Spszfp6 encoding protein of sand willow spsrlck1 upstream regulatory factor and application thereof

Abstract

The application discloses a Salix psylvestris SpsRLCK1 upstream regulatory factor SpsZFP6 coding protein and application thereof, and through a yeast single-hybrid library screening experiment, an upstream regulatory factor SpsZFP6 of a SpsRLCK1 promoter is successfully identified. Under drought stress conditions, transgenic Arabidopsis thaliana overexpressing the SpsZFP6 shows significantly enhanced stress resistance. Specifically, the transgenic plants are superior to wild-type plants in root length, fresh weight and plant height under drought treatment, the cell membrane integrity is better, the antioxidant enzyme activity is significantly improved, and the ROS accumulation is reduced. It is shown that the SpsZFP6 can specifically improve the stress resistance of plants under drought conditions, and provides an important theoretical basis and practical foundation for forest molecular breeding. The application not only helps to cultivate a transgenic new variety with higher drought resistance, but also provides a new direction for further research on plant stress resistance mechanism.

Description

TECHNICAL FIELD

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

[0002] Salix psammophila is a shrub of Salicaceae Salix, which is originally from the west of China. It has developed root system, grows rapidly, and is drought-tolerant, cold-tolerant and wind-tolerant. It is one of the important tree species of the three major protective forest belts in China, and is one of the tree species with the largest artificial afforestation area in sandy land in China. It has great value for the ecological environment construction and social and economic benefits in the west region. Researching the mechanism of resistance-related of Salix psammophila has important significance for ecological restoration, land improvement and economic benefits in arid and semi-arid regions.

[0003] Zinc finger proteins as a class of transcription factor family studied widely, this kind of protein contains highly conserved domain, according to the position and number of histidine (His) and cysteine (Cys) residues can be divided into ten categories: C2H2, C2C2, C3H, C3HC4, C2HC5, C4HC3, C2HC, C4, C6 and C8. Among them, C2H2 type zinc finger protein (C2H2-ZFPS) constitutes one of the largest transcriptional regulatory factor families in plants. The function research of C2H2 type zinc finger protein mainly includes: (1) regulating plant growth and development, morphogenesis and biochemical metabolism, such as flower development, floral organogenesis, leaf initiation, etc.; (2) participating in various signal transduction pathways, which can be positive or negative regulatory factors, and may interact with plant hormones, effectively enhancing the resistance of plants to adversity. At present, C2H2 type zinc finger proteins have been found and identified in many species such as Arabidopsis, rice and potato, which proves that they can regulate plant growth and development and improve the resistance of plants to adversity. Overexpression of ZAT18 Arabidopsis has stronger tolerance to drought stress, while ZAT18 mutant is more sensitive to drought, indicating that the gene is involved in the response to drought stress. ZFP245 can improve the drought resistance of rice by increasing the ability to remove ROS, such as peroxidase and superoxide dismutase. Similarly, when the OsDRZ1 gene in rice is overexpressed, the drought resistance of seedlings is improved. SUMMARY

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

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

[0006] SpsZFP6 gene encoding the SpsZFP6 protein of Salix pschegiana described above.

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

[0008] Expression vector containing the SpsZFP6 gene described above.

[0009] The SpsZFP6 gene or expression vector described above in improving the drought tolerance of plants.

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

[0011] Further, the plant is Salix or Arabidopsis.

[0012] Further, the Salix plant is Salix pschegiana.

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

[0014] The present application carries out yeast one-hybrid library screening experiment on the promoter SpsRLCK1, and successfully obtains the upstream regulatory factor SpsZFP6 thereof. By observing and analyzing the growth state and physiological indexes of SpsZFP6 transgenic and wild type Arabidopsis under drought stress, it is found that the growth state of transgenic Arabidopsis under drought stress is stronger than that of wild type Arabidopsis. At the same time, the physiological index results also confirm that the drought resistance of SpsZFP6 overexpressing Arabidopsis is obviously better than that of wild type Arabidopsis. This finding illustrates that overexpression of SpsZFP6 can specifically enhance the drought resistance of plants. Therefore, the C2H2 type zinc finger protein transcription factor SpsZFP6 is isolated and identified from Salix pschegiana, and its stress resistance function is analyzed, which provides important theoretical and practical significance for cultivating new drought-resistant transgenic varieties in the field of molecular breeding of forest trees. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 The SpsRLCK1 upstream yeast one-hybrid library screening result provided for example 1 of the present application;

[0016] Figure 2 The luciferase experiment result for detecting the interaction between the SpsRLCK1 promoter and the upstream regulatory factor SpsZFP6 provided for example 1 of the present application, A is the luciferase imaging determination of tobacco leaves, and B is the luciferase activity (LUC) analysis;

