Application of rice OsCLE-W gene in improving plant salt and alkali tolerance
By cloning and overexpressing the rice OsCLE-W gene, the problem of inhibited growth and development of rice under high pH alkaline stress was solved, and the tolerance and biomass maintenance of rice in saline-alkali environment were improved, thus enhancing the stress resistance and yield of rice.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- HUNAN AGRI UNIV
- Filing Date
- 2026-04-24
- Publication Date
- 2026-06-02
AI Technical Summary
Existing genetic resources are not sufficiently effective in regulating alkaline stress with high pH levels, which leads to stunted growth and development of rice, reduced yield, and often accompanied by side effects.
The atypical W-type CLE gene OsCLE-W in rice was cloned and overexpressed to regulate rice salt tolerance. The OsCLE-W gene was transferred into rice via Agrobacterium-mediated transformation and its expression was driven by the Ubi promoter.
It significantly improves the tolerance of rice to salt and alkali stress, maintains good biomass, and enhances the growth and yield of rice under adverse conditions.
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Figure CN122128352A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of plant genetic engineering technology, and particularly to rice. OsCLE-W Application of genes in improving plant salt and alkali tolerance. Background Technology
[0002] With the increasing severity of global climate change and soil salinization, salt-alkali stress has become one of the main environmental factors limiting the yield and quality of rice. Ion toxicity, osmotic stress, and oxidative damage caused by high salinity severely inhibit root development and photosynthetic efficiency in rice, leading to a significant decrease in yield. Although some genes regulating plant salt tolerance have been identified, the regulatory efficacy of existing gene resources is still insufficient when dealing with alkaline stress at high pH levels, and often accompanied by the side effect of stunted growth and development.
[0003] The CLE (CLAVATA3 / Embryo Surrounding Region-related) family consists of important small secretory peptides in plants, regulating growth, development, and stress responses through intercellular signaling. Based on the sequence characteristics of their conserved C-terminal domain (CLE domain), most known CLE members belong to the highly conserved R-type CLEs (whose conserved motif typically contains arginine (Arg) at the 12th amino acid position), primarily influencing plant development by regulating stem cell homeostasis. In contrast, atypical CLE members characterized by W-type (tryptophan (Trp) at the 12th amino acid position) are more unique in sequence evolution and, due to their structural variations, often exhibit biological functions and signal transduction mechanisms distinctly different from typical R-type members. Currently, research on W-type CLE members, especially in rice... OsCLE-W Research on its role in abiotic stress responses remains extremely limited. Summary of the Invention
[0004] The purpose of this invention is to provide rice OsCLE-W This invention addresses the problems of existing technologies by applying genes to improve the salt and alkali tolerance of plants. Specifically, it identifies and clones the atypical W-type CLE gene in rice. OsCLE-W The study found that this gene significantly enhances rice's tolerance to salt-alkali stress and helps it maintain good biomass under adverse conditions. OsCLE-W The biological potential of this atypical member not only expands our understanding of the functional diversity of the CLE family, but also provides a new and excellent genetic resource for cultivating high-yield, salt-tolerant rice varieties using bioengineering technology. This has significant practical implications for developing and utilizing saline-alkali marginal land and ensuring national food security.
[0005] To achieve the above objectives, the present invention provides the following solution: One of the technical solutions of this invention, OsCLE-W The application of genes or their encoded secretory peptides in improving salt and alkali tolerance in rice. OsCLE-W The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence of the secreted peptide is shown in SEQ ID NO.2.
[0006] The second technical solution of the present invention is an expression vector for improving the salt and alkali tolerance of plants, wherein the expression vector contains the... OsCLE-W Genes, and OsCLE-W Genes are operatively linked to promoters.
[0007] The third technical solution of the present invention is a recombinant Agrobacterium, wherein the recombinant Agrobacterium contains the expression vector described above.
[0008] The fourth technical solution of the present invention is a method for improving the salt and alkali tolerance of rice, comprising: OsCLE-W Genes are transferred into plants and enable... OsCLE-W Gene overexpression.
[0009] Based on the above technical solution, the present invention has the following technical effects: 1. This invention is the first to clone and analyze atypical CLE family genes in rice that respond to salt-alkali stress. OsCLE-W The nucleotide sequence, amino acid sequence, and promoter sequence of this gene were published, which is of great significance for elucidating the molecular mechanism of salt-alkali tolerance regulation in rice and for rice salt-alkali tolerance breeding.
