Application of GmSCEc gene in improvement of soybean salt tolerance

Abstract

The invention discloses application of a GmSCEc gene in improvement of soybean salt tolerance, and belongs to the technical field of plant stress resistance. The purpose of the invention is to improve the salt tolerance of soybeans. According to the application of the GmSCEc gene protein in improving the salt tolerance and the oxidation resistance of the soybeans, the amino acid sequence of the GmSCEc gene protein is shown as SEQ ID NO.4. The invention further discloses a preparation method of the GmSCEc gene protein. The overexpression of the GmSCEc can improve the antioxidant enzyme activity of soybeans under salt stress, reduce oxidative damage and more effectively protect soybean cells from being damaged by salt stress, so that the salt tolerance of the soybeans is improved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant stress resistance, and particularly relates to application of a GmSCEc gene in improving salt tolerance of soybeans. BACKGROUND

[0002] Soybean (Glycine max) is an important food, oil and feed crop in the world, and plays an important role in agricultural production. However, with the population growth and the shortage of arable land resources, many marginal lands (such as saline-alkali land) are considered for agricultural planting. These lands usually have a high salt content, which constitutes a serious stress to most crops, including soybeans. Therefore, it is of important practical significance and strategic necessity to study and improve the salt stress tolerance of soybeans. SUMMARY

[0003] The application aims to improve the salt tolerance of soybeans.

[0004] The application provides application of a GmSCEc protein in improving the salt tolerance of soybeans, wherein the amino acid sequence of the GmSCEc protein is shown as SEQ ID NO. 4.

[0005] The application provides application of a GmSCEc gene in improving the salt tolerance of soybeans, wherein the nucleic acid sequence of the GmSCEc gene is shown as SEQ ID NO. 3.

[0006] The application provides application of a recombinant vector containing the nucleic acid sequence shown as SEQ ID NO. 3 in improving the salt tolerance of soybeans.

[0007] Further limitation, the starting vector is pFGC5941.

[0008] The application provides application of a recombinant host cell containing the nucleic acid sequence shown as SEQ ID NO. 3 in improving the salt tolerance of soybeans.

[0009] Further limitation, the host cell is a prokaryotic cell or a eukaryotic cell.

[0010] Further limitation, the salt-tolerant condition is 200 mM NaCl.

[0011] The application provides a soybean breeding method for salt stress tolerance, and the method is as follows: Step 1: connecting a GmSCEc gene with a pFGC5941 vector to obtain a recombinant vector; Step 2: transforming the recombinant vector obtained in step 1 into Agrobacterium to obtain a recombinant Agrobacterium; Step 3: transfecting the recombinant Agrobacterium obtained in step 2 into soybeans to obtain transgenic soybeans.

[0012] Further limited, the sequence of the gene amplified in step 1 is shown as SEQ ID NO. 1 and SEQ ID NO. 2.

[0013] The application provides a method for improving salt tolerance of soybean, overexpressing the gene shown in SEQ ID NO. 3 in soybean, and treating under 200 mM NaCl conditions.

