Sugarcane ScGIP1 gene, encoded protein and application

Abstract

The invention provides a sugarcane ScGIP1 gene, an encoded protein and application, and belongs to the technical field of genetic engineering. The nucleotide sequence of the ScGIP1 gene is as shown in SEQ ID NO. 1. The invention preliminarily reveals that the sugarcane aspartic protease ScGIP1 as a novel plant disease-resistant factor has potential application value in improvement of plant disease resistance, enriches functional cognition of aspartic protease in plant immune response, provides potential gene resources for disease prevention and control of sugarcane and other crops, and has broad application prospects. Good application prospects and popularization values are realized.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to a sugarcane ScGIP1 gene, its encoded protein, and its applications. Background Technology

[0002] Sugarcane (Saccharum spp.) is the most important sugar crop and biomass fuel crop, and a major international agricultural crop. Sugarcane pokkah boeng disease (PBD) is a significant global fungal disease of sugarcane caused by various Fusarium species. In my country's major sugarcane-growing areas, Fusarium sacchari is the primary pathogen. This disease is now widespread in sugarcane-growing countries and regions, severely impacting sugarcane yield and quality, causing substantial economic losses. Developing disease-resistant varieties is the most economical and effective means of controlling this disease. Elucidating the molecular mechanisms of Fusarium sacchari pathogenicity and its molecular network of interaction with the host, and identifying target genes, is of great significance for improving sugarcane pokkah boeng resistance using modern biotechnology.

[0003] Aspartic proteases (APs) are a class of acidic hydrolases widely distributed in organisms, primarily involved in protein processing and degradation, playing a crucial role in various physiological and pathological processes. Their catalytic activity depends on the highly conserved Asp-Thr / Ser-Gly motif. In plants, most APs belong to the A1 type of the ClanAA subfamily in the MEROPS database. The amino acid sequence of plant APs typically consists of three parts: an N-terminal signal peptide, a plant-specific insert (PSI), and a C-terminus (carboxy-terminal). During protein processing, the N-terminal signal peptide is cleaved at the endoplasmic reticulum, followed by the removal of the propeptide and PSI sequence during protein maturation, ultimately forming a protein composed of an active site domain and a C-terminus.

[0004] Plant abiotic proteins (APs) play multifaceted biological functions in growth and development, organ senescence, and responses to abiotic stress. During plant development, APs primarily participate in the degradation of storage proteins and nutrient transport. For example, in wheat, aspartic proteases WAP1 and WAP2 hydrolyze storage proteins such as gluten, which are expressed in the radicle, scutellum, and aleurone layer, facilitating storage protein transport and early seedling growth. In Arabidopsis, ASPG1 influences seed dormancy, vigor, and germination by regulating the gibberellin (GA) signaling pathway, while simultaneously enhancing the expression of key transcription factors in the ABA signaling pathway (such as ABF2), promoting stomatal closure, and thus improving drought resistance. Furthermore, AP1 in wheat can regulate protein degradation in chloroplasts, delaying leaf senescence and prolonging the duration of photosynthesis, thereby increasing grain yield. In rice, OsAP65 contributes to pollen germination and pollen tube growth; its loss of function leads to impaired male gametophyte transmission, severely affecting fertilization. Besides their role in developmental regulation, APs also play a crucial role in plant resistance to biotic stress. For example, the nuclear-localized APCB1 in Arabidopsis can enhance plant resistance to Botrytis cinerea by promoting the degradation of BAG6 and activating the expression of various defense-related genes. Introducing the potato AP member StAP-PSI into Arabidopsis plants enhanced their resistance to Botrytis cinerea infection by regulating the expression of defense genes through the jasmonic acid (JA) signaling pathway. These results demonstrate that plant aspartic proteases play important functions in the plant life cycle and exert regulatory effects in responses to biotic and abiotic stresses, making them key molecules for plant adaptation to complex environmental conditions.

