Receptor-like cytoplasm kinase gene from sugarcane, expression cassette and application of receptor-like cytoplasm kinase gene

Abstract

The invention relates to the technical field of plant genes, in particular to a receptor-like cytoplasm kinase gene derived from sugarcane, an expression cassette and application of the receptor-like cytoplasm kinase gene. The nucleotide sequence of the receptor-like cytoplasm kinase gene ScRLCK1 is as shown in SEQ ID NO. 1. By constructing ScRLCK1 overexpressed sugarcane and arabidopsis thaliana materials and carrying out related experiments, the salt stress resistance of the ScRLCK1 overexpressed plants is obviously enhanced, and an effective way can be provided for realizing high-salt-tolerance sugarcane variety cultivation and industrial sustainable development.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant genes, and particularly relates to a receptor-like cytoplasmic kinase gene from sugarcane, an expression cassette and application thereof. BACKGROUND

[0002] Saccharum officinarum L. is an important economic crop, and the sucrose produced from sugarcane accounts for 70% of the world's sugar production. China's sugar production accounts for 90% of the world's sugar production, and is the most important sugar crop in China. The yield of sugarcane is affected by many factors, which can be simply divided into biological stress and abiotic stress, and the abiotic stress mainly includes drought, high temperature, salt stress, etc. The research results show that salt stress can delay the bud germination of sugarcane, affect the bud growth, reduce the germination rate, and reduce the fresh weight and dry weight of sugarcane. Therefore, improving the salt tolerance of sugarcane is of great significance to the production of sugarcane.

[0003] Receptor-like cytoplasmic kinases (RLCKs) are a class of protein kinases widely existing in plants, which play a key role in the growth and development of plants and the response to biological and abiotic stresses. RLCKs are similar to receptor-like kinases (RLKs), but lack extracellular ligand binding domains, so they mainly regulate the physiological processes of plants through intracellular signal transduction. RLCKs regulate the physiological processes of plants such as growth, signal transduction, abiotic stress and biological stress response by phosphorylating downstream target proteins. In plant immunity, RLCKs, as a core signal transduction component, receive signals from pattern recognition receptors (PRRs) to activate pattern-triggered immunity (PTI), including reactive oxygen species (ROS) production, Ca 2+ influx, mitogen-activated protein kinase (MAPK) cascade, cellulose synthesis, phosphatidic acid (PA) production, hormone synthesis and signaling, and transcription remodeling. In addition, RLCKs are also involved in effector-triggered immunity (ETI) and the interaction between ETI and PTI.

[0004] In recent years, the role of RLCKs in plant salt tolerance has gradually attracted attention. Studies have shown that RLCKs help plants maintain normal growth and development in high-salt environments by regulating intracellular signal transduction, gene expression, and protein activity. For example, the overexpression of OsRLCK1 in rice can enhance the tolerance of plants to salt stress, which is mainly achieved by improving the ability of reactive oxygen species (ROS) clearance, increasing the accumulation of osmotic adjustment substances, and regulating the expression of stress response genes. OsRLCK241 OsRLCK311 ​Under salt stress, it can interact with the plasma membrane aquaporin AtPIP2;1, regulating the stomatal response to ABA, thereby achieving a balance between maintaining plant water balance and gas exchange, and promoting rice growth under salt stress. In willow (Salix psammophila), SpRLCK1 Overexpression of these genes enhances plant tolerance to drought and salt stress, primarily by increasing the activity of antioxidant enzymes and regulating the expression of stress-related genes. These findings indicate that RLCKs play a crucial role in plant salt tolerance, regulating plant responses to salt stress through multiple mechanisms and serving as key molecules for plant adaptation to high-salt environments.

[0005] Although RLCKs have shown good results in salt stress resistance in various plants, their salt tolerance in sugarcane has not yet been reported.

[0006] Based on this, the present invention is proposed. Summary of the Invention

[0007] The purpose of this invention is to provide a receptor-like cytoplasmic kinase gene derived from sugarcane. ScRLCK1, Another purpose is application ScRLCK1 Genes enhance plant salt tolerance 。

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a receptor-like cytoplasmic kinase gene derived from sugarcane. ScRLCK1 The receptor-like cytoplasmic kinase gene ScRLCK1 The nucleotide sequence is shown in SEQ ID NO.1.

[0009] This invention also provides overexpression of the aforementioned receptor-like cytoplasmic kinase gene. ScRLCK1 Application in improving plant salt stress tolerance; The plant in question is Arabidopsis thaliana or sugarcane.

[0010] The present invention also provides the aforementioned receptor-like cytoplasmic kinase gene. ScRLCK1 The encoded protein sequence is shown in SEQ ID NO.2.

[0011] The present invention also provides the application of the protein sequence in improving plant salt stress tolerance; The plant in question is Arabidopsis thaliana or sugarcane.

