Shatg18a gene, protein and application

Abstract

The application provides a ShATG18a gene, protein and application, and belongs to the technical field of biology. The sequence of the gene is shown in SEQ ID NO. 1, and the encoded protein sequence is shown in SEQ ID NO. 2. After overexpression of the ShATG18a gene in sugarcane and rice, the cold tolerance of the sugarcane and rice is enhanced, and the transcription level of a cold tolerance related gene is also improved, which indicates that the gene can positively regulate the cold tolerance of the sugarcane and rice, is an important candidate gene in genetic improvement of the cold tolerance of the sugarcane, and has certain application value for cultivation of a cold tolerance sugarcane variety.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically plant stress biology and molecular breeding, and particularly relates to a ShATG18a gene, protein and its application. Background Technology

[0002] Sugarcane is an important sugar crop and a typical C4 high-temperature crop. Below 20℃, sugarcane's photosynthesis, root activity, and cell membrane integrity all decline significantly, leading to substantial losses in yield and quality. Improving sugarcane's cold resistance is a key technological requirement for expanding its planting range and ensuring yields in northern and high-altitude regions.

[0003] Autophagy is a key protective mechanism in plants against low-temperature stress. By degrading damaged organelles and proteins, autophagy helps cells maintain energy balance and scavenge reactive oxygen species (ROS), thereby mitigating membrane damage and oxidative stress caused by low temperatures. Previous studies have shown that ATG series genes are significantly upregulated under stresses such as heat, salinity, and drought, participating in the regulation of stress resistance. ATG18, responsible for autophagosome formation and membrane bending in yeast and model plants, is an important component of the PI3-kinase complex; its deficiency leads to decreased autophagy activity and a significant reduction in cellular tolerance to environmental stress. Therefore, ATG18 is considered a "hub gene" in the autophagy pathway. Research on ATG18 and its autophagy mechanisms and functions in sugarcane is still scarce. Therefore, studying the role of ATG18a in sugarcane cold tolerance can not only reveal the molecular mechanisms of sugarcane's low-temperature adaptation but also provide potential target genes for molecular breeding of cold-resistant sugarcane, possessing significant scientific and applied value. Summary of the Invention

[0004] To address the above problems, this invention provides a ShATG18a gene, protein, and its applications.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] A ShATG18a gene, the sequence of which is shown in SEQ ID NO: 1;

[0007] The ShATG18a gene is positively correlated with plant cold resistance.

[0008] A ShATG18a protein, wherein the ShATG18a protein is the protein encoded by the aforementioned ShATG18a gene, and its amino acid sequence is shown in SEQ ID NO: 2.

[0009] A recombinant vector containing the ShATG18a gene, wherein the recombinant vector containing the ShATG18a gene is obtained by ligating the aforementioned ShATG18a gene into an overexpression vector.

[0010] A strain carrying the ShATG18a gene, wherein the strain carrying the ShATG18a gene is an Agrobacterium containing the ShATG18a gene.

[0011] The sequence of the ShATG18a gene is shown in SEQ ID NO: 1.

[0012] An application of the ShATG18a gene in improving plant cold resistance, wherein the application is to overexpress or overexpress the ShATG18a gene in sugarcane or rice to improve cold resistance;

[0013] The sequence of the ShATG18a gene is shown in SEQ ID NO: 1.

[0014] Furthermore, the application involves transforming sugarcane or rice with a recombinant vector containing the ShATG18a gene or a strain carrying the ShATG18a gene, thereby overexpressing or overexpressing the ShATG18a gene in sugarcane or rice to improve cold resistance.

[0015] Furthermore, the application increases the content of proline and superoxide dismutase and decreases the content of malondialdehyde in sugarcane or rice by overexpressing or overexpressing the ShATG18a gene.

[0016] A method for improving the cold resistance of plants, wherein the method is to overexpress or overexpress the ShATG18a gene in sugarcane or rice to improve the cold resistance of plants;

[0017] The sequence of the ShATG18a gene is shown in SEQ ID NO: 1.

[0018] Furthermore, the method involves overexpressing or overexpressing the ShATG18a gene in sugarcane or rice to increase proline and superoxide dismutase content and decrease malondialdehyde content, thereby improving the plant's cold resistance.