[0005] Although APs have shown good results in the control of various plant diseases, there are no research reports on their control of sugarcane top rot (PBD). Summary of the Invention

[0006] The purpose of this invention is to provide a sugarcane ScGIP1 gene, its encoded protein, and its applications.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides a sugarcane ScGIP1 gene, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0009] The present invention provides a protein encoded by the ScGIP1 gene, the amino acid sequence of which is shown in SEQ ID NO.2.

[0010]

[0011] SEQ ID NO.2: MWNPKLLVFISLCISVLSSPCTAASGGGKPLVTAVTKDASTSLYTAPL KDGHPLLLDLSSPVISLTTCASKNGTVATLSANTTDGQNPLFPVSFSAVTSCRPQAKVLAGAVSVTSLSPSSQSFPAHVVRTHKVANKIALCLPSDGKSTSGNSVGVAIFGGDPLFFIPADRGDFTTMLAGTAPLHGFNGSPGYYMSSTGVAVEQNRV STSGGMLVVGLSSTIPYTAVRSDVYVTLLRAFYAAASGPNFSWMSRVIAVAPFERCYDSTMLPQSLLGYSMPQIDVMLEGGQNFTVLGGNSMVQVNSNTACLGFVKAAAGQAPAAVIGGFQLENHLLVLDVEKKQLGFTTFLNAIGLSCSNFNFTLAA

[0012] This invention provides a primer pair for amplifying the ScGIP1 gene, the nucleotide sequence of the forward primer is shown in SEQ ID NO.3, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.4.

[0013] This invention provides an overexpression vector for overexpressing the ScGIP1 gene.

[0014] The present invention provides an engineered bacterium containing the aforementioned overexpression vector.

[0015] This invention provides the application of the ScGIP1 gene, the protein, the primer pair, the overexpression vector, or the engineered bacteria in improving sugarcane resistance to sugarcane top rot.

[0016] This invention provides the application of the ScGIP1 gene, or the protein, or the primer pair, or the overexpression vector, or the engineered bacteria in improving plant resistance to gray mold, wherein gray mold is caused by the fungus Botrytis cinerea.

[0017] This invention provides a method for improving sugarcane resistance to sugarcane top rot. The engineered bacteria are used to infect sugarcane callus tissue, which is then differentiated and rooted before being transplanted into the field for further cultivation to obtain transgenic sugarcane plants with improved resistance to sugarcane top rot.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] This invention cloned an aspartic protease family gene, ScGIP1, from sugarcane. Induced expression pattern analysis showed that ScGIP1 expression was significantly upregulated during Fusarium solani infection of sugarcane, reaching a peak at 96 hours post-inoculation. Tobacco leaves transiently expressing ScGIP1 exhibited enhanced resistance to Botrytis cinerea. Given that ScGIP1 can induce a plant immune response, this study constructed a 35S promoter-driven ScGIP1 overexpression vector and obtained transgenic sugarcane (OE-ScGIP1), with enhanced resistance to Fusarium solani. This invention provides a theoretical basis for cultivating new germplasm resistant to Fusarium solani and for the sustainable control of top rot.

[0020] This invention initially reveals that sugarcane aspartic protease ScGIP1, as a novel plant disease resistance factor, has potential application value in improving plant disease resistance, enriches the functional understanding of aspartic protease in plant immune response, and provides potential gene resources for disease control in sugarcane and other crops, with good application prospects and promotion value. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0022] Figure 1 This is the primary structure of the ScGIP1 protein; it encodes 365 amino acids, with a 24-amino acid signal peptide at the N-terminus, and the aspartic protease domain is located at 69-349 amino acids.

[0023] Figure 2 The results show the relative expression level of the ScGIP1 gene during Fusarium canaliculata infection in Example 2.

[0024] Figure 3 The structural map of the ScGIP1-PVX plasmid in Example 3;

[0025] Figure 4 The structural diagram of the GFP-PVX plasmid in Example 3 is shown.