[0012] The present invention also provides an expression cassette for improving plant salt stress tolerance, comprising any one of the following: (1) Recombinant vector; (2) Recombinant bacteria; The recombinant vector contains the receptor-like cytokinase gene.ScRLCK1 Cloned into an overexpression vector driven by the 35S promoter; The overexpression vector was pCambia2300; The recombinant bacteria contains the aforementioned receptor-like cytokinase gene. ScRLCK1 Alternatively, the recombinant vector can be transformed into Agrobacterium EHA105.

[0013] The present invention also provides the application of the expression cassette in improving the salt stress tolerance of plants; The plant in question is Arabidopsis thaliana or sugarcane.

[0014] The present invention also provides a method for detecting the receptor-like cytoplasmic kinase gene. ScRLCK1 The reagent for expressing the expression level is a primer pair, as shown in SEQ ID NO.7~8.

[0015] The present invention also provides the application of the reagent described herein in the detection of salt tolerance in plants.

[0016] The present invention also provides a method for improving the salt stress tolerance of plants, comprising the following steps: The aforementioned receptor-like cytoplasmic kinase gene ScRLCK1 Or the expression cassette may be transferred into the plant; The plant in question is Arabidopsis thaliana or sugarcane.

[0017] This invention also provides a method for constructing highly salt-tolerant transgenic sugarcane, comprising the following steps: 1) Transfer sugarcane explants to callus induction medium and culture in the dark for 16-20 days to obtain induced callus; 2) Place the induced callus tissue into the bacterial suspension of recombinant bacteria and incubate for 30-45 min to obtain infected induced callus tissue; 3) Transfer the infected induced callus to a co-culture medium and culture for 2-4 days. Then transfer it to a callus culture medium and culture for 2-3 weeks to obtain qualified callus. 4) Transfer qualified callus tissue to differentiation medium and culture for 28-34 days to obtain seedlings containing adventitious buds; 5) Transplant seedlings containing adventitious buds into rooting medium and culture for 28-34 days to obtain sugarcane seedlings; transplant the sugarcane seedlings into the field and culture to obtain transgenic sugarcane; The recombinant bacteria contains the receptor-like cytoplasmic kinase gene. ScRLCK1 The strain or the recombinant bacteria in the expression cassette; The callus induction culture medium, using water as a solvent, comprises the following components at the following concentrations: 4–5 g / L MS powder, 27–28 g / L sucrose, 0.4–0.6 g / L hydrolyzed casein, 0.05–0.06 g / L L-cysteine, 0.1–0.2 g / L citric acid, 0.08–0.12 g / L inositol, 2.5–3.5 mL / L 1 mg / mL 2,4-D solution, and 2.8–3.2 g / L Phytagel plant gel. The co-culture medium, using water as a solvent, comprises the following components at the following concentrations: 4–5 g / L MS powder, 0.8–1.2 g / L glucose, 3–5 g / L sucrose, 0.4–0.6 g / L hydrolyzed casein, 0.05–0.06 g / L L-cysteine, 0.1–0.2 g / L citric acid, 0.08–0.12 g / L inositol, 2.5–3.5 mL / L 2,4-D solution (1 mg / mL), 0.8–1.2 mL / L KT solution (1 mg / mL), and 4.5–5.0 g / L agar powder. The callus culture medium uses water as a solvent and includes the following components at the following concentrations: 4–5 g / L MS powder, 2–3 g / L glucose, 7–8 g / L sucrose, 0.4–0.6 g / L hydrolyzed casein, 0.05–0.06 g / L L-cysteine, 0.1–0.2 g / L citric acid, 0.08–0.12 g / L inositol, 2.5–3.5 mL / L 2,4-D solution (1 mg / mL), 0.8–1.2 mL / L KT solution (1 mg / mL), and 4.5–5.0 g / L agar powder. Differentiation and rooting media, using water as a solvent, independently comprise the following components at the following concentrations: 4–5 g / L MS powder, 28–32 g / L sucrose, 0.4–0.6 g / L hydrolyzed casein, 0.05–0.06 g / L L-cysteine, 0.1–0.2 g / L citric acid, 0.08–0.12 g / L inositol, 0.8–1.2 mL / L 1 mg / mL 6-BA solution, and 2.8–3.2 g / L Phytagel plant gel.

[0018] The present invention has the following advantages: (1) This invention is the first to clone and identify a receptor-like cytoplasmic kinase gene in sugarcane. ScRLCK1 The functional study of this gene in the process of treating sugarcane with NaCl provides new evidence for plant salt tolerance and serves as a key gene for providing new genetic resources for sugarcane breeding.

[0019] (2) The receptor-like cytokinase gene cloned in this invention enriches the research on salt-tolerant functional genes in sugarcane.

