PnGST gene of the island plant Psoralea corylifolia and its application in plant salt tolerance breeding

Abstract

The present invention discloses the application of PnGST gene of PnGST gene of PnGST gene of PnGST gene in cultivating varieties with high tolerance to plant salt stress, and belongs to the field of gene engineering technology. The nucleotide sequence of PnGST gene is shown in SEQ ID NO.1, or is completely complementary to the sequence shown in SEQ ID NO.1, or is a nucleotide sequence encoding an amino acid sequence shown in SEQ ID NO.2. The present invention can improve the tolerance of plants to salt stress by improving the expression of PnGST, can be applied to plant genetic engineering breeding, and provide a new method for the creation or improvement of new germplasm for plants to resist salt stress.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and specifically relates to the application of the PnGST gene of Psoralea corylifolia in regulating the salt stress ability of plants or salt stress-related breeding. Background Art

[0002] Affected by high-salt environments such as sea wave invasion or salt spray deposition, plant survival and growth are greatly threatened, and vegetation degradation is serious, which increases the difficulty of protecting and building island vegetation and ecosystems. According to incomplete statistics from UNESCO and FAO, the area of ​​saline-alkali land in the world is 954.38 million hectares, of which 99.13 million hectares are in my country. Breeding excellent salt-resistant plant varieties is an important way to solve island environmental protection and provide food production. However, the genetic regulatory network of plant salt resistance is intricate, and the molecular regulatory mechanisms of plants with different genetic backgrounds vary greatly. Although some genes that regulate plant salt resistance have been reported, discovering and cloning more new genes that have the function of regulating plant salt resistance in different genetic backgrounds, as well as developing new methods for breeding salt-resistant plant varieties, are still technical problems that need to be solved urgently.

[0003] Widely distributed in tropical and subtropical coastal areas, Phyla nodiflora is a perennial vine of the genus Phyla in the family Verbenaceae. Phyla nodiflora is mostly distributed in coastal areas with stronger alkalinity. The soil water retention capacity in such areas is weak, and there are phenomena such as salt fog deposition and seawater backflow. However, Phyla nodiflora still shows tenacious vitality and growth, indicating that it has strong resistance to stress and is a dominant species of native vegetation on tropical islands. In addition, Phyla nodiflora has strong covering ability and strong resistance to disease and foreign invasion. It is not easy to die or degenerate during the vegetation construction process on the island. There are also fewer harmful plants in the area where it is located, and it is relatively easy to prevent and control. It can be used to construct windbreak and sand fixation green space, and is a key species for vegetation restoration on tropical coral islands. The exploration and application of salt-resistance-related genetic resources from the dominant species of native vegetation on tropical islands, the Eupatorium, is of great significance for guiding the screening and cultivation of island stress-resistant plant resources. It also provides an important theoretical basis for the restoration and construction of vegetation and ecosystems on tropical coral islands. It can also provide new genetic resources for the salt-tolerant breeding of tropical coral island plants and provide guidance for the creation and improvement of new island salt-resistant germplasm. Summary of the invention

[0004] The purpose of the present invention is to provide an application of a PnGST gene of Psoralea corylifolia or its orthologous gene in regulating the response of plants to salt stress and / or cultivating salt stress-resistant plant varieties.

[0005] The present invention clones the PnGST gene of PnGST, constructs and compares the phenotype and survival rate of PnGST overexpression strains under salt stress. The PnGST overexpression material and the control are treated with high concentration of NaCl, and it is found that the PnGST transgenic Arabidopsis shows a higher seedling rate and a stronger growth phenotype than the control; at the same time, compared with the control material, a higher level of GST enzyme activity and a lower level of MDA, H2O2 and O2- content are detected in the PnGST transgenic Arabidopsis. The above results show that PnGST transgenic plants have stronger tolerance in the process of dealing with salt stress. Since the PnGST gene comes from PnGST, those skilled in the art have reason to believe that reducing the expression of the PnGST gene in the PnGST plant can reduce the tolerance of PnGST to salt stress, making it more sensitive to salt stress. The nucleotide sequence of the PnGST gene is shown in SEQ ID NO.1, or is completely complementary to the sequence shown in SEQ ID NO.1, or is a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.2. This gene is expected to be applied to genetic engineering breeding of plants, providing a theoretical basis for the creation or improvement of new germplasms that can resist salt stress and are sensitive to salt. Considering the degeneracy of codons, the modification of the bases of the above nucleotide sequence without changing the amino acid sequence also falls within the scope of protection of the present invention.