[0019] A plant breeding method, wherein the breeding method is to obtain sugarcane or rice with enhanced cold resistance by overexpressing or overexpressing the ShATG18a gene in sugarcane or rice;

[0020] The sequence of the ShATG18a gene is shown in SEQ ID NO: 1.

[0021] The beneficial effects of the ShATG18a gene, protein, and applications of this invention are as follows:

[0022] The research of this invention shows that overexpression of ShATG18a in rice and sugarcane can significantly improve the cold tolerance of seedlings and alter key indicators such as proline, superoxide dismutase, and malondialdehyde. In addition, ShATG18a also affects the expression of cold response marker genes in rice, suggesting that it is involved in the regulation of low temperature signaling pathways. Therefore, ShATG18a is a cold tolerance functional gene with important application prospects.

[0023] This invention clones the sugarcane ShATG18a gene, constructs an overexpression vector, and improves the cold resistance of plants by overexpressing this single gene (sugarcane ShATG18a gene). This demonstrates that the ShATG18a gene can positively regulate the cold resistance of sugarcane and rice seedlings, and is an important candidate gene in molecular breeding of plant cold resistance, with potential application value for the genetic improvement of plant cold resistance.

[0024] In practical applications, the ShATG18a gene can be introduced into target plants to obtain cold-resistant sugarcane and rice plants, providing a new method for molecular breeding of sugarcane and rice. Attached Figure Description

[0025] Figure 1 This is a map of the pCAMBIA3300-ShATG18a plant expression vector in Example 1 of this invention;

[0026] Figure 2 This describes the expression of ShATG18a in PCR-positive transgenic sugarcane plants overexpressing ShATG18a in Example 4 of the present invention. Specifically, the expression level of ShATG18a in sugarcane leaves was analyzed using RT-qPCR, with GAPDH as an internal reference gene, and the data were normalized. Figure 2 In the data, #1, #2, and #3 represent three stable expression lines ShATG18a#1, ShATG18a#2, and ShATG18a#3, respectively, and WT-XTT22 represents sugarcane XTT22. The data represent mean ± SE (n=3). One-way ANOVA using the t-test showed significant differences among all different letter representations (P<0.05).

[0027] Figure 3 This refers to the phenotypic characteristics of sugarcane leaves under cold stress in Example 4 of the present invention; wherein, ShATG18a-OE is a sugarcane plant overexpressing ShATG18a, and WT-XTT22 is wild-type sugarcane XTT22; the scale bar of the wild-type sugarcane XTT22 plant phenotype under cold stress is 5 cm, and the scale bar of the ShATG18a transgenic sugarcane leaf phenotype under cold stress is 1 cm;

[0028] Figure 4This describes the expression status of ShATG18a overexpressing transgenic rice in Example 4 of this invention. The expression status of ShATG18a overexpressing transgenic rice was verified using semi-quantitative RT-PCR, with OsACTIN as an internal reference. In ShATG18a-OE, #2, #3, and #4 represent three ShATG18a overexpressing transgenic rice varieties, and in WT-ZH11, #2, #3, and #4 represent three ZH11 rice varieties.

[0029] Figure 5 This refers to the phenotypic characteristics of rice leaves under cold stress in Example 4 of the present invention; wherein, WT-ZH11 is wild-type rice ZH11, and ShATG18a-OE is ShATG18a gene-positive integrated rice; the scale bar for the plant phenotype of wild-type rice ZH11 leaves under cold stress is 10 cm, and the scale bar for the phenotype of ShATG18a gene-positive integrated rice leaves under cold stress is 1 cm;

[0030] Figure 6 This is the effect of ShATG18a overexpression on the cold resistance parameters of rice in Example 4 of the present invention; wherein, Figure A shows the change in proline content before and after cold treatment, Figure B shows the change in malondialdehyde content before and after cold treatment, and Figure C shows the change in superoxide dismutase (SOD) activity before and after cold treatment.

[0031] Proline content (a), malondialdehyde content (b), and SOD activity (c) in wild-type rice ZT11 and ShATG18a transgenic rice under cold stress. Data represent mean ± SE (n=3). All different letters indicate significant differences in one-way ANOVA using a t-test (P<0.05).

