Suaeda heteroptera SsWRKY30 gene and application thereof

Abstract

The invention relates to the technical field of molecular biology, and particularly provides a suaeda heteroptera SsWRKY30 gene and application thereof, and the nucleotide sequence of the suaeda heteroptera SsWRKY30 gene is as shown in SEQ ID NO. 1. According to the invention, the SsWRKY30 gene is transferred into tobacco by means of molecular biology and biotechnology, so that a subcellular localization result of the SsWRKY30 gene is obtained; the SsWRKY30 gene is transferred into arabidopsis thaliana to obtain an overexpressed arabidopsis thaliana plant, and comparative analysis proves that the tolerance of the overexpressed arabidopsis thaliana to salt and drought stress is obviously improved.

Description

Technical Field

[0001] The invention relates to the technical field of molecular biology, and in particular provides a Suaeda salsa L. SsWRKY30 gene and application thereof. Background Art

[0002] Suaeda salsa (L.) Pall., a Suaeda salsa of the family Ricoeaceae, is an annual herb with fleshy leaves. It is a typical indicator plant for saline-alkali soil. It has strong salt and drought tolerance, and the ability to repair saline-alkali and contaminated soil. The discovery of its stress-resistant genes is of great significance for improving plant stress resistance and improving saline-alkali soil.

[0003] Transcription factors are generally composed of several relatively independent functional domains, including DNA binding domains, transcriptional regulatory domains, signal molecule binding domains, and nuclear localization signal domains. Transcription factors, also known as trans-acting factors, regulate the expression of downstream target genes by binding to cis-acting elements in gene promoter regions or enhancer regions and interacting with other proteins. They play an important role in different tissues, cells, or environmental conditions of plants, and enhance the ability of plants to resist biotic and abiotic stresses. WRKY transcription factors are one of the largest families of transcriptional regulators in higher plants. The name WRKY comes from the domain with the amino acid sequence characteristic of WRKY in the gene, which consists of about 60 amino acid residues, a highly conserved WRKYGQ heptapeptide sequence at the N-terminus, and a zinc finger motif at the C-terminus. The zinc finger motif can be C2H2 type (CX4~5CX22~23HX1H) or C2HC (CX7CX23HX1-C). The conserved heptapeptide motif of WRKYGQK in some plants changes into various variants such as WRKYGEK, WRKYGKK, WRKYGSK, WRKYSGQR, WRKYDQK, WRKYHQK, WRKYGHK, WRKYHQK, etc. According to the number of WRKY domains and the characteristics of zinc finger motifs, WRKY proteins can be divided into three different families, Group Ⅰ, Group Ⅱ and Group Ⅲ. The Group Ⅱ family is further divided into five subfamilies, namely Ⅱa, Ⅱb, Ⅱc, Ⅱd and Ⅱe, according to the primary structure of amino acids. The GroupⅠ family contains two WRKY domains and a C2H2 (C-X4–C–X22–23–HXH) zinc finger structure, the GroupⅡ family contains a WRKY domain and a C2H2 (C-X4–5–C–X23–HXH) zinc finger structure, and the GroupⅢ family consists of a WRKY domain and a C2H2 (C-X7–C–X23–HXC) zinc finger structure. In addition, the GroupⅡ family can be further divided into three branches: IIa + IIb, IIc, and IId + IIe. WRKY TFs show selective recognition and binding ability to the W-box (C / T) TGAC (T / C) region in the promoter region of the target gene. This enables them to regulate gene expression at the transcriptional level. Acting as an activator or inhibitor of gene expression depending on the specific regulatory environment.

[0004] In 1994, Ishiguro and Nakamura cloned the first WRKY protein SPF1 from sweet potato. Since then, WRKY genes have been discovered in many plants, including Arabidopsis (74), tobacco (164), rice (102), corn (119), sorghum (94), tomato (81), and wheat (171). However, there is a lack of research on WRKY transcription factor genes and their functions in Suaeda salsa. Therefore, this application uses Suaeda salsa as research material to identify WRKY transcription factor genes and explore their biological functions. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a Suaeda salsa L. SsWRKY30 gene and application thereof.

[0006] The present invention is implemented by providing a Suaeda salsa SsWRKY30 gene, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0007] Provided is a protein encoded by a Suaeda salsa SsWRKY30 gene, wherein the amino acid sequence of the protein is shown as SEQ ID NO.2.