[0026] Figure 5 The area of ​​lesions in tobacco plants transiently expressing pGR107:ScGIP1 and pGR107:GFP after inoculation with Botrytis cinerea in Example 3;

[0027] Figure 6 This is a schematic diagram illustrating the construction of the overexpression vector OE-ScGIP1 in Example 4;

[0028] Figure 7 The structural map of the overexpression vector OE-ScGIP1 (pCambia2300-ScGIP1) constructed in Example 4 is shown below.

[0029] Figure 8 The results show the relative expression levels of ScGIP1 in wild-type sugarcane and transgenic sugarcane plants in Example 5.

[0030] Figure 9 The images show the lesion patterns (scale bar = 1 cm) on the leaves of wild-type sugarcane and transgenic sugarcane plants in Example 5, and the statistical results of the lesion area (numerical values ​​are expressed as mean ± SD, n = 12).

[0031] Figure 10 The growth of mycelium from leaves of wild-type sugarcane and transgenic sugarcane plants after in vitro inoculation in Example 5 is shown. The error bars represent the standard error of three biological and technical replicates, and ** indicates extremely significant differences (P<0.01). Detailed Implementation

[0032] 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.

[0033] Example 1

[0034] 1. Sugarcane RNA extraction and reverse transcription

[0035] Total RNA was extracted from fresh leaves of the sugarcane variety “Zhongzhe 9” susceptible to top rot using the TaKaRa MiniBEST Universal RNA Extraction Kit, and the integrity of the RNA was detected by agarose gel electrophoresis.

[0036] Using a reverse transcription kit ( In One-Step gDNA Removal and cDNA Synthesis SuperMix, the gDNA eraser removes DNA. The reaction system consists of 2.0 μL 5×gDNA Eraser Buffer, 1.0 μL gDNA Eraser, 2.0 μg RNA, and 6.0 μL L Nase-free ddH2O. After mixing, incubate at 42°C for 2 min.

[0037] Prepare a reverse transcription premix. The reaction mixture consists of: 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 to a final volume of 20 μL. After gentle mixing and centrifugation, proceed with the reverse transcription reaction: 37℃ for 15 min; 85℃ for 5 s; and cool at 4℃. Store the resulting cDNA at -20℃ for later use.

[0038] 2. Obtaining the ScGIP1 gene

[0039] Using the sugarcane genome database as a reference (Bao Y, Zhang Q, Huang J, et al. A chromosomal-scale genome assembly of modern cultivated hybrid sugarcane provides insights into origination and evolution[J]. Nature Communications, 2024, 15(1), 3041.), forward primer ScGIP1-F (SEQ ID NO.3) and reverse primer ScGIP1-R (SEQ ID NO.4) were designed.

[0040] ScGIP1-F: 5'-ATGTGGAATCCCAAACTCCTCGTC-3'

[0041] ScGIP1-R: 5'-TTACTAGGCGGCAAGAGTGAAATTG-3'

[0042] 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×PhantaMasterMix, 1 μL Forward Primer (10 μM), 1 μL Reverse Primer (10 μM), 2 μL cDNA, and ddH2O added to a final volume of 50 μL. The PCR reaction program was as follows: 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, for 32 cycles; final extension at 72℃ for 5 min.

[0043] The product was ligated into a TA cloning vector, transformed into *E. coli* DH5α, and positive clones were screened and confirmed by sequencing. The open reading frame (ORF) of the ScGIP1 gene was obtained, and its nucleotide sequence is shown in SEQ ID NO.1, with a full length of 1095 bp. The 365 amino acids corresponding to the ORF of the ScGIP1 gene were determined using BlastX (http: / / www.ncbi.nlm.nih.gov), leading to the deduction of the amino acid sequence of the protein encoded by the ScGIP1 gene as shown in SEQ ID NO.2, with a 24-aa signal peptide at the N-terminus and no transmembrane region. Conserved domains were analyzed using Conserved domains (https: / / www.ncbi.nlm.nih.gov / Structure / cdd / wrpsb.cgi). Domain prediction revealed that ScGIP1 belongs to the plant aspartic proteases (APs) family. Figure 1 ).