[0032] Figure 7 This refers to the transcriptional level of cold-resistance-related genes in rice after cold stress in Example 5 of this invention. Specifically, the transcriptional level of cold-resistance-related genes in rice with positive ShATG18a gene integration was analyzed using RT-qPCR, with OsACTIN as the internal reference gene, and the data were normalized. In ShATG18a-OE, #2, #3, and #4 represent three rice plants with positive ShATG18a gene integration, and WT-ZH11 represents wild-type rice ZH11. Data represent mean ± SE (n=3). All different letters indicate significant differences in one-way ANOVA using a t-test (P<0.05). Detailed Implementation

[0033] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention can also be implemented in other ways different from those described herein. Those skilled in the art can make similar extensions without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below. The present invention will be further described in detail below with reference to specific embodiments to enable those skilled in the art to understand it.

[0034] Plant materials:

[0035] The sugarcane variety XTT22 and the rice variety ZH11 were planted at the Institute of Tropical Biotechnology, Chinese Academy of Tropical Agricultural Sciences.

[0036] Cold stress treatment method: Cold stress was applied to an artificial climate incubator at -5℃ for 7 hours.

[0037] Example 1: Construction of ShATG18a overexpression vector

[0038] 1) Cloning of the full-length sequence of the sugarcane ShATG18a gene

[0039] The CDS sequence of ShATG18a was obtained from the sugarcane XTT22 genome database (ShATG18a gene sequence as shown in SEQ ID NO: 1, ShATG18a protein sequence as shown in SEQ ID NO: 2), and specific primers were designed for PCR amplification. Total DNA was extracted from young sugarcane XTT22 leaves using the CTAB method and used as a template for PCR amplification of the full-length ShATG18a sequence. The full-length amplification primers for ShATG18a were: ShATG18a-F: ATGGCGACGCCCGCCGC, ShATG18a-R: CTACGGTTGTTCCGACG. PCR reaction system:

[0040] Table 1 PCR amplification system

[0041]

[0042] PCR amplification program: 95℃ for 5 min; 95℃ for 30 s, 60℃ for 30 s, 72℃ for 30 s, 35 cycles; extension at 72℃ for 5 min. PCR amplification products were subjected to 1% agarose gel electrophoresis, gel excision and recovery, and sent to Sangon Biotech Co., Ltd. for sequencing verification.

[0043] 2) Construction of ShATG18a overexpression vector

[0044] Following the same construction method as the GFP fusion expression vector, a 3×FLAG and nuclear localization NLS sequence were first added to the 5' end of the ORF sequence via PCR amplification, and a 3×His tag was added to the 3' end. BamHI and SacI restriction sites were added to both ends of the tagged ShATG18a reading frame sequence, respectively. The reading frame sequence was then integrated into the pCAMBIA3300 plant expression vector, which already contained the downstream of the Ubi promoter, via restriction enzyme digestion and ligation. The primers for adding the restriction sites were: ShATG18a-OE-F: 5'-GGGGATCCATGGCGACGCCCGCCGC-3', ShATG18a-OE-R: 5'-GGGAGCTCCTACGGTTGTTCCGACG-3'. The PCR product with added restriction sites and the vector were double-digested, ligated, and transformed. Positive clones were then sequenced to verify the inserted fragment. The correctly sequenced ligation vector was verified by double enzyme digestion, and the pCAMBIA3300-ShATG18a plant expression vector was successfully constructed. Figure 1 This refers to a recombinant vector containing the ShATG18a gene. The constructed pCAMBIA3300-ShATG18a plant expression vector was transformed into Agrobacterium strain EHA105 using the freeze-thaw method, resulting in Agrobacterium carrying the pCAMBIA3300-ShATG18a vector, which was then used for the next step of genetic transformation.