[0008] Provided is an application of a Suaeda salsa L. SsWRKY30 gene for subcellular localization in tobacco.

[0009] Provided is an application of a Suaeda salsa L. SsWRKY30 gene for improving the tolerance of plants to salt and drought.

[0010] Provided are primers for amplifying the Suaeda salsa SsWRKY30 gene, which are pCAMBIA1300- SsWRKY30-F and pCAMBIA1300- SsWRKY30-R, and the nucleotide sequences are as follows:

[0011] pCAMBIA1300-SsWRKY30-F:

[0012] 5'-GAGCTCGGTACCCGGGGATCCATGGAACTCAATGGTTGCAC-3'

[0013] pCAMBIA1300-SsWRKY30-R:

[0014] 5'-GCCCTTGCTCACCATGTCGACTTCTTTATCTTGAGAAAAGCTAGTAG-3';

[0015] The first 6 bases at the 5' end of the primer are protective bases, and the following 6 bases are restriction site sequences. These 11 bases do not belong to the SsWRKY30 gene sequence, but are necessary for constructing a recombinant plant overexpression vector.

[0016] Provided is a plant overexpression vector containing the Suaeda salsa SsWRKY30 gene, which is pCAMBIA1300-35S-GFP-SsWRKY30.

[0017] Provided is an application of a plant overexpression vector containing the Suaeda salsa SsWRKY30 gene, which is an application of the plant overexpression vector pCAMBIA1300-35S-GFP-SsWRKY30 in subcellular localization in tobacco.

[0018] Provided is an application of a plant overexpression vector containing the Suaeda salsa SsWRKY30 gene, which is an application of the plant overexpression vector pCAMBIA1300-35S-GFP-SsWRKY30 in improving the tolerance of plants to salt and drought.

[0019] Compared with the prior art, the advantages of the present invention are:

[0020] The present invention cloned a transcription factor SsWRKY30 gene from Suaeda salsa for the first time. The SsWRKY30 gene was transferred into tobacco by molecular biology and biotechnology to obtain the subcellular localization results of the SsWRKY30 gene; at the same time, it was transferred into Arabidopsis thaliana to obtain Arabidopsis thaliana plants overexpressing the SsWRKY30 gene. Comparative analysis showed that the tolerance of the overexpressed Arabidopsis thaliana to salt and drought stress was significantly improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The present invention is further described in detail below in conjunction with the accompanying drawings and embodiments:

[0022] Figure 1 The subcellular localization of SsWRKY30 in Nicotiana benthamiana, where GFP: green fluorescent protein; mCherry: nuclear localized protein; Bright: bright field; Merged: overlay of GFP, mCherry and Bright;

[0023] Figure 2-1 The effect of salt stress on the root growth of Arabidopsis thaliana, where A: Scanning image of Arabidopsis thaliana roots under salt stress; B: Total root length; C: Root surface area; WT: Wild-type plant; OE: Overexpression plant; Control: Normal growth conditions;

[0024] Figure 2-2The effect of drought stress on the root growth of Arabidopsis thaliana, where A: Scanning image of Arabidopsis thaliana root system under drought stress; B: Total root length; C: Root surface area; WT: Wild-type plant; OE: Overexpression plant; Control: Normal growth conditions;

[0025] Figure 2-3 shows the effects of salt and drought stress on stomatal aperture of Arabidopsis leaves, where WT: wild-type plants; OE: overexpression plants; Control: normal growth conditions;

[0026] Figure 2-4 Effects of salt and drought stress on the relative water content of Arabidopsis plants, where A: phenotypes of wild type and transgenic lines under salt stress; B: phenotypes of wild type and transgenic lines under drought stress; C: relative water content of wild type and transgenic lines under salt stress; D: relative water content of wild type and transgenic lines under drought stress; WT: wild type plants; OE: overexpression plants; Control: normal growth conditions;

[0027] Figure 2-5 The effects of salt and drought stress on ion permeability of Arabidopsis plants;

[0028] Figure 2-6 Effects of salt and drought stress on the soluble protein content in Arabidopsis plants. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0030] Example 1: Cloning of the Suaeda salsa transcription factor SsWRKY30 gene

[0031] Suaeda salsa was used as the test material; the total RNA of mycelium was extracted using the EASYspinPlus plant RNA rapid extraction kit, and the first chain of cDNA was synthesized by reverse transcription;

[0032] Gene cloning: Using the first strand of reverse transcribed cDNA as a template, using primers

[0033] pCAMBIA1300-SsWRKY30-F:

[0034] 5'-GAGCTCGGTACCCGGGGATCCATGGAACTCAATGGTTGCAC-3'

[0035] pCAMBIA1300-SsWRKY30-R:

[0036] 5'-GCCCTTGCTCACCATGTCGACTTCTTTATCTTGAGAAAAGCTAGTAG-3';

[0037] PCR amplification was performed, and the PCR product was recovered to obtain a target fragment of 894 bp.