[0044] Example 2: Analysis of the expression pattern of the ScGIP1 gene in sugarcane infected by pathogens

[0045] Select the first leaf of a 5-leaf stage sugarcane (Zhongzhe 9 variety), ensuring uniform growth. Make incisions on both sides of the petiole using a sterile needle. Using a 0.5cm diameter punch, cut agar blocks from a vigorous area of ​​mycelial growth (Fusarium sacchari CNO-1) and inoculate them onto the leaf wounds. Secure the blocks with sealing film and maintain humidity at 25℃ to prevent the mycelial cake from drying out. Collect samples every 12 hours for 7 days after inoculation.

[0046] Total RNA was extracted from the collected samples and reverse transcribed into cDNA (using the same method as in Example 1) to serve as a template for subsequent experiments. ScGAPDH was selected as the internal control gene. The reaction was performed according to the TB Green Premix Ex Taq protocol. TM The instructions for using the II (TaKaRa) kit were as follows: Real-time quantitative PCR (RT-qPCR) was performed using the following primers. The reaction mixture consisted of: 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 to a final volume of 20 μL. The PCR program was as follows: 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. Each reaction was performed in triplicate. Two biological replicates were used. -ΔΔCt Calculation and analysis.

[0047] qScGAPDH-F: 5'-CACGGCCACTGGAAGCA-3' (SEQ ID NO.5)

[0048] qScGAPDH-R: 5'-TCCTCAGGGTTCCTGATGCC-3' (SEQ ID NO.6)

[0049] qScGIP1-F: 5'-TGCATCTCGGTGCTGTCGT-3' (SEQ ID NO.7)

[0050] qScGIP1-R: 5'-CGTGTAGAGGGAGGTGGACG-3' (SEQ ID NO.8)

[0051] The results are as follows Figure 2 As shown, during Fusarium canaliculata infection of sugarcane, the expression level of ScGIP1 was continuously upregulated and maintained at a high level, reaching a peak at 96 h post-infection. This indicates that the expression level of ScGIP1 is significantly upregulated during the Fusarium canaliculata infection stage of sugarcane, and may play a role in the pathogenicity of Fusarium.

[0052] Example 3

[0053] 1. Construction of transient expression of ScGIP1 in tobacco

[0054] Using ScGIP1 cDNA as a template, specific primers ScGIP1-pGR107-F and ScGIP1-pGR107-R were designed for amplification;

[0055] ScGIP1-pGR107-F: 5'-GCACCAGCTAGCATCGATATGTGGAATCCCAAAC TCC-3' (SEQ IDNO.9)

[0056] ScGIP1-pGR107-R: 5'-GTATGGGTACGCGGCCGCGGCGGCAAGAGTGAA ATTG-3' (SEQ IDNO.10)

[0057] Reaction system: 25 μL 2×Phanta Master Mix, 1 μL Forward Primer (10 μM), 1 μL Reverse Primer (10 μM), 1 μL ScGIP1-TA plasmid, and ddH2O added to a final volume of 50 μL. The PCR reaction program was as follows: pre-denaturation at 95℃ for 30 s; denaturation at 95℃ for 15 s, annealing at 56℃ for 15 s, extension at 72℃ for 30 s, 30 cycles; final extension at 72℃ for 5 min. The amplified product was then ligated into a ClaⅠ and NotⅠ linearized PVX vector (i.e., pGR107 vector) using a seamless cloning method. The reaction mixture consisted of: 1 μL 5×CEII Buffer, 0.5 μL Exnase II, 100 ng ScGIP1 purified product, 80 ng linearized PVX vector, and ddH2O added to a final volume of 6 μL. After reacting at 37℃ for 30 min, the mixture was transformed into *E. coli* DH5α using a heat shock method. Following transformation, the bacterial culture was plated on LB agar containing kanamycin (50 mg / L) and incubated at 37℃ for 14 h. Single clones were selected for colony PCR to screen for positive clones. The selected positive clones were then sequenced for verification, and the correctly sequenced positive plasmid (ScGIP1-PVX) was preserved. The structural map of the obtained ScGIP1-PVX plasmid is shown below. Figure 3 As shown.

