Application of GhSPX9 gene in improving cold tolerance of cotton
By overexpressing the GhSPX9 gene in cotton, the cold tolerance of cotton was regulated, the problem of low temperature stress in cotton seedlings was solved, and the cold tolerance of cotton was enhanced and genetically improved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- INST OF COTTON RES CHINESE ACAD OF AGRI SCI
- Filing Date
- 2024-04-08
- Publication Date
- 2026-05-19
AI Technical Summary
Cotton seedlings are susceptible to low-temperature stress, which can lead to bud and root rot, affecting growth, development, and yield. There is a lack of highly cold-resistant germplasm resources, and existing research is insufficient.
By overexpressing the GhSPX9 gene in cotton, the cold tolerance of cotton can be regulated, cold-resistant cotton varieties can be bred, and varieties with high GhSPX9 gene expression levels are identified as cold-resistant varieties.
To enhance the cold resistance of cotton, prevent cold damage, promote cotton genetic improvement and molecular breeding, and improve the application effect of cotton cold resistance genes.
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Figure CN118166028B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of plant molecular biology, and in particular to the application of the GhSPX9 gene in improving the cold resistance of cotton. Background Technology
[0002] Xinjiang is influenced by continental air masses year-round, resulting in scarce rainfall, a dry climate, and abundant sunshine. Its unique geographical location makes it my country's largest cotton production base. However, cotton sowing and seedling emergence occur in April and May each year, a period characterized by variable weather and susceptibility to late spring frosts. This can cause widespread root rot, bud rot, and death of cotton seedlings, resulting in gaps in the rows and impacting cotton growth, yield, and quality assurance.
[0003] SPX proteins are classified into four families based on their structure: the SPX family, the SPX-EXS family, the SPX-MES family, and the SPX-RING family. In Arabidopsis thaliana, four members of the SPX family—AtSPX1-AtSPX4—have been identified. Overexpression of AtSPX1 in Arabidopsis enhances the expression of genes such as ACP5, PAP2, and RNSI, suggesting that AtSPX1 has a potential transcriptional regulatory role in phosphorus starvation. AtSPX3 RNAi materials altered the plant's response to phosphorus starvation, leading to increased phosphorus concentration in Arabidopsis leaves and decreased phosphorus concentration in roots. Therefore, AtSPX3 may function as a negative regulator in plant phosphorus starvation signaling. Currently, most research on SPX genes focuses on phosphorus-related aspects; their functions and applications related to cold tolerance remain unknown.
[0004] Cotton is a warm-season crop. Low temperatures before and after emergence slow down germination and root development. Prolonged low-temperature stress can cause widespread seedling and root rot, resulting in large-scale seedling loss and reduced seedling numbers. In severe cases, replanting may be necessary, further delaying the cotton's growth process. Continuous low-temperature stress can also induce seedling diseases such as damping-off. However, current research on cold damage control is very limited, and there is a lack of cotton germplasm resources with high cold tolerance. Therefore, to improve the cold tolerance of cotton seedlings and promote the healthy development of cotton production in Xinjiang, it is imperative to work on improving cotton cold tolerance and identifying genetic regulatory sites and key genes related to cold tolerance. Summary of the Invention
[0005] The technical problem to be solved by this invention is to provide a gene related to the cold resistance of cotton and its application.
[0006] The technical solution of the present invention is: the application of the GhSPX9 gene or an expression vector containing the GhSPX9 gene in regulating the cold resistance of cotton, wherein the nucleotide sequence of the GhSPX9 gene is as shown in SEQ ID No. 1 or a degenerate sequence encoding the same protein as the sequence shown in SEQ ID No. 1.
[0007] Furthermore, the regulation of cotton cold resistance refers to overexpressing the GhSPX9 gene in cotton to improve its cold resistance.
[0008] A method for breeding cold-resistant cotton varieties involves detecting the expression level of the GhSPX9 gene in cotton. Varieties with higher GhSPX9 gene expression levels are considered to be more cold-resistant cotton varieties.
[0009] Compared with the prior art, the present invention has the following beneficial effects:
[0010] (1) This invention is the first to clearly define the functional role of the gene GhSPX9 in the cold resistance of cotton, providing a target gene for enhancing the cold resistance of cotton.