[0014] Beneficial effects: In this study, the activities of POD, SOD and APX in the roots and leaves of overexpression lines and wild type lines at different time points under 200 mM NaCl treatment were determined, and the results showed that the activities of the three enzymes in leaves and roots reached a peak after 12 hours of salt treatment, and then decreased, and the enzyme activities of GmSCEc-OE were significantly higher than those of WT. Consistent with the results of DAB and NBT staining, it can be proved that overexpression of GmSCEc can improve the antioxidant enzyme activity of soybean under salt stress, reduce oxidative damage, and more effectively protect soybean cells from salt stress, thereby improving the salt tolerance of soybean. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 For GmSCEc Figure 1 is a diagram of the acquisition of OE T1 generation plants and the PCR detection results; A: genetic transformation process of overexpression soybean; B: PCR detection of overexpression soybean resistance gene bar gene; Figure 2 Figure 2 is a diagram of GmSCEc-OE soybean T3 expression detection and SUMO level detection results; A: T3 generation overexpression soybean GmSCEc gene expression detection; B: T3 generation overexpression soybean FLAG antibody detection GmSCEc-3FLAG protein expression detection; C: T3 generation overexpression soybean SUMO level WB detection, bar represents SD, and t-test analysis is used for significance analysis, n=3; Figure 3 Figure 3 is a diagram of salt stress phenotype analysis results of GmSCEc-OE and wild type soybean; Figure 4 Figure 4 is a diagram of the determination results of phenotype related indexes of GmSCEc-OE and wild type soybean under salt stress; A: salt injury index of GmSCEc overexpression and wild type soybean under salt stress for 3 days; B: chlorophyll content; C: MDA content; D: Na+ / K+, bar represents SD, and ANOVA variance analysis is used for significance analysis, n=3; Figure 5 Figure 5 is a diagram of salt stress phenotype results of GmSCEc-OE and wild type soybean at different times; Figure 6 Figure 6 is a diagram of DAB and NBT staining results of GmSCEc-OE and wild type soybean after salt stress; Figure 7 Figure 1 shows the determination of GmSCEc-OE and wild-type soybean salt stress antioxidant enzyme activity; the line graph shows the SOD (A, B), POD (C, D) and APX (E, F) activities in leaves and roots at different time points under 200 mM NaCl treatment; Figure 8 Figure 2 shows the results of pFGC5941-GmSCEc-3FLAG plant gene expression vector construction; A: GmSCEc gene high-fidelity PCR; B: pFGC5941-GmSCEc-3FLAG transformation DH5a bacterial liquid PCR detection; C: pFGC5941-GmSCEc-3FLAG transformation EHA105 bacterial liquid PCR detection. DETAILED DESCRIPTION

[0016] Example 1. GmSCEc Construction of overexpression vector 1. The constructed vector plasmid pCAMBIA35S-GmSCEc-eGFP was used as a template for amplification GmSCEc - eGFP (pCAMBIA35S vector linking GmSCEc and eGFP) as a template for amplification GmSCEc - eGFP The fragment, and the primer sequence is shown in Table 1.

[0017] Table 1 GmSCEc Primer sequence for plant expression vector construction

[0018] The specific reaction system is shown in Table 2: Table 2

[0019] The amplification program is as follows:

[0020] Using Asc I restriction endonuclease to cut the prokaryotic expression vector pFGC5941- 3FLAG (pFGC5941 vector linking 3xFLAG tag) enzyme system as shown in Table 3, and recovered by agarose gel reagent kit; Table 3

[0021] Using the method of homologous recombination to construct prokaryotic expression vector pFGC5941- GmSCEc-eGFP-3FLAG , the system is as shown in Table 4: Table 4

[0022] After 30 min of ligation, the E. coli competent DH5α was transformed by the conventional freeze-thaw method, and the plasmid was extracted for PCR identification. The PCR system and conditions were the same as above, and the primers used were GmSCEc-F: TTACAATTACCATGGGGCGCGCCATGTCTGGTGGTATC (SEQ ID NO. 11); GmSCEc-R: GGTATCGATAAGCTTGGCGCGCCAGAGAAGAGGTGGGTA (SEQ ID NO. 12). The results are shown in Figure 2. GmSCEc -F and eGFP -R, and the correct recombinant plasmid was identified by PCR and sequencing. GmSCEc-F: TTACAATTACCATGGGGCGCGCCATGTCTGGTGGTATC (SEQ ID NO. 11); GmSCEc-R: GGTATCGATAAGCTTGGCGCGCCAGAGAAGAGGTGGGTA (SEQ ID NO. 12). Figure 8 The results are shown in Figure 2.

[0023] GmSCEc gene (SEQ ID NO. 3): ATGTCTGGTGGTATCGCCCGTGGACGCCTCACCGAGGAGCGCAAGTCGTGGCGGAAGAACCATCCCCATGGTTTTGTTGCAAACCCGGAAACCCTGCCCCATGGAACCGTGAACTTGATGGGGGGGCATTGCACTATTCCCGGGAAAACTGGGACTGATTGGGAGGGTGGCTACTTCCCGCTTACGCTGCACTTTAGTGAAGACTACCCAAGCAAGCCTCCAAAGTGTAAATTCCCACAAGGTTTCTTCCACCCTAATGTTTATCCTTCTGGGACTGTTTGCTTGTCTATACTTAATGAGGATAGTGGGTGGAGACCAGCCATAACAGTTAAGCAAATTCTTGTGGGCATCCAAGACTTACTTGATCAGCCAAATCCTGCTGATCCTGCCCAGACAGAAGGCTATCATCTATTCATCCAGGATGCAGCAGAGTACAAGAGAAGGGTCCGGCAGCAGGCAAAGCAATACCCACCTCTTCTCTAG; GmSCE protein SEQ ID NO. 4: MSGGIARGRLTEERKSWRKNHPHGFVANPETLPHGTVNLMGGHCTIPGKTGTDWEGGYFPLTLHFSEDYPSKPPKCKFPQGFFHPNVYPSGTVCLSILNEDSGWRPAITVKQILVGIQDLLDQPNPADPAQTEGYHLFIQDAAEYKRRVRQQAKQYPPLL.

