Application of OseIF3l gene in regulating and controlling salt tolerance of rice

Abstract

The invention discloses application of an OseIF3l gene in regulating and controlling salt tolerance of rice, and belongs to the technical field of biology. The regulation and control of the salt tolerance specifically refers to the overexpression of the OseIF3l gene, the salt tolerance of the rice is enhanced, and the expression quantity of the OseIF3l gene is reduced, so that the salt tolerance of the rice is reduced. Biological experiments prove that overexpression of the OseIF3l can significantly improve the salt tolerance on the premise of not sacrificing the growth performance, a new target is provided for rice salt tolerance breeding, the application potential of the OseIF3l gene is verified through multiple dimensions, and an innovative solution is provided for breaking through the bottleneck of the existing salt tolerance breeding technology.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to application of the OseIF31 gene in regulating salt tolerance of rice. Background Art

[0002] Salt stress is one of the key abiotic stress factors that restrict rice growth, development, and yield. Currently, strategies to improve rice salt tolerance rely primarily on traditional breeding and genetic engineering techniques, but these approaches have significant limitations. Traditional breeding, which involves hybridization and selection of salt-tolerant materials obtained through natural variation or artificial mutagenesis, is relatively safe, but suffers from long breeding cycles (typically 8-10 years) and low trait selection efficiency, and is limited by the limited genetic variation for salt tolerance in existing germplasm resources.

[0003] In the field of genetic engineering, current research focuses mainly on two types of functional genes: one is genes that regulate ion homeostasis (such as sodium ion transporter genes such as SOS1 and NHX), and the other is genes involved in osmotic regulation (such as genes related to osmotic substance synthesis such as P5CS and BADH). Although overexpression of these genes can improve rice salt tolerance to a certain extent, they often only target a single stress response pathway and are prone to negative effects such as slowed plant growth. More importantly, existing targets are mostly concentrated on effector genes downstream of stress, while key genes that regulate salt stress signal perception, transduction, and multi-pathway synergy have not been fully explored, resulting in a bottleneck in the improvement of salt tolerance.

[0004] In recent years, the role of translation initiation regulators in plant stress responses has gained increasing attention. Eukaryotic translation initiation factor 3 (eIF3), a core regulatory complex for protein synthesis, has multiple subunits that have been shown to participate in abiotic stress responses. However, there is currently no definitive conclusion regarding whether individual eIF3 subunits (particularly eIF3l) regulate salt tolerance through translation-independent mechanisms, or whether they influence cross-regulation between stress signaling pathways and other physiological processes. Therefore, discovering novel regulatory genes that integrate multiple stress response pathways and elucidating their molecular mechanisms are crucial for overcoming the current technological bottlenecks in salt-tolerance breeding. Summary of the Invention

[0005] The present invention aims to provide the application of the OseIF31 gene in regulating salt tolerance in rice to address the aforementioned problems in the prior art. Through biological experiments, the present invention demonstrates that overexpression of OseIF31 can significantly improve salt tolerance without sacrificing growth performance, providing a new target for salt-tolerance breeding in rice. The application potential of the OseIF31 gene has also been verified in multiple dimensions, offering an innovative solution to overcome the bottlenecks in existing salt-tolerance breeding techniques.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides an application of the OseIF31 gene in regulating the salt tolerance of rice. Overexpression of the OseIF31 gene enhances the salt tolerance of the rice, while reduction of the expression level of the OseIF31 gene reduces the salt tolerance of the rice.

[0008] The CDS sequence of the OseIF31 gene is shown in SEQ ID NO.2.

[0009] Optionally, the rice is japonica rice.

[0010] The present invention also provides a method for enhancing salt tolerance of rice, comprising the step of overexpressing the OseIF31 gene in the rice.

[0011] Optionally, the rice is japonica rice.

[0012] The present invention also provides the use of the OseIF31 gene in cultivating high salt-tolerant rice.

[0013] Optionally, the rice is japonica rice.

[0014] The present invention also provides a method for cultivating highly salt-tolerant rice, comprising the steps of introducing the OseIF31 gene into the rice to obtain plants that stably and highly express the OseIF31 gene.

[0015] Optionally, the rice is japonica rice.