[0011] (2) Through various experiments, this invention found that silencing the GhSPX9 gene reduces the cold tolerance of the plant; overexpression of the GhSPX9 gene enhances the cold tolerance of the plant. The results show that GhSPX9 plays a positive regulatory role in the cold tolerance of cotton.
[0012] (3) The gene GhSPX9 discovered in this invention can be applied to prevent and control cold damage in cotton. Specifically, it can be used to enhance the genetic improvement or molecular breeding of cotton cold resistance, such as to cultivate cold-resistant cotton varieties. Attached Figure Description
[0013] Figure 1 SPX family gene evolutionary tree;
[0014] Figure 2 Analysis of the gene structure and domains of the SPX family genes;
[0015] Figure 3 SPX family gene expression heatmap under cold stress;
[0016] Figure 4 Silencing the SPX9 gene reduces the cold resistance of cotton.
[0017] Figure 5 Overexpression of the SPX9 gene in cotton enhances its cold resistance. Detailed Implementation
[0018] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were all purchased from commercial sources.
[0019] According to one aspect of the present invention, the application of the GhSPX9 gene in enhancing the cold resistance of cotton is provided, wherein the GhSPX9 gene has a nucleotide sequence as shown in SEQ ID NO:1, or has a nucleotide sequence that is more than 90% identical to the nucleotide sequence shown in SEQ ID NO:1 and has the same function.
[0020] This invention, through extensive research, has discovered that the GhSPX9 gene has a special function in cotton cold resistance. Cotton plants with silenced GhSPX9 gene expression have significantly reduced cold resistance. The proposed application provides a target gene and theoretical support for enhancing cotton cold resistance.
[0021] It should be noted that "having" means that the GhSPX9 gene nucleotide sequence may only have the nucleotide sequence shown in SEQ ID NO:1, or it may be composed of the nucleotide sequence shown in SEQ ID NO:1 and other nucleotide sequences, such as nucleotide sequences encoding functional units for protein purification tags, fluorescent protein markers, and DNA binding sites, or encoding elements that regulate gene transcription and expression, including but not limited to promoters, strong promoters, enhancers, or transcription factor binding sites; "having" may also mean that the nucleotide sequence shown in SEQ ID NO:1 is not continuous in the GhSPX9 gene, but can produce cDNA with the nucleotide sequence shown in SEQ ID NO:1.
[0022] "Identity" here refers to the similarity to the nucleotide sequence shown in SEQ ID NO:1. Differences from the nucleotide sequence shown in SEQ ID NO:1 may be caused by alterations, deletions, or insertions of one or more nucleotides, as well as codon degeneracy. These alterations make the gene sequence not completely identical to the nucleotide sequence shown in SEQ ID NO:1, but it has at least 90% identity, for example, but not limited to 90%, 95%, 98%, 99%, or higher. Furthermore, genes with more than 90% identity to the nucleotide sequence shown in SEQ ID NO:1 have the same function as genes with the nucleotide sequence shown in SEQ ID NO:1. Identity can be evaluated visually or using computer software, such as comparison using conventional BLAST software in the art. When the GhSPX9 gene has the nucleotide sequence shown in SEQ ID NO:1, it is more effective in enhancing the cold resistance of cotton.
[0023] It is understood that, in addition to the GhSPX9 gene itself, biological materials containing the aforementioned GhSPX9 or proteins encoding the GhSPX9 gene can also be used to enhance the cold resistance of cotton. These biological materials may include, but are not limited to, expression cassettes, vectors, or transgenic cell lines.
[0024] Low-temperature stress is specifically divided into two categories: damage caused by temperatures above 0 degrees Celsius is called chilling injury; damage caused by temperatures below 0 degrees Celsius is called frost injury. These two types of stress have different response mechanisms. When plants suffer frost injury, ice crystals form inside and between cells, causing irreversible mechanical damage to plant cells and dehydration of the plant protoplast, potentially leading to plant death. When plants suffer chilling injury, it primarily inhibits the activity of enzymes involved in various metabolic pathways and cellular processes, causing metabolic imbalance in plant cells and thus affecting normal growth and development. The first signs of cold stress in plants are physiological and biochemical changes. For example, low temperatures cause stomata to shrink or even close, reducing transpiration, photosynthesis, and respiration, thus affecting the absorption of water and nutrients from the soil by the roots. Therefore, after cold stress occurs, the first visible physiological changes are wilting, yellowing, and the appearance of lesions on the leaves.