[0017] Figure 3 The real-time fluorescent quantitative PCR analysis result of the expression level of the Salix pschegiana SpsZFP6 gene under drought stress provided for example 3 of the present application;

[0018] Figure 4 Quantitative data of transgenic Arabidopsis thaliana expression provided for example 5 of the present application;

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

[0020] Figure 6 Phenotype of transgenic Arabidopsis thaliana and wild type Arabidopsis thaliana in soil provided for example 6 of the present application;

[0021] Figure 7 Relative conductivity of transgenic Arabidopsis thaliana and wild type Arabidopsis thaliana after drought treatment provided for example 6 of the present application;

[0022] Figure 8 Relative water content of transgenic Arabidopsis thaliana and wild type Arabidopsis thaliana after drought treatment provided for example 6 of the present application;

[0023] Figure 9 Leaf NBT and DAB staining of transgenic Arabidopsis thaliana and wild type Arabidopsis thaliana after drought treatment provided for example 6 of the present application, A is NBT staining, B is DAB staining;

[0024] Figure 10 MDA content of transgenic Arabidopsis thaliana and wild type Arabidopsis thaliana after drought treatment provided for example 6 of the present application;

[0025] Figure 11 POD content of transgenic Arabidopsis thaliana and wild type Arabidopsis thaliana after drought treatment provided for example 6 of the present application;

[0026] Figure 12 CAT content of transgenic Arabidopsis thaliana and wild type Arabidopsis thaliana after drought treatment provided for example 6 of the present application;

[0027] Figure 13 SOD content of transgenic Arabidopsis thaliana and wild type Arabidopsis thaliana after drought treatment provided for example 6 of the present application;

[0028] Figure 14 AsA content of transgenic Arabidopsis thaliana and wild type Arabidopsis thaliana after drought treatment provided for example 6 of the present application. DETAILED DESCRIPTION

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

[0030] Example 1 Screening and verification of SpsZFP6, an upstream regulatory gene of SpsRLCK1 in Salix pschlagii

[0031] 1.1 Screening of upstream regulatory gene of SpsRLCK1 in Salix pschlagii by yeast one-hybrid

[0032] After the SpsRLCK1 promoter was amplified by PCR, it was incorporated into the phis2 vector between the EcoRI and SacI enzyme cutting sites through homologous recombination. The bait vector, which includes the SpsRLCK1 promoter attached to the pHis2 reporter gene, and the prey vector, which contains the Salix cDNA library fused to the GAL4 activation domain, were co-transformed into Y187 yeast cells. Monoclonal colonies were grown in DDO (DO / -Leu / -Trp) and TDO (DO / -His / -Leu / -Trp) media, with pHis2-p53 and pGADT7-Rec2-p53 as positive controls and pHis2-p53 and pGADT7-Rec2-SpsZFP6 as negative controls. As shown in FIG. 1, the results showed that both pHis2-SpsRLCK1 Figure 1 pro +pGADT7-Rec2-SpsZFP6 could grow on DDO and 60mM 3AT TDO media, while the negative controls could only be cultured on DDO. This indicates that SpsZFP6 interacts with the promoter of SpsRLCK1.

[0033] 1.2 Dual luciferase experiment

[0034] ​The SpsZFP6 sequence was cloned into the pCAMBIA1302 vector by homologous recombination technology. The SpsRLCK1 promoter was inserted into the pGreenII0800-LUC vector to obtain 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 by heat shock method, spread inoculated on LB solid medium containing three antibiotics (kanamycin 0.05 g / L, rifampicin 0.34 g / L and gentamicin 0.01 g / L), and incubated in a 28°C constant temperature incubator until bacterial plaques were formed. After verification, the positive bacterial liquid was stored for use. The stored positive colonies were inoculated in LB liquid medium containing antibiotics and cultured in a 28°C shaking incubator for 6 hours. Then 20 mL of LB culture solution containing the same antibiotics was added to a 50 mL centrifuge tube, 200 μl of small shaking bacterial liquid was added, and after 6 hours, centrifugation was performed at 6000 rpm for 10 min, the bacterial bodies were collected, resuspended twice with 1 / 2MS liquid medium, and then the collected bacterial bodies were diluted to OD 600 = 1.0 using Infiltration buffer solution. The diluted bacterial liquid was mixed at a ratio of 1:1, and then dark incubated for 2 hours. The bacterial liquid combinations were as follows: I: 35S::SpsZFP6 + SpsRLCK1 pro -Luc, II: 35S::SpsZFP6 + pGreen II 0800-Luc, III: pCAMBIA1302 + SpsRLCK1 pro -Luc, IV: pCAMBIA1302 + pGreen II 0800-Luc. Well-grown tobacco leaves were selected, and the mixed bacterial liquid was injected from the back of the tobacco into different parts of the same tobacco leaf using a 1 ml syringe. After 2 days of infection, 1 mm D-fluorescein potassium salt was uniformly sprayed on the back of the tobacco leaf using a small spray bottle, and then treated at 37°C in the dark for 7 min. After treatment, the fluorescence signal intensity of the tobacco leaf was observed by scanning and imaging using PlantView 100 (BLT, China). The results are shown in Figure 2 , the test group 35S::SpsZFP6 + SpsRLCK1 pro -Luc had a strong fluorescence signal intensity, which was significantly higher than that of the control group, indicating that SpsZFP6 combined with the SpsRLCK1 promoter, thereby stimulating the expression of SpsRLCK1.