[0024] 2. GmSCEc overexpression soybean genetic transformation process The successfully constructed pFGC5941- GmSCEc - eGFP - 3FLAG The expression vector was transformed into Agrobacterium tumefaciens GV3101 using a conventional freeze-thaw method. Results are as follows: Figure 8 As shown in Figure C, this is Agrobacterium containing the pFGC5941-GmSCEc-eGFP-3FLAG vector. Soybeans germinated for one day were used as material, and explants were placed in a petri dish containing 50 mL of Agrobacterium suspension. Approximately 150 explants were treated within two hours and incubated at room temperature for 30 min. After infection, the Agrobacterium suspension was discarded, and the explants were placed in a co-culture medium and co-cultured in the dark at 23°C for 3 days. After co-culture, the embryos were transferred to adventitious bud induction medium and cultured under light at 25°C for 7 days. Then, they were placed on adventitious bud selection medium and cultured for three weeks to induce resistant buds. Finally, they were transferred to elongation medium and cultured under light for 6-9 weeks. The regenerated and elongated seedlings were then rooted.

[0025] 3. GmSCEc Positive identification of overexpressed soybean Select healthy soybean seedlings, cut a small amount of leaves for DNA extraction, grind them into a fine powder in liquid nitrogen in a 2 mL centrifuge tube, add 1 mL of CTAB extraction buffer preheated to 65℃, and vortex to mix. Then incubate in a 65℃ water bath for 30-60 minutes, gently inverting to mix every 10 minutes. After cooling to room temperature, add an equal volume of chloroform:isoamyl alcohol (24:1), gently invert to mix for 10 minutes, centrifuge at 12000 rpm for 10 minutes, and transfer the supernatant to a new tube. Add 1 / 10 volume of 3 M sodium acetate (pH 5.2) and 0.6 volumes of pre-chilled isopropanol to the supernatant, gently invert to mix, incubate at -20℃ for 30 minutes, centrifuge at 12000 rpm for 10 minutes, discard the supernatant, wash the precipitate twice with 1 mL of 70% ethanol, centrifuge, and invert. Finally, add 50-100 μL of TE buffer (containing 1 μL of RNase A), incubate at 37°C for 30 minutes to degrade RNA, then incubate at 55°C for 1 hour. After centrifugation, collect the supernatant and store at -20°C for long-term storage. The DNA is used for PCR detection of glufosinate resistance genes. BAR Genetic testing, primer sequences are shown in Table 5.

[0026] Table 5 BAR Gene detection primer sequence

[0027] Further from the transcription level detection of overexpression plants expression quantity, take soybean different tissue parts material each about 200 mg, quickly put in 2 mL RNase Free tube with porcelain bead, use sample quick grinding instrument to grind the sample, the extraction steps are described in Ultrapure RNA Kit (CW0581) instruction.

[0028] Synthesis of cDNA first strand: prepare 200 μL RNase free centrifuge tube on ice, add 700 ng template RNA, 2 µL 4×gDNA wiper Mix, RNase Free Water to 8 µL, mix well by blowing, 42℃ in PCR instrument for 2 min; directly add 2 µL 5×HiScriptⅡ Select qRT SuperMixⅡ to the reaction solution, mix well by blowing, 50℃ for 15 min, 85℃, 2 min after termination reaction.

[0029] qRT-PCR analysis GmSCEc Gene expression pattern.

[0030] Using TransStart Tip Green qPCR SuperMix kit, fluorescence quantitative PCR instrument, the relative expression of target gene changes fold using 2 -ΔΔCt Method calculation. Primer sequence see table 6 below.