[0025] The present invention will be further illustrated by the following examples.
[0026] The experimental materials used in the embodiments of this invention are described as follows:
[0027] The transgenic cotton recipient material used in this embodiment is upland cotton (Gossypium hirsutum) "ZM113" (Zhongmian 113).
[0028] The vectors used in this embodiment are as follows: pTRV2: a gene silencing vector used for the transformation of silencing materials; pCambia2300-GFP: an overexpression vector used for the transformation of overexpression materials.
[0029] The strains used in this embodiment are as follows: Escherichia coli competent cells DH5α and Agrobacterium competent cells GV3101.
[0030] Unless otherwise specified, the materials or reagents used in the examples were prepared according to existing methods or were purchased directly from the market.
[0031] Example 1: Evolutionary analysis of the GhSPX gene
[0032] Downloaded upland cotton and Arabidopsis data were used to establish a local database using BLAST. The amino acid sequences of Arabidopsis SPX were used as query sequences, and a combination of BLASTP and tBLASTN alignment methods was employed to obtain candidate SPX genes in upland cotton. Then, the amino acid sequences of four Arabidopsis SPX sequences were submitted to the online Pfam database for analysis to obtain conserved domains of Arabidopsis SPX. The online Pfam database was used to determine whether the candidate sequences contained the conserved domains SPX_1, SPX_2, and SPX_3, thereby obtaining the cotton SPX family genes.
[0033] Using the amino acid sequence of the SPX gene from upland cotton, a phylogenetic tree was constructed using MEGA (version 7.0) software. Based on the genome annotation file obtained from the cottongen website, the Visualize Gene Structure tool in TBtools was used to analyze and visualize the cotton gene structure. Annotation information for the cotton SPX gene family was compiled into a gff3 file, and the start positions and key structures of all full-length cotton SPX genes were compiled into a text file. These compiled files were imported into TBtools, and the software was run to obtain the gene structure diagram.
[0034] Fourteen SPX genes were ultimately identified in upland cotton. Phylogenetic analysis of the SPX genes from Arabidopsis thaliana and upland cotton revealed that these SPX genes were divided into four branches: five GhSPX genes in branch one, two GhSPX genes in branch two, three GhSPX genes in branch three, and four GhSPX genes in branch four. Figure 1 ).
[0035] Gene structure and domain analysis were performed on the 14 GhSPX genes. Gene structure analysis revealed that 12 GhSPX genes contained 3 exons and 2 introns, GhSPX6 contained 2 exons and 2 introns, and GhSPX14 contained 2 exons and 1 intron. Domain analysis showed that 12 GhSPX genes contained three SPX domains, GhSPX6 contained only an SPX_1 domain, and GhSPX14 lacked an SPX domain. Figure 2 ).
[0036] Example 2: Transcriptome Analysis of Cold-Treated Cotton Materials
[0037] Using the radicles of the control group and the 4℃ low-temperature treatment group of each variety as materials, three biological replicates were made for each group. Total RNA was extracted using the RNA extraction kit (DP441) from Tiangen Biotech, and after passing quality testing, the transcriptome was sequenced by Wuhan Paisennong Co., Ltd. using the Illumina high-throughput sequencing platform.
[0038] The raw sequencing data obtained from the Illumina platform were filtered to remove paired reads with primer adapters and low-quality paired reads, resulting in clean data. These clean reads were then aligned with the upland cotton reference genome and assembled. The sequences were then compared with seven commonly used databases using BLAST software for annotation, and gene expression levels were calculated using the FPKM algorithm.
[0039] The p-values and p-adj values for differential expression were calculated. The smaller the corrected p-values and p-adj values, the more significant the difference in gene expression. Differentially expressed genes between the treatment group and the control group were screened using p-adj < 0.05 and |log2 Fold Change| > 1 as screening criteria.
