic Arabidopsis thaliana strains OE-1 and OE-2 were cultured on normal 1 / 2MS medium for 10 days and then transferred to 1 / 2MS medium without or with 150 mM mannitol (mannitol simulates drought). Figure 5 The results showed that under drought stress, the root length of transgenic SpsZFP6 Arabidopsis was slightly longer than that of wild-type Arabidopsis seedlings, the number of lateral roots was significantly increased, and the fresh weight of transgenic SpsZFP6 Arabidopsis was heavier than that of wild-type Arabidopsis. The root length and fresh weight of transgenic Arabidopsis were significantly affected compared with wild-type Arabidopsis, indicating that overexpression of SpsZFP6 enhanced the drought resistance of Arabidopsis, giving it a growth advantage over wild-type plants.

[0067] 6.2 Phenotypic Analysis of Transgenic Arabidopsis

[0068] The wild-type Arabidopsis thaliana WT and two homozygous transgenic lines OE-1 and OE-2 of the T3 generation were cultured in normal 1 / 2MS medium for 10 days and then transplanted to peat soil: vermiculite = 3:1 soil for growth. After one month of growth, plants with basically the same growth conditions were selected. Figure 6 The Arabidopsis seedlings were grown under normal watering for 13 days. After 13 days, the phenotypes of the Arabidopsis seedlings were observed, revealing no significant differences between the WT and OE-1 and OE-2 lines. Irrigation was then stopped, and the Arabidopsis seedlings were subjected to drought treatment. After 10 days of drought, the phenotypes of the Arabidopsis seedlings were observed, revealing that the leaves of the wild-type Arabidopsis WT plants exhibited more severe wilting and yellowing than the transgenic Arabidopsis. After 10 days of drought, watering was resumed, and the phenotypes of the Arabidopsis plants were further observed three days later. Growth had resumed in the transgenic Arabidopsis OE-1 and OE-2 plants, with reduced yellowing, while the WT Arabidopsis plants showed no obvious signs of growth. This indicates that the SpsZFP6-transgenic Arabidopsis plants possess greater drought resistance than the wild-type Arabidopsis.

[0069] 6.3 Determination of relative electrical conductivity of transgenic Arabidopsis

[0070] Take the WT and SpsZFP6 transgenic Arabidopsis leaves under normal and drought treatment in 6.2, wash away the dust with clean water, rinse them with double distilled water, and use filter paper to absorb the surface moisture. Then put the Arabidopsis leaves with different treatments into triangular flasks respectively, and add 50ml of ultrapure water to the triangular flasks and vacuum them for 20 minutes. Then use a conductivity meter to measure the conductivity value after vacuuming as R1. Then put the triangular flask into a water bath and boil it. After cooling, the conductivity value is measured as R2. The calculation formula of relative conductivity can be obtained as follows: relative conductivity (%) = R1 / R2×100%. The results are as follows Figure 7 As shown in the figure: Under normal treatment, there is no difference in the relative conductivity between WT Arabidopsis 0 and the transgenic SpsZFP6 gene strain. Under drought stress, the relative conductivity of the strain overexpressing the SpsZFP6 gene is much smaller than that of the WT, and there are significant differences with the WT strain, indicating that the transgenic SpsZFP6 Arabidopsis is more drought-resistant than the WT Arabidopsis, and the SpsZFP6 gene plays a very important role in drought resistance in Arabidopsis plants.

[0071] 6.4 Determination of relative water content in transgenic Arabidopsis

[0072] The relative water content of Arabidopsis leaves was determined by the drying and weighing method. First, the leaves of WT and SpsZFP6 transgenic Arabidopsis after normal and drought treatment in 6.2 were washed with running water and dried with filter paper, and weighed on a balance, recorded as FW. Then the leaves were placed in a self-sealing bag filled with tap water, and the leaves were soaked for 6 hours to reach saturation, and the weight was weighed. Recorded as TW. The weighed leaves were then placed in an electric blast drying oven at 80°C for drying until the leaves were dried to a constant weight, and the weight of the leaves at this time was weighed on a balance, recorded as DW. At this time, the calculation formula for the relative water content of Arabidopsis leaves can be obtained as follows: Relative water content of leaves (%) = (FW-DW) / (TW-DW)×100%. The results show that Figure 8Under normal conditions, the relative water content of wild-type Arabidopsis and transgenic Arabidopsis is not much different and remains at a high level. However, under drought conditions, the relative water content of the plant leaves decreases, and the relative water content of the wild-type Arabidopsis is even lower, indicating that the SpsZFP6 gene can enable Arabidopsis to retain more water, thereby making the transgenic Arabidopsis more drought-resistant.

[0073] 6.5 NBT and DAB analysis of transgenic Arabidopsis

[0074] First, prepare 3,3′-diaminobenzidine (DAB) staining solution. Use a balance to weigh 0.788g of Tris-HCl, add ultrapure water to dissolve, and dilute to 100mL. Adjust the pH to 5.5, then add 0.1g of DAB powder and shake well to a concentration of 1mg / mL. Next, prepare nitroblue tetrazolium (NBT) staining solution. Use a balance to weigh 0.5g of NBT, add 22.87mL of phosphate buffer A and 21.25mL of buffer B to a total volume of 100mL, and prepare a destaining solution. Mix acetic acid, glycerol, and anhydrous ethanol in a ratio of 1:1:3.