[0045] Example 2: Genetically transformed sugarcane with ShATG18a overexpression

[0046] In this embodiment, tender shoot tips and leaves of sugarcane XTT22 were used as explants. Agrobacterium-mediated transformation was used to obtain transgenic, resistant, positive sugarcane seedlings induced by the ShATG18a-Flag fusion vector. The ShATG18a-Flag positive resistant plants in the tissue culture bottles were allowed to harden off for one week before being transplanted into crystal soil to acclimate to the microbial growth environment. Once their root systems were stable, they were transplanted into seedling trays for further cultivation, as detailed below:

[0047] 1) Selection and processing of sugarcane materials

[0048] (1) Sugarcane variety XTT22 was used as the genetic transformation recipient material. Healthy plants without disease or pests were selected, and the outer old leaves were removed layer by layer. Young leaves about 12cm away from the sugarcane stem tip growth point were selected as explants for inducing callus culture.

[0049] (2) Place the selected explants in 75% alcohol for 5-8 minutes, then place them in 0.1% mercuric chloride for 8-10 minutes. After disinfection, wash them 2-3 times with sterile ddH2O and blow them dry.

[0050] (3) Remove the outermost leaf sheath of the explant, then cut it into thin slices about 0.2-0.5 mm thick, place them on M1 solid medium, and culture them in the dark at 22℃ for 15-20 days. Change the medium and subculture until embryogenic callus grows. Select the most viable embryogenic callus as material for genetic transformation.

[0051] 2) Activation of Agrobacterium strains containing plant expression vectors

[0052] (1) Agrobacterium strain carrying the pCAMBIA3300-ShATG18a vector was streaked in YEP solid medium containing 50 µg / mL rifampicin, 10 µg / mL streptomycin and 100 µg / mL kanamycin, and cultured upside down at 28 °C for 2 days. Single colonies were picked and inoculated into YEP liquid medium containing 50 µg / mL rifampicin, 10 µg / mL streptomycin and 100 µg / mL kanamycin, and cultured at 28 °C and 225 rpm for 12-16 h.

[0053] (2) Take the above bacterial culture and place it in 80 mL of YEP liquid medium containing 50 µg / mL rifampin, 10 µg / mL streptomycin and 100 µg / mL kanamycin. Incubate at 28 °C and 230 rpm until OD. 600 When the concentration reaches 0.4~0.6, the bacterial solution is obtained.

[0054] (3) Transfer the bacterial culture to a new sterile centrifuge tube, centrifuge at 4℃ and 4000rpm for 5min, discard the supernatant, aspirate the remaining culture medium, add 120mL of MR liquid medium (containing 150µmol / L AS), resuspend the bacterial cells, and incubate at 28℃ and 200rpm for 2h to obtain Agrobacterium infection solution.

[0055] 3) Agrobacterium infection of sugarcane embryonic callus

[0056] (1) Select embryogenic callus with good growth from sugarcane callus, and transfer it to sterilized filter paper to dry.

[0057] (2) Transfer the above sugarcane embryonic callus into Agrobacterium infection solution and place for 30 min.

[0058] (3) Filter out the Agrobacterium infection solution and place the infected callus on sterile filter paper to dry.

[0059] (4) The dried infected callus tissue was placed on MS solid medium and incubated in the dark at 22°C for 5 days.

[0060] (5) After dark culture, the tissue was washed once in sterile water containing 200 mg / L carbenicillin (Car), then washed 2 to 4 times with sterile water without Car, and washed once with liquid MS. The tissue was then placed on sterile callus filter paper and dried with a strainer.

[0061] (6) After drying, place it on M2 solid medium containing 200 mg / L Carb and culture it in a constant temperature incubator at 28°C for about three weeks until the embryogenic callus tissue slowly differentiates into green seedlings.

[0062] 4) Screening Agrobacterium-transformed plants using PPT

[0063] The callus-differentiated seedlings were transferred to a selection medium containing PPT (i.e., MS medium with 2 mg / L 6-benzylaminopurine and 2.0 mg / L herbicide PPT, which is phosphatidylin) and cultured for about 20 days. During this process, the medium should be changed in a timely manner until the transformed seedlings are screened out.

[0064] 5) Transformed seedling rooting culture and hardening-off

[0065] The selected transgenic seedlings were transferred to M3 solid medium for rooting culture. After the seedlings had fully developed their roots, they were removed from the medium, cleaned, old leaves were removed, disinfected, and then transplanted into crystal soil. The seedlings were marked and hardened off for one week. After they grew well, they were planted in flower pots to obtain ShATG18a overexpressing transgenic plants.