[0040] Using transcriptome data, the expression levels of all GhSPX genes were analyzed and a heatmap was generated. The results showed that 12 GhSPX genes were regulated by cold stress. Specifically, the expression levels of GhSPX3-6 genes decreased under cold stress, while the expression levels of GhSPX1-2 and GhSPX7-12 genes increased. It is speculated that these genes play an important role in plant cold tolerance. Figure 3 ).
[0041] The following is an experiment on the effect of the GhSPX9 gene on the cold resistance of cotton. The nucleotide sequence of the GhSPX9 gene is shown in SEQ ID No. 1.
[0042] Example 3: Recovery of linearized carrier and target fragment
[0043] (1) Linearization of expression vector: The gene silencing vector pTRV2 and the overexpression vector pCambia2300-GFP were incubated at 37℃ for 1 h according to the following reaction system to obtain the linearized vector:
[0044]
[0045] (2) Obtaining the target fragment: Using the cDNA obtained in the previous step as a template, the sequence was amplified using the high-fidelity enzyme 2×Phanta Max Master Mix (Dye Plus) from Novizan. The PCR reaction system is as follows:
[0046]
[0047] The PCR amplification system is as follows:
[0048]
[0049]
[0050] Primer information
[0051]
[0052] (3) The above PCR products and linearized expression vector were purified using the FastPureGel DNA Extraction Mini Kit (DC301) from Novizan. The specific steps are as follows:
[0053] (a) Quickly cut the agarose gel containing the target band under UV light to avoid prolonged exposure to UV light that could damage the DNA. Cut the gel into small pieces and place them into a clean 1.5 mL centrifuge tube for subsequent gel dissolution.
[0054] (b) Weigh the gel using an electronic balance. Add 3 times the volume of BufferGDP according to the formula 100mg equals 100μL. Incubate in a 55°C water bath for 15 minutes, inverting and mixing 2-3 times during the process to completely dissolve the gel.
[0055] (c) Place the FastPure DNA Mini Columns-G adsorption column in a 2 mL collection tube, carefully transfer the sol liquid cooled to room temperature into the adsorption column, and centrifuge at 12000 rpm for 1 min.
[0056] (d) Discard the filtrate and then return the adsorption column to the collection tube. Add 300 μL of Buffer GDP to the adsorption column, let it stand at room temperature for 3 min, and then centrifuge at 12000 rpm for 1 min.
[0057] (e) Discard the filtrate and then return the adsorption column to the collection tube. Add 700 μL of Buffer GW (anhydrous ethanol has been added as indicated on the bottle) and centrifuge at 12,000 rpm for 1 min.
[0058] (f) Repeat step (5).
[0059] (g) After discarding the filtrate, put the adsorption column back into the collection tube and centrifuge at 12000 rpm for 2 min.
[0060] (h) Place the adsorption column in a sterile 1.5 ml centrifuge tube, add 40 μL of ElutionBuf fer (preheated to 55 °C in a water bath) to the center of the adsorption column, and incubate at room temperature for 5 min. Elute the DNA by centrifugation at 12000 rpm for 1 min, and store at -20 °C for later use.
[0061] Example 4: Construction of the vector and transformation of Escherichia coli
[0062] Using Nanjing Novizan Company The Ultra One Step Cloning Kit performs homologous recombination of linearized vectors and target fragments to construct vectors, which are then prepared in the following liquid systems on ice:
[0063]
[0064]
[0065] The reaction was carried out at 50°C for 10 minutes, and then immediately cooled on ice. The recombinant product was then transformed into competent E. coli cells.
[0066] (1) Add all 10 μL of the recombinant ligation product to 100 μL of freshly thawed DH5α competent cells and let stand on ice for 30 min.
[0067] (2) Heat shock in a 42℃ water bath for 45 seconds, then immediately place on ice for 2 minutes.
[0068] (3) Add 500 μL of LB liquid culture medium without antibiotics and place it on a shaker at 37°C for 1 h.