[0038] Example 2: Construction of plant expression vector

[0039] The purified gel recovery product SsWRKY30 gene was connected to the pCAMBIA1300-35S-GFP linear large fragment using In-FusionHDCloningkit. The reaction system was: 5μL2×CloningMix, 3μL linear large fragment, 2μL target gene fragment, 50℃ for 30min. The ligation product was transformed into E. coli competent TOP10, and the plant expression vector pCAMBIA1300-35S-GFP-SsWRKY30 with the target gene was obtained by shaking the bacteria and extracting the plasmid after colony PCR and sequencing. Then, Agrobacterium GV3101 competent cells were transformed, and colony PCR was performed to identify the Agrobacterium containing the recombinant plasmid for the transformation of tobacco leaves.

[0040] Example 3: Subcellular localization detection of tobacco leaves transformed with recombinant plasmids

[0041] Agrobacterium GV3101 positive clones containing pCAMBIA1300-35S-GFP and pCAMBIA1300-35S-GFP-FvFTR1 recombinant plasmids were first inoculated into 10 mL of 50 μg mL -1 Rif (rifampicin) and 50 μg mL -1 Kan (kanamycin) in YEP liquid medium, shake culture at 28 °C for 16 h, and then inoculate in 50 mL of 50 μg mL -1 Rif (rifampicin) and 50 μg mL -1 Kan (kanamycin) in YEP liquid medium, shake culture at 28℃ for 3h until OD600 is 0.8-1, centrifuge at 4000rpm for 10min to collect the cells, and use 0.5mol·L -1 MES+1mol·L -1 MgCl2+100mmol·L -1 Suspend the bacteria in AS (acetosyringone) aqueous solution to an OD600 of 0.6-0.8, and let stand at 28°C in the dark for 3 hours; inject the bacterial suspension into the tobacco leaf surface with a needle-free syringe; culture in the dark for 24 hours, and then culture in the light for 72 hours.

[0042] Subcellular localization detection: Using tobacco leaves injected with pCAMBIA1300-35S-GFP Agrobacterium as control, laser confocal microscopy was used to detect the expression of pCAMBIA1300-35S-GFP-SsWRKY30 fusion protein in tobacco protoplasts. The fluorescence signal of pCAMBIA1300-35S-GFP was detected in multiple subcellular compartments such as plasma membrane, nucleus and cytoplasm, while the fluorescence signal of pCAMBIA1300-35S-GFP-SsWRKY30 was mainly detected in the plasma membrane, with a small amount detected in the cytoplasm, indicating that SsWRKY30 in Suaeda salsa was mainly localized in the nucleus, which is consistent with its transcription factor function. For related results, please refer to the attached Figure 1 .

[0043] Example 4: Functional verification of recombinant plasmid transformation into Arabidopsis thaliana

[0044] 100 μl of pCAMBIA1300-SsWRKY30-GFP Agrobacterium culture fluid stored in -80℃ ultra-low temperature refrigerator was taken out in the bacterial clean bench and added to 10mL YEB (containing Kana and Rif) liquid culture medium, and then placed in a 28℃ constant temperature shaker incubator at 180rpm for 12h; 3mL of the cultured bacterial fluid was added to 30mL YEB (containing Kan and Rif) liquid culture medium, and then placed in a 28℃ constant temperature shaker incubator at 180rpm for culturing. The ultraviolet spectrophotometer measured the bacterial solution OD600 = 0.8-1.0; centrifuged at 4500rpm for 10min at 4℃, discarded the supernatant, and resuspended the collected bacteria with 10mLddH2O; the resuspended bacteria were centrifuged at 4500rpm for 10min at 4℃, discarded the supernatant, and resuspended again with 5% sucrose solution, and the ultraviolet spectrophotometer measured the bacterial solution concentration OD600 = 0.6-0.8; the bacterial solution was placed in a 28℃ constant temperature incubator and cultured for 3h in the dark. Add 3μl SilwetL-77 to the bacterial suspension and mix it by turning the centrifuge tube upside down. Select wild-type Arabidopsis plants with white buds and no pods for inoculation. Water them the day before inoculation, and dip the flower buds into the suspended bacterial solution for 45s during inoculation. The infected Arabidopsis plants were placed flat and cultured in the dark for 24 hours, and then moved to normal light for culture. Every 5-7 days, the plants were again infected, about 3-4 times. The infected Arabidopsis plants were cultured until the seeds were harvested.

