The PnCAT2 gene of the island plant Psoralea corylifolia and its application in plant salt tolerance breeding

Abstract

The present invention discloses the application of PnCAT2 gene of PnCAT2 gene of PnCAT2 gene in cultivating varieties with high tolerance to plant salt stress, and belongs to the field of gene engineering technology. The nucleotide sequence of PnCAT2 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 PnCAT2, 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 PnCAT2 gene of Psoralea corylifolia in regulating the salt stress ability of plants or salt stress-related breeding. Background Art

[0002] The South China Sea Islands are located at the southernmost tip of my country. Due to their location in the center of the ocean and their small area, they are vulnerable to high-salt environments such as wave invasion or salt fog 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. Cultivating 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 cultivating 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 PnCAT2 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 PnCAT2 gene of PnCAT2, constructs and compares the phenotype and survival rate of PnCAT2 overexpression strains under salt stress. The PnCAT2 overexpression material and the control are treated with high concentration of NaCl, and it is found that the PnCAT2 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 CAT2 enzyme activity and a lower level of MDA, H2O2 and O2-content are detected in the PnCAT2 transgenic Arabidopsis. The above results show that PnCAT2 transgenic plants have stronger tolerance in the process of dealing with salt stress. Since the PnCAT2 gene comes from PnCAT2, those skilled in the art have reason to believe that reducing the expression of the PnCAT2 gene in the PnCAT2 plant can reduce the tolerance of the PnCAT2 to salt stress, making it more sensitive to salt stress. The nucleotide sequence of the PnCAT2 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 a plant to cope with salt stress, wherein the protein sequence is as 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] In a preferred embodiment, the ability of the plant to resist salt stress refers to a higher germination rate of plant seeds under salt stress conditions.

[0021] In another preferred embodiment, the ability of the plant to resist salt stress refers to a higher seedling rate of plant seedlings under salt stress conditions.

[0022] In another preferred embodiment, the ability of the plant to resist salt stress refers to the plant having a longer root length and / or a higher plant height under salt stress conditions.

[0023] In a preferred embodiment, the ability of the plant to resist salt stress is produced by reducing the content of MDA, H2O2 and / or O2-.

[0024] Compared with the prior art, the present invention has the following advantages:

[0025] 1) The present invention provides a novel protein capable of regulating the ability of plants to cope with salt stress.

[0026] 2) The present invention provides new genes that can regulate the ability of plants to cope with salt stress.

[0027] 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 PnCAT2 gene or the protein encoded by it, or increasing the sensitivity of plants to salt stress by reducing the expression of PnCAT2 gene or the protein encoded by it.

[0028] 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 PnCAT2 gene or the protein encoded by it, and / or cultivating plant varieties sensitive to salt stress by reducing the expression of PnCAT2 gene or the protein encoded by it. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The beneficial effects of the present invention are described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0030] Figure 1 Shown is the identification of 35S-PnCAT2-GFP transgenic lines.

[0031] Figure 2 Shown is the gene expression level analysis of PnCAT2 in Arabidopsis transgenic lines.

[0032] Figure 3 Shown is the protein expression level analysis of PnCAT2 in Arabidopsis transgenic lines.

[0033] Figure 4 Shown is the salt sensitivity analysis of PnCAT2 overexpression lines. NaCl sensitivity analysis. (a) Phenotypes of wild type (WT) and PnCAT2 overexpression materials grown on 1 / 2MS and 1 / 2MS plates supplemented with 165mM NaCl for 5 days; (b) Germination rate analysis of wild type and PnCAT2 overexpression materials grown on 1 / 2MS and 1 / 2MS plates supplemented with 165mM NaCl for 5 days; (c) Seedling rate analysis of wild type and PnCAT2 overexpression materials grown on 1 / 2MS and 1 / 2MS plates supplemented with 165mM NaCl for 5 days; (d) Root length phenotypes of wild type and PnCAT2 overexpression materials grown on 1 / 2MS medium for 5 days and then transferred to 1 / 2MS and 1 / 2MS plates supplemented with 120mM NaCl for 5 days; (e) Phenotypic changes of each plant material after continuous spraying of clean water and 300mM NaCl for 5 days in wild type and PnCAT2 overexpression Arabidopsis grown in soil for 4 weeks.

[0034] Figure 5Analysis of physiological indicators of PnCAT2 overexpression strains under salt stress. (ad) Wild-type and PnCAT2 overexpression materials grown on 1 / 2MS medium for 7 days were sprayed with water and 165 mM NaCl, and CAT enzyme activity, MDA, H2O2 and O2- contents of each plant material were measured after 12 h of treatment. DETAILED DESCRIPTION

[0035] 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.

[0036] 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.

