Application of Oseif3l gene in regulating salt tolerance of rice

Abstract

The application discloses application of an OseIF3l gene in regulating salt tolerance of rice and belongs to the technical field of biotechnology.The regulation of salt tolerance is specifically overexpression of the OseIF3l gene, the salt tolerance of the rice is enhanced, the expression amount of the OseIF3l gene is reduced, and the salt tolerance of the rice is reduced.It is proved by biological experiments that overexpression of OseIF3l can significantly improve the salt tolerance without sacrificing the growth performance, a new target is provided for salt-tolerant breeding of rice, the application potential of the OseIF3l gene is verified in multiple dimensions, and an innovative solution is provided for breaking through the bottleneck of existing salt-tolerant breeding technology.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to the application of the OseIF3l gene in regulating salt tolerance in rice. Background Technology

[0002] Salt stress is one of the key abiotic stress factors restricting the growth, development, and yield of rice. Currently, strategies to improve rice salt tolerance mainly rely on traditional breeding and genetic engineering techniques, but these methods all have significant limitations. Traditional breeding involves screening for salt-tolerant materials obtained through natural variations or artificial mutagenesis and then hybridizing them. Although this method is relatively safe, it has disadvantages such as a long breeding cycle (usually 8-10 years) and low efficiency in trait selection. Furthermore, it is limited by the limited salt-tolerant genetic variations in existing germplasm resources.

[0003] In the field of genetic engineering, current research mainly focuses on two types of functional genes: one type is genes that regulate ion homeostasis (such as sodium ion transporter genes like SOS1 and NHX), and the other type is genes involved in osmotic regulation (such as genes related to the synthesis of osmotic substances like P5CS and BADH). Although overexpression of these genes can improve rice salt tolerance to some extent, it often targets only a single stress response pathway and easily leads to negative effects such as stunted plant growth. More importantly, existing targets are mostly concentrated on downstream effector genes under stress, while the key genes regulating salt stress signal perception, transduction, and multi-pathway synergistic effects are insufficiently explored, resulting in a bottleneck in salt tolerance improvement.

[0004] In recent years, the role of translation initiation regulators in plant stress responses has gradually attracted attention. Eukaryotic translation initiation factor 3 (eIF3), as a core regulatory complex of protein synthesis, has multiple subunits that have been shown to participate in abiotic stress responses. However, whether the various eIF3 subunits (especially eIF3l) regulate salt tolerance through translation-independent mechanisms, and whether they affect the cross-regulation of stress signaling pathways with other physiological processes, remains unclear. Therefore, discovering novel regulatory genes that can integrate multiple stress response pathways and elucidating their molecular mechanisms is of great significance for overcoming the current technical bottlenecks in salt-tolerant breeding. Summary of the Invention

[0005] The purpose of this invention is to provide the application of the OseIF3l gene in regulating salt tolerance in rice, thereby addressing the problems existing in the prior art. This invention demonstrates through biological experiments that overexpression of OseIF3l can significantly improve salt tolerance without sacrificing growth performance, providing a new target for salt-tolerant rice breeding. Furthermore, the application potential of the OseIF3l gene has been verified through multi-dimensional analysis, providing an innovative solution to overcome the bottlenecks in existing salt-tolerant breeding technologies.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides the application of the OseIF3l gene in regulating salt tolerance in rice. Overexpression of the OseIF3l gene enhances the salt tolerance of rice, while reducing the expression level of the OseIF3l gene decreases the salt tolerance of rice.

[0008] The CDS sequence of the OseIF3l 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 the salt tolerance of rice, comprising the step of overexpressing the OseIF3l gene in the rice.

[0011] Optionally, the rice is japonica rice.

[0012] The present invention also provides the application of the OseIF3l gene in the breeding of highly 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 step of introducing the OseIF3l gene into the rice to obtain plants that stably and highly express the OseIF3l gene.

[0015] Optionally, the rice is japonica rice.