[0066] 6) PCR detection of the integration fragment in ShATG18a overexpressing transgenic plants

[0067] When the seedlings planted in the plug trays have grown 3-4 leaves, take 0.1g of tender leaves and grind them into powder with liquid nitrogen. Extract the total DNA from the leaves using the CTAB method. Use the extracted DNA as a detection template and dilute it 10 times before use.

[0068] First, the integration of the herbicide-resistant bar gene into the transgenic plants was detected. Primers for bar gene detection: Bar409-F: 5'-CGAGACAAGCACGGTCAACT-3'; Bar409-R: 5'-CTGCCAGAAACCCACGTCAT-3'. Primers for PCR detection of ShATG18a overexpressing transgenic plants: ShATG18a-F: 5'-ATGGCGACGCCCGCCGC-3'; ShATG18a-R: 5'-CTACGGTTGTTCCGACG-3'. The PCR reaction system is as follows:

[0069] Table 2 PCR reaction system

[0070]

[0071] PCR amplification program: 95℃ for 5 min; 95℃ for 30 s, 60℃ for 30 s, 72℃ for 30 s, 35 cycles; extension at 72℃ for 5 min. PCR amplification products were subjected to 1% agarose gel electrophoresis, gel excision and recovery, and sent to Sangon Biotech Co., Ltd. for sequencing verification.

[0072] First, PCR detection of bar gene integration was performed on the ShATG18a overexpressing transgenic plants. Then, using the DNA of the successfully transformed plants with the Bar gene as templates, the integration of the overexpressing fragment was detected to determine the integrity of the integrated fragment region, thus obtaining PCR-positive ShATG18a overexpressing transgenic sugarcane plants.

[0073] Example 3: Genetically transformed rice with ShATG18a overexpression

[0074] To further verify the function of the ShATG18a gene, transgenic rice materials with the ShATG18a gene in the ZH11 background were studied and obtained, as follows:

[0075] 1) Culture of callus tissue

[0076] (1) Rice variety ZH11 was used as the genetic transformation recipient material. Full rice seeds were selected, the husks were removed, and blackened seeds were discarded.

[0077] (2) Soak the clean and intact seeds in 75% ethanol solution for 4-6 minutes for sterilization, then sterilize with 0.1% mercuric chloride for 15 minutes (can be done on a shaker), and finally rinse with sterile water 3-4 times.

[0078] (3) Place the rinsed seeds on sterile filter paper and let the water dry. Then place them in MS medium containing 1 mg / L abscisic acid (ABA) and culture them in the dark for 12-15 days. Then place the seeds in MS+ABA medium containing 3 mg / L 2,4-dichlorophenoxyacetic acid (2,4-D) and culture them for 12-15 days to induce callus formation. Select high-quality callus tissue for subculture.

[0079] 2) Cultivation of Agrobacterium tumefaciens in bacterial suspension

[0080] (1) Agrobacterium strain carrying the pCAMBIA3300-ShATG18a vector was streaked in YEP solid medium containing 50 µg / mL rifampicin, 10 µg / mL streptomycin and 100 µg / mL kanamycin, and cultured upside down at 28 °C for 2 days. Single colonies were picked and inoculated into YEP liquid medium containing 50 µg / mL rifampicin, 10 µg / mL streptomycin and 100 µg / mL kanamycin, and cultured at 28 °C and 220 rpm for 12-16 h.

[0081] (2) Place the above bacterial culture in 10 mL of YEP liquid medium containing 50 µg / mL rifampin, 10 µg / mL streptomycin and 100 µg / mL kanamycin, and incubate at 28 °C and 220 rpm until OD. 600 It reached 0.5.

[0082] (3) The bacterial culture was transferred to 100 mL of YEP liquid medium containing 50 µg / mL rifampin, 10 µg / mL streptomycin and 100 µg / mL kanamycin, and cultured at 28 °C and 220 rpm for 6-7 h with shaking until OD was reached. 600 It is approximately 0.5.

[0083] (4) Transfer the bacterial culture to a new sterile centrifuge tube, centrifuge at 4000 rpm for 5 min at 4℃, discard the supernatant, aspirate the remaining culture medium, add 120 mL of MR liquid medium (containing 90 µmol / L AS), resuspend the bacterial cells, and incubate at 28℃ and 200 rpm for 2 h to obtain Agrobacterium infection solution.