[0016] The present invention discloses the following technical effects:

[0017] The present invention reveals the potential of the OseIF31 gene to significantly improve salt tolerance by studying its role in rice salt tolerance. The OseIF31 gene-edited strain constructed by CRISPR / Cas9 technology exhibited a phenotype of dwarfed plants, smaller grains, and significantly reduced salt tolerance, confirming the key role of OseIF31 in regulating rice growth and development and stress resistance. The OseIF31 overexpression strain showed a stronger survival rate under salt stress, and the agronomic traits were not significantly different from those of the wild type, indicating that overexpression of OseIF31 can significantly improve salt tolerance without sacrificing growth performance. Further hormone content and transcriptome analysis showed that OseIF31 integrates multiple stress response pathways by regulating the accumulation of hormones such as auxin, gibberellins, abscisic acid, and salicylic acid, as well as the expression of salt stress-related genes, thereby achieving efficient regulation of salt tolerance.

[0018] This invention not only provides a new target for salt-tolerance breeding in rice, but also verifies the application potential of the OseIF31 gene through multiple dimensions, providing an innovative solution for breaking through the bottleneck of existing salt-tolerance breeding technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0020] Figure 1 This is the evolutionary tree analysis diagram of eIF31 protein;

[0021] Figure 2 The expression analysis of eIF31 gene in different parts of rice at the booting stage (A) and flowering stage (B) detected by qRT-PCR;

[0022] Figure 3 The expression level of eIF31 gene in rice seedling leaves was detected by qRT-PCR at different time points after salt stress treatment;

[0023] Figure 4 Comparison and analysis of sequencing results of eIF31 gene-edited strain materials;

[0024] Figure 5 Figure 5. Phenotypic observation and agronomic trait statistical analysis of eIF3l gene-edited lines. AC: Photographs of wild-type (A), oseif3l-1 (B), and oseif3l-2 (C) plants, bar = 10 cm; DF: Photographs of wild-type (D), oseif3l-1 (E), and oseif3l-2 (F) panicles, bar = 2 cm; G: Observation of grain length, width, and thickness of seeds of Nip and eIF3l gene-edited lines, scalebar = 1 cm; H, plant height statistics; I, tillering statistics; J, panicle length statistics; KM: Statistical analysis of phenotypic differences in grain length (K), grain width (L), and grain thickness (M), n = 10; NP, Scanning electron microscopy observation of glume epidermal cells of rice wild-type (N), oseif3l-1 (O), and oseif3l-2 (P), bar = 50 μm; Q: glume cell width statistics;

[0025] Figure 6 Phenotypic observation and statistics of the eIF31 gene-edited strains at the seedling stage and after salt stress treatment. A: Phenotypic observation of the Nip and eIF31 gene-edited strains before salt treatment; B: Phenotypic observation of the Nip and eIF31 gene-edited strains 7 days after recovery from salt treatment; C: Survival statistics of the Nip and eIF31 gene-edited strains 7 days after recovery from salt treatment;

[0026] Figure 7Figure 2: Expression level identification and phenotypic observation of eIF31 gene overexpression plants. Figures A and C show the phenotypic observations of the wild type (A), eIF31 gene overexpression plants OE-1 (B), and OE-2 (C); D: Observation of grain length and width of seeds of Nip and eIF31 gene overexpression lines; E: Expression level detection of eIF31 gene overexpression plants; F: Plant height statistics; G: Tillering statistics; HJ: Statistical analysis of phenotypic differences in grain length (H), grain width (I), and grain thickness (J);

[0027] Figure 8 Observation (A) and statistics (B) of the phenotypes of eIF31 gene overexpression lines at the seedling stage and under salt stress treatment;

[0028] Figure 9 It is the detection and analysis of hormone content after salt treatment at the seedling stage, among which AL stands for indole-3-acetic acid, indolepropionic acid, indolebutyric acid, indolecarboxylic acid, GA3, GA4, GA7, abscisic acid, jasmonic acid, jasmonic acid-isoleucine, dihydrojasmonic acid and salicylic acid;

[0029] Figure 10 Transcriptome analysis and qRT-PCR validation of the eIF31 gene-edited strain and the wild type after salt stress treatment, where A is the principal component analysis (PCA) of the transcriptome data; B is the Venn diagram analysis of genes with significantly different expression levels in the transcriptome data; C is the heat map analysis of salt stress-related genes; D is the qRT-PCR validation of salt stress-related genes;

[0030] Figure 11 This is a map of pYLsgRNA related vectors;

[0031] Figure 12 This is the map of the pYLCRISPR vector. DETAILED DESCRIPTION

[0032] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0033] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0034] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0035] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0036] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0037] The present invention conducts biological experiments using the OseIF31 gene and protein sequence of the japonica rice variety Nipponbare as research objects, wherein the eIF31 protein amino acid sequence is shown in SEQ ID NO.1.

