Saline-alkali tolerant hub gene as well as application and acquisition method thereof

Abstract

The invention is applicable to the technical field of agricultural biology, and provides a saline-alkaline tolerant hub gene as well as application and an acquisition method thereof, and particularly, the saline-alkaline tolerant hub gene comprises at least one of the following genes: GHA01G1528, GHA08G2688, GHD08G2683, GHD01G1620 and GHA10G0617; the nucleotide sequence of the GHA01G1528, the nucleotide sequence of the GHA08G2688, the nucleotide sequence of the GHD08G2683, the nucleotide sequence of the GHD01G1620 and the nucleotide sequence of the GHA10G0617 are shown as SEQ ID NO.1-5 in a sequence table respectively. The saline-alkaline tolerant hub gene is determined by combining saline-alkaline stress and rehydration treatment with transcriptome sequencing analysis and weighted gene co-expression network analysis, can be applied to cotton breeding, provides a theoretical basis for exploring a saline-alkaline tolerant molecular mechanism of cotton, and provides a new gene resource for saline-alkaline tolerant breeding of cotton.

Description

Technical Field

[0001] The present invention belongs to the field of agricultural biotechnology, and particularly relates to a salt-tolerant hub gene, its uses, and a method for obtaining the same. Background Art

[0002] Saline-alkali soil is a general term for different saline soils, including saline soil, alkali soil, alkalized soil, and other soils showing different degrees of salinization. It has been found that saline soil contains a high content of neutral salts, mainly including NaCl and Na2SO4, which mainly affect crop growth and development by influencing osmotic balance, ion toxicity, nutrient absorption and synthesis, and respiration.

[0003] Plants face various challenges when growing in a high-salinity environment, such as ion toxicity, water stress, and inhibited photosynthesis. To adapt to these conditions, plants have developed various adaptive strategies. Salt-tolerant plants may increase leaf thickness and root development under salt stress to improve water and nutrient element absorption. At the same time, plants may also regulate the composition and structure of the cell wall to maintain cell stability and integrity.

[0004] Plants may redistribute ions and soluble compounds under salt stress to mitigate the negative impact of salt on cell functions and metabolism. For example, plants may accumulate potassium ions and reduce sodium ion absorption to maintain intracellular ion balance. Plants may also synthesize antioxidants to counteract damage caused by oxidative stress. Plants may change gene expression patterns under salt stress, such as genes that directly protect plants from environmental stress and genes that regulate the expression of target genes in response to stress.

[0005] Cotton ( Gossypium hirsutumL.) belongs to the Malvaceae family. It is a crop with good drought resistance, salt tolerance and barrenness tolerance. It is a "pioneer crop" for the development and utilization of saline-alkali land. Under the influence of high salt stress, chlorophyll synthesis is slow, seedlings grow slowly and grow malformed, and the two cotyledons are difficult to flatten. When cotton seedlings are in the two-leaf and three-leaf stages, flower bud differentiation has begun in the body, which is a period that is more sensitive to salt. Usually, cotton plants under salt stress are similar to those under drought stress, showing symptoms such as leaf edge scorching, darkening of leaf color, cotyledon shedding and plant wilting. This is due to the increase in osmotic pressure in the soil solution, which leads to physiological drought and ion accumulation, hindering the absorption of nutrients and water. Previous studies have done a lot of research on cotton salt tolerance. Peng et al. used RNA-Seq technology to analyze the expression of transcription factor families and transcripts in the leaves of seedlings of two cotton germplasms after 200mM NaCl stress for 4h and 24h, and speculated that 11 highly expressed transcription factor genes may be closely related to the salt tolerance of upland cotton. Zhang Binglei et al. discovered the specifically expressed alkali-tolerant candidate gene GhA12G2168 (cystathionine γ-synthase gene ChMGL11) by subjecting upland cotton to different saline-alkali treatments. Zhang Yuexin et al. found that melatonin can improve the salt tolerance of cotton. Cui Ruifeng et al. showed that the symbiotic metabolite inositol galactose under the interaction of cotton bacteria and salt is converted into raffinose to participate in plant osmotic regulation and scavenge free radicals to alleviate salt stress. These findings provide new ideas for salt-tolerant breeding of cotton and are expected to be further applied in related fields in the future.