[0006] In one aspect, the present invention provides a protein for regulating the ability of plants to cope with salt stress, characterized in that the protein sequence is shown in SEQ ID NO.2.

[0007] Furthermore, the present invention provides a gene for regulating the ability of plants to cope with salt stress, characterized in that the gene sequence comprises a nucleotide sequence as shown in SEQ ID NO.1, or comprises a nucleotide sequence that is completely complementary to the sequence shown in SEQ ID NO.1, or comprises a nucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO.2.

[0008] In another aspect, the present invention provides a use of the aforementioned protein or the aforementioned gene in regulating the ability of plants to cope with salt stress.

[0009] Furthermore, the regulating the ability of plants to cope with salt stress is to increase the ability of plants to resist salt stress by overexpressing the aforementioned protein or the aforementioned gene, or to reduce the ability of plants to resist salt stress by reducing the expression of the aforementioned protein or the aforementioned gene.

[0010] In a preferred embodiment, the plant is a dicotyledonous plant.

[0011] Furthermore, the plant is a plant of the family Cruciferae or Verbenaceae.

[0012] Furthermore, the plant is a plant of the genus Arabidopsis or the genus Cyperus.

[0013] Furthermore, the plant is Arabidopsis thaliana or Psoralea corylifolia.

[0014] In another aspect, the present invention provides a use of the aforementioned protein or the aforementioned gene in breeding plant varieties with high tolerance to salt stress and / or sensitivity to salt stress.

[0015] Furthermore, the cultivation of plant varieties with high tolerance to salt stress is to obtain plant varieties with high resistance to salt stress by overexpressing the aforementioned protein or the aforementioned gene, and the cultivation of plant varieties sensitive to salt stress is to obtain plant varieties sensitive to salt stress by reducing the expression of the aforementioned protein or the aforementioned gene. The sensitivity to salt stress means reducing the tolerance of Rhizoma Cibotii to salt stress.

[0016] In a preferred embodiment, the plant is a dicotyledonous plant.

[0017] Furthermore, the plant is a plant of the family Cruciferae or Verbenaceae.

[0018] Furthermore, the plant is a plant of the genus Arabidopsis or the genus Cyperus.

[0019] Furthermore, the plant is Arabidopsis thaliana or Psoralea corylifolia.

[0020] Compared with the prior art, the present invention has the following advantages:

[0021] 1) The present invention provides a novel protein capable of regulating the ability of plants to cope with salt stress.

[0022] 2) The present invention provides new genes that can regulate the ability of plants to cope with salt stress.

[0023] 3) The present invention provides a new method for regulating plant resistance to salt stress, namely, improving the ability of plants to resist salt stress by increasing the expression of PnGST genes or proteins encoded by them, or increasing the sensitivity of plants to salt stress by reducing the expression of PnGST genes or proteins encoded by them.

[0024] 4) The present invention provides a new method for cultivating plant varieties resistant to salt stress, namely, cultivating plant varieties with high resistance to salt stress by increasing the expression of PnGST genes or proteins encoded by them, and / or cultivating plant varieties sensitive to salt stress by reducing the expression of PnGST genes or proteins encoded by them. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The beneficial effects of the present invention are described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0026] Figure 1 Shown is the identification of 35S-PnGST-GFP transgenic lines.

[0027] Figure 2 Shown is the gene expression level analysis of PnGST in Arabidopsis transgenic lines.

[0028] Figure 3 Shown is the protein expression level analysis of PnGST in Arabidopsis transgenic lines.