[0038] SEQ ID NO.1:

[0039] MASSAAAAFYRDREDAAPPSSGPGGGGAMAAYDPSYVPDSVKTFVSHLYRHIRDRNVYETHQMYEGGFTRLSDRHFRDTPWPPAEAVAAHCDGDHVFLLLYRELWFRHAHARVQGLTPAQRAESWDNYCSLFSVVLQGVVNMQLPNQWLWDMVDEFVYQFQSFCQYRAKLKNKTHEEIALLKQYDQAWSVYGVLNYLKALVEKSMIGEILEREKVGLEQFTATDGYDYEGGSNVLKMLGYYSMIGLLRVHCLLGDYHTGLKCLAPIDISQQGVYTTVIGSHISTIYHYGFASLMMRRYIDGIREFNKILLYILKCKQYHQNSPQYDQLLKKNEQMYALLAICLSLCPQDKLIDENVGTQLKEKYGDKMTKMHRYDDEAYAIYDELFSYACPKFITASPPVLREPYTNYNQDAYRLQLKLFLYEVKQQQLLSGIRSYLKLYSTITIGKLAKYMDVDEVTLRTILMTYKHKMHSIDSDGKVISSADFDFYIDEDIIHVVESKLTKNHGDYFLRQILQFEEMITQLDKVQFD。

[0040] The CDS sequence of the OseIF3l gene is shown in SEQ ID NO.2.

[0041] SEQ ID NO.2:

[0042]

[0043] The full-length sequence of the OseIF31 gene is shown in SEQ ID NO.3.

[0044] SEQ ID NO.3:

[0045]

[0046] The vectors pYL-U3-gRNA, pYL-U6a-gRNA and pYLCRISPR used in the construction of the OseIF31 gene editing strain in the embodiment of the present invention were provided by Academician Liu Yaoguang of the Rice Research Institute of Guangdong Academy of Agricultural Sciences. Figure 11 and Figure 12 , those skilled in the art can also use other vectors with the same function to construct the OseIF31 gene editing strain.

[0047] Example 1 Analysis of Sequence Conservation and Expression Pattern of Rice eIF31 Gene

[0048] 1. Conservation analysis of eIF31 protein sequence

[0049] The rice (Oryza sativa L.ssp. Japonica) eIF31 protein sequence (SEQ ID NO.1) was used as the query sequence, and the Blast tool was used to perform homologous sequence alignment in the NCBI database to screen eIF31 homologous protein sequences of representative species.

[0050] From the alignment results, eIF31 protein sequences from various representative species were downloaded and subjected to multiple sequence alignment. The aligned sequences were imported into MEGA 6.0 software, and a phylogenetic tree was constructed using the neighbor-joining (NJ) method. The results showed that the eIF31 sequences of japonica and indica rice were completely identical (100% similarity), with no differences, and were highly conserved between species ( Figure 1 ).

[0051] 2. Expression patterns of the OseIF31 gene in different rice tissues and growth stages

[0052] (1) Plant material cultivation:

[0053] Rice variety Nipponbare seeds were sterilized and sown on 1 / 2MS solid culture medium (containing 3% sucrose, pH 5.8) and cultured at 28°C with 16 h light / 8 h dark to the target growth stage.

[0054] (2) OseIF31 gene expression detection:

[0055] Rice leaf, stem, root and panicle tissues were collected at the booting stage and flowering stage, respectively, and stored at -80℃ after quick freezing in liquid nitrogen.

[0056] Total RNA was extracted from the samples using TRIzol reagent (Invitrogen), treated with DNase I, and then reverse transcribed into cDNA (PrimeScript RT kit, TaKaRa). The expression levels of the eIF31 gene in different parts of rice at different stages were analyzed by qRT-PCR.

[0057] The results showed that OseIF31 was constitutively expressed in rice ( Figure 2 A and B).

[0058] 3. Analysis of OseIF31 response to salt stress

[0059] Salt treatment experiment:

[0060] Three-leaf rice seedlings were transferred to 1 / 2MS liquid medium containing 200mM NaCl. Leaf tissues were collected at 0, 1, 2, 3, 4, 5, 6, 8, 10, 12, 24, and 36 hours, and the expression of the eIF31 gene was detected and analyzed by qRT-PCR, with three biological replicates per group. The results showed that the expression of eIF31 was significantly upregulated after salt stress, reaching a peak at 12 hours of treatment, indicating that OseIF31 is closely related to the salt stress response of rice ( Figure 3 ).