[0020] (3) Breeding salt-tolerant varieties is the most effective strategy to alleviate the impact of salt stress on sugarcane yield. This can be achieved by constructing... ScRLCK1 Overexpression of sugarcane and Arabidopsis materials and related experiments were conducted. ScRLCK1 The plants exhibit significantly enhanced resistance to salt stress, providing an effective approach for cultivating highly salt-tolerant sugarcane varieties and promoting sustainable industrial development. Attached Figure Description

[0021] Figure 1 for ScRLCK1 -TA plasmid; Figure 2 The prediction results for the ScRLCK1 domain are shown in the figure. Figure 3 For sugarcane leaves and roots under salt stress ScRLCK1 Gene expression levels (left image shows root system, right image shows leaf system); Figure 4 For the recombinant vector OE- ScRLCK1; Figure 5 In transgenic Arabidopsis thaliana ScRLCK1 Gene expression levels; Figure 6 In genetically modified sugarcane ScRLCK1 Gene expression levels; Figure 7 The growth of transgenic Arabidopsis thaliana under salt stress; Figure 8 This shows the growth of genetically modified sugarcane under salt stress. Detailed Implementation

[0022] receptor cytoplasmic kinase gene ScRLCK

[0023] receptor cytoplasmic kinase gene ScRLCK1 The encoded protein sequence is shown in SEQ ID NO.2, SEQ IDNO.2:MGCCGSSLRAGTHPEKKPPGRAAGALPPHRSSYSLNQHQAPAPSAARAGAGGRQVPPLKEFSLAELRAATGGFAAENIVSESGEKAPNFVYKGRLEASRRAIAVKKFTKMAWPDPKQFAEEAK GVGKLRHCRLANLIGYCCDGDERLLVAEFMPNDTLAKHLFHWENQTIEWAMRLRVAYYIAEALEYCSTEGRPLYHDLNAYRVLFDENGPRLSCFGLMKNSRDGKSYSTNLAYTPPEYLRNGRVTSESVIF SFGTILLDLLSGKRIPPSRVLDMIKGNNIQVLMDSHLEGNYSTDEATTLVDLASQCLQYEPRDRPNTKKLVSILEPLQIKSEVPSYEMLGIPKYEEEEAPPPPQPQHPLSPMGEACSRMDLTAIHQILVNT HYRDEGSNELSFQEWTQQMRDMLEARKRGDFAFRDKDFKAAIDCYTQFVDVGTMVSPTVYARRSLCHLMCDQPDAALRDAMQAQCVYPDWPTAFYMQAVALSKLNMQSDATDMLNEASQLEEKRQNTKP.

[0024] The primer pairs for amplifying the nucleotide sequence described in SEQ ID NO.1 are shown in SEQ ID NO.3~4, with the forward primer SEQ ID NO.3: ATGGGCTGCTGCGGCTCCTC and the reverse primer SEQ ID NO.4: TCAAGGTTTTGTGTTCTGCC.

[0025] Real-time quantitative PCR detection of receptor cytoplasmic kinase genes ScRLCK1 The internal reference gene for expression level is ScGAPDH, Primers are shown in SEQ ID NO. 5-6: Forward primer SEQ ID NO. 5: CACGGCCACTGGAAGCA, Reverse primer SEQ ID NO. 6: TCCTCAGGGTTCCTGATGCC; Real-time quantitative PCR was used to detect receptor-like cytokinase genes. ScRLCK1The primer pairs for the expression level are shown in SEQ ID NO.7~8, with the forward primer SEQ ID NO.7: AGTTTGTGCCACCTCATGT and the reverse primer SEQ ID NO.8: GTGTTCTGCCGCTTCTCTT.