[0069] (4) Centrifuge at 5,000 r / min for 2 min, discard 400 μL of supernatant in a clean bench, leave 100 μL of bacterial culture and spread it evenly on LB solid medium containing the corresponding antibiotic, blow dry and invert in an incubator at 37℃ overnight.
[0070] (5) On the second day, pick single clones from the plate and shake them in 500 μL of LB liquid medium containing the corresponding antibiotic until the bacterial culture is turbid. Perform bacterial culture PCR and send positive clones to Shanghai Sangon Biotech Co., Ltd. for sequencing. Compare the sequencing results returned by the company with the target sequence. If the comparison results are completely correct, preserve the bacteria.
[0071] Example 5: Extraction of Escherichia coli plasmids
[0072] Using Beijing Quanshi Gold The Plasmid MiniPrep Kit is used for the extraction of E. coli plasmids. The specific steps are as follows:
[0073] (1) Take 20 mL of Escherichia coli culture that has been cultured overnight on a shaker at 37℃ and put it into a 50 mL centrifuge tube. Centrifuge at 8,000 r / min for 8 min and discard the supernatant.
[0074] (2) Add 1,000 μL of colorless solution RB (containing RNase A), and shake the vortex apparatus vigorously to precipitate and suspend the bacteria. No small bacterial clumps should remain.
[0075] (3) Add 1,000 μL of blue solution LB, gently invert and mix 6-8 times to fully lyse the cells until the solution turns a clear blue color, indicating complete lysis. The operation should be completed within 5 minutes.
[0076] (4) Add 1,400 μL of yellow solution NB, gently mix by turning it upside down 6-8 times until the solution color changes from blue to yellow and forms a firm yellow aggregate, indicating that the neutralization is complete. Let it stand at room temperature for 5 minutes.
[0077] (5) After centrifuging at 8,000 r / min for 10 min, carefully aspirate the supernatant into the centrifuge column in multiple portions, 800 μL each time, centrifuge at 10,000 r / min for 1 min, and discard the liquid in the collection tube.
[0078] (6) Add 700 μL of rinsing solution WB (add the corresponding amount of anhydrous ethanol before the first use), centrifuge at 10,000 r / min for 1 min, discard the liquid in the collection tube, centrifuge the empty tube at 10,000 r / min for 2 min, and place it on a clean bench to dry for 5 min to remove residual rinsing solution.
[0079] (7) Place the centrifuge column in a clean 1.5 mL centrifuge tube, add 100 μL of 65 °C preheated Elution Buffer to the center of the centrifuge column, let it stand at room temperature for 5 min, and then centrifuge at 10,000 r / min for 2 min to elute the plasmid DNA.
[0080] (8) The plasmid DNA concentration was measured using NanoDrop 2000, and the obtained plasmid was stored at -20℃.
[0081] Example 6 Agrobacterium transformation
[0082] (1) Take 2 μL of plasmid and add it to 100 μL of Agrobacterium competent cells in a freeze-thaw state (5 min is best), and gently ripple the tube wall with your fingers to mix.
[0083] (2) Perform the following operations in sequence: stand on ice for 5 minutes, in liquid nitrogen for 5 minutes, in a 37°C water bath for 5 minutes, and stand on ice for 5 minutes.
[0084] (3) Add 600 μL of LB liquid culture medium to the clean bench and place it in a shaker at 200 rpm at 28°C for 3 h to recover and culture.
[0085] (4) Centrifuge at 3000 rpm for 3 min, collect the bacterial cells, discard part of the supernatant in a clean bench, and evenly spread 150 μL of bacterial solution onto LB solid medium containing Kan and Rif resistance. Incubate upside down in an incubator at 28℃ for 2 days.
[0086] (5) Positive clone detection: Select single clone strains and culture them in 600 μL of LB liquid medium containing Kan and Rif resistance for 12 h at 28℃ and 200 rpm.
[0087] (6) Use bacterial culture as a template for PCR verification.
[0088] Example 7: Silencing the GhSPX9 gene reduces the cold tolerance of cotton.