[0029] Figure 4 The salt sensitivity analysis of PnGST overexpression lines is shown. (a) Phenotypes of wild-type and PnGST overexpression materials grown on 1 / 2MS medium with or without 165mM NaCl for 5 days; (b) Seedling rate statistics of wild-type and PnGST overexpression materials grown on 1 / 2MS medium with or without 165mM NaCl for 5 days; (c) Wild-type and transgenic lines grown for 4 weeks were treated with 300mM NaCl for 5 consecutive days, and the pictures show the phenotypic changes of each plant on the sixth day.

[0030] Figure 5 The figure shows the changes of physiological indicators of PnGST overexpression lines in response to salt stress. (a) GST enzyme activity of wild type and transgenic materials grown on 1 / 2MS medium for 7 days with or without 165mM NaCl for 12h; (b) MDA content of wild type and transgenic materials grown on 1 / 2MS medium for 7 days with or without 165mM NaCl for 12h; (c) H2O2 content of wild type and transgenic materials grown on 1 / 2MS medium for 7 days with or without 165mM NaCl for 12h; (d) O2- content of wild type and transgenic materials grown on 1 / 2MS medium for 7 days with or without 165mM NaCl for 12h. DETAILED DESCRIPTION

[0031] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by ordinary technicians in the technical field to which this application belongs.

[0033] The nucleotide sequence of the PnGST gene of the genus Psoralea corylifolia in the embodiment is shown in SEQ ID NO.1:

[0034]

[0035] The amino acid sequence of the PnGST protein of Psoralea corylifolia is shown in SEQ ID NO.2:

[0036] MRRASGLAAIRRAVGGVAAESSATGNDFQLQHRVFQAALFSTNSGNSSSSARSHWFSYNRIADDRSGRASARAVAGTMLFS

[0037] MAATTLVEEVHAKEPVQSRFRPKDVILYQYEACPFCNKVKAFLDYYDIPYKIVEVNPISKKEIKWSEYKKVPILVVDGEQ

[0038] MVDSSDIINRLTNKIYPDAPTDSSGEESEEKKWRSWVDSHLVHLLSPNIYRSSSEALESFDYITSHGNFSFTERLTAKYA

[0039] GAVAMYFVSKKLKKKYNITDERAALYEAAEAWVDALKGQDFLGGTKPNLADLAVYGVLRPIRYLRSGRDMIENTRIGDWY

[0040] SRMENAVGAPSRIQA

[0041] Example 1

[0042] Construction and identification of transgenic materials

[0043] First, RNA was extracted from the leaves of P. sutchuenensis using an RNA extraction kit (Vazyme), and cDNA was synthesized according to the operating instructions of the first-strand reverse transcription kit of Imagene. The cDNA obtained by reverse transcription of P. sutchuenensis RNA was used as a template, and the cDNA sequence of the PnCAT2 gene was obtained by PCR amplification using F primer (ACCATGGTAGATCTGACTAGTATGAGGAGGGCTAGCGGACTC) and R primer (AAGTTCTTCTCCTTTACTAGTAGCCTGGATTCTTGAGGGTGC) and KOD FX high-fidelity enzyme (ToYoBo). The reaction system was: 2×KOD buffer 25μL, 2mM dNTPs 8μL, F primer and R primer 1.5μL each, cDNA template 2μL, KOD FX (1U / μl) 0.5μL, and water was added to make up to 50μL. The reaction procedure was: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 sec, annealing at 56°C for 30 sec, extension at 68°C for 60 sec, 32 cycles; final extension at 68°C for 5 min. After the reaction, the PCR product was recovered by a gel recovery kit (Magen). The pCAMBIA1302 binary vector with a GFP tag was then linearized using the restriction endonuclease SpeI (NEB). The enzyme digestion system was: 10 μg of vector, 2 μL of SpeI endonuclease, 5 μL of 10× buffer, and water was added to 50 μL. The digestion conditions were: 37°C for 2 hours. After the reaction, the digestion product was recovered by a gel recovery kit (Magen).