[0045] Stress resistance experiment: After sterilizing the wild-type and transgenic Arabidopsis seeds, they were sown on 1 / 2MS solid culture medium for normal growth. When they grew to the four-leaf stage, they were transplanted into sterilized nutrient soil and cultured in an environment of 16h light / 8h dark and 22±1℃. When they were cultured for 4 weeks, they were irrigated with 100mM and 200mM NaCl solutions for 14 days. The drought treatment was for four-week-old Arabidopsis seedlings, which were naturally drought-stricken for 14 days. Each treatment had a control group with normal watering and an experimental group with stress treatment. At least 100 plants were transplanted for each strain to measure their growth, physiological and biochemical indicators. Table 1 shows the effect of salt stress on the total root length of Arabidopsis, Table 2 shows the effect of salt stress on the root surface area of ​​Arabidopsis, and the external manifestations of the effect of salt stress on Arabidopsis root growth are shown in Table 2. Figure 2-1 Table 3 shows the effect of drought on the total root length of Arabidopsis thaliana, Table 4 shows the effect of drought on the root surface area of ​​Arabidopsis thaliana, and the effect of drought stress on the root growth of Arabidopsis thaliana is shown in Figure 2-2 When plants are subjected to salt and drought stress, the roots are the primary sensing part, so the roots usually suffer more damage earlier than stems and leaves. The results of the Arabidopsis root scanning experiment showed that ( Figure 2-1 , Figure 2-2 ), under salt and drought stress, the total root length and root surface area of ​​wild-type and transgenic Arabidopsis decreased, but the reduction of transgenic Arabidopsis was significantly lower than that of wild-type, especially the total root length and root surface area of ​​transgenic material OE11 under stress were basically the same as those of wild-type plants under untreated conditions. These results indicate that the SsWRKY30 gene improves the root development ability of Arabidopsis under salt and drought stress.

[0046] Table 1

[0047]

[0048] Table 2

[0049]

[0050] Table 3

[0051]

[0052] Table 4

[0053]

[0054] Table 5 shows the effects of salt and drought stress on stomatal aperture, Figures 2-3 show the effects of salt and drought stress on stomatal aperture of Arabidopsis leaves, and Table 6 shows the effects of salt and drought stress on relative water content of Arabidopsis plants. Figure 2-4 The effect of salt and drought stress on the relative water content of Arabidopsis plants; under salt and drought stress, the stomatal opening of the leaves decreases. This is for plants to adapt to the stress environment, reduce plant transpiration, and maintain water and carbon balance in the body. Figure 2-3 As shown in Table 5, under salt and drought stress, the stoma of Arabidopsis leaves decreased, and the stomatal opening of transgenic Arabidopsis was significantly smaller than that of transgenic plants. With the increase of salt concentration, the stomatal opening showed a downward trend. Figure 2-4 , Table 6) The results showed that with the increase of salt and drought stress intensity, the water content of the plant body had a downward trend. Under the same stress treatment conditions, the water content of the transgenic plants was significantly higher than that of the wild plants. The rehydration results after 14 days of drought treatment showed that the transgenic Arabidopsis resumed growth after rehydration, while the wild-type Arabidopsis died. This indicates that the expression of the SsWRKY30 gene improves the tolerance of Arabidopsis to salt and drought by regulating the water content and stomatal aperture under salt and drought stress.