[0089] I. Target gene cloning and vector construction
[0090] (1) Select self-pollinated ZM113 seeds preserved in the laboratory, soak the seeds in sterile water overnight at 37°C, select the seeds with white sprouts, plant them with the radicle facing down in a 1:1 mixture of nutrient soil and vermiculite, cover with a transparent lid, and inject Agrobacterium after the cotyledons have fully unfolded.
[0091] (2) Take 40 mL of Agrobacterium tumefaciens culture containing gene silencing vector that has been cultured overnight into a 50 mL clean centrifuge tube, centrifuge at 4000 rpm for 10 min to enrich the bacterial cells, and discard the supernatant.
[0092] (3) Use the prepared resuspension solution to resuspend the bacteria and adjust OD600 = 1.0.
[0093] (4) Incubate at room temperature in the dark for 3 hours.
[0094] (5) Carefully scratch the back of the cotton cotyledon with the needle of a 1mL sterile syringe (do not penetrate the leaf), and inject the bacterial solution into the leaf through the wound until the entire leaf is soaked.
[0095] About two weeks after inoculation, the true leaves of the positive control TRV:PDS plants turned white. Figure 4 (A). Subsequently, RNA was extracted from the leaves of TRV:00 and TRV:GhSPX9 plants, reverse transcribed, and quantitative fluorescence identification was performed. The results showed that the expression of the GhSPX9 gene was significantly reduced. Figure 4 (Middle B). These results confirm that the experimental procedure was correct and the GhSPX9 gene was silenced. TRV:00 and TRV:GhSPX9 plants were transferred to a light incubator and cultured at 6℃, with a 16-hour light-8-hour dark cycle and 60% relative humidity. Phenotypic observation was performed after 3 days of treatment, and the results showed that the leaves of TRV:GhSPX9 plants were severely wilted. Figure 4 (D). The survival rates of TRV:00 and TRV:GhSPX9 plants were statistically analyzed, and it was found that the survival rate of TRV:GhSPX9 plants was significantly lower than that of TRV:00 plants. Figure 4 (C)
[0096] Example 8: Overexpression of the GhSPX9 gene enhances the cold resistance of cotton.
[0097] In a clean bench, sterile seedlings were cut into small segments and transferred to Agrobacterium infection medium containing the GhSPX9 gene overexpression vector. Infection was carried out for 10 minutes at 200 rpm. After infection, the hypocotyls were transferred to sterilized large culture dishes and allowed to dry. Then, the hypocotyls were transferred to co-culture medium and cultured in the dark for 48 hours. After co-culture, they were transferred to induction medium. After about 30 days of culture, loose callus tissue began to grow at both ends of the hypocotyls. Subculture was continued 1-2 times. Finely granular embryogenic callus tissue was selected and transferred to differentiation medium for further culture until it differentiated into seedlings.
[0098] Leaves of T0 generation seedlings were collected, DNA was extracted, and specific fragment primers were designed for PCR amplification to verify positive plants. The negative control showed no target band, while the positive control showed a clear band at the target location. Figure 5 (A) These positive T0 generation plants were further propagated in the field. WT and T4 generation cotton seeds were selected, soaked in sterile water overnight at 37℃, and seeds with emerging white hairs were planted with the radicle facing down in a 1:1 mixture of nutrient soil and vermiculite. A transparent lid was placed over the seeds, and removed after the cotyledons unfolded. The plants were cultured in a greenhouse until the 6-leaf stage, then transferred to a light incubator with a temperature of 6℃, a 16-hour light-8-hour dark cycle, and a relative humidity of 60%. Phenotypic observation was performed after 3 days. The results showed that the leaves of plants overexpressing the GhSPX9 gene exhibited slight wilting, while WT plants showed severe wilting, demonstrating that plants overexpressing the GhSPX9 gene had higher cold tolerance than WT plants. Figure 5 (B)
Claims
1. The application of the GhSPX9 gene or an expression vector containing the GhSPX9 gene in regulating the cold resistance of cotton, wherein the nucleotide sequence of the GhSPX9 gene is as shown in SEQ ID No. 1 or a degenerate sequence encoding the same protein as the sequence shown in SEQ ID No. 1, and the regulation of cotton cold resistance refers to overexpressing the GhSPX9 gene in cotton to improve the cold resistance of cotton.