[0035] Example 2 Cloning of SpsZFP6 gene of Salix pschlagii

[0036] 2.1 Obtaining the target gene sequence

[0037] According to the sequence number in Populus trichocarpa in Phytozome database, the cDNA sequence homologous to SpsZFP6 was screened, and then the primer was designed by using primer 5 software according to the screened sequence, and finally the complete gene sequence was amplified by PCR technology. The primer is shown in Table 1.

[0038] Table 1 Cloning primer

[0039]

[0040] Based on the high-fidelity PCR reaction system, the total RNA extracted from the leaves of Salix pschegiana was reverse transcribed to obtain cDNA as a template for gene cloning. The specific reaction system (20 μl) is as follows: PrimeSTAR, 10 μl; Cloning F, 1 μl; Cloning R, 1 μl; S. pschegiana cDNA, 2 μl; ddH2O, 6 μl.

[0041] The PCR cloning reaction program is as follows: 95 ℃ for 5 minutes; 95 ℃ for 10 seconds, 57 ℃ for 30 seconds, 72 ℃ for 50 seconds, 40 cycles; 72 ℃ for 2 minutes. The DNA fragment was recovered by using a DNA product purification kit (Norgen, Nanjing).

[0042] 2.2 Purpose gene ligation T vector

[0043] The recovered target fragment was ligated to the T vector, and the reaction system was composed of: T vector, 1 μl; the above DNA product, 30 ng; Solution I, 5 μl; finally, the total volume was adjusted to 10 ul with ddH2O, and the ligation was performed at 16 ℃ overnight. After the completion of the ligation reaction, the product was transformed into E. coli, and single colony was selected for bacterial liquid PCR verification, and the positive colonies were sequenced for confirmation. After sequencing verification, the nucleotide sequence is shown in SEQ ID No. 1, and the gene fragment is named as SpsZFP6, which consists of 753 bp of bases, encoding 250 amino acids (SEQ ID No. 2).

[0044] Example 3 Expression characteristics analysis of SpsZFP6 gene of Salix pschegiana

[0045] 3.1 Primer design

[0046] According to the obtained complete gene sequence, the quantitative primer was designed by using Primer3 software, and the quantitative fluorescent PCR was performed. The primer is shown in Table 2.

[0047] Table 2 Quantitative primer

[0048]

[0049] 3.2 Drought stress treatment on Salix tissue culture seedlings

[0050] The normal growing Salix pschlagii tissue culture seedlings were transferred to the medium containing 100 mM Mannitol for treatment. The total RNA was extracted from the roots and reverse transcribed into cDNA. Then the qRT-PCR reaction was performed, and the reaction system was as follows (20 μl): SYBR qPCR Master Mix, 10 μl; Quantitative F, 0.4 μl; Quantitative R, 0.4 μl; Salix cDNA, 2 μl; ddH2O, 7.2 μl.

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

[0052] The obtained data were analyzed as shown in Figure 3 The results showed that the expression amount of SpsZFP6 gene in Salix was increased under drought stress treatment, and therefore it was speculated that the gene might be induced by drought stress.

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

[0054] 4.1 Primer design

[0055] The plant overexpression vector was constructed, and the primer with pCAMBIA1302 homologous arm was designed. The primer is shown in Table 3.

[0056] Table 3 Overexpression primer

[0057]

[0058] 4.2 Construction of pCAMBIA1302-SpsZFP6 overexpression vector

[0059] The SpsZFP6 gene was amplified with overexpression F and overexpression R as primers, the product was recovered, the pCAMBIA1302 vector was double-digested, the linearized vector was used, and the homologous recombination enzyme was used to construct the target gene into the vector. The reaction system was (10 μl): target gene, 15 ng; vector, 105 ng; 5 × CE II Buffer, 2 μl; Exnase II, 1 μl; and finally ddH2O was added to 10 μl.