[0006] Rewatering plants under salt stress is an effective measure to restore their phenotype, physiology and gene expression. Rewatering can reduce the extent to which plants are affected by salinity, but there is limited research on how rewatering affects plant response to salt stress. By gaining a deeper understanding of the physiological and molecular mechanisms of salt stress and rewatering, water resources can be better protected and used efficiently. No research has been published on the response of cotton to rewatering after salt stress. Therefore, salt tolerance in cotton remains to be explored. Summary of the invention

[0007] The purpose of the embodiments of the present invention is to provide a salt-alkali tolerance hub gene, aiming to solve the problems raised in the background technology.

[0008] In response to the above problems, the present invention provides a salt-alkali tolerance hub gene, including at least one of the following genes: GH_A01G1528, GH_A08G2688, GH_D08G2683, GH_D01G1620, GH_A10G0617; the nucleotide sequences of the GH_A01G1528, GH_A08G2688, GH_D08G2683, GH_D01G1620, GH_A10G0617 are shown in the sequence table SEQ ID NO.1-5 respectively.

[0009] Another object of the present invention is to provide a use of the above-mentioned salt-tolerant and alkali-tolerant hub gene to improve the salt and alkali tolerance of cotton.

[0010] Another object of the present invention is to provide a use of the above-mentioned salt-tolerant and alkali-tolerant hub gene to improve the adaptability of cotton after rehydration under salt and alkali stress.

[0011] After the cotton containing the salt-tolerant and alkali-tolerant hub gene is rehydrated under salt and alkali stress, the concentration of malondialdehyde decreases, and the chlorophyll content, superoxide dismutase activity and peroxidase activity all increase.

[0012] Preferably, compared with the cotton without the salt-tolerant and alkali-tolerant hub gene, the cotton containing the salt-tolerant and alkali-tolerant hub gene has fewer differentially expressed genes after rehydration under salt and alkali stress.

[0013] Another object of the present invention is to provide a use of the above-mentioned salt-tolerant and alkali-tolerant hub gene in cotton breeding.

[0014] Another object of the present invention is to provide a method for obtaining the above-mentioned salt-tolerant and alkali-tolerant hub gene, comprising the following steps: Select different types of cotton varieties, and collect samples under salt and alkali stress and after rehydration treatment respectively; Extract RNA from the samples, and perform transcriptome sequencing analysis to obtain expression data; Screen differentially expressed genes from the expression data to obtain differentially expressed genes of different types of cotton varieties under salt and alkali stress and after rehydration treatment; Construct a weighted gene co-expression network according to the differentially expressed genes of different types of cotton varieties under salt and alkali stress and after rehydration treatment to obtain a cotton salt and alkali tolerance co-expression network; Divide the cotton salt and alkali tolerance co-expression network into multiple co-expression modules; Take the salt and alkali tolerance phenotype of the sample as a trait input, perform specific identification on multiple co-expression modules, determine the module related to the rehydration recovery ability under salt and alkali stress, and obtain the grey module; Calculate the connectivity of the genes in the grey module, screen highly connected genes to obtain the salt-tolerant and alkali-tolerant hub gene.

[0015] Preferably, the cotton varieties include salt-tolerant cotton and salt-sensitive cotton.

[0016] Preferably, the step of constructing a weighted gene co-expression network according to the differentially expressed genes of different types of cotton varieties under salt and alkali stress and after rehydration treatment to obtain a cotton salt and alkali tolerance co-expression network specifically includes: Based on the differentially expressed genes of different types of cotton varieties under saline-alkali stress and after rewatering treatment, the soft threshold was determined through scale-free topology analysis, and a weighted gene co-expression network was constructed to obtain the cotton saline-alkali tolerance co-expression network.

[0017] Preferably, the method for dividing the cotton saline-alkali tolerance co-expression network into multiple co-expression modules is the dynamic tree cutting algorithm. The saline-alkali tolerance hub genes provided by the present invention include at least one of the following genes: GH_A01G1528, GH_A08G2688, GH_D08G2683, GH_D01G1620, GH_A10G0617; the above-mentioned saline-alkali tolerance hub genes are determined by combining transcriptome sequencing analysis and weighted gene co-expression network analysis under saline-alkali stress and rewatering treatment, and can be applied to cotton breeding, providing a theoretical basis for exploring the molecular mechanism of cotton saline-alkali tolerance and providing new gene resources for cotton saline-alkali tolerance breeding. Brief Description of the Drawings

[0018] Figure 1 It shows the growth conditions of cotton seedlings at different time periods under salt stress.

