Salt-tolerant protein from populus euphratica and application of salt-tolerant protein

Abstract

The invention relates to a salt-tolerant protein derived from populus euphratica and application of the salt-tolerant protein. The amino acid sequence of the salt-tolerant protein is as shown in SEQ ID No. 4. The salt-tolerant protein gene is transferred into a poplar, and after the poplar expresses the protein, the poplar can show the salt-tolerant characteristic, so that the protein can be expressed in a plant, and the plant can obtain the salt-tolerant characteristic.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of functional proteins and genes, in particular to a salt-tolerant protein, a gene encoding the same and applications thereof. BACKGROUND

[0002] Soil salinization is one of the severe challenges faced by global agricultural production and ecological environment. According to statistics, the area of salinized soil accounts for about 10% of the total land area in the world, and is expanding year by year. The saline-alkali land in China is mainly distributed in the northeast, north China, northwest and coastal areas, with a total area of more than 500 million mu. Salt-alkali stress can cause a series of physiological metabolic disorders such as dehydration, ion imbalance and oxidative damage in plant cells, seriously inhibiting plant growth and development, and even leading to plant death.

[0003] Poplar is an important fast-growing timber tree species and ecological protection forest tree species, widely used in industrial timber, landscaping and ecological restoration of saline-alkali land. However, most poplar varieties have weak salt tolerance, grow slowly and have low survival rate in salinized soil, which seriously limits their popularization and application in saline-alkali areas. Traditional poplar salt-tolerant breeding methods have long cycle and low efficiency, which is difficult to meet the urgent needs of production practice for salt-tolerant poplar varieties. SUMMARY

[0004] The present application provides a salt-tolerant protein, the amino acid sequence of which is shown in SEQ ID No. 4.

[0005] The present application provides a nucleic acid encoding the salt-tolerant protein of claim 1.

[0006] In one embodiment, the base sequence of the nucleic acid is shown in SEQ ID No. 3.

[0007] The present application provides the use of the salt-tolerant protein of the present application or the nucleic acid of the present application in plant salt tolerance.

[0008] In one embodiment, the plant is one of the Salicaceae family.

[0009] In one embodiment, the plant is one of the Populus genus. Populus ).

[0010] In one embodiment, the plant is Populus tomentosa Populus alba )and / or Populus glandulosus Populus glandulosa .

[0011] In one embodiment, the plant is 84K poplar.

[0012] In one embodiment, the nucleic acid as described in the second aspect of the present application is introduced into the plant to make the plant express the protein as described in one of the aspects of the present application, to obtain a transgenic plant, which exhibits salt tolerance.

[0013] The present application has the following beneficial effects: the protein having the amino acid sequence as shown in SEQ ID No. 4 is found to have salt tolerance, and when the gene is introduced into a poplar (e.g. 84K poplar) to make the poplar express the protein, the poplar can exhibit salt tolerance, and thus the protein can be used to express in a plant to make the plant have salt tolerance. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 PCR identification of PeA salt tolerance gene in transgenic seedlings PeA1 to PeA10 is shown, with wild type 84K poplar seedlings as negative control.

[0015] Figure 2 Relative transcription level of PeA salt tolerance gene in wild type 84K poplar seedlings and transgenic seedlings PeA1 to PeA10 is shown, with wild type 84K poplar seedlings as negative control.

[0016] Figure 3 Phenotype, relative conductivity, net photosynthetic rate, stomatal conductance, intercellular CO2 concentration, transpiration rate and dry weight of whole plant of 2-month-old seedlings are shown, with wild type 84K poplar seedlings as control.

[0017] Figure 4 Phenotype, relative conductivity, net photosynthetic rate, stomatal conductance, intercellular CO2 concentration, transpiration rate and dry weight of whole plant of 4-month-old seedlings are shown, with wild type 84K poplar seedlings as control.

[0018] Figure 5 Contents of MDA, POD, CAT and APX in 4-month-old seedlings are shown, with wild type 84K poplar seedlings as negative control.

[0019] Figure 6 Na + content, K + content and Na + / K + ratio in roots, stems and leaves of 4-month-old seedlings are shown, with wild type 84K poplar seedlings as control. DETAILED DESCRIPTION

[0020] The above content of the present application is further explained in detail in the form of preferred embodiments, which do not constitute limitation to the present application.