[0075] Prepare a 10mL centrifuge tube, add the staining solution and the leaves to be tested, so that the staining solution can completely submerge the leaves, vacuum the test tube for 30 minutes to allow the leaves to sink in the staining solution, then wrap the test tube with tin foil, place the test tube in a 37℃ incubator, and stain under dark conditions for 8 hours. After observing that the staining is successful, pour out the staining solution, add decolorizing solution, and decolorize in a water bath at 95℃. After the decolorization is successful, cool the leaves to room temperature, clean them with anhydrous ethanol, and gently absorb excess water on the surface of the leaves with filter paper to observe the color development of active oxygen on the leaves. The results are as follows Figure 9 As shown in the figure, under normal treatment, there was no significant difference between wild-type Arabidopsis and transgenic Arabidopsis. However, under drought stress, DAB and NBT showed a darker blue coloring effect on the leaves of wild-type Arabidopsis, while the coloring effect of SpsZFP6 transgenic plants was lighter. The results showed that overexpression of SpsZFP6 can reduce the intracellular hydrogen peroxide H2O2 and superoxide anion O2 - content, thereby improving the drought resistance of Arabidopsis.

[0076] 6.6 Malondialdehyde content analysis

[0077] The content of malondialdehyde (MDA) was detected using the thiobarbituric acid method. Samples from different groups were taken and ground into a homogenate with 1.5 mL of 10% TCA in an ice bath. Centrifuged at 10,000 rpm / min for 15 minutes, 1 mL of supernatant was taken and added with 1 mL of 0.6% TBA, mixed and boiled in a boiling water bath for 15 minutes, immediately placed on ice to cool for 10 minutes and then centrifuged at 12,000 rpm / min for 20 minutes, 2 mL of supernatant was taken and its absorbance was measured at 450 nm, 532 nm and 600 nm. The control tube contained 1 mL of 0.6% TBA and 1 mL of water. The content of malondialdehyde in the sample can be calculated according to the formula: MDA (μmol / g) = [(6.45×OD 532 -0.56×OD 450 )×v] / w. The result is as follows Figure 10 As shown in the data, there was no significant difference in MDA content between wild-type Arabidopsis and transgenic Arabidopsis under normal growth conditions. However, after drought treatment, the MDA content of both wild-type and transgenic Arabidopsis increased, but the MDA content of transgenic Arabidopsis was lower than that of wild-type Arabidopsis, and the difference between the two was very significant, indicating that the SpsZFP6 gene has a very important drought resistance function in Arabidopsis plants.

[0078] 6.7 Antioxidant Enzyme Activity Analysis

[0079] Superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) were detected using a Solebol kit under a UV spectrophotometer. Figure 11 、 12 As shown in Figures 13 and 14, under normal conditions, there were no significant differences in POD, SOD, and CAT activity between SpsZFP6 transgenic plants and wild-type plants. However, after drought stress, the POD, SOD, and CAT activities of transgenic plants were significantly higher than those of wild-type plants, indicating that the drought resistance of SpsZFP6 transgenic Arabidopsis was significantly stronger than that of wild-type Arabidopsis.

[0080] 6.8 Ascorbic acid content analysis

[0081] The ascorbic acid (AsA) content was determined using the 2,6-dichloroindophenol (DCIP) method. The slurry was ground with 2 mL of 10% TCA and centrifuged at 10,000 rpm / min for 15 minutes at 4°C. 200 μl of the supernatant was added sequentially with 150 mmol / L NaH2PO4 solution, 200 μl of distilled water, 10% TCA solution, 44% H3PO4 solution, 4% C10 400 μl of H8N2 solution and 200 μl of 3% FeCl3 solution were added. The mixture was placed at 37°C for 1 hour. The absorbance at 525 nm was measured using a UV spectrophotometer. Each process was repeated 3 times and the results were averaged. Figure 14 As shown in the results, after drought stress, the AsA content of the plants increased, but the AsA content of the transgenic Arabidopsis was significantly higher than that of the wild-type Arabidopsis, which indicates that the SpsZFP6 gene can enhance the drought resistance of the plant.

[0082] In summary, plants transgenic for the SpsZFP6 gene can participate in drought stress by maintaining a stable state of reactive oxygen species (ROS), thereby improving the drought tolerance of plants.

Claims

1. Salix psammophila SpsZFP6 protein, the amino acid sequence of which is shown in SEQ ID No.

2.

2. The SpsZFP6 gene encoding the Salix psammophila SpsZFP6 protein according to claim 1.

3. The SpsZFP6 gene according to claim 2, characterized in that The nucleotide sequence of the SpsZFP6 gene is shown in SEQ ID No.

1.

4. An expression vector containing the SpsZFP6 gene according to claim 2 or 3.

5. Use of the SpsZFP6 gene according to claim 2 or 3 or the expression vector according to claim 4 in improving plant drought tolerance.

6. The use according to claim 5, characterized in that By overexpressing the Salix psammophila SpsZFP6 gene in plants, the drought tolerance of plants is improved.

7. The use according to claim 5 or 6, characterized in that The plant is a Willow plant or Arabidopsis thaliana.

8. The use according to claim 7, characterized in that The Salix plant is Salix psammophila.

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