[0044] We inserted the gene fragment into the pCAMBIA1302 binary vector by homologous recombination. The recombination system was: 40 ng of the target fragment, 240 ng of the vector fragment, 2.5 μL of 2×MultiF Seamless Assembly Mix (Abclonal), and a total volume of 5 μL. The recombination conditions were: 50°C, 15 min. The constructed recombinant plasmid was transferred into 50 μL of E. coli competent cells DH5α and spread on LB solid medium containing kanamycin (working concentration of 50 mg / L), inverted in a 37°C incubator for overnight culture, and then a single clone was picked for PCR and sequencing (Qingke Biotechnology). The sequencing primers were 1302-CEXU-R: TCACCTTCACCCTCTCCACT and W1R1114-W1040-F05: TGTTCTCCATCAACCACGAGT. The clones with correct sequencing were positive recombinant plasmids. We then transferred it into 100 μL of Agrobacterium competent cells GV3101, and transferred the PnGST gene into Arabidopsis by the floral dipping method. The harvested T1 generation seeds were spotted on a 1 / 2MS solid medium containing 50 μg / L hygromycin to screen out transgenic positive seedlings (positive seedlings grew normally, non-positive seedlings grew abnormally or even did not grow). The positive seedlings were cultured until the T2 generation seeds were harvested, and the seeds were continued to be spotted on a 1 / 2MS solid medium containing hygromycin. If all the seeds grew normally, it was proved that the strain was a homozygous plant.

[0045] We then used primers F (AGGAGGTTCACGCGAAAGAG) and R (TCACCTTCACCCTCTCCACT) to identify multiple 35S-PnGST-GFP transgenic lines by PCR ( Figure 1 ). The reaction system was: 2×Taq mix (Sangon Biotech Co., Ltd.) 10 μL, F primer and R primer 1 μL each, template 2 μL, water was added to make up to 20 μL. The reaction procedure was: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 sec, annealing at 56°C for 30 sec, extension at 72°C for 60 sec, 32 cycles; final extension at 72°C for 5 min.

[0046] Example 2

[0047] Analysis of PnGST expression levels in Arabidopsis transgenic lines

[0048] RNA was extracted from the leaves of P. sutchuenensis using an RNA extraction kit (Vazyme), and cDNA was synthesized according to the instructions of the first-strand reverse transcription kit of Imagene. The primer pairs of PnGST-qF (CACGGCAACTTCAGCTTCAC) and PnGST-qR (CACGGCAACTTCAGCTTCAC) and the primer pairs of AtUBQ5-qF (GACGCTTCATCTCGTCC) and AtUBQ5-qR (CCACAGGTTGCGTTAG) of the housekeeping gene AtUBQ5 of Arabidopsis thaliana were used as internal references. qRT-PCR was performed using the Taq Pro Universal SYBR qPCR Master Mix (Vazyme) kit to detect gene expression. The reaction system was: 2×qPCR Master Mix 5 μL, qF primer and qR primer 1 μL each, cDNA template 2 μL, and water was added to 10 μL. The qRT-PCR amplification reaction was completed by a BIO-RAD CFX96 fluorescence quantitative PCR instrument, and the program settings were as follows: 95℃30sec; 95℃5sec, 68℃30sec, 45 cycles. The qRT-PCR fluorescence quantitative experiment found that the gene expression level of PnGST in the Arabidopsis transgenic lines was greatly improved ( Figure 2 ).