[0026] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0027] The culture medium formulation used in the genetic transformation of sugarcane (salt-tolerant transgenic sugarcane) in this embodiment of the invention is as follows: Preparation of 1 mg / mL 2,4-dichlorophenoxyacetic acid (2,4-D) solution: Weigh 100 mg 2,4-D, add 1.0 mL 1 M KOH solution, shake to dissolve, then dilute to 100 mL with distilled water and store at 4℃ for later use; Preparation method of 1 mg / mL kinetin (KT) solution: Weigh 100 mg KT, add 1.0 mL of 1 M KOH solution, shake to dissolve, and then make up to 100 mL with distilled water. Store at 4℃ for later use. Preparation method of 1 mg / mL 6-benzylaminopurine (6-BA) solution: Weigh 100 mg 6-BA, add 1.0 mL 1 M KOH solution, shake to dissolve, and then make up to 100 mL with distilled water. Store at 4℃ for later use. Preparation of 100 μM acetylsuccinone (AS) solution: Weigh 0.196 g of AS, add 10 mL of DMSO to dissolve, filter through a 0.22 μm filter membrane, dispense into 1.5 mL centrifuge tubes, and store at 4℃ for later use; Preparation of 1 L callus induction medium (SCIM3): using water as solvent, including 4.74 g MS powder, 27.25 g sucrose, 0.5 g hydrolyzed casein, 0.054 g L-cysteine, 0.15 g citric acid, 0.1 g inositol, 3 mL of 1 mg / mL 2,4-D solution, and 3 g Phytagel plant gel; Preparation of 1 L co-culture medium (SCCM): using water as solvent, including 4.74 g MS powder, 1 g glucose, 4 g sucrose, 0.5 g hydrolyzed casein, 0.054 g L-cysteine, 0.15 g citric acid, 0.1 g inositol, 3 mL 1 mg / mL 2,4-D solution, 1 mL 1 mg / mL KT solution, and 4.8 g agar powder; Preparation of 1 L callus culture medium (CPMS): using water as solvent, including 4.74 g MS powder, 2.5 g glucose, 7.5 g sucrose, 0.5 g hydrolyzed casein, 0.054 g L-cysteine, 0.15 g citric acid, 0.1 g inositol, 3 mL of 1 mg / mL 2,4-D solution, 1 mL of 1 mg / mL KT solution, and 4.8 g agar powder; Preparation of 1L differentiation medium (CD): using water as solvent, including 4.74 g MS powder, 30 g sucrose, 0.5 g casein, 0.054 g L-cysteine, 0.15 g citric acid, 0.1 g inositol, 1 mL of 1 mg / mL 6-BA solution, and 3.0 g Phytagel; The rooting medium is the same as the differentiation medium.

[0028] Example 1

[0029] ScRLCK1 Gene identification

[0030] Sugarcane RNA extraction and reverse transcription

[0031] Total RNA was extracted from fresh leaves of the sugarcane variety "Zhongzhe 9," which is susceptible to shoot rot, using the TaKaRa MiniBEST Universal RNA Extraction Kit. The integrity of the RNA was assessed by agarose gel electrophoresis. DNA was removed using the gDNA eraser in the TransScript® One-Step gDNA Removal and cDNA Synthesis SuperMix kit. The reaction mixture consisted of 2.0 μL 5×gDNA Eraser Buffer, 1.0 μL gDNA Eraser, 2.0 μg RNA, and 6.0 μL RNase-free ddH2O. After mixing, the mixture was incubated at 42°C for 2 min. A reverse transcription premix was prepared with the following reaction mixture: 2.0 μL 5×Prime Script Buffer, 4.0 μL RNase-Free ddH2O, 4.0 μL RT Primer Mix, 1.0 μL Prime Script RT Enzyme Mix I, and ddH2O was added to a final volume of 20 μL. After gently mixing and centrifuging, proceed with the reverse transcription reaction: 37℃ for 15 min; 85℃ for 5 s, then cool at 4℃. Store at -20℃ for later use.

[0032] ScRLCK1 Gene sequence acquisition

[0033] Using the sugarcane genome database (NCBI accession number GWHEQVP00000000) as a reference, forward primers were designed. ScRLCK1 -F(5'ATGGGCTGCTGCGGCTCCTC 3' (SEQ ID NO.3)); reverse primer ScRLCK1 -R(5'TCAAGGTTTTGTGTTCTGCC 3' (SEQ ID NO.4)). Using the cDNA obtained above as a template, PCR amplification was performed using the high-fidelity enzyme Phanta Max Super-Fidelity DNA Polymerase (Vazyme) according to the following system: 25 μL 2×Phanta Master Mix, 1 μL Forward Primer (10 μM), 1 μL Reverse Primer (10 μM), 2 μL cDNA, and ddH2O was added to a final volume of 50 μL. The PCR reaction program was: pre-denaturation at 95℃ for 30 s; denaturation at 95℃ for 15 s, annealing at 60℃ for 15 s, extension at 72℃ for 30 s, 32 cycles; final extension at 72℃ for 5 min. The product was then ligated into the TA cloning vector to obtain... ScRLCK1 -TA plasmid ( Figure 1 ), and transform E. coli DH 5α, screen for positive clones and confirm with sequencing. Obtain the required... ScRLCK1 The open reading frame (ORF) of the gene, with its nucleotide sequence shown in SEQ ID NO: 1, was determined using BlastX (http: / / www.ncbi.nlm.nih.gov). ScRLCK1 The 515 amino acids corresponding to the open reading frame (ORF) of a gene can be used to infer... ScRLCK1 The protein sequence encoded by the gene is shown in SEQ ID NO: 2. Conserved domains were determined using Conserveddomains (https: / / www.ncbi.nlm.nih.gov / Structure / cdd / wrpsb.cgi), and the domain prediction results are as follows: Figure 2 As shown.