[0061] Example 2 Construction of OseIF31 gene-edited strain and salt tolerance analysis

[0062] 1. Construction of OseIF31 gene-edited strain

[0063] The OseIF31 gene was edited using CRISPR / Cas9 gene editing technology.

[0064] (1) Target design and vector construction

[0065] Two CRISPR / Cas9 target sites were designed for the rice OseIF31 gene (LOC_Os05g13950, SEQ ID NO.3) (target 1: 5′-AGACGCACCAGATGTACGAGGGCGG-3′, SEQ ID NO.4; target 2: 5′-AATGATTGGAGAGATCCTGGAGAGGGA-3′, SEQ ID NO.5), and SEQ ID NO.4 was ligated into the pYL-U3-gRNA vector ( Figure 11 ), SEQ ID NO.5 was connected to the pYL-U6a-gRNA vector ( Figure 11), using the above two vector plasmids as templates, amplify the pYL-U3-gRNA and pYL-U6a-gRNA fragments, and further connect the pYL-U3-gRNA and pYL-U6a-gRNA fragments to the pYLCRISPR vector by the cutting and ligating method ( Figure 12 ) (containing the hygromycin resistance selection marker).

[0066] (2) Genetic transformation and positive plant screening

[0067] The recombinant vector was transformed into embryonic callus of the japonica rice variety Nipponbare using the Agrobacterium-mediated method (EHA105 strain). Resistant calli were selected by hygromycin (50 mg / L) and T0 generation plants were obtained after regeneration. The target site was amplified by PCR and verified by sequencing. Two stably inherited homozygous edited lines (oseif3l-1 and oseif3l-2) were screened, with frameshift mutations in exon 1 and exon 2, respectively ( Figure 4 ).

[0068] 2. Phenotypic Analysis of Gene-Edited Strains

[0069] (1) Plant growth phenotype

[0070] When T2 homozygous plants were planted in the field, the biomass of oseif3l-1 and oseif3l-2 plants was significantly less than that of the wild type (WT). Figure 5 AC), the ears were also significantly smaller than those of the wild type ( Figure 5 DF), further statistical analysis of agronomic traits showed that the plant heights of oseif3l-1 and oseif3l-2 were significantly shorter than those of the wild type ( Figure 5 of H), while the tiller number was significantly greater than that of the wild type ( Figure 5 I), the spike length was significantly shorter than that of the wild type ( Figure 5 J).

[0071] (2) Abnormal grain development

[0072] The seeds of oseif3l-1 and oseif3l-2 were observed and their size was statistically analyzed. The results showed that the length and width of the seeds of oseif3l-1 and oseif3l-2 were significantly smaller than those of the wild type ( Figure 5 G, K, L), while there was no significant difference in grain thickness ( Figure 5 G and M of the seeds). The hull cells of the seeds were further observed using a scanning electron microscope. The results showed that the width of the seed cells of oseif3l-1 and oseif3l-2 was significantly larger than that of the wild type ( Figure 5This indicates that the OseIF31 gene may affect the development of rice seeds by affecting the number of glume cells.

[0073] 3. Salt tolerance analysis

[0074] Three-leaf stage wild-type rice (Nip) and mutant (oseif3l-1 and oseif3l-2) seedlings were treated with 200 mM NaCl solution for 7 days and then recovered for 7 days. The survival rate of mutants was significantly lower than that of wild-type ( Figure 6 ).

[0075] The results showed that the loss of OseIF3l resulted in dwarf plants, smaller grains, and reduced salt tolerance, suggesting that this gene is involved in rice growth and stress resistance by regulating cell development and stress response.

[0076] Example 3 Construction of OseIF31 gene overexpression strain and salt tolerance analysis

[0077] 1. Construction and transformation of overexpression vector

[0078] (1) Vector construction

[0079] The full-length CDS of OseIF31 (SEQ ID NO. 2) was amplified using Nipponbare cDNA as a template and cloned into the pCAMBIA1300 vector (containing a 35S promoter and a hygromycin resistance marker).

[0080] (2) Genetic transformation and identification

[0081] T0 generation plants were obtained by Agrobacterium transformation, and two high-expressing strains (OE-1 and OE-2) were screened by qRT-PCR ( Figure 7 E).