[0084] 3) Agrobacterium infects callus tissue and co-cultures it with callus tissue.

[0085] (1) Take out the embryogenic callus that has been subcultured for 12 days, put it into a sterilized culture dish, add the cultured Agrobacterium infection solution, and shake it continuously to make the callus fully contact with Agrobacterium and soak for 15 minutes.

[0086] (2) Remove the callus tissue and blot the surface moisture with sterile filter paper. Finally, inoculate the callus tissue into a solid co-culture medium (the solid co-culture medium is MS medium with 2 mg / L 2,4-dichlorophenoxyacetic acid, 0.6 g / L hydrolyzed casein, 0.6 g / L L-proline, 25 g / L sucrose and 5 g / L agar added, pH 5.5) and incubate in the dark at 28℃ for 3 days to obtain the co-cultured callus tissue.

[0087] 4) Screening of resistant callus and obtaining transgenic plants

[0088] The co-cultured callus tissue was placed in MS medium containing 150 mg / L carbenicillin for resistance screening. Then, it was placed in differentiation medium (containing 300 mg / L hydrolyzed casein, 500 mg / L proline, 500 mg / L glutamine, 0.5 mg / L naphthaleneacetic acid, 2 mg / L 6-benzylaminopurine, 12 g / L agar powder, and 30 g / L sucrose) for differentiation culture. After the green shoots developed into seedlings 3 cm long, they were transferred to rooting medium (1 / 2 MS medium containing 5 mg / L naphthaleneacetic acid and 30 g / L sucrose) for further culture. Rooting was observed after 15-16 days. When the roots reached approximately 2 cm, the seedlings were transplanted into pots and cultured in a greenhouse to obtain ShATG18a overexpressing transgenic rice plants.

[0089] Example 4: Expression level analysis of ShATG18a and cold-resistance genes

[0090] 1) Method

[0091] Approximately 100 mg of fresh leaves from sugarcane and rice were taken and thoroughly ground into powder using liquid nitrogen. Total RNA was extracted using a plant total RNA extraction kit (Omega), and genomic DNA was digested using the kit's built-in DNase. The extracted RNA was then used to synthesize cDNA via reverse transcription using a Fermentas cDNA first-strand reverse transcription kit. The reaction system is as follows:

[0092] Table 3 Reaction System

[0093]

[0094] 65℃ for 5 minutes, then cool on ice.

[0095] Table 4 Reaction System

[0096]

[0097] The reaction was terminated at 42℃ for 60 min and 70℃ for 5 min. Store at -20℃ for later use.

[0098] Primers for detecting ShATG18a expression: ShATG18a-RT-F: 5'-GGGGCAGATTAGGGTGGAAC-3'; ShATG18a-RT-R: 5'-GCTAGCAGTTGCAATGAGCC-3'; Primers for detecting OsICE1 expression (cold-resistant gene): OsICE1-RT-F: 5'-AGCAAGATGGACAGGGCTTC-3'; OsICE1-RT-R: 5'-TTGTGGGTGTTGGAGGCAAT-3'; Primers for detecting sugarcane internal reference gene expression: GADPH-F: 5'-CACGGCCACTGGAAGCA-3'; GADPH-R: 5'-TCCTCAGGGTTCCTGATGCC-3'. Primers for detecting rice internal control gene expression: OsACTIN-F: 5'-CAACACCCCTGCTATGTACG-3'; OsACTIN-R: 5'-CATCACCAGAGTCCAACACAA-3'. qRT-PCR reaction system:

[0099] Table 5 qRT-PCR reaction system

[0100]

[0101] qRT-PCR amplification program: 95℃ for 3 min; 95℃ for 10 s, 58℃ for 30 s, 72℃ for 30 s; 40 cycles. After amplification, the ct values ​​of the internal reference gene and the target gene were obtained separately. The method calculates the relative expression level of the target gene.