[0010] 2. This invention is the first to demonstrate, through transgenic means, the efficacy of rice. OsCLE-W Genes involved in rice salt and alkali tolerance. Overexpression of these genes... OsCLE-W Genetic modification can significantly improve the salt and alkali tolerance of rice, indicating that... OsCLE-W Positive regulation of rice salt and alkali tolerance. Attached Figure Description
[0011] Figure 1 The CLE genes in rice are shown in (A). Among them, (A) shows the gene sequences of all CLE genes in rice and the W-type CLE, with red pentagrams representing W-type CLE genes; (B) shows the expression levels of OsCLE-W under different salt concentration stresses; and (C) shows the expression levels of OsCLE-W under different alkali treatment times.
[0012] Figure 2 The diagram shows the sequence and structure of OsCLE-W. In (A), the blue area represents the intracellular region, the purple area represents the transmembrane region, and the green area represents the extracellular region. In (B), the diagram shows the subcellular localization of OsCLE-W in tobacco.
[0013] Figure 3 for OsCLE-W Schematic diagrams of vectors for overexpressing transgenic materials and their expression levels. (A) Schematic diagram of the OsCLE-W overexpression vector; (B) Expression level diagram of the transgenic overexpression material.
[0014] Figure 4 For overexpression OsCLE-W Phenotypic diagram of genes under salt-alkali stress. Among them, (A) OsCLE-W Phenotypic images of overexpressed transgenic materials and wild-type Zhonghua 11 before and after salt-alkali stress treatment, (B) Survival rate of wild-type and overexpressed materials under salt-alkali stress, (C) Fresh weight of aboveground and underground parts of wild-type and overexpressed materials under salt-alkali stress. Detailed Implementation
[0015] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.
[0016] The embodiments of the present invention provide OsCLE-W The application of genes or their encoded secretory peptides in improving salt and alkali tolerance in rice. OsCLE-W The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence of the secreted peptide is shown in SEQ ID NO.2.
[0017] In some specific implementations, overexpression of the rice OsCLE-W gene or increasing the level of the secreted small peptide can improve the rice's tolerance to salt and alkali stress.
[0018] This invention also provides an expression vector for improving plant salt tolerance, the expression vector containing the aforementioned... OsCLE-W Genes, and OsCLE-W Genes are operatively linked to promoters.
[0019] In some specific implementations, the promoter is the Ubi promoter.
[0020] This invention also provides a recombinant Agrobacterium, which contains the aforementioned expression vector.
[0021] This invention also provides a method for improving the salt and alkali tolerance of rice, including the aforementioned OsCLE-W Genes are transferred into plants and enable... OsCLE-W Gene overexpression.
[0022] In some specific implementations, the transfer is carried out via Agrobacterium-mediated transformation.
[0023] This invention utilizes rice cloning OsCLE-WAfter sequencing the gene encoding sequence, an overexpression vector was constructed, which was then transformed into the rice variety Zhonghua 11. The salt tolerance of the transgenic rice and the wild-type Zhonghua 11 was evaluated. Experiments showed that... OsCLE-W It has a positive regulatory effect on the salt and alkali tolerance of rice, and overexpression of this gene can significantly improve the salt and alkali tolerance of rice.
[0024] Gene source, cloning, and vector construction: Using total RNA from rice Zhonghua 11 seedlings (two weeks after sowing) as a template, cDNA was reverse transcribed. Using the cDNA as a template, specific primers were designed for the start and stop codon regions, and PCR was performed to amplify the full-length OsCLE-W gene of this invention. The upstream primer was OsCLE-W-CDS-F (SEQ ID NO.4), and the downstream primer was OsCLE-W-CDS-R (SEQ ID NO.5). The gene was then cloned into the ZL035 subcellular localization vector. The upstream primer was OsCLE-W-ZL035-F (SEQ ID NO.6), and the downstream primer was OsCLE-W-ZL035-R (SEQ ID NO.7). Using Zhonghua 11 seedling DNA (two weeks after sowing) as a template, PCR was performed... OsCLE-W Specific primers were designed for the first 2kb of the start codon and the start codon region of the gene. PCR reaction was performed to amplify the OsCLE-W gene promoter of the present invention. The primers were: front primer: OsCLE-W-promoter-F (SEQ ID NO.8) and back primer: OsCLE-W-promoter-R (SEQ ID NO.9).