[0021] Unless otherwise specified, the strains, plasmids, and reagents used in the embodiments of this invention can all be purchased commercially.

[0022] RNA Extraction Kit: RN38-EASYspin Plus Plant (Polysaccharide and Polyphenol) RNA Rapid Extraction Kit, purchased from Adley Biotechnology Co., Ltd., catalog number RN3801.

[0023] Reverse transcription kit: PrimeScript™ RT reagent Kit with gDNA Eraser, purchased from Takara Bio Engineering (Dalian) Co., Ltd. (Dalian), catalog number: RR047A.

[0024] TransStart Top Green qPCR SuperMix was purchased from TransGen Biotech Ltd., product number: AQ131-04.

[0025] The cloning vector pMD18-T was purchased from Takara in Dalian.

[0026] The plant expression vector pCAMBIA1300-35S was purchased from Wuhan Miaoling Biotechnology Co., Ltd., product number: P17949.

[0027] Agrobacterium competent cells EHA105 were purchased from Shanghai Weidi Biotechnology Co., Ltd., WEIDI, catalog number: AC1013.

[0028] LB solid medium: tryptone 10.0 g / L, yeast extract 5.0 g / L, NaCl 10.0 g / L, agar 15 g / L, sterilized at 121 degrees Celsius for 20 min.

[0029] LB liquid medium: tryptone 10.0 g / L, yeast extract 5.0 g / L, NaCl 10.0 g / L, sterilized at 121 degrees Celsius for 20 min.

[0030] The wild-type poplar variety used in this invention is 84K poplar, a hybrid clonal line of the poplar family, with its female parent being Populus tomentosa (white poplar). Populus alba The father is *Populus glomeratus* (Glandularia spp.) Populus glandulosa ). Example 1: Cloning of the PeA salt tolerance gene in Populus euphratica

[0031] Select healthy potted poplar trees ( Populus euphratica Leaf samples were taken from *Populus euphratica* seedlings and ground in liquid nitrogen. Total RNA was extracted from the leaves using an RNA extraction kit. The integrity and purity of the RNA were assessed by agarose gel electrophoresis and a Nanodrop 2000 UV spectrophotometer (Thermo Fisher Scientific) to ensure that the RNA was undegraded and of acceptable purity.

[0032] The total RNA of the extracted leaves was used as a template to synically the first strand of cDNA by using a reverse transcription kit, and then the cDNA was used as a template, PeAF1 (the sequence of which is shown in SEQ ID No. 1) and PeAR1 (the sequence of which is shown in SEQ ID No. 2) were used as a primer pair, and high-fidelity DNA polymerase was used for PCR (polymerase chain reaction) amplification to obtain a PCR product. After purification of the PCR product, it was connected to the pMD18-T vector, and the E. coli DH5a competent cells were transformed, and then cultured on LB solid medium containing ampicillin for 8 to 10 hours, and then a single colony was picked and identified by PCR with PeAF1 and PeAR1 as a primer pair, and a positive recombinant bacteria was screened and named as DH5a / pMD18-PeA, wherein the positive recombinant plasmid is pMD18-PeA. The positive bacteria / DH5a / pMD18-PeA was sent to Beijing Lihe Huada Gene Technology Co., Ltd. for sequencing, and the obtained sequence is shown in SEQ ID No. 3, and the amino acid sequence of the encoded protein is shown in SEQ ID No. 4. Example 2: Obtaining Populus euphratica salt-tolerant gene PeA poplar seedlings

[0033] The plant expression vector pCAMBIA1300-35S was double digested with restriction enzymes BamH I and Hind III, and the enzyme digestion product was recovered by agarose gel electrophoresis and agarose gel recovery kit. The pMD18-PeA plasmid was used as a template, PeAF2 (the sequence of which is shown in SEQ ID No. 5) and PeAR2 (the sequence of which is shown in SEQ ID No. 6) were used as a primer pair, and high-fidelity DNA polymerase was used for PCR (polymerase chain reaction) amplification to obtain a PCR product for homologous recombination. The PCR product for homologous recombination was purified and connected to the pCAMBIA1300-35S vector backbone by using a universal one-step seamless cloning kit (Jiangsu Kangweishijisuo Biological Technology Co., Ltd., CW3036M). The recombinant product was transformed into E. coli DH5a competent cells, and then cultured on LB solid medium containing kanamycin for 8 to 10 hours, and then a single colony was picked and identified by PCR with PeAF1 and PeAR1 as a primer pair, and a positive recombinant bacteria was screened and named as DH5a / pCAMBIA1300-35S-PeA, wherein the positive recombinant plasmid is pCAMBIA1300-35S-PeA.