[0034] ScRLCK1 Expression pattern analysis in sugarcane treated with NaCl

[0035] To verify ScRLCK1Whether genes are involved in regulating salt stress. This invention selected sugarcane (variety Zhongzhe 9) at the 1-2 leaf stage and cultured it in 1 / 2 MS liquid medium containing 200 mmol / L NaCl. Sugarcane grown in 1 / 2 MS liquid medium served as a control group. Root and leaf samples were collected from sugarcane at 0 h, 6 h, 12 h, 24 h, 48 h, 72 h, 96 h, 120 h, and 144 h after treatment. Total RNA was extracted from the collected samples and reverse transcribed into cDNA, which was used as template for subsequent experiments. ScGAPDH As an internal reference gene. qScGAPDH -F forward primer (5'CACGGCCACTGGAAGCA 3' (SEQ ID NO.5)), qScGAPDH-R Reverse primer (5'TCCTCAGGGTTCCTGATGCC 3' (SEQ ID NO.6)). Design ScRLCK1 Specific primers qScRLCK1 -F(5'AGTTTGTGCCACCTCATGT 3'(SEQ ID NO.7)) and qScRLCK1 -R(5'GTGTTCTGCCGCTTCTCTT 3' (SEQ ID NO. 8)). Use TB Green Premix Ex Taq TM The instructions for using the II (TaKaRa) kit were to perform real-time quantitative PCR (RT-qPCR), with three biological replicates per reaction. Two [reactions were performed]. -ΔΔCt The method involves calculation and analysis. The results are as follows: Figure 3 As shown.

[0036] result: ScRLCK1 The full-length ORF is 1545 bp, encoding 515 amino acids, with no signal peptide and no transmembrane region. Domain prediction revealed that ScRLCK1 belongs to the plant receptor-like kinase (RLK) family.

[0037] Figure 3 It can be seen that, ScRLCK1 The expression level of [a specific substance] was upregulated under NaCl stress, reaching a peak and then gradually decreasing to normal levels; the peak expression was observed in leaves after 72 h of treatment and in roots after 120 h of treatment. This indicates that during the process of sugarcane under salt stress, [the following is a description of the regulation of [a specific substance]: ScRLCK1 Transcription of this substance is significantly activated, and it may play an important regulatory role in sugarcane's response to salt stress.

[0038] Example 2

[0039] Genetic transformation of sugarcane and Arabidopsis

[0040] Construction of recombinant vectors

[0041] Constructed using Example 1 ScRLCK1 -Using TA plasmid as a template, design specific primers ScRLCK1 -2300-F (5'AACACGGGGGACGAGCTCATGGGCTGCTGCGGCTCCTC 3' (SEQ ID NO.9)), ScRLCK1 -2300-R (5'CTGCAGGTCGACTCTAGATCAAGGTTTTGTGTTCTGCC 3' (SEQ ID NO.10)). The reaction system and procedure were the same as in Example 1, and the amplified product was ligated to the culture medium obtained by seamless cloning. Sac I and Xba I The pCambia2300 overexpression vector, driven by a linearized 35S promoter, was digested with enzymes to obtain the recombinant vector OE-. ScRLCK1 ( Figure 4 ), transforming E. coli DH 5α, screen for positive clones and confirm them by sequencing.

[0042] Agrobacterium-mediated genetic transformation in Arabidopsis thaliana

[0043] OE- ScRLCK1 The recombinant vector was transformed into Agrobacterium EHA105 strain to obtain recombinant bacteria, which were then used for Arabidopsis genetic transformation experiments. The specific steps of the Agrobacterium-mediated transformation method are as follows: (1) Preparation of receptor materials Wild-type Arabidopsis thaliana with good growth and no bolting was selected as the transformation recipient.

[0044] (2) Prepare Agrobacterium-containing liquid

[0045] A single colony of recombinant bacteria containing the target gene was inoculated into LB liquid medium, activated, and cultured to the logarithmic growth phase. After harvesting the bacterial suspension, the bacterial cells were resuspended in osmotic buffer (1 / 2 MS + 10% sucrose + 400 μl / L polycyclic ether modified polydimethylsiloxane Silwet-77) to achieve an OD600 value of 0.8, thus obtaining the bacterial suspension of recombinant bacteria.

[0046] (3) Infection

[0047] Immerse the inflorescence of Arabidopsis thaliana in a bacterial suspension of recombinant bacteria, gently shake to ensure full contact between the inflorescence and the bacterial suspension, and soak for 1 minute.

[0048] (4) Cultivation

[0049] Cover the plant with plastic wrap after dipping it in flowers to maintain humidity, and incubate it in the dark for 1 day. Then place it under normal incubation conditions. After 2 days, remove the plastic wrap and continue incubation until the plant matures.