[0058] Simultaneously, following the above construction method, a PVX plasmid linked to fluorescent protein (GFP) was constructed, yielding GFP-PVX (structural diagram shown below). Figure 4 (As shown).

[0059] 2. Identification of plant resistance to Botrytis cinerea induced by ScGIP1

[0060] The preserved Botrytis cinerea strain (B. cinerea strain B05.10, preserved in our laboratory) was inoculated onto PDA medium for activation and incubated at 28°C for 5 days. After the colonies grew well, agar blocks were cut from the areas with vigorous mycelial growth using a 0.5 cm diameter punch and transferred to another PDA plate for further incubation for 6 days. When the colonies reached a good growth state, agar blocks containing mycelia were cut using the same method described above.

[0061] The correctly sequenced positive plasmid ScGIP1-PVX was transformed into Agrobacterium GV3101 via a freeze-thaw method. Positive transformants were picked and cultured in LB medium containing 50 mg / L Kan and 20 mg / L Rifampicin at 28°C with shaking for 2 days. The bacterial cells were collected by centrifugation at 4000 rpm for 5 min. The bacterial concentration was adjusted to an OD600 of 0.4–0.5 using Agrobacterium resuspension (10 mmol / L MgCl2, 10 mmol / L 2-(N-morpholine)ethanesulfonic acid, 150 μmol / L 3',5'-dimethoxy-4'-hydroxyacetophenone). Tobacco leaves that were 5–6 leaf-age, uniformly grown, and in good condition were selected. ScGIP1-PVX was injected into the underside of the tobacco leaves using a sterile syringe, ensuring that the injected leaves were of similar size. Tobacco leaves injected with GFP-PVX served as a control. The tobacco leaves were returned to the plant culture chamber for further cultivation. After 24 hours of protection from light, the injected leaves were removed, and the petioles were wrapped with moistened absorbent cotton to maintain appropriate humidity. They were then placed in a petri dish containing moistened filter paper. 5mm diameter circular Botrytis cinerea cakes were inoculated onto the tobacco leaves, and the leaves were then incubated in the dark at 28℃ for 5 days. During this period, the changes in the area of ​​leaf lesions were observed and photographed.

[0062] The results are as follows Figure 5 As shown, when botrytis cinerea was inoculated on tobacco leaves transiently expressing pGR107:ScGIP1, the lesion area in the pGR107:ScGIP1 expression region was significantly smaller than that in the pGR107:GFP treatment group, and the growth of botrytis cinerea was inhibited. This indicates that ScGIP1 may play a broad-spectrum disease resistance role in plant immunity.

[0063] Example 4 Sugarcane genetic transformation experiment

[0064] 1. Construction of overexpression vectors (construction diagram as shown in the figure) Figure 6 (As shown)

[0065] This study constructed a 35S promoter-driven ScGIP1 overexpression vector. Using the ScGIP1-TA plasmid as a template, specific primers were designed: the forward primer ScGIP1-2300-F and the reverse primer ScGIP1-2300-R. The amplification reaction system and procedure were the same as in Example 3. Simultaneously, following the method in Example 3, the amplification product was ligated into the linearized pCambia2300 vector digested with SacⅠ and XbaⅠ using a seamless cloning method. This vector was then transformed into *E. coli* DH5α, and positive clones were screened and confirmed by sequencing to obtain the overexpression vector OE-ScGIP1 (pCambia2300-ScGIP1, structural diagram shown below). Figure 7 (As shown).