[0031] Table 6 GmSCEc qPCR primer sequence

[0032] The specific reaction system is shown in table 7 as follows: Table 7

[0033] Reaction program as follows:

[0034] 4. Results: GmSCEc Obtaining of overexpression soybean In order to further explore GmSCEc The physiological function of soybean under abiotic stress, the vector of transformed soybean hairy root was transferred into Agrobacterium tumefaciens, and cotyledon node was used as explant for genetic transformation, and GmSCEcRegenerated soybean plants overexpressing the gene (Figure 1A). First, DNA was extracted from the overexpressing soybean, and resistance genes were... bar Perform testing (such as) Figure 2 B), the results proved that the tested transgenic lines GmSCEc -OE#7, GmSCEc -OE#8 and GmSCEc -OE#12 were all positive. T3 generation soybeans were screened, and transcriptional analysis was performed first. GmSCEc Differences in gene expression folds (e.g.) Figure 2 A). Soybean RNA was extracted, reverse transcribed into cDNA, and analyzed using RT-qPCR. GmSCEc -OE T3 generation genetically modified soybeans GmSCEc Expression fold. The results showed that, compared to the wild type, the expression folds of the three transgenic lines were significantly higher. GmSCEc Gene expression levels were all increased, ranging from 10 to 30 times that of the wild type. Next, the expression of GmSCEc-3FLAG was detected at the protein level (e.g., Figure 2 B). Total protein was extracted from soybeans and detected by Western blotting using FLAG antibody. Results showed that FLAG antibody signals were detectable in all three transgenic lines compared to the wild type. Since GmSCEc is a SUMO-binding enzyme, the SUMOylation level in the three overexpressing soybeans was further examined (e.g., ...). Figure 2 C). The results showed that the SUMOylation levels of the three overexpressing soybeans were increased compared with those of the wild type, indicating that the overexpressed GmSCEc expressed E2-binding enzyme activity in soybeans.

[0035] Example 2. GmSCEc Overexpression of soybean under salt stress treatment and detection of physiological indicators 1. GmSCEc Overexpressing soybean and wild-type soybean seeds were planted in 7 cm × 7 cm black square pots, with 4 seedlings in each pot. They were cultured normally until stage V2.

[0036] Salt stress was applied in two ways: soil culture and hydroponics. The soil culture treatment method was as follows: After normal cultivation to stage V2, 200 mM NaCl treatment was initiated every two days, with photographs taken and phenotypes observed. Soybean seedlings were subjected to stress treatment with 1 / 4 Hoagland solution containing 200 mM NaCl. At 0 h, 6 h, 12 h, and 24 h after treatment, the second trifoliate leaf of the soybean seedlings was collected and stored in liquid nitrogen at -80°C in the dark.

[0037] For detailed procedures on the determination of MDA, PRO, SOD, POD, and APX, please refer to "Experimental Tutorial of Plant Physiology".

[0038] The determination method of salt injury index: all plants were observed and counted, and the salt injury index of each treatment of each plant was calculated. The salt injury symptom identification standard is shown in Table 8.

[0039] Table 8 Salt injury symptom identification standard

[0040] The salt-alkali stress index (SAI) was calculated:

[0041] Wherein N0, N1, N2, N3, N4 and N5 are the number of plants with scores of 0, 1, 2, 3, 4 and 5 levels respectively, and N is the total number of plants tested for each genotype.

[0042] 2. GmSCEc Phenotype and physiological index analysis of overexpression soybean under salt treatment In order to study the function of GmSCEc under salt stress, the phenotype of GmSCEc overexpression soybean under salt stress was first analyzed. Soybeans were germinated in a medium of 1:1 soil and vermiculite and cultured to V2 stage, and 200 mM NaCl solution was irrigated daily, and water was irrigated in the control group. The phenotype was recorded by taking pictures. The leaves of WT began to wilt the next day after salt stress, while the leaves of GmSCEc overexpression lines remained green and did not wilt during the treatment, as shown in Figure 3 This proves that overexpression of GmSCEc can improve the salt tolerance of soybean plants.