[0050] (5) Screening

[0051] Seeds of T0 generation Arabidopsis thaliana were collected and sown on 1 / 2 MS medium containing kanamycin for growth selection. Positive plants were obtained and transplanted into potting soil for further growth. After maturity, T1 generation seeds were collected for further selection. Positive plants from lines with a segregation ratio of 3:1 were selected and transplanted into potting soil for continued growth. Seeds were harvested individually from each plant and then screened again on 1 / 2 MS medium containing kanamycin. Lines that no longer segregated were identified as homozygous positive lines and transplanted into potting soil for further growth. The harvested seeds were the T3 generation homozygous positive Arabidopsis thaliana seeds. RNA was extracted from all homozygous lines, and cDNA was obtained through reverse transcription. ScRLCK1 Expression levels were detected (using primer pairs as shown in SEQ ID NO. 7-8), and lines with expression levels meeting expectations were selected for further studies. This study ultimately selected two homozygous Arabidopsis lines with significantly increased expression levels (At-). OE-ScRLCK1-4 and At- OE-ScRLCK1-15 () Figure 5 (To conduct subsequent experiments)

[0052] Genetic transformation of sugarcane by Agrobacterium (construction of salt-tolerant transgenic sugarcane)

[0053] OE- ScRLCK1 The recombinant vector was transformed into Agrobacterium EHA105 strain to obtain recombinant bacteria, which were then used for sugarcane genetic transformation experiments. The specific steps of the Agrobacterium-mediated transformation method are as follows: (1) Culture of sugarcane somatic embryos Disease-free sugarcane stem segments were selected as explants and their surfaces were disinfected with 75% alcohol in a clean bench. The epidermis and leaf sheaths were removed, and young stem segments (8 cm) from the middle section were cut. Using a sterile scalpel, the stem segments were cut into 2 mm thick pieces, with the growth point facing down on each piece, and transferred to SCIM3 medium. The pieces were incubated in the dark at 25°C for 18 days to induce callus formation. During this process, the growth of the material was checked regularly to prevent contamination.

[0054] (2) Agrobacterium culture

[0055] Will carry ScRLCK1 Recombinant strain EHA105 of Agrobacterium was selected, and positive monoclonal samples were inoculated into LB liquid medium containing 50 mg / L Kan and 20 mg / L Rif antibiotics. The culture was then placed in a shaker and incubated until the bacterial concentration reached OD500. 600To ensure that Agrobacterium is in the logarithmic growth phase and in good growth condition, 100 μL of 100 μM AS solution was added to prepare an Agrobacterium suspension (a suspension of recombinant bacteria).

[0056] (3) Infection and co-cultivation

[0057] Select granular, dry induced callus tissue. Gently peel off the swollen portion with sterile forceps and place it in an Agrobacterium suspension. Incubate at 28°C for 40 min, gently shaking the culture flask every 15 min to ensure uniform mixing of Agrobacterium and induced callus tissue and complete infection. After infection, discard the bacterial suspension and transfer the induced callus tissue to sterile filter paper. Allow it to air dry for 4 h, removing excess Agrobacterium suspension. When transferring the fully dried callus tissue to SCCM co-culture medium, use sterile filter paper to separate the callus tissue from the medium to avoid potential bacterial overgrowth or medium contamination from direct contact. This ensures effective infection of the callus tissue by Agrobacterium on the filter paper and maintains nutrient supply. Incubate the culture dishes at 20°C for 3 days.

[0058] (4) Resistance screening and culture

[0059] After co-culturing, the induced callus tissue was transferred to a beaker containing sterile water and gently rinsed with a pipette for 2 minutes. The rinsing solution was then discarded, and the sterile water was replaced. This rinsing step was repeated twice. The induced callus tissue was then allowed to air dry until no obvious moisture was visible on the surface. It was then transferred to CPMS medium containing 30 mg / L Geneticin (G418) for selection. This selection process lasted for 3 weeks, during which callus growth was regularly observed, and untransformed callus tissue was removed promptly.

[0060] (5) Callus differentiation

[0061] Selected callus tissues were transferred to differentiation medium (CD) containing the screening antibiotics and cultured at 28°C with a photoperiod of 14 h light (3000 Lux) followed by 10 h darkness. The medium needed to be changed regularly to maintain nutrient supply to the callus tissues and prevent the accumulation of metabolites from affecting tissue differentiation. After 30 days of culture, embryogenic callus tissues gradually formed green adventitious shoots.

[0062] (6) Rooting culture

[0063] Carefully remove the seedlings differentiated from the differentiation medium, taking only one seedling from each callus tissue, and inoculate them into rooting culture bottles. Under suitable light conditions of 25°C, regularly observe the plant growth, ensuring the plants have sufficient water and nutrients. After 30 days of culture, sugarcane seedlings with fully developed roots and leaves can be transplanted to the field for long-term growth observation.