[0019] Figure 2 It is a comparison chart of the changes in MDA content of cotton seedlings under salt stress and after rewatering.

[0020] Figure 3 It is a microscopic structure diagram of cotton leaves after salt stress and rewatering.

[0021] Figure 4 In it: (A) is a clustering dendrogram and Pearson correlation coefficient heat map based on the normalized FPKM values of expressed genes. In the figure, the abscissa and ordinate are both sample numbers, and their order is determined by the sample correlation clustering result. The clustering trees are at the top and left of the figure, and the color reflects the correlation size between samples; (B) is the principal component analysis of the identified genes.

[0022] Figure 5 It is the qRT-PCR results of 10 genes. The relevant significance levels (p values) are added above each histogram. Ns indicates no significant effect at the p < 0.05 level, * indicates a significant effect at the p < 0.05 level; the black bars are ST; the gray bars are SS.

[0023] Figure 6 It is the RNA-seq heat map of 10 genes.

[0024] Figure 7 In it: (A) is the Venn diagram of gene expression in ST leaves; (B) is the Venn diagram of gene expression in SS leaves.

[0025] Figure 8In: (A) is a bar graph of differential gene expression among different varieties under the same treatment; (B) is a Venn diagram of differential gene expression among different varieties under the same treatment.

[0026] Figure 9 In: (a) is the GO analysis of differentially expressed genes under normal conditions (ST-CK vs SS-CK); (b) is the GO analysis of differentially expressed genes under salt stress (ST-NaCl vs SS-NaCl); (c) is the GO analysis of differentially expressed genes under re-watering (ST-RW vs SS-RW).

[0027] Figure 10 In: (a) is the KEGG analysis of differentially expressed genes under normal conditions (ST-CK vs SS-CK); (b) is the KEGG analysis of differentially expressed genes under salt stress (ST-NaCl vs SS-NaCl); (c) is the KEGG analysis of differentially expressed genes under re-watering (ST-RW vs SS-RW).

[0028] Figure 11 In: (a) is a bar graph of differential gene expression between treatments; (b) is a Venn diagram of differential gene expression between treatments.

[0029] Figure 12 In: (a) is the GO analysis of differentially expressed genes in salt-tolerant ST under salt stress (ST-CK vs ST NaCl); (b) is the GO analysis of differentially expressed genes in salt-tolerant ST under re-watering (ST-CK vs ST-RW); (c) is the GO analysis of differentially expressed genes in salt-sensitive SS under salt stress (SS-CK vs SS-NaCl); (d) is the GO analysis of differentially expressed genes in salt-sensitive SS under re-watering ((SS-CK vs SS-RW).

[0030] Figure 13 In: (a) is the KEGG analysis of differentially expressed genes in salt-tolerant ST under salt stress (ST-CK vs ST NaCl); (b) is the KEGG analysis of differentially expressed genes in salt-tolerant ST under re-watering (ST-CK vs ST-RW); (c) is the KEGG analysis of differentially expressed genes in salt-sensitive SS under salt stress (SS-CK vs SS-NaCl); (d) is the KEGG analysis of differentially expressed genes in salt-sensitive SS under re-watering (SS-CK vs SS-RW).

[0031] Figure 14 In: (a) is sample clustering analysis; (b) is soft threshold selection; (c) is gene clustering tree analysis; (d) is a heatmap of the correlation between modules and treatments.

[0032] Figure 15 The results screened by the cytohubba plugin are shown. Specific implementation manners

[0033] In order to make the objectives, technical solutions and advantages of the present invention more clear and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0034] Weighted gene co-expression network analysis (WGCNA) is very fast and efficient in mining functionally related genes in co-expression modules and has been widely applied to various crops to mine corresponding candidate genes. Based on this, in the embodiments of the present invention, the response of cotton to salt stress and its adaptability after rewatering were explored through morphology, physiology and microstructure, and salt-tolerant genes of cotton were mined through transcriptome sequencing (RNA-seq) technology. The embodiments of the present invention can provide a theoretical basis for further exploring the molecular mechanism of cotton salt tolerance and provide new gene resources for cotton salt-tolerant breeding.