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

[0017] This invention investigates the role of the OseIF3l gene in rice salt tolerance, revealing its potential to significantly enhance salt tolerance. OseIF3l gene-edited lines constructed using CRISPR / Cas9 technology exhibited dwarfing, smaller grains, and significantly reduced salt tolerance, confirming the crucial role of OseIF3l in regulating rice growth, development, and stress resistance. OseIF3l-overexpressing lines showed stronger survival rates under salt stress, with no significant difference in agronomic traits compared to the wild type, indicating that OseIF3l overexpression can significantly improve salt tolerance without sacrificing growth performance. Further hormone content and transcriptome analyses revealed that OseIF3l integrates multiple stress response pathways by regulating the accumulation of hormones such as auxin, gibberellin, 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-tolerant rice breeding, but also verifies the application potential of the OseIF3l gene from multiple dimensions, providing an innovative solution to break through the bottleneck of existing salt-tolerant breeding technology. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 Phylogenetic tree diagram of eIF3l protein;

[0021] Figure 2 The graph shows the expression level analysis of the eIF3l gene in different parts of rice during the booting stage (A) and flowering stage (B) using qRT-PCR.

[0022] Figure 3 To detect the expression level of the eIF3l gene in rice seedling leaves at different time points after salt stress treatment using qRT-PCR.

[0023] Figure 4 Comparative analysis of sequencing results for eIF3l gene-edited strains;

[0024] Figure 5 Phenotypic observation and agronomic trait statistical analysis of eIF3l gene-edited lines were conducted. Specifically, 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 seed length, width, and thickness of Nip and eIF3l gene-edited lines (Scale bar = 1 cm); H: plant height statistics; I: tiller statistics; J: panicle length statistics; KM: phenotypic difference statistical analysis of grain length (K), grain width (L), and grain thickness (M) (n = 10); NP: scanning electron microscopy observation of glumes epidermal cells of wild-type (N), oseif3l-1 (O), and oseif3l-2 (P) grains (Bar = 50 μm); Q: glume cell width statistics.

[0025] Figure 6 This study aimed to observe and statistically analyze the seedling phenotypes and salt stress phenotypes of eIF3l gene-edited lines. The results included: A: Phenotypic observation of seedlings of Nip and eIF3l gene-edited lines before salt treatment; B: Phenotypic observation of Nip and eIF3l gene-edited lines after 7 days of recovery following salt treatment; and C: Survival rate statistics of Nip and eIF3l gene-edited lines after 7 days of recovery following salt treatment.

[0026] Figure 7This study aims to identify the expression level and phenotypic characteristics of plants overexpressing the eIF3l gene. Specifically, AC represents photographic observations of the phenotypes of wild-type (A), eIF3l-overexpressing plants OE-1 (B), and OE-2 (C); D shows observations of seed length and width in Nip and eIF3l-overexpressing lines; E shows the detection of eIF3l gene expression level in overexpressing plants; F shows plant height statistics; G shows tillering statistics; and HJ presents statistical analysis of phenotypic differences in seed length (H), seed width (I), and seed thickness (J).

[0027] Figure 8 Seedling phenotypes and salt stress phenotypes of eIF3l gene overexpression lines (A) and statistical analysis (B);

[0028] Figure 9 The study analyzed the hormone content after salt treatment during the seedling stage. The AL values ​​were in the following order: 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 eIF3l gene-edited lines and wild-type lines after salt stress treatment were performed. A represents principal component analysis (PCA) of the transcriptome data; B represents Venn diagram analysis of genes with significant differences in transcriptome expression levels; C represents heatmap analysis of salt stress-related genes; and D represents qRT-PCR validation of salt stress-related genes.

[0030] Figure 11 Map of pYLsgRNA-related vectors;

[0031] Figure 12 The pYLCRISPR vector map. Detailed Implementation

[0032] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of 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 terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0034] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0035] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

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

[0037] This invention uses the OseIF3l gene and protein sequence of the japonica rice variety Nipponbare as the research object to carry out biological experiments, wherein the amino acid sequence of the eIF3l protein 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 OseIF3l 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 to construct the OseIF3l gene-editing line in this embodiment of the invention were all provided by Academician Liu Yaoguang of the Rice Research Institute of Guangdong Academy of Agricultural Sciences. Related vector maps are also available. Figure 11 and Figure 12 Those skilled in the art can also use other vectors with the same function to construct OseIF3l gene-editing lines.