[0060] The reaction program was: 37 °C for 30 min, then reduced to 4 °C or immediately placed on ice for cooling.

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

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

[0063] The constructed pCAMBIA1302-SpsZFP6 overexpression vector was transformed into Agrobacterium GV3101 by heat shock method. Then the SpsZFP6 gene was transformed into Arabidopsis thaliana by Agrobacterium mediation. The transformation steps were as follows: select Arabidopsis thaliana which was cultured for 1 month, grew healthily, was 4-6 cm high, and had a large number of half-opened flower buds and a small number of fruit pods, the culture temperature was 23-25°C, the light was 16 / 8h (day / night), the flower buds were infected in the bacterial solution containing pCAMBIA1302-SpsZFP6 expression vector for 45-60s, a watering can was used to spray water, the plants were cultured in the dark for 12h, then were placed in normal conditions, the same method was used for secondary infection after 7d, when the transformed fruit pods matured, the seeds were collected and evenly spread on the solid 1 / 2MS medium containing antibiotics, and the positive strains were screened. About 10 days later, the seedlings which grew normally in the selection medium were positive strains. After the seedlings grew two true leaves, they were transplanted into the soil for cultivation. The cDNA of the rooted plants was extracted by CTAB method, and the DNA of the leaf was extracted for PCR verification. The RNA of the 20 positive seedlings screened was extracted, and the transgenic expression amount was verified by qRT-PCR. The results are shown in Figure 4 , the OE-1 and OE-2 plants with suitable expression amount were selected for the following drought resistance phenotype identification analysis.

[0064] Example 6 Drought resistance analysis of Arabidopsis thaliana SpsZFP6 transgenic plants

[0065] 6.1 Root length and fresh weight of transgenic Arabidopsis thaliana

[0066] The wild type Arabidopsis thaliana WT and the transgenic Arabidopsis thaliana of two strains OE-1 and OE-2 were cultured on normal 1 / 2MS medium for 10d, then were transferred to 1 / 2MS medium without and with 150mM mannitol (mannitol simulating drought) for growth, and the results are shown in Figure 5 . The results showed that the root length of the transgenic Arabidopsis thaliana SpsZFP6 was slightly longer than that of the wild type Arabidopsis thaliana seedlings under drought stress, the lateral roots also increased significantly, and the fresh weight of the transgenic Arabidopsis thaliana was heavier than that of the wild type Arabidopsis thaliana, the root length and fresh weight of the transgenic Arabidopsis thaliana and the wild type Arabidopsis thaliana had significant influence, which indicated that overexpression of SpsZFP6 enhanced the drought resistance of Arabidopsis thaliana, and made it more advantageous than the wild type plants in growth.

[0067] 6.2 Phenotype analysis of transgenic Arabidopsis thaliana

[0068] The wild type Arabidopsis thaliana WT and the two homozygous transgenic strains OE-1 and OE-2 of T3 generation were transplanted to the soil with a ratio of grass carbon soil to vermiculite of 3:1 for growth after being cultured on normal 1 / 2MS medium for 10 days, and plants with basically consistent growth were selected after one month of growth.Figure 6 After 13 days of normal watering, no obvious difference was observed between WT and OE-1, OE-2 lines. Then the watering was stopped and drought stress was applied. After 10 days of drought stress, the WT plants showed more severe wilting and yellowing than the transgenic plants. After 10 days of drought stress, watering was resumed and the plants were observed 3 days later. The transgenic plants OE-1 and OE-2 had recovered and the yellowing was reduced, while the WT plants showed no obvious signs of recovery. This indicates that the transgenic plants have stronger drought tolerance than the WT plants.

[0069] 6.3 Measurement of relative conductivity of transgenic Arabidopsis plants

[0070] The leaves of WT and SpsZFP6 transgenic Arabidopsis plants from 6.2 were washed with clean water to remove dust, then rinsed with flowing double-distilled water, and the surface water was absorbed with filter paper. The leaves were then placed in a triangular flask and 50 ml of ultrapure water was added. The flask was vacuumed for 20 min, then the conductivity was measured to be R1. The flask was then placed in a water bath and boiled, and the conductivity was measured after cooling to be R2. The relative conductivity was calculated as follows: relative conductivity (%) = R1 / R2 x 100%. The results are shown in Table 6.3. Figure 7 As shown in Table 6.3, under normal conditions, there was no difference in the relative conductivity between WT and SpsZFP6 transgenic plants. Under drought stress, the relative conductivity of the SpsZFP6 transgenic plants was much lower than that of the WT plants, and there was a significant difference between the WT and SpsZFP6 transgenic plants. This indicates that the SpsZFP6 transgenic plants have stronger drought tolerance than the WT plants, and the SpsZFP6 gene plays an important role in drought tolerance in Arabidopsis plants.