[0055] Table 5

[0056]

[0057] Table 6

[0058]

[0059] Table 7 shows the effects of salt and drought stress on the ion permeability of Arabidopsis plants. Figure 2-5 The effects of salt and drought stress on ion permeability of Arabidopsis plants. Under normal growth conditions, there was no significant difference in IL values ​​between SsWRKY30 transgenic Arabidopsis and wild-type Arabidopsis (p>0.05). -1 After NaCl treatment, the ion permeability of wild-type Arabidopsis increased by 2.63 times compared with that in normal state, and the ion permeability of transgenic lines decreased by 0.733 times compared with that of wild-type Arabidopsis after stress. -1 When the ion permeability value of wild-type Arabidopsis increased by 4.10 times compared with the ion permeability value under normal conditions, the ion permeability value of transgenic lines decreased by 0.80 times compared with wild-type Arabidopsis after stress. Under drought stress, the ion permeability value of wild-type Arabidopsis increased by 3.91 times compared with the ion permeability value under normal conditions, and the ion permeability value of transgenic lines decreased by 0.80 times compared with wild-type Arabidopsis after stress. SsWRKY30 gene expression can improve the tolerance of transgenic Arabidopsis to salt and drought by reducing the maintenance of ion osmotic balance.

[0060] Table 7

[0061]

[0062] Table 8 shows the effects of salt and drought stress on the soluble protein content of Arabidopsis plants. Figure 2-6To investigate the effects of salt and drought stress on the soluble protein content in Arabidopsis plants. The accumulation of soluble protein is an important strategy for plants to cope with salt and drought stress. Figure 2-6 As shown in Table 8, there was no significant difference in the soluble protein (SP) content between transgenic Arabidopsis and Arabidopsis under no stress conditions. Under salt and drought stress, the SP content in the plant gradually increased with the increase of salt concentration. Under 100 mmol·L-1 NaCl treatment, the SP content of the transgenic lines increased by an average of 1.43 times compared with that of the wild-type Arabidopsis. When the salt concentration was 200 mmol·L-1, the SP content of the transgenic lines increased by an average of 1.36 times compared with that of the wild-type Arabidopsis. Under drought stress, the SP content of the transgenic lines increased by 1.3 times compared with that of the wild-type Arabidopsis. In summary, the SsWRKY30 gene can increase the content of soluble proteins of osmotic regulating substances in transgenic Arabidopsis, effectively reduce the cytoplasmic osmotic potential, maintain the water absorption of cells under salinity or drought conditions, and avoid dehydration, thereby improving the tolerance of transgenic Arabidopsis under salt stress.

[0063] The embodiments of the present invention are described in detail above with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge scope of ordinary technicians in this field without departing from the purpose of the present invention.

Claims

1. A Suaeda salsa SsWRKY30 gene, characterized in that: The nucleotide sequence of the Suaeda salsa SsWRKY30 gene is shown in SEQ ID NO.

1.

2. A protein encoded by the SsWRKY30 gene of Suaeda salsa, characterized in that: The amino acid sequence of the protein is shown in SEQ ID NO.

2.

3. An application of the SsWRKY30 gene of Suaeda salsa, characterized in that: For subcellular localization in tobacco.

4. An application of the SsWRKY30 gene of Suaeda salsa, characterized in that: Used to improve plant tolerance to salt and drought.

5. A primer for amplifying the SsWRKY30 gene of Suaeda salsa, characterized in that: For pCAMBIA1300-SsWRKY30-F and pCAMBIA1300-SsWRKY30-R, the nucleotide sequences are as follows: pCAMBIA1300-SsWRKY30-F: 5'-GAGCTCGGTACCCGGGGATCCATGGAACTCAATGGTTGCAC-3' pCAMBIA1300-SsWRKY30-R: 5'-GCCCTTGCTCACCATGTCGACTTCTTTATCTTGAGAAAAGCTAGTAG-3'; The first 6 bases at the 5' end of the primer are protective bases, and the following 6 bases are restriction site sequences. These 11 bases do not belong to the SsWRKY30 gene sequence, but are necessary for constructing a recombinant plant overexpression vector.

6. A plant overexpression vector containing the Suaeda salsa SsWRKY30 gene, characterized in that: is pCAMBIA1300-35S-GFP-SsWRKY30.

7. An application of a plant overexpression vector containing the Suaeda salsa SsWRKY30 gene, characterized in that: The invention relates to the use of the plant overexpression vector pCAMBIA1300-35S-GFP-SsWRKY30 described in claim 4 for subcellular localization in tobacco.

8. An application of a plant overexpression vector containing the Suaeda salsa SsWRKY30 gene, characterized in that: The invention relates to an application of the plant overexpression vector pCAMBIA1300-35S-GFP-SsWRKY30 as claimed in claim 4 in improving the tolerance of plants to salt and drought.

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