[0082] 2. Phenotypic Analysis of Overexpression Strains

[0083] The phenotypes of the overexpression lines were further observed and agronomic traits were statistically analyzed. The results showed that the agronomic traits of OE-1 and OE-2, such as plant height, tiller number, grain length, and grain width, had no significant differences from those of wild-type rice Nip ( Figure 7 AD, FJ), indicating that OseIF31 overexpression does not affect the normal growth and development of rice.

[0084] 3. Salt tolerance analysis

[0085] After 7 days of treatment with 200 mM NaCl, the leaves of the three-leaf stage wild-type rice (Nip) and overexpression lines (OE-1 and OE-2) wilted less severely than those of the wild type, and the survival rate after 7 days of recovery was significantly higher than that of the wild type ( Figure 8 ).

[0086] These results indicate that overexpression of OseIF31 can significantly enhance salt tolerance in rice without causing negative growth effects, and has potential for breeding applications.

[0087] Example 4 Hormone Content and Transcriptome Analysis

[0088] 1. Analysis of changes in hormone content under salt stress

[0089] The hormone content of the materials before and after salt stress treatment was further tested, and the following hormone contents were determined using high performance liquid chromatography-mass spectrometry (HPLC-MS / MS):

[0090] Auxins: indole-3-acetic acid (IAA), indolepropionic acid (IPA), indolebutyric acid (IBA), indolecarboxylic acid (IC A).

[0091] Gibberellins: GA3, GA4, GA7.

[0092] Stress-related hormones: abscisic acid (ABA), jasmonic acid (JA), jasmonic acid-isoleucine (JA-Ile), dihydrojasmonic acid (DHJA), and salicylic acid (SA).

[0093] The results showed that indole-3-acetic acid ( Figure 9 A), gibberellins G3, G4 and G7 ( Figure 9 EG), abscisic acid ( Figure 9 H) and salicylic acid ( Figure 9 The content of L) was significantly increased, indicating that OseIF31 may respond to salt stress through a hormone-mediated pathway.

[0094] 3. Transcriptome analysis and verification

[0095] Transcriptome sequencing was performed on the wild-type Nip, gene-edited strains oseif3l-1 and oseif3l-2 treated with salt stress, and the expression levels of salt stress-related pathway genes were verified by qRT-PCR, with three biological replicates in each group.

[0096] Differentially expressed genes were screened according to the conditions of Log2Fold change|>1; P-adjust<0.05. The results showed that a total of 20 significantly differentially expressed genes related to rice salt stress were obtained after screening, of which 5 genes were down-regulated (OsBIERF1, OsRB D1, OsARD1, OsDO, OsLSK1), and 15 genes were up-regulated (OsMDH12.1, OsARD2, SRWD3, Os-NADP-ME2, OsPEX5, BIP130, OsPP2A-3, OsPR4c, OsALDH10A5, OsSIRP3, OsD-LDH 2, OsbZIP20, OsLG3, OsbHLH035, OsCHR726). The cluster heat map of differentially expressed genes is shown in Figure 3. Figure 10 .

[0097] Conclusion: OseIF3l participates in the regulation of salt tolerance by regulating the accumulation of hormones (IAA, GA, ABA, SA) and the expression of salt-responsive genes.

[0098] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. Application of the OseIF31 gene in regulating salt tolerance in rice, characterized in that: Overexpression of the OseIF31 gene enhances the salt tolerance of the rice, while reduction of the expression level of the OseIF31 gene reduces the salt tolerance of the rice; The CDS sequence of the OseIF31 gene is shown in SEQ ID NO.

2.

2. The use according to claim 1, characterized in that The rice is japonica rice.

3. A method for enhancing salt tolerance of rice, characterized in that: The method comprises the step of overexpressing the OseIF31 gene in the rice; the CDS sequence of the OseIF31 gene is shown as SEQ ID NO.

2.

4. The method according to claim 3, characterized in that The rice is japonica rice.

5. Application of the OseIF31 gene in cultivating high salt-tolerant rice, characterized in that: The CDS sequence of the OseIF31 gene is shown in SEQ ID NO.

2.

6. The use according to claim 5, characterized in that The rice is japonica rice.

7. A method for cultivating high salt-tolerant rice, characterized in that: The method comprises the steps of introducing the OseIF31 gene into the rice to obtain a plant stably and highly expressing the OseIF31 gene; the CDS sequence of the OseIF31 gene is shown in SEQ ID NO.

2.

8. The method according to claim 7, characterized in that The rice is japonica rice.

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