[0025] Verification of salt and alkali tolerance in transgenic plants: OsCLE-W After two weeks of co-culturing with wild-type Zhonghua 11 transgenic rice seedlings, overexpressing the gene, the seedlings were treated with 20 mM Na2CO3 (pH=9) Kimura B nutrient solution and normal nutrient solution, respectively. Ten days after treatment, the growth status of the rice was photographed and the survival rate was calculated. The aboveground and underground parts of the rice seedlings were weighed after washing. The results showed that... OsCLE-W The aboveground biomass of the overexpressing transgenic rice plants was significantly higher than that of the wild-type Zhonghua 11 after alkali treatment, and the survival rate was also significantly higher.
[0026] The primer sequences involved in the following examples are shown in Table 1.
[0027] Table 1
[0028] Example 1 rice CLE Genome-wide identification of gene families and OsCLE-WExpression pattern analysis Bioinformatics identification of the CLE gene family in rice: Using 32 known CLE protein sequences from Arabidopsis thaliana as query sequences, BLASTP and TBLASTN searches were performed in rice genome databases (such as the Rice Annotation Project Database, RAP-DB, or MSU Rice Genome Annotation Project). Candidate sequences containing characteristic CLE domains were screened using conserved domain analysis software (such as HMMER, Pfam, or SMART).
[0029] Screening and sequence analysis of W-type CLE genes: Multiple sequence alignment was performed on the identified rice CLE members. ClustalW or Muscle software was used to analyze the conserved motifs of amino acids 12-14 at the C-terminus. Classification was based on the type of amino acid at position 12 of the conserved motif. R-type: The 12th amino acid is arginine (Arg); W-type: The 12th amino acid is tryptophan (Trp). Through comparison and identification, the gene involved in this invention... OsCLE-W (Gene ID: LOC_Os05g48740 ) belongs to a typical atypical W-type member, such as Figure 1 As shown in A.
[0030] Cloning of the OsCLE-W gene: Total RNA was extracted from the roots of rice variety Zhonghua 11 (ZH11) and reverse transcribed into cDNA. PCR amplification was performed using primers OsCLE-W-CDS-F (SEQ ID NO.4) and OsCLE-W-CDS-R (SEQ ID NO.5), and the target fragment was obtained using high-fidelity DNA polymerase. The product was ligated into a cloning vector and sequenced for verification, yielding the full-length OsCLE-W sequence (SEQ ID NO.1), which is completely identical to the target sequence, its encoded protein sequence (SEQ ID NO.2), and its promoter sequence (SEQ ID NO.3).
[0031] SEQ ID NO.1:ATGAGGCCTCGTGTTGTCCAGGTATGGTGCCTCATTGTGCTCGCCATGATCGTCGTCTTCGCGGCGACGCCGGCAATGGCGGCCCGCGACGGCCGCCGGCTTCACCCGCCGGCGCCGGCGGCTCGCGGCGGCGGGGCGTGGAACAGGGTGAGTGTGACGGCGGAGATCGTCGGCGGCGGCGGCAAGTGGGAAGTCCCCGGCGGACCTGACCCGCAGCACCACCACTAA。
[0032] SEQ ID NO.2:MRPRVVQVWCLIVLAMIVVFAATPAMAARDGRRLHPPAPAARGGGAWNRVSVTAEIVGGGGKWEVPGGPDPQHHH。
[0033]
[0034] The above PCR reaction program is as follows: 95℃ for 5 min, 95℃ for 30 s, 60℃ for 30 s, 72℃ for 20 s, 72℃ for 5 min, 35 cycles.
[0035] The PCR reaction system is shown in Table 2.