[0102] 2) Genetic transformation of sugarcane using the Ubi-ShATG18a-Flag overexpression vector

[0103] Three stable expression lines, ShATG18a#1, ShATG18a#2, and ShATG18a#3, were randomly selected from PCR-positive transgenic sugarcane plants overexpressing ShATG18a, with sugarcane XTT22 as a negative control. The expression level of ShATG18a was detected by qRT-PCR quantitative PCR in step 1). The results are as follows: Figure 2 As shown, ShATG18a was successfully overexpressed in the three transgenic sugarcanes, and the expression level of ShATG18a in sugarcane leaves can be analyzed by RT-qPCR.

[0104] 3) ShATG18a overexpression enhances sugarcane cold resistance.

[0105] To investigate the function of the ShATG18a gene, sugarcane plants overexpressing ShATG18a and wild-type sugarcane XTT22 were subjected to cold stress treatment. The results are as follows: Figure 3As shown, the leaves of sugarcane plants overexpressing ShATG18a showed no damage, while the leaves of wild-type XTT22 plants showed more obvious damage. These results indicate that overexpression of the ShATG18a gene enhances the cold resistance of sugarcane.

[0106] 4) Genetic transformation of rice using the ShATG18a overexpression vector

[0107] When the ShATG18a overexpressing transgenic rice seedlings in the flowerpots had produced a large number of leaves, three ShATG18a overexpressing transgenic rice plants (ShATG18a#2, ShATG18a#3, and ShATG18a#4) were randomly selected, with rice ZH11 as a negative control. RNA was extracted from the tender leaves, and the expression of the ShATG18a gene was detected using semi-quantitative RT-PCR as described in step 1). The results are as follows: Figure 4 As shown, the three transgenic rice strains ShATG18a #2, ShATG18a #3, and ShATG18a #4 are ShATG18a gene-positive integrated rice.

[0108] 5) ShATG18a overexpression enhances cold resistance in rice.

[0109] After obtaining ShATG18a gene-positive integrated rice, in order to study the effect of the ShATG18a gene on the cold resistance of rice, wild-type rice ZH11 and ShATG18a gene-positive integrated rice were subjected to cold stress treatment. The results are as follows: Figure 5 As shown, after 7 hours of cold stress treatment, the leaves of rice with ShATG18a gene positive integration showed no damage, while the leaves of wild-type rice ZH11 showed more significant damage. These results indicate that overexpression of the ShATG18a gene in rice enhances its cold resistance.

[0110] 6) Determination of relevant components

[0111] This invention subjects ShATG18a gene-positive integrated rice and wild-type rice ZH11 to low-temperature stress treatment at -5℃, and measures the proline and malondialdehyde content and SOD activity before and after stress. The specific methods are as follows:

[0112] 61) Determination of proline content

[0113] (1) Mix 2 mL ddH2O, 2 mL glacial acetic acid and 2 mL acidic ninhydrin reagent (add 1.25 g ninhydrin to a system of 30 mL glacial acetic acid and 20 mL 6 mol / L phosphoric acid).

[0114] (2) Heat the mixture at 100°C for 30 min.

[0115] (3) After the temperature drops to room temperature, add 4 mL of toluene and mix thoroughly. Centrifuge at 5000 rpm for 5 min to separate the organic phase.

[0116] (4) Detect the OD520 of the organic phase and compare it with a standard curve constructed using known proline concentration (Sigma) to determine the concentration of proline in the sample.

[0117] 62) Determination of malondialdehyde content

[0118] (1) Add 6 mL of 10% TCA (v / v) to 0.5 g of rice leaves, grind the leaves, and centrifuge at 11000 g for 8 min at room temperature. The resulting supernatant is the required extract.

[0119] (2) Take 3 mL of extract (add 2 mL of ddH2O to the control group) and mix it evenly with 3 mL of 0.6% TBA (v / v) solution. Heat the mixture in a boiling water bath in a glass test tube for 15 min.

[0120] (3) Place the sample on ice to cool it down immediately, centrifuge at 3000g for 8min, and measure the absorbance of the supernatant at wavelengths of 450nmol, 532nmol and 600nmol.

[0121] (4) The concentration of MDA is calculated using the following formula:

[0122] MDA concentration (µmol / L) = 6.45 × (OD) 532 -OD 600 -0.56×OD450

[0123] (5) The reagent formula used in this experiment:

[0124] 10% Trichloroacetic Acid (TCA) (w / v): Dissolve 10g of TCA in 100mL of distilled water.