[0066] ScGIP1-2300-F: 5'-AACACGGGGGACGAGCTCATGTGGAATCCCAAACT CCT-3' (SEQ IDNO.11)

[0067] ScGIP1-2300-R: 5'-CTGCAGGTCGACTCTAGACTAGGCGGCAAGAGTGA AAT-3' (SEQ IDNO.12)

[0068] 2. Agrobacterium-mediated genetic transformation of sugarcane

[0069] The OE-ScGIP1 recombinant vector was transformed into Agrobacterium tumefaciens strain EHA105 for sugarcane genetic transformation experiments. The specific steps of the Agrobacterium-mediated transformation method are as follows:

[0070] (1) Culture of sugarcane somatic embryos

[0071] Select disease-free sugarcane stem segments and sterilize their surface with 75% alcohol in a clean bench. Remove the epidermis and leaf sheaths, and cut off the tender central stem segment (approximately 8 cm). Using a sterile scalpel, cut the stem segment into small pieces approximately 2 mm thick, with the growth point facing down on each piece. Transfer these pieces to SCIM3 medium. Incubate in the dark at 25°C for 18 days to induce callus formation. Regularly check the growth of the material during this process to prevent contamination.

[0072] (2) Agrobacterium culture

[0073] Positive single clones of the EHA105 Agrobacterium strain carrying the ScGIP1 gene were selected and inoculated into LB liquid medium containing 50 mg / L Kan and 20 mg / L Rif antibiotics. The culture was placed in a shaker and cultured until the bacterial concentration OD600 reached 0.5-1.0, ensuring that Agrobacterium was in the logarithmic growth phase and its growth status was good. 100 μL LAS (acetylsuccinone) was added to prepare Agrobacterium resuspension.

[0074] (3) Infection and co-cultivation

[0075] Select granular, dry callus tissue, gently peel off the swollen part with sterile forceps, place it in Agrobacterium resuspension, and incubate at 28°C for 45 min. Gently shake the culture flask every 15 min to ensure that the Agrobacterium and callus tissue are evenly mixed and that infection is complete. After infection, pour off the bacterial solution, transfer the callus tissue to sterile filter paper, and let it air dry for 4 h to remove excess Agrobacterium suspension.

[0076] Fully dried callus tissue was transferred to SCCM medium for co-culture. To avoid potential bacterial overgrowth or medium contamination from direct contact, sterile filter paper was used to separate the callus tissue from the medium, ensuring that Agrobacterium could effectively infect the callus tissue on the filter paper and maintain nutrient supply. The culture dishes were incubated at 20°C for 3 days.

[0077] (4) Resistance screening and cultivation

[0078] After co-culturing, the 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 process was repeated three times. The callus tissue was then allowed to air dry until no visible moisture remained on the surface. It was then transferred to CPMS medium containing 30 mg / L Geneticin (G418) for selection. The selection process lasted for 3 weeks, during which callus growth was regularly monitored, and untransformed callus tissue was promptly removed.

[0079] (5) Callus differentiation

[0080] Selected callus tissues were transferred to differentiation medium (CD) containing the screening antibiotic (30 mg / L Geneticin) and cultured at 28°C under 14-hour light (3000 Lux) / 10-hour dark conditions. The medium was changed regularly to maintain nutrient supply to the callus tissues and prevent the accumulation of metabolites from affecting tissue differentiation. After one month of culture, embryogenic callus tissues gradually formed green adventitious shoots.

[0081] (6) Rooting culture

[0082] Carefully remove the seedlings differentiated from the differentiation medium, taking only one seedling from each callus tissue, and inoculate them into rooting culture bottles. After the roots and leaves have initially developed, gently remove the sugarcane seedlings with tweezers, wash away any remaining culture medium from the roots, and then transplant them into the soil to continue growing. Under suitable light conditions at 25℃, regularly observe the plant growth and ensure the plants have sufficient water and nutrients. After one month, the sugarcane seedlings with fully developed roots and leaves can be transplanted to the field for long-term growth observation.