[0036] Table 2
[0037] Example 2 OsCLE-W Analysis of gene expression patterns under salt and alkali stress To verify OsCLE-W The expression pattern of the OsCLE-W gene was analyzed by quantitative real-time q-PCR under different time treatments following salt and alkali stress. Specifically, rice seedlings aged 11 to 2 weeks were cultured as described in Example 1. Samples were taken from the roots after salt stress (50 mM, 100 mM treatment for two days) and alkali stress treatment at 0 h, 3 h, 6 h, and 9 h, with three biological replicates for each. Rice RNA (Novitamin RC411) was extracted after flash freezing in liquid nitrogen. The concentration of the extracted RNA was determined using Nanodrop, and 500 ng was used as the concentration for reverse transcription. The RNA was then reverse transcribed into cDNA. Primers OsCLE-W-CDS-F (SEQ ID NO. 4) and OsCLE-W-CDS-R (SEQ ID NO. 5), along with the housekeeping gene... Actin The gene expression pattern was detected. The methods for determining gene expression levels at different time points under salt-alkali stress are described above.
[0038] Table 3
[0039] The results are as follows Figure 1 As shown, OsCLE-W Expression was significantly upregulated under different salt stress concentrations. Figure 1 (of B), and salt-alkali stress can also significantly induce the expression of the OsCLE-W gene in a short period of time (of B). Figure 1 (C).
[0040] Example 3 OsCLE-W Gene structure and subcellular localization analysis Obtained through the above Example 1 OsCLE-W After the gene sequence ( Figure 2(A) The transmembrane structure of this sequence was predicted using TMHMM-2.0 (https: / / services.healthtech.dtu.dk / services / TMHMM-2.0 / ). The results showed that the gene has a significant transmembrane structure and extracellular region, indicating that this gene is consistent with other CLE genes and has extracellular secretory activity. Figure 2 (A)
[0041] To investigate the subcellular localization of OsCLE-W, the inventors constructed a subcellular localization vector for OsCLE-W and transformed it into Agrobacterium competent cells EHA105, where it was expressed in Nicotiana benthamiana. The results showed that OsCLE-W was localized in the apoplastic region (…). Figure 2 (B) Specific implementation method: Using the cloned OsCLE-W coding region as a template, amplification was performed using primers OsCLE-W-ZL035-F (SEQ ID NO. 6) and OsCLE-W-ZL035-R (SEQ ID NO. 7), and the obtained PCR products were recovered. 5 μg of the subcellular localization vector ZL035 was digested with the restriction endonuclease SalI (the digestion reaction was carried out at 37℃ for 30 min), and the resulting digestion products were recovered. Homologous recombination (Novozymes C112) was used to... OsCLE- W The cloned Agrobacterium was inserted into the ZL035 vector, and after successful sequencing, it was transformed into Agrobacterium strain EHA105. A single transformed Agrobacterium colony was cultured overnight in 5 ml LB broth until OD reached... 600 The concentration was 1.0, and the cells were centrifuged at 5000 rpm. The collected bacterial cells were then resuspended in tobacco injection solution to OD0. 600 The concentration was 0.8, and after standing at room temperature for 3 hours, it was mixed with an equal volume of Agrobacterium strain (AtPIP2A-RFP) for tobacco cell membrane localization and injected into tobacco leaves. After incubation in an incubator for 48 hours, images were taken using a laser confocal microscope (Zeiss LSM 980).
[0042] The tobacco injection solution system is shown in Table 4.
[0043] Table 4
[0044] Example 4 OsCLE-W Obtaining overexpression materials of genes First, according to the results obtained in Example 1 OsCLE-WThe full-length gene sequence was obtained, and specific primers containing specific restriction endonuclease sites were designed: OsCLE-W-Ubi-F (SEQ ID NO.10) and OsCLE-W-Ubi-R (SEQ ID NO.11). PCR amplification was performed using ZH11 root cDNA as a template, and the target fragment was recovered. A plant overexpression vector (pCAMBIA1300-Ubi) driven by the Ubi promoter was selected. The vector and the target fragment were digested with the restriction endonuclease BamHI. Subsequently, the OsCLE-W gene fragment was directionally inserted downstream of the Ubi promoter via homologous recombination to construct the recombinant expression plasmid Ubi::OsCLE-W (…). Figure 3 (A) The verified recombinant plasmid was introduced into Agrobacterium strain EHA105 via electroporation, and embryogenic callus induced from mature embryos of rice variety Zhonghua 11 (ZH11) was transformed using Agrobacterium-mediated transformation. After co-culture, hygromycin B selection culture, differentiation, and rooting culture, regenerated plants were obtained and transplanted to a greenhouse. Genomic DNA was extracted from the leaves of the T0 generation regenerated plants, and the hygromycin resistance gene HPT was used to analyze the DNA. OsCLE-W Gene-specific primers were used for PCR identification to screen for positive transgenic plants. Subsequently, total RNA was extracted from the roots of the positive lines and analyzed by qRT-PCR to detect... OsCLE-W The overexpression level of [the substance / organization]. Experimental results showed that in the obtained transgenic lines... OsCLE-W The expression levels of both were significantly increased compared to wild-type ZH11. Figure 3 The samples were identified as OE-OsCLE-W-1 and OE-OsCLE-W-2 (subject to further phenotypic analysis).