[0125] 0.6% Thiobarbituric acid (TBA) (w / v): Dissolve it first with NaOH, then bring it to a final volume using a 10% trichloroacetic acid (TCA) (v / v) solution.

[0126] 63) Detection of SOD activity

[0127] (1) Take about 0.2g of fresh rice leaves (fresh weight) and grind them under liquid nitrogen.

[0128] (2) Add 2 mL of 50 mM phosphate buffer (pH 7.8) and mix well. Centrifuge at 4°C and 8000 rpm for 15 min. Collect the supernatant to obtain the crude enzyme extract.

[0129] (3) Add 1.5 mL of phosphate buffer to a 1.5 mL centrifuge tube, then add NBT, methionine, riboflavin, and EDTA in sequence, and mix gently. The system should be pale yellow at this point.

[0130] (4) Add 0.1 mL of sample enzyme extract and adjust the total volume to 3 mL with deionized water.

[0131] (5) Place the reaction tube in a light intensity of about 4000 lx for 10 min, and set up a dark control and a blank control for calibration.

[0132] (6) After the reaction is complete, the absorbance (A) is read at 560 nm using a spectrophotometer. 560 And record it.

[0133] (7) Inhibition rate (%) = (A 暗 -A 样品 ) / (A 暗 -A 空白 ) × 100%

[0134] (8) Definition of enzyme activity: 1 unit (U) SOD = the amount of enzyme that can inhibit 50% NBT photoreduction under the above conditions.

[0135] (9) Actual active expression: Ug -1 FW = U / dry weight of sample.

[0136] The results showed that under suitable and stress-free growth conditions, the proline content of ZH11 in ShATG18a gene-positive integrated rice and wild-type rice ( Figure 6 Figure a), malondialdehyde content ( Figure 6 (Figure b in the image) and SOD activity ( Figure 6 (Figure c) showed no significant difference. However, after stress treatment, the proline content and SOD activity of the ShATG18a gene-positive rice increased to varying degrees, while the malondialdehyde content was significantly lower than that of wild-type rice ZH11. These results indicate a close association between the ShATG18a gene and cold tolerance, and its overexpression enhances the rice's tolerance to low-temperature environments, consistent with the phenotypic observations of this invention.

[0137] 7) ShATG18a overexpression enhances cold resistance in rice

[0138] The transcriptional level of the rice cold resistance-related gene OsICE1 was detected by RT-qPCR in step 1), and the results are as follows: Figure 7 As shown, after cold stress, the transcription level of OsICE1 in rice with positive integration of the ShATG18a gene was significantly higher than that in wild-type rice ZH11, which is consistent with the phenotype, indicating that overexpression of the ShATG18a gene enhances the cold resistance of rice.

[0139] All other parts not described in detail are existing technologies. Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. Those skilled in the art can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An application of the ShATG18a gene in improving plant cold resistance, characterized in that, The application involves overexpressing the ShATG18a gene in sugarcane or rice to improve cold resistance. The sequence of the ShATG18a gene is shown in SEQ ID NO:

1.

2. The application according to claim 1, characterized in that, The application involves transforming sugarcane or rice with a recombinant vector containing the ShATG18a gene or a strain carrying the ShATG18a gene, thereby overexpressing the ShATG18a gene in the sugarcane or rice to improve cold resistance.

3. The application according to claim 2, characterized in that, The recombinant vector containing the ShATG18a gene is obtained by ligating the ShATG18a gene into an overexpression vector.

4. The application according to claim 2, characterized in that, The strain carrying the ShATG18a gene is Agrobacterium containing the ShATG18a gene.

5. A method for improving the cold resistance of plants, characterized in that, The method involves overexpressing the ShATG18a gene in sugarcane or rice to improve the plant's cold resistance. The sequence of the ShATG18a gene is shown in SEQ ID NO:

1.

6. A method for breeding plants, characterized in that, The breeding method involves overexpressing the ShATG18a gene in sugarcane or rice to obtain sugarcane or rice with enhanced cold resistance. The sequence of the ShATG18a gene is shown in SEQ ID NO: 1.

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