[0047] Example 1: Sequence conservation and expression pattern analysis of the rice eIF3l gene

[0048] 1. Conservation analysis of the eIF3l protein sequence

[0049] Using the rice (Oryza sativa L.ssp. Japonica) eIF3l protein sequence (SEQ ID NO.1) as the query sequence, homologous sequence alignment was performed in the NCBI database using the Blast tool to screen for eIF3l homologous protein sequences of representative species.

[0050] eIF3l protein sequences from different representative species were downloaded from the alignment results and multiple sequence alignment was performed. 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 eIF3l sequences of japonica and indica rice were completely identical (100% similarity), with no differences, and exhibited high conservation among different species. Figure 1 ).

[0051] 2. Expression patterns of the OseIF3l gene in different tissues and growth stages of rice

[0052] (1) Plant material culture:

[0053] After sterilization, rice cultivar Nipponbare seeds were sown on 1 / 2 MS solid medium (containing 3% sucrose, pH 5.8) and cultured at 28°C under 16h light / 8h darkness until the target growth period.

[0054] (2) OseIF3l gene expression detection:

[0055] Rice leaves, stems, roots, and young panicle tissues were collected at the booting and flowering stages, respectively, and then flash-frozen in liquid nitrogen and stored at -80℃.

[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 eIF3l gene at different growth stages and in different parts of rice were analyzed by qRT-PCR.

[0057] The results showed that OseIF3l was constitutively expressed in rice. Figure 2 (A and B).

[0058] 3. Response analysis of OseIF3l to salt stress

[0059] Salt treatment experiment:

[0060] Three-leaf stage rice seedlings were transferred to 1 / 2 MS liquid medium containing 200 mM NaCl. Leaf tissues were collected at 0, 1, 2, 3, 4, 5, 6, 8, 10, 12, 24, and 36 h. The expression level of the eIF3l gene was detected and analyzed by qRT-PCR, with three biological replicates per group. The results showed that the expression level of eIF3l was significantly upregulated after salt stress, reaching a peak at 12 h of treatment, indicating that eIF3l is closely related to the salt stress response of rice. Figure 3 ).

[0061] Example 2: Construction and Salt Tolerance Analysis of OseIF3l Gene-Edited Lines

[0062] 1. Construction of OseIF3l gene-editing lines

[0063] The OseIF3l gene was edited using CRISPR / Cas9 gene editing technology.

[0064] (1) Target design and vector construction

[0065] Two CRISPR / Cas9 targets were designed for the rice OseIF3l 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). SEQ ID NO.4 was ligated into the pYL-U3-gRNA vector. Figure 11 ), SEQ ID NO.5 was ligated into the pYL-U6a-gRNA vector ( Figure 11Using the two vector plasmids mentioned above as templates, pYL-U3-gRNA and pYL-U6a-gRNA fragments were amplified, and then ligated into the pYLCRISPR vector using a cut-and-ligate method. Figure 12 (Contains hygromycin resistance selection markers).

[0066] (2) Genetic transformation and screening of positive plants

[0067] The recombinant vector was transformed into embryogenic callus of the japonica rice variety Nipponbare using Agrobacterium-mediated transformation (EHA105 strain). Resistant callus was obtained through screening with hygromycin (50 mg / L), and T0 generation plants were obtained after regeneration. Two stable homozygous edited lines (oseif3l-1 and oseif3l-2) were screened by PCR amplification of the target site and sequencing verification, with frameshift mutations occurring in exon 1 and exon 2, respectively. Figure 4 ).

[0068] 2. Phenotypic analysis of gene-edited lines

[0069] (1) Plant growth phenotype

[0070] In field planting, T2 generation homozygous plants showed significantly lower biomass for oseif3l-1 and oseif3l-2 compared to the wild-type (WT) Nipponbare. Figure 5 The AC type, and the spikelets are also significantly smaller than those of the wild type. Figure 5 Further statistical analysis of agronomic traits (DF) revealed that the plant height of oseif3l-1 and oseif3l-2 was significantly shorter than that of the wild type. Figure 5 The number of tillers was significantly higher than that of the wild type (H), while the number of tillers was significantly higher than that of the wild type. Figure 5 The spike length of the I type is significantly shorter than that of the wild type. Figure 5 J).