[0037] The nucleotide sequence of the PnCAT2 gene of the embodiment is shown in SEQ ID NO.1:

[0038]

[0039] The amino acid sequence of PnCAT2 protein from Psoralea corylifolia is shown in SEQ ID NO.2:

[0040] MDPYKYRPSTSFNTPYMTTNSGLPVWNDNNSQTIGNRGPILLEDYHLVEKISNFTRERIPERVVHARGATAKGFFEVTHD

[0041] ITHLTCADFLRAPGVQTPVLVRFSTVIHERGSPETMRDPRGFATKFYTREGNFDLVGNNFPVFFVRDPMKFPDVIHAFKP

[0042] NPKTNIQENWRIFDFLCHHPESLSTFTFFYDDVGIPTDYRHIEGSSVQAYTMINKAGKVHYVMFSWRPSLGVKCLTDEEA

[0043] IRVGGSNHSHATKDLYDAIAAGNYPEWKLYIQVMDPEHEDRYDFDPLDTTKTWPEDQLPLIPVGRLVLNKNIDNFFQENE

[0044] MVAMNPAHIVPGIHYSDDKMLQGRVFAYGDTQRYRLGPNYLQLPVNAPKCPHHNNHHDGAMSFLHRDEEVNYFPSKYDTC

[0045] RHAERFPIPSVVLKGRRERRIIDKENDFKEPGERYRSWDPARQERFIKRLADGLSDPRLTPEIRSIWVGYWSQADRSLGQ

[0046] KLASRLNMRPMM

[0047] Example 1 Construction and identification of transgenic materials

[0048] 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 (ACCATGGTAGATCTGACTAGTATGGATCCCTACAAGTACCGT) and R primer (AAGTTCTTCTCCTTTACTAGTCATCATCGGCCTCATGTTGAG) 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).

[0049] 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 Agrobacterium competent cells GV3101, and transferred the PnCAT2 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.

[0050] We then used primers F (TCCCAGAGGATTCGCCACTA) and R (TCACCTTCACCCTCTCCACT) to identify multiple 35S-PnCAT2-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.

[0051] Example 2 Analysis of the expression level of PnCAT2 in Arabidopsis transgenic lines

[0052] 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 PnCAT2-qF (CGGGATTCACTACTCGGACG) and PnCAT2-qR (CCGGGCTCCTTGAAGTCATT) 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 PnCAT2 in the Arabidopsis transgenic lines was significantly increased ( Figure 2 ).

[0053] Subsequently, we analyzed the protein expression level of PnCAT2 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, the transfer membrane mainly uses the 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 PnCAT2#3, PnCAT2#14, and PnCAT2#20 overexpression lines for subsequent experiments.

[0054] Example 3: Phenotypic response of PnCAT2 transgenic lines to high salt stress

[0055] We simultaneously planted seeds of wild-type WT and PnCAT2 transgenic lines on 1 / 2MS culture plates containing or without NaCl, and observed and counted the seed germination rate and seedling rate of the materials for 5 consecutive days. The results showed that on the culture medium without NaCl, there was no significant difference in germination and growth between the overexpression line and the wild type; however, under the condition of NaCl concentration of 165mM, the PnCAT2 overexpression line had a higher germination rate and seedling rate than the wild type ( Figure 4 ac). Subsequently, we observed the taproot growth of wild-type and PnCAT2 overexpression lines in vertical culture on 1 / 2MS plates. The results showed that there was no significant difference in taproot growth of plant materials under normal conditions; next, we transferred seedlings grown on 1 / 2MS medium for 5 days to 1 / 2MS plates supplemented with 120mM NaCl. After 5 days of growth, we found that the root length of the PnCAT2 overexpression line was significantly longer than that of the control ( Figure 4 d), indicating that the PnCAT2 overexpression strain has a strong salt tolerance.

[0056] In addition, we also tested the tolerance of PnCAT2 overexpression lines grown in soil for 4 weeks to salt stress. We sprayed NaCl for 5 consecutive days on transgenic and wild-type seedlings, and used water spraying as a control. The results showed that there was no significant difference in the growth of transgenic and wild-type lines sprayed with water, but after continuous spraying of 300mM NaCl, the seedling height of the PnCAT2 overexpression line was significantly higher than that of the control, and the degree of leaf wilting and curling was significantly lower than that of the control ( Figure 4 e), the above results indicate that PnCAT2 overexpression can enhance Arabidopsis resistance to high salt treatment.

[0057] Example 4: Analysis of physiological indicators of PnCAT2 transgenic lines in response to high salt stress

[0058] Next, we analyzed the activity of CAT enzymes and the accumulation of reactive oxygen species in PnCAT2 Arabidopsis transgenic lines under salt stress. We first measured the CAT activity and the contents of MDA, H2O2, and O2- in seedlings grown on 1 / 2MS plates for 7 days. The results showed that there were no significant differences in the physiological indicators of the various plant materials. Subsequently, we sprayed 165mM NaCl on seedlings grown on 1 / 2MS plates for 7 days. After culturing for 12 hours, we measured various indicators of transgenic seedlings and wild-type seedlings and found that the contents of MDA, H2O2, and O2- in PnCAT2 overexpression lines were significantly lower than those in the control, while the activity of CAT enzymes in PnCAT2 overexpression lines was significantly higher than that in the control ( Figure 5 ).

[0059] Based on the above results, it is confirmed that the PnCAT2 gene can improve the tolerance of plants to salt stress, and can therefore be used in genetic breeding to improve the salt tolerance of island plants.

[0060] 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 sequence of the protein 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 according to claim 1 or the gene according to 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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