[0045] Example 5 OsCLE-W Phenotypic identification of gene overexpression materials under alkaline stress Seeds from two independent overexpression homozygous lines (OE-1 and OE-2) and the wild-type (ZH11) identified in the above examples were selected, disinfected, and germinated before being sown in 1 / 2 KB nutrient solution for hydroponics until they reached the two-leaf-one-heart stage. Seedlings with uniform growth and development were selected for alkali stress treatment. The treatment group had 15 mM Na2CO3 added to the 1 / 2 KB nutrient solution to adjust the pH to 9.0. To ensure the stability of the environmental stress, the corresponding nutrient solution or treatment solution was changed every 2 days during the treatment period.
[0046] After 10 days of continuous treatment, the growth status of each strain was observed and data were collected. The results showed that under alkaline stress, the wild-type ZH11 exhibited obvious alkali damage symptoms, with a significantly lower survival rate than the overexpression material. Symptoms included rapid leaf dehydration and curling, widespread yellowing and scorching, and severely inhibited plant growth; some weaker plants even showed signs of complete wilting and lodging. In contrast, the overexpression lines OE-1 and OE-2 demonstrated significant alkali resistance, with less damage; most leaves remained green and open, and their growth vigor was significantly better than the wild type.
[0047] For quantitative assessment OsCLE-W To assess the contribution to biomass accumulation, the aboveground fresh weight and underground (root) fresh weight of each line were measured and recorded. Statistical analysis showed that under alkaline stress, the biomass of wild-type ZH11 decreased significantly, while the aboveground fresh weight of overexpression lines OE-1 and OE-2 was significantly higher than that of the wild type, with no significant change in underground fresh weight. These results strongly demonstrate that the atypical W-type gene can be driven by the Ubi promoter. OsCLE-W Overexpression in rice can significantly alleviate the growth inhibition caused by alkaline environment on plant tissues, effectively maintain the accumulation of substances under adversity, and has significant potential for increasing yield and improving stress resistance.
[0048] In summary, OsCLE-W It exhibits a positive regulatory effect on rice salt and alkali tolerance; increasing its expression level can enhance rice's salt and alkali tolerance.
[0049] Obtain the present invention OsCLE-W For overexpression of transgenic materials, please contact Professor Wu Dezhi of the College of Agriculture, Hunan Agricultural University, at 3-348, Building 3, South Gate of Yuelushan Laboratory, No. 246 Hongqi Road, Donghu Street, Furong District, Changsha, Hunan Province, 410128, China.
[0050] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.
Claims
1. OsCLE-W The application of a gene or its encoded secretory peptide in improving the salt and alkali tolerance of rice is characterized by, The OsCLE-W The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence of the secretory peptide is shown in SEQ ID NO.
2.
2. The application according to claim 1, characterized in that, Overexpression of the rice OsCLE-W gene or increasing the level of the secreted small peptide can improve the rice's tolerance to salt and alkali stress.
3. An expression vector for enhancing plant salt and alkali tolerance, characterized in that, The expression vector contains the content described in claim 1. OsCLE-W Genes, and OsCLE-W Genes are operatively linked to promoters.
4. The expression vector according to claim 3, characterized in that, The promoter is the Ubi promoter.
5. A recombinant Agrobacterium, characterized in that, The recombinant Agrobacterium contains the expression vector described in claim 3 or 4.
6. A method for improving the salt and alkali tolerance of rice, characterized in that, Includes the method described in claim 1 OsCLE-W Genes are transferred into plants and enable... OsCLE-W Gene overexpression.
7. The method according to claim 6, characterized in that, The transfer was performed using Agrobacterium-mediated transformation.