[0064] (7) Screening and identification

[0065] RNA was extracted from transgenic sugarcane plants, and cDNA was obtained through reverse transcription. Real-time quantitative PCR (RT-qPCR) was then performed to detect the cDNA. ScRLCK1 The expression level (primer pairs as shown in SEQ ID NO.7~8) was determined, and the reaction was performed according to the instructions of the TB Green® Premix Ex Taq™ II (TaKaRa) kit.

[0066] sugarcane ScGAPDH Genes and Arabidopsis At Actin 2 Genes were used as internal controls, with 2 -ΔΔCT Methods: The relative expression levels of the target gene were calculated and analyzed. Reaction system: 10 μL 2×TB Green Premix Ex Taq, 1 μL Forward primer (2 μM), 1 μL Reverse primer (2 μM), 2 μL cDNA (200 ng / μL), and ddH2O added to a final volume of 20 μL. PCR reaction program: pre-denaturation 95℃ for 30 s; amplification 95℃ for 15 s; 60℃ for 30 s, 45 cycles; melting curve program: 95℃ for 15 s, 95℃ for 60 s, 97℃ for 1 s; cooling: 37℃ for 30 s. Finally, two transgenic sugarcane lines with significantly increased expression levels were identified for further research. Sc-OE-ScRLCK1-4-9 and Sc-OE-ScRLCK1-12 () Figure 6 After 9 months of growth (with 15-20 sugarcane buds on the sugarcane stalk), the positive single plants are cut into single bud segments and planted in the field. The sugarcane seedlings that have been propagated are used for subsequent experiments such as resistance identification.

[0067] ScGAPDH Gene primers are shown in SEQ ID NO.5~6; At Actin 2 The primers for the internal reference gene are shown in SEQ ID NO.11~12, SEQ ID NO.11 (upstream primer): AAATCACAGCACTTGCACCAAGC; SEQ ID NO.12 (downstream primer): GGCCTTGGAGATCCACATCTGC.

[0068] Example 3

[0069] ScRLCK1 Resistance identification of transgenic Arabidopsis and sugarcane materials

[0070] wild type and ScRLCK1 Seeds of transgenic homozygous Arabidopsis thaliana lines were first disinfected with 75% alcohol for 1 min, then with 50% 84 disinfectant for 2 min, followed by washing four times with sterile ddH2O water for 3 min each time. Finally, sterile 0.1% agarose was added to suspend the seeds. After vernalization at 4℃ for 3 days, the disinfected seeds were sown on 1 / 2 MS medium (control group) and 1 / 2 MS medium containing 200 mmol / L NaCl (treatment group), and physiological indicators such as seed germination and growth were observed. The results are as follows: Figure 7 As shown.

[0071] Harvesting wild type and ScRLCK1 Sugarcane stalks from transgenic sugarcane lines were cut into single-bud segments. Plump, insect-free buds were selected for subsequent experiments, with at least 20 buds from each line (including wild type). The buds were evenly placed in vermiculite, thoroughly watered with 1 / 2 MS nutrient solution, and then covered with a film to promote bud germination. After two true leaves had emerged, the vermiculite on the root surface was rinsed off, and healthy, uniformly growing buds were selected for salt stress treatment. The control group was hydroponically cultured in 1 / 2 MS nutrient solution, while the control group was hydroponically cultured in 1 / 2 MS nutrient solution containing 200 mmol / L NaCl. The growth and development of the sugarcane were observed. Results are as follows: Figure 8 As shown.

[0072] Figure 7 The results showed that in 200 mM NaCl medium, ScRLCK1 The transgenic Arabidopsis thaliana showed significantly better growth than the wild type; indicating that... ScRLCK1 It can improve the tolerance of Arabidopsis thaliana to salt stress.

[0073] Figure 8 The results showed that under 200 mM NaCl treatment, the growth and green retention of the transgenic sugarcane material were superior to those of the wild type, indicating that... ScRLCK1 It can improve the sugarcane's tolerance to salt stress.

[0074] As can be seen from the above embodiments, this study is the first to reveal the properties of sugarcane. ScRLCK1 The function of genes in salt tolerance has expanded our understanding of the RLCK signaling network and provided important genetic resources for the genetic improvement of salt tolerance in sugarcane and other crops. Under salt stress, ScRLCK1 The expression of [specific ingredient] was significantly upregulated, and its overexpression significantly enhanced the salt tolerance of transgenic plants, as shown in: OE- ScRLCK1 Transgenic Arabidopsis thaliana and transgenic sugarcane grew better under salt stress than wild-type varieties. This provides new strategies and targets for crop stress resistance breeding.

[0075] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A receptor-like cytokinase gene derived from sugarcane ScRLCK1 Its characteristics are, The receptor-like cytoplasmic kinase gene ScRLCK1 The nucleotide sequence is shown in SEQ ID NO.

1.