[0071] 6.4 Measurement of relative water content of transgenic Arabidopsis plants

[0072] The relative water content of Arabidopsis leaves was measured by the oven-drying method. The leaves of WT and SpsZFP6 transgenic Arabidopsis plants from 6.2 were washed with flowing water and the water was absorbed with filter paper. The leaves were weighed using a balance and recorded as FW. The leaves were then placed in a self-sealing bag and filled with tap water to saturate the leaves for 6 h. The leaves were weighed and recorded as TW. The leaves were then placed in an electric heating air-drying oven at 80°C until the leaves reached a constant weight. The weight of the leaves was measured and recorded as DW. The relative water content of the leaves was calculated as follows: relative water content (%) = (FW-DW) / (TW-DW) x 100%. The results are shown in Table 6.4. Figure 8Under normal conditions, the relative water content of wild-type Arabidopsis and transgenic Arabidopsis had little difference and remained at a high level, while under drought conditions, the relative water content of plant leaves decreased, and the relative water content of wild-type Arabidopsis was lower, indicating that the SpsZFP6 gene can make Arabidopsis retain more water, thereby making the transgenic Arabidopsis more drought-resistant.

[0073] 6.5 NBT and DAB analysis of transgenic Arabidopsis

[0074] First, 3,3'-diaminobenzidine (DAB) staining solution was prepared by weighing 0.788 g of Tris-HCl on a balance, dissolving and diluting to 100 mL with ultrapure water, adjusting the pH to 5.5, and then adding 0.1 g of DAB powder and shaking to prepare a liquid with a concentration of 1 mg / mL. Then, nitroblue tetrazolium (NBT) staining solution was prepared by weighing 0.5 g of NBT on a balance, adding 22.87 mL of phosphate buffer A and 21.25 mL of phosphate buffer B to make up to 100 mL, and preparing a liquid with a concentration of 1 mg / mL. Finally, the decolorizing solution was prepared by mixing acetic acid, glycerol and anhydrous ethanol at a ratio of 1:1:3.

[0075] A 10 mL centrifuge tube was prepared, and the staining solution and the leaves to be tested were added so that the staining solution could completely submerge the leaves. The test tube was vacuumed for 30 min, the leaves were submerged in the staining solution, and the test tube was wrapped with tin foil. The test tube was placed in a 37°C incubator and stained in the dark for 8 h. After successful staining, the staining solution was poured out, the decolorizing solution was added, and the decolorizing was performed in a water bath at 95°C. After successful decolorizing, the leaves were cooled to room temperature, cleaned with anhydrous ethanol, and the excess water on the surface of the leaves was gently absorbed with filter paper to observe the coloration of active oxygen on the leaves. The results are shown in Figure 9 There was no significant difference between wild-type Arabidopsis and transgenic Arabidopsis under normal conditions, while under drought stress, DAB and NBT showed blue coloration in wild-type Arabidopsis leaves, and the coloration effect was deeper, while the coloration effect of SpsZFP6 transgenic plants was shallower. The results showed that overexpression of SpsZFP6 can reduce the content of intracellular hydrogen peroxide H2O2 and superoxide anion O2 - , 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 H8N2 solution 400 μl and 200 μl 3% FeCl3 solution. The mixture was placed at 37°C for 1 h. The absorbance value at 525 nm was measured by UV spectrophotometer. Each process was repeated 3 times, and the results were averaged. The results are shown in Table 2. Figure 14 As shown in Table 2, the AsA content of the plants was increased after drought stress, but the AsA content of the transgenic Arabidopsis was significantly higher than that of the wild-type Arabidopsis, which indicated that the SpsZFP6 gene could enhance the drought resistance of the plants.

[0082] In summary, the SpsZFP6 gene-transferred plants can participate in drought stress by maintaining the stability of reactive oxygen species (ROS), thereby improving the drought resistance of the plants.

Claims

1. SpsZFP6 gene or a nucleic acid molecule comprising SpsZFP6 The use of a gene or a nucleic acid molecule comprising By overexpressing Salix ps SpsZFP6 Gene in plants, the plants are Salix or Arabidopsis, the SpsZFP6 The gene is a nucleic acid fragment encoding Salix ps SpsZFP6 protein shown in SEQ ID No. 2.

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