[0083] The formulations of the culture media used in this embodiment are as follows:

[0084] 2,4-D stock solution (1 mg / mL): Weigh 100 mg of 2,4-D, add 1.0 mL of 1 M KOH solution, shake to dissolve, and then dilute to 100 mL with distilled water. Store at 4 °C for later use.

[0085] KT stock solution (1 mg / mL): Weigh 100 mg KT, add 1.0 mL of 1 M KOH solution, shake to dissolve, and then dilute to 100 mL with distilled water. Store at 4 °C for later use.

[0086] 6-BA stock solution (1 mg / mL): Weigh 100 mg 6-BA, add 1.0 mL of 1 M KOH solution, shake to dissolve, and then dilute to 100 mL with distilled water. Store at 4 °C for later use.

[0087] AS stock solution (100μM): Weigh 0.196g of AS, dissolve it in 10mL of DMSO, filter it through a 0.22μm filter membrane, dispense it into 1.5mL centrifuge tubes, and store it at 4℃ for later use.

[0088] SCIM3 (callus induction medium) 1L: MS powder 4.74g, sucrose 27.25g, hydrolyzed casein 0.5g, L-cysteine ​​0.054g, citric acid 0.15g, inositol 0.1g, 2,4-D 3mL (stock solution concentration 1mg / mL), Phytagel 3g;

[0089] SCCM (co-culture medium) 1L: MS powder 4.74g, glucose 1g, sucrose 4g, hydrolyzed casein 0.5g, L-cysteine ​​0.054g, citric acid 0.15g, inositol 0.1g, 2,4-D 3mL (stock solution concentration 1mg / mL), KT 1mL (stock solution concentration 1mg / mL), agar powder 4.8g;

[0090] CPMS (Callus Pre-medium) 1L: MS powder 4.74g, glucose 2.5g, sucrose 7.5g, hydrolyzed casein 0.5g, L-cysteine ​​0.054g, citric acid 0.15g, inositol 0.1g, 2,4-D 3mL (stock solution concentration 1mg / mL), KT 1mL (stock solution concentration 1mg / mL), agar powder 4.8g;

[0091] CD (differentiation medium) 1L: MS powder 4.74g, sucrose 30g, hydrolyzed casein 0.5g, L-cysteine ​​0.054g, citric acid 0.15g, inositol 0.1g, 6-BA 1mL (stock solution concentration 1mg / mL), Phytagel 3.0g; rooting medium is the same as CD.

[0092] Example 5: Detection and Disease Resistance Identification of Transgenic Materials

[0093] 1. RT-qPCR detection of transgenic plants

[0094] The expression level of the target gene in transgenic plants was analyzed using the real-time quantitative PCR (RT-qPCR) detection method described in Example 2. The detection results are as follows: Figure 8 As shown, compared with wild-type plants (WT), the expression level of ScGIP1 in each transgenic plant was significantly upregulated. Three OE-ScGIP1 transgenic lines (OE-18, OE-21, and OE-26) with significantly upregulated ScGIP1 expression were selected for subsequent studies.

[0095] 2. Disease resistance identification

[0096] 1. Activated strains

[0097] A drop of 5 μL of Fusarium sacchari CNO-1 spores was placed in the center of a PDA medium and cultured for 7 days. Using a 0.5 cm diameter punch, agar blocks were cut from the edge of the mycelial growth and transferred to another PDA plate for further culture for 6 days. When the colonies reached a good growth stage, agar blocks with consistent mycelial growth were punched from the edge of the colonies.