[0043] The results are shown in Figure 4 A, the salt injury index of the overexpression lines was less than 30% at 3 days of salt stress, while that of WT was 90%. After salt stress, GmSCEc The chlorophyll content of the overexpression lines remained above 3.17 mg / g, while that of WT decreased to 0.96 mg / g Figure 4 B). This indicates that the overexpression lines can better maintain photosynthetic function under salt stress. The MDA content of each line increased after salt stress, but the increase in the overexpression lines was lower than that in WT, as shown in Figure 4 C, indicating that the overexpression lines can more effectively protect cell membranes from oxidative damage. This further supports the strong antioxidant capacity and membrane lipid stability of the overexpression lines. 3. In order to study the effect of different times of salt stress on GmSCEc overexpression soybean, overexpression soybean and wild type soybean were treated with 200 mM NaCl hydroponics for 0, 6, 12 and 24 hours. It can be seen that wild type began to wilt at 12 hours, and the leaves were obviously wilted at 24 hours. While GmSCEc overexpression soybean OE#7 and OE#12 lines did not show obvious wilting, Figure 5From the phenotype, it can be seen that... GmSCEc Overexpression of this substance significantly enhances the salt tolerance of soybeans.

[0044] 4. First, DAB and NBT staining methods were used to detect oxidative damage in soybean leaves after salt treatment, such as... Figure 6 Under untreated conditions, leaves of all lines stained with NBT and DAB showed lighter staining, indicating lower ROS levels and no significant difference in staining area between wild-type and overexpression. After treatment with 200 mM NaCl, overexpression soybean leaves showed smaller staining area compared to the wild-type, which showed deeper and larger staining, indicating... GmSCEc Gene overexpression lines exhibited lower accumulation of H2O2 and superoxide anions under salt stress.

[0045] 5. In order to study GmSCEc To investigate the antioxidant capacity of overexpressed soybeans under salt stress, this study measured the activities of POD, SOD, and APX in the roots and leaves of overexpressed and wild-type lines at different time points after treatment with 200 mM NaCl. Figure 7 The results showed that the activities of the three enzymes in leaves and roots reached their peak after 12 hours of salt treatment, and then declined. GmSCEc The enzyme activity of -OE was significantly higher than that of WT. This is consistent with the results of DAB and NBT staining, all of which demonstrate... GmSCEc Overexpression can increase the activity of antioxidant enzymes in soybeans under salt stress, reduce oxidative damage, and more effectively protect soybean cells from salt stress, thereby improving the salt tolerance of soybeans.

Claims

1. Application of GmSCEc protein in improving salt tolerance and antioxidant capacity of soybean, characterized in that, The amino acid sequence of the GmSCEc gene protein is shown as SEQ ID NO.

4.

2. Application of GmSCEc gene in improving salt tolerance and antioxidant capacity of soybean, characterized in that, The nucleic acid sequence of the GmSCEc gene is shown as SEQ ID NO.

3.

3. Application of a recombinant vector containing the nucleic acid sequence shown as SEQ ID NO. 3 in improving salt tolerance and antioxidant capacity of soybean.

4. Use according to claim 3, characterized in that, The starting vector is pFGC5941.

5. Application of a recombinant host cell containing the nucleic acid sequence shown as SEQ ID NO. 3 in improving salt tolerance and antioxidant capacity of soybean.

6. Use according to claim 5, characterized in that, The host cell is a prokaryotic cell or a eukaryotic cell.

7. The use according to any one of claims 1 to 6, characterized in that, The salt-tolerant condition is 200 mM NaCl.

8. A method of breeding salt-stress tolerant and antioxidant soybean, characterized by, The method is as follows: Step 1: connecting the GmSCEc gene with the pFGC5941 vector to obtain a recombinant vector; Step 2: transforming the recombinant vector obtained in Step 1 into Agrobacterium to obtain a recombinant Agrobacterium; Step 3: transfecting the recombinant Agrobacterium obtained in Step 2 into soybean to obtain transgenic soybean.

9. The method of claim 8, wherein, The sequence of the amplified gene in Step 1 is shown as SEQ ID NO. 1 and SEQ ID NO.

2.

10. A method for improving salt tolerance and antioxidant capacity of soybean, characterized by, After overexpression of the gene shown as SEQ ID NO. 3 in soybean, the soybean is treated under the condition of 200 mM NaCl.

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