[0034] 1 microgram of pCAMBIA1300-35S-PeA expression vector was added to 50 microliters of Agrobacterium EHA105 competent cells for transformation, then 800 microliters of LB liquid medium was added and mixed, transferred to a 2 milliliter centrifuge tube, cultured at 28 degrees Celsius, 220 rpm for 1.5 hours; the bacterial solution was centrifuged at 12000 rpm for 1 minute, and the supernatant was discarded; 100 microliters of LB liquid medium was added to suspend thoroughly, and then uniformly coated on LB solid medium containing kanamycin and rifampicin, and cultured at 28 degrees Celsius for 3 to 4 days, and a single colony was picked and identified by PCR and sequencing to obtain a positive clone, and a positive strain EHA105 / pCAMBIA1300-35S-PeA was obtained.

[0035] Sterile tissue culture seedlings of 84K poplar were cultured, and 84K poplar sterile leaves were cut into small pieces of 0.5 cm x 0.5 cm, and the leaf pieces were immersed in OD 600 =0.5 EHA105 / pCAMBIA1300-35S-PeA bacterial solution for 10 min, then the surface bacterial solution was absorbed with sterile filter paper, and transferred to co-culture medium (MS medium + 0.05 mg / L naphthalene acetic acid + 0.5 mg / L 6-benzylaminopurine) for 2 to 3 days of culture. The callus after co-culture was transferred to screening medium (MS + 0.05 mg / L naphthalene acetic acid + 0.50 mg / L 6-benzylaminopurine + 200 mg / L timentin + 5 mg / L hygromycin), and the resistant callus was subcultured every 2 weeks. When the resistant callus differentiated into buds, and the buds grew to 3 to 5 cm, they were transferred to rooting medium (1 / 2 MS + 0.05 mg / L indole butyric acid + 0.01 mg / L indole acetic acid + 200 mg / L timentin + 3 mg / L hygromycin) to induce rooting, and 10 transgenic seedlings for molecular identification were obtained, and were marked as PeA1 to PeA10. Wild type 84K poplar seedlings (WT) were used as negative controls for culture.

[0036] Genomic DNA was extracted from the leaves of the transgenic seedlings PeA1 to PeA10 for molecular identification and wild type 84K poplar seedlings, and PCR identification was performed with each DNA as a template and PeAF1 and PeAR1 as primer pairs. If the target fragment was amplified, the transgenic seedlings for molecular identification were positive plants, and the results are shown in Figure 1 . Figure 3 The results show that the 10 transgenic seedlings PeA1 to PeA10 for molecular identification are all positive.

[0037] Total RNA was extracted from leaves of transgenic seedlings PeA1 to PeA10 and wild-type 84K poplar seedlings. Using the total RNA from transgenic seedlings PeA1 to PeA10 as templates, first-strand cDNA was synthesized using a reverse transcription kit. Then, using the cDNA from each seedling as templates, primer pairs qRT-PeAF (sequence shown in SEQ ID No. 7) and qRT-PeAR (sequence shown in SEQ ID No. 8) were used; and Actin7-F (sequence shown in SEQ ID No. 9) and Actin7-R (sequence shown in SEQ ID No. 10) were used to amplify the actin 7 gene as an internal control. actin7 Primer pairs were used for RT-qPCR with TransStart Top Green qPCR SuperMix, as follows: Figure 2 As shown, the expression level of PeA in the three transgenic seedlings PeA1, PeA2 to PeA3 was significantly higher than that in other transgenic seedlings.