[0098] 2. Comparative analysis of disease resistance

[0099] Transgenic sugarcane (OE-18, OE-21, OE-26) at the 5-leaf stage with uniform growth and wild-type 'Zhongzhe 9' (WT) were selected. The leaves were first rinsed with deionized water, and after absorbing excess moisture, equally sized abrasions were made on both sides of the veins. The leaves were then preserved in petri dishes lined with moist filter paper. CNO-1 agar blocks were inoculated onto the abrasion sites of the transgenic sugarcane leaves and 'Zhongzhe 9' leaves, respectively. After inoculation, the leaves were kept under suitable humidity and incubated in a 28℃ dark incubator for 6 days. Changes in lesions were observed and recorded during this period. Each treatment was replicated at least 15 times, and the lesion area was measured using ImageJ software. Results are as follows: Figure 9 As shown, the lesion area on the leaves of the OE-ScGIP1 transgenic line was significantly smaller compared to the wild-type line.

[0100] Seven days after inoculation, the leaves were cleaned with 75% alcohol. Samples were taken from leaf areas at the same location as the initial inoculation wound using a perforator. Leaf samples were disinfected in 0.1% mercuric chloride solution for 10 minutes, rinsed three times with sterile water, inoculated onto PDA solid medium, air-dried, sealed, and incubated in a 28℃ dark incubator for 4 days. Mycelial growth was observed and photographed. Results are as follows: Figure 10 As shown, compared with wild-type leaves, the mycelial area of ​​OE-ScGIP1 transgenic sugarcane leaves in recovery culture was significantly reduced. This indicates that ScGIP1 overexpression can enhance sugarcane resistance to Fusarium canis.

[0101] As demonstrated by the above examples, this invention identified a novel aspartic protease gene, ScGIP1, in sugarcane and analyzed its disease resistance function. Leafy tobacco plants pretreated with ScGIP1 exhibited stronger resistance to *Botrytis cinerea*, indicating that ScGIP1 may have a broad-spectrum role in regulating plant disease resistance. During *Fusarium solani* infection of sugarcane, the ScGIP1 gene showed a clear induced expression trend, reaching a peak expression level 96 hours after inoculation, indicating that the ScGIP1 gene participates in the infection process of *Fusarium solani*. Both in vitro inoculation and recovery culture of sugarcane leaves showed that overexpression of ScGIP1 in sugarcane significantly enhances resistance to *Fusarium solani*.

[0102] The above description is merely a preferred embodiment of the present invention. It should be noted that, for those skilled in the art, the following are preferred embodiments of the present invention.

[0103] For those skilled in the art, without departing from the principles of this invention, further modifications can be made.

[0104] Several improvements and refinements are also considered to be within the scope of protection of this invention.

Claims

1. A sugarcane ScGIP1 gene, characterized in that, The nucleotide sequence of the ScGIP1 gene is shown as SEQ ID NO.

1.

2. A protein encoded by the ScGIP1 gene according to claim 1, characterized in that, The amino acid sequence of the protein is shown as SEQ ID NO.

2.

3. A primer pair for amplifying the ScGIP1 gene of claim 1, characterized in that, The nucleotide sequence of the forward primer is shown as SEQ ID NO. 3, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO.

4.

4. An overexpression vector overexpressing the ScGIP1 gene of claim 1.

5. An engineered bacterium containing the overexpression vector of claim 4.

6. Use of the ScGIP1 gene of claim 1, the protein of claim 2, the primer pair of claim 3, the overexpression vector of claim 4, or the engineered bacterium of claim 5 in improving the resistance of sugarcane to sugarcane top rot.

7. The use of the ScGIP1 gene of claim 1, or the protein of claim 2, or the primer pair of claim 3, or the overexpression vector of claim 4, or the engineering bacteria of claim 5 in improving the resistance of plants to gray mold, characterized in that, The gray mold is caused by Botrytis cinerea.

8. A method of increasing resistance of sugar cane to sugarcane smut disease, characterised by, The engineered bacterium of claim 5 is used to infect sugarcane callus, which is then cultured for differentiation and rooting, and transplanted into the field for culture, to obtain transgenic sugarcane plants with improved resistance to sugarcane top rot.

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