2. Overexpression of the receptor-like cytokinase gene as described in claim 1 ScRLCK1 Application in improving plant salt stress tolerance; The plant in question is Arabidopsis thaliana or sugarcane.

3. The receptor-like cytokinase gene of claim 1 ScRLCK1 The encoded protein sequence is characterized by, As shown in SEQ ID NO.

2.

4. The application of the protein sequence described in claim 3 in improving plant salt stress tolerance; The plant in question is Arabidopsis thaliana or sugarcane.

5. An expression cassette for enhancing plant salt stress tolerance, characterized in that, Including any of the following: (1) Recombinant vector; (2) Recombinant bacteria; The recombinant vector is the receptor-like cytokinase gene described in claim 1. ScRLCK1 Cloned into an overexpression vector driven by the 35S promoter; The overexpression vector was pCambia2300; The recombinant bacteria contains the receptor-like cytokinase gene described in claim 1. ScRLCK1 Alternatively, the recombinant vector can be transformed into Agrobacterium EHA105.

6. The application of the expression cassette according to claim 5 in improving plant salt stress tolerance; The plant in question is Arabidopsis thaliana or sugarcane.

7. A method for detecting the receptor-like cytokinase gene of claim 1 ScRLCK1 The reagent for expressing the amount of [expression] is characterized by, The reagent is a primer pair, as shown in SEQ ID NO.7~8.

8. The application of the reagent according to claim 7 in the detection of salt tolerance in plants.

9. A method for improving the salt stress tolerance of plants, characterized in that, Includes the following steps: The receptor-like cytoplasmic kinase gene as described in claim 1 ScRLCK1 Or the expression cassette as described in claim 5 can be transferred into a plant; The plant in question is Arabidopsis thaliana or sugarcane.

10. A method for constructing highly salt-tolerant transgenic sugarcane, characterized in that, Includes the following steps: 1) Transfer sugarcane explants to callus induction medium and culture in the dark for 16-20 days to obtain induced callus; 2) Place the induced callus tissue into the bacterial suspension of recombinant bacteria and incubate for 30-45 min to obtain infected induced callus tissue; 3) Transfer the infected induced callus to a co-culture medium and culture for 2-4 days. Then transfer it to a callus culture medium and culture for 2-3 weeks to obtain qualified callus. 4) Transfer qualified callus tissue to differentiation medium and culture for 28-34 days to obtain seedlings containing adventitious buds; 5) Transplant seedlings containing adventitious buds into rooting medium and culture for 28-34 days to obtain sugarcane seedlings; transplant the sugarcane seedlings into the field and culture to obtain transgenic sugarcane; The recombinant bacteria contains the receptor-like cytoplasmic kinase gene as described in claim 1. ScRLCK1 The strain or the recombinant bacteria in the expression cassette of claim 5; The callus induction medium, using water as a solvent, comprises the following components at the following concentrations: 4–5 g / L MS powder, 27–28 g / L sucrose, 0.4–0.6 g / L hydrolyzed casein, 0.05–0.06 g / L L-cysteine, 0.1–0.2 g / L citric acid, 0.08–0.12 g / L inositol, 2.5–3.5 mL / L 1 mg / mL 2,4-D solution, and 2.8–3.2 g / L Phytagel plant gel. The co-culture medium, using water as a solvent, comprises the following components at the following concentrations: 4–5 g / L MS powder, 0.8–1.2 g / L glucose, 3–5 g / L sucrose, 0.4–0.6 g / L hydrolyzed casein, 0.05–0.06 g / L L-cysteine, 0.1–0.2 g / L citric acid, 0.08–0.12 g / L inositol, 2.5–3.5 mL / L 2,4-D solution (1 mg / mL), 0.8–1.2 mL / L KT solution (1 mg / mL), and 4.5–5.0 g / L agar powder. The callus culture medium uses water as a solvent and includes the following components at the following concentrations: 4–5 g / L MS powder, 2–3 g / L glucose, 7–8 g / L sucrose, 0.4–0.6 g / L hydrolyzed casein, 0.05–0.06 g / L L-cysteine, 0.1–0.2 g / L citric acid, 0.08–0.12 g / L inositol, 2.5–3.5 mL / L 2,4-D solution (1 mg / mL), 0.8–1.2 mL / L KT solution (1 mg / mL), and 4.5–5.0 g / L agar powder. Differentiation and rooting media, using water as a solvent, independently comprise the following components at the following concentrations: 4–5 g / L MS powder, 28–32 g / L sucrose, 0.4–0.6 g / L hydrolyzed casein, 0.05–0.06 g / L L-cysteine, 0.1–0.2 g / L citric acid, 0.08–0.12 g / L inositol, 0.8–1.2 mL / L 1 mg / mL 6-BA solution, and 2.8–3.2 g / L Phytagel plant gel.

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