[0049] Subsequently, we analyzed the protein expression level of PnGST in Arabidopsis transgenic lines by western blot technology ( Figure 3 ). Protein SDS-PAGE gel electrophoresis is mainly used Tetra electrophoresis tank (BIO-RAD) and its supporting equipment, transfer membrane mainly uses small Mini Transfer tank (BIO-RAD) and its supporting equipment. The total protein of transgenic plants was extracted according to the plant total protein extraction kit of Sangon Biotechnology. After adding the corresponding amount of 5×SDS loading buffer to the protein sample, it was electrophoresed at 130V, 350mA, 1.5h. Then the protein separated in the SDS-PAGE gel was transferred to the PVDF membrane at 130V, 350mA, 1.5h. The PVDF membrane containing the protein band was blocked with 5% skim milk (TBST) for 1h, and then incubated with Actin or GFP primary antibody (Abcam, dilution ratio of 1:5000) at 4℃ overnight, and the corresponding secondary antibody (Abcam, dilution ratio of 1:5000) was incubated for 2h. Finally, the protein was detected with ECL chemiluminescent solution (Bio-RAD) on the ChemiDoc TM MP Imaging System (Bio-RAD), and finally a stable transgenic strain that can be used for subsequent functional verification was identified. We selected PnGST#18 and PnGST#20 overexpression lines for subsequent experiments.

[0050] Example 3: Phenotypic response of PnGST transgenic lines to high salt stress

[0051] The salt stress sensitivity analysis of wild-type and transgenic lines grown on 1 / 2MS medium containing 165mM NaCl (1 / 2MS medium without NaCl added as control) for five days showed that the seedling rates of WT and overexpression lines on 1 / 2MS medium were basically the same, while under salt stress conditions, the seedling rate of overexpression lines was higher than that of control ( Figure 4 a, b). In addition, we treated Arabidopsis plants that had grown normally for 4 weeks with 300 mM NaCl. After 5 consecutive days of treatment, we observed the phenotypic changes of the plants and found that compared with the control (treated with the same amount of deionized water), the NaCl-treated materials showed different degrees of growth inhibition. Compared with the wild type, the phenotypes of delayed seedling development and leaf wilting in PnGST transgenic Arabidopsis were milder ( Figure 4 c) The above results show that overexpression of PnGST can improve the salt tolerance of plants.

[0052] Example 4: Analysis of physiological indicators of PnGST transgenic lines in response to high salt stress

[0053] Next, we analyzed the changes in physiological indicators of wild-type and PnGST Arabidopsis transgenic lines under salt stress. The results showed that there was no significant difference in the GST enzyme activity, MDA, H2O2 and O2- contents in the wild-type control and overexpression lines grown normally on 1 / 2MS plates for 7 days; however, after adding 165mM NaCl to the 1 / 2MS plate and continuing to culture for 12h, the GST enzyme activity of the overexpression PnGST gene plate seedlings increased to a certain extent compared with the control, while the MDA, H2O2 and O2- contents were lower than the control, and the O2- content was significantly reduced compared with the control ( Figure 5 ). The results showed that PnGST overexpression could increase the activity of GST enzyme in Arabidopsis thaliana, reduce the accumulation of reactive oxygen toxic substances, and thus improve the plant's resistance to high salt stress.

[0054] Based on the above results, it is confirmed that the PnGST gene can improve the tolerance of plants to salt stress, and can therefore be used in the genetic breeding of island salt-tolerant plants.

[0055] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to the above embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A protein for regulating the ability of plants to cope with salt stress, characterized in that: The protein sequence is shown in SEQ ID NO.

2.

2. A gene that regulates a plant's ability to cope with salt stress, characterized in that: The gene sequence encodes the amino acid sequence shown in SEQ ID NO.

2.

3. Use of the protein according to claim 1 or the gene according to claim 2 in regulating the ability of plants to cope with salt stress, characterized in that: The method for regulating the ability of a plant to cope with salt stress is to improve the ability of a plant to resist salt stress by overexpressing the protein described in claim 1 or the gene described in claim 2, and the plant is Arabidopsis thaliana.

4. Use of the protein according to claim 1 or the gene according to claim 2 in cultivating plant varieties with high tolerance to salt stress, characterized in that: The plant variety cultivated with high tolerance to salt stress is obtained by overexpressing the protein described in claim 1 or the gene described in claim 2 to obtain the plant variety with high tolerance to salt stress, and the plant is Arabidopsis thaliana.

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