[0038] PeA1, PeA2, and PeA3 seedlings, along with wild-type seedlings, were propagated by micro-insertion into subculture medium (1 / 2 MS + 0.02 mg / L naphthaleneacetic acid + 0.05 mg / L indolebutyric acid), yielding multiple seedlings from each line. Wild-type 84K poplar seedlings (WT) were used as a negative control. Example 3: Salt tolerance test of transgenic poplar seedlings

[0039] Six identical transgenic seedlings and six wild-type seedlings were selected from each line in Example 2 and transplanted into pots containing substrate. The six seedlings from each line were divided into two groups: one group was cultured for two months (2 months old), and the other for four months (4 months old). Then, the 2-month-old and 4-month-old seedlings were watered with a 200 mM NaCl aqueous solution, 500 mL per pot, twice a week. Each group had three replicates, with one seedling constituting one replicate. After two weeks of treatment, the phenotypes of seedlings at both ages were photographed and recorded. Relative conductivity was measured using a conductivity meter, and net photosynthetic rate, stomatal conductance, intercellular CO2 concentration, and transpiration rate were measured using a LI-6400 photosynthesis system. For 4-month-old seedlings, malondialdehyde (MDA) content was determined using the thiobarbituric acid method, and peroxidase (POD) activity was determined using the guaiacol method. Catalase (CAT) activity was determined using ultraviolet absorption spectrometry, and ascorbate peroxidase (APX) activity was determined using the ascorbate oxidation method. Finally, the dry weight of the entire plant, including roots, was measured for both 2-month-old and 4-month-old seedlings. Na+ content in the dry roots, stems, and leaves of 4-month-old seedlings was determined using atomic absorption spectrometry (AAS). + and K + Content, and calculate Na+ / K + Compare. See results. Figures 3 to 6 .

[0040] Figure 3 and Figure 4 The results showed that under salt stress, the growth of both 2-month-old and 4-month-old transgenic seedlings was significantly better than that of wild-type seedlings. For example, in 2-month-old and 4-month-old seedlings treated with salt, the electrical conductivity of wild-type seedlings reached 0.77%, while that of transgenic seedlings was only 0.44% to 0.49%. The net photosynthetic rate, stomatal conductance, intercellular CO2 concentration, and transpiration rate of transgenic seedlings were all significantly higher than those of wild-type seedlings, and the dry weight of transgenic seedlings was significantly higher than that of wild-type seedlings. These results indicate that PeA can improve plant growth, photosynthetic capacity, and cell membrane stability under salt stress.

[0041] Figure 5 The results showed that the MDA (oxidative damage) content in transgenic seedlings under salt stress decreased by 24.6% to 43.6% compared to the wild type. Regarding antioxidant enzymes, the CAT content in transgenic seedlings increased by 1.4 to 1.7 times compared to the wild type, the APX content increased by 1.5 to 2.4 times, and the POD content increased by 1.7 to 2.0 times. In summary, this indicates that the PeA transcription factor significantly enhances plant salt tolerance by regulating reactive oxygen species balance.

[0042] Figure 6 The results showed that Na in transgenic seedlings under salt stress + The content was reduced by 38% to 63% compared to the wild type; K + The content increased by 1.3 to 3.5 times compared to the wild type; the Na content of the transgenic type was 1.3 to 3.5 times higher. + / K + The reduction rate ranged from 11.4% to 55.1% compared to the wild type. These differences indicate that... PeA Overexpression can significantly reduce Na + Accumulate and improve K + The content of Na will then decrease + / K + The ratio enhances the plant's salt tolerance by maintaining ion homeostasis.

Claims

1. A salt-tolerant protein having the amino acid sequence shown in SEQ ID No.

4.

2. The nucleic acid encoding the salt-tolerant protein of claim 1.

3. The nucleic acid according to claim 2, characterized in that, The base sequence of the nucleic acid is shown in SEQ ID No.

3.

4. The application of the salt-tolerant protein according to claim 1 or the nucleic acid according to claim 2 or 3 in the application of salt tolerance in plants.

5. The application according to claim 4, characterized in that, The plant in question is a species of the Salicaceae family.

6. The application according to claim 5, characterized in that, The plant in question belongs to the genus *Populus*. Populus One of them.

7. The application according to claim 5, characterized in that, The plant in question is Populus tomentosa (white poplar). Populus alba ) and / or glandular poplar ( Populus glandulosa ).

8. The application according to claim 5, characterized in that, The plant in question is an 84K poplar.

9. The application according to any one of claims 4 to 8, characterized in that, The nucleic acid as described in claim 2 or 3 is transferred into the plant to make the plant express the protein as described in claim 1, thereby obtaining a transgenic plant that exhibits salt tolerance.

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