[0071] (2) Abnormal grain development

[0072] Observation and size statistics of oseif3l-1 and oseif3l-2 seeds showed that the seed length and width of oseif3l-1 and oseif3l-2 seeds were significantly smaller than those of the wild type. Figure 5 The G, K, L values ​​showed no significant difference in particle thickness. Figure 5 (G and M). Further observation of the seed glume cells using scanning electron microscopy revealed that the width of seed cells in oseif3l-1 and oseif3l-2 was significantly larger than that in the wild type (G and M). Figure 5The OQ result indicates that the OseIF3l gene may affect rice seed development by influencing the number of glume cells.

[0073] 3. Salt tolerance analysis

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

[0075] The results indicate that OseIF3l deficiency leads to dwarfing of plants, smaller grains, and reduced salt tolerance, suggesting that this gene participates in rice growth and stress resistance by regulating cell development and stress response.

[0076] Example 3: Construction and Salt Tolerance Analysis of OseIF3l Gene Overexpression Lines

[0077] 1. Construction and transformation of overexpression vectors

[0078] (1) Carrier construction

[0079] Using Nipponbare cDNA as a template, the full-length CDS of OseIF3l (SEQ ID NO.2) was amplified and cloned into the pCAMBIA1300 vector (containing the 35S promoter and hygromycin resistance marker).

[0080] (2) Genetic transformation and identification

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

[0082] 2. Phenotypic analysis of overexpression lines

[0083] Further observation of the phenotypes of the overexpression lines and statistical analysis of agronomic traits showed that the plant height, tiller number, grain length, and grain width of OE-1 and OE-2 were not significantly different from those of wild-type rice Nip. Figure 7 The results showed that OseIF3l overexpression does not affect the normal growth and development of rice.

[0084] 3. Salt tolerance analysis

[0085] After treatment with 200 mM NaCl for 7 days, wild-type rice (Nip) seedlings at the three-leaf stage and overexpression lines (OE-1 and OE-2) showed less leaf wilting in the OE lines than in the wild-type, and their survival rate was significantly higher after 7 days of recovery. Figure 8 ).

[0086] This indicates that OseIF3l overexpression can significantly enhance rice salt tolerance 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 levels under salt stress

[0089] Further analysis of hormone content in the materials before and after salt stress treatment was conducted using high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS) to determine the following hormone contents:

[0090] Auxins: Indole-3-acetic acid (IAA), indolepropionic acid (IPA), indolebutyric acid (IBA), indolecarboxylic acid (ICA).

[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 analysis 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 significantly increased levels of OseIF3l (L) suggest that OseIF3l may respond to salt stress through a hormone-mediated pathway.

[0094] 3. Transcriptome analysis and validation

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

[0096] Differentially expressed genes were screened according to the criteria of Log2Fold change|>1 and 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. Among them, 5 genes were downregulated (OsBIERF1, OsRB D1, OsARD1, OsDO, OsLSK1), and 15 genes were upregulated (OsMDH12.1, OsARD2, SRWD3, Os-NADP-ME2, OsPEX5, BIP130, OsPP2A-3, OsPR4c, OsALDH10A5, OsSIRP3, OsD-LDH2, OsbZIP20, OsLG3, OsbHLH035, OsCHR726). The clustering heatmap of the differentially expressed genes is shown below. Figure 10 .

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

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

Claims

1. Use of OseIF3l gene in regulating salt tolerance of rice, characterized in that, The salt tolerance of the rice is enhanced by overexpressing the OseIF3l gene. The CDS sequence of the OseIF3l gene is shown as SEQ ID NO.

2.

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

3. A method for enhancing salt tolerance in rice, characterized by, The step of overexpressing the OseIF3l gene in the rice is included; the CDS sequence of the OseIF3l gene is shown as SEQ ID NO.

2.

4. The method of claim 3, wherein, The rice is japonica rice.

5. A method for breeding high salt-tolerant rice, characterized by, The step of introducing the OseIF3l gene into the rice to obtain a plant stably overexpressing the OseIF3l gene is included; the CDS sequence of the OseIF3l gene is shown as SEQ ID NO.

2.

6. The method of claim 5, wherein, The rice is japonica rice.

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