[0035] Specifically, in the embodiments of the present invention, a salt-alkali-tolerant hub gene is provided, which includes at least one of the following genes: GH_A01G1528, GH_A08G2688, GH_D08G2683, GH_D01G1620, GH_A10G0617; the nucleotide sequences of the GH_A01G1528, GH_A08G2688, GH_D08G2683, GH_D01G1620, GH_A10G0617 are respectively shown as SEQ ID NO.1-5 in the sequence listing.

[0036] Among them, GH_A01G1528 encodes a non-specific lipid transfer protein LTP4; GH_A08G2688 encodes a long-chain alkane synthesis-related protein CER3; GH_D08G2683 encodes a long-chain alkane synthesis-related protein CER3; GH_D01G1620 encodes a non-specific lipid transfer protein LTP1; GH_A10G0617 encodes an inorganic pyrophosphatase PEPC1.

[0037] In another embodiment of the present invention, a use of the above-mentioned salt-alkali-tolerant hub gene for improving the salt-alkali tolerance of cotton is also provided.

[0038] In another embodiment of the present invention, a use of the above-mentioned salt-alkali-tolerant hub gene for improving the adaptability of cotton after rewatering under salt-alkali stress is also provided.

[0039] Specifically, after the cotton containing the salt-alkali-tolerant hub gene is rewatered under salt-alkali stress, the concentration of malondialdehyde decreases, and the chlorophyll content, superoxide dismutase activity and peroxidase activity all increase.

[0040] In addition, compared with cotton without the salt-tolerant hub gene, cotton containing the salt-tolerant hub gene has a smaller number of differentially expressed genes after rehydration under salt-alkali stress.

[0041] In another embodiment of the present invention, there is also provided a use of the above-mentioned salt-tolerant hub gene in cotton breeding.

[0042] In another embodiment of the present invention, there is also provided a method for obtaining the above-mentioned salt-tolerant hub gene, comprising the following steps: S1. Select different types of cotton varieties (such as salt-tolerant cotton and salt-sensitive cotton), and collect samples respectively under salt-alkali stress and after rehydration treatment; S2. Extract RNA from the samples and perform transcriptome sequencing analysis to obtain expression data; S3. Screen differentially expressed genes from the expression data to obtain differentially expressed genes of different types of cotton varieties under salt-alkali stress and after rehydration treatment; S4. According to the differentially expressed genes of different types of cotton varieties under salt-alkali stress and after rehydration treatment, determine the soft threshold through scale-free topology analysis, construct a weighted gene co-expression network, and obtain a cotton salt-tolerant co-expression network; S5. Use the dynamic tree cutting algorithm to divide the cotton salt-tolerant co-expression network into multiple co-expression modules; S6. Input the salt-tolerant phenotype of the samples as a trait, perform specific identification on multiple co-expression modules, determine the module related to the rehydration recovery ability under salt-alkali stress, and obtain the grey module; S7. Calculate the connectivity of genes within the grey module, screen highly connected genes, and obtain the salt-tolerant hub gene.

[0043] For the experimental methods without specific experimental conditions noted in the following examples, they are generally carried out according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. Unless otherwise specified, the reagents, raw materials, and equipment used in the present invention can be obtained commercially.

[0044] Example 1: In this example of the present invention, two cotton materials with extremely different salt tolerances were used: salt-sensitive 52-128 (SS) and salt-tolerant Jing Simian (ST). A natural saline-alkali land experiment was conducted in Shawan, China from 2018 to 2019. The growth traits and salt damage rates of different cotton materials under salt stress are shown in Table 1. The results show that there are significant differences in salt tolerance between these two cotton materials at the seedling stage.

[0045] Cotton seeds were disinfected with 5% sodium hypochlorite (NaClO) for 20 minutes, rinsed three times with sterile water, sown in germination boxes (12.4 cm × 17.5 cm), and placed under controlled conditions (day / night temperature 26°C / 18°C, relative humidity 65%, day / night duration 8h / 16h, light intensity 12000 lux. Stored in a phytotron). After the cotyledons were fully expanded, they were transferred to 1 / 2 Hoaglands nutrient solution until the three-leaf stage. Cotton seedlings at the three-leaf stage were grown in 200 mM NaCl solution for 48 h, and then cultured in 1 / 2 Hoagland nutrient solution for 48 h (RW, re-watering). Samples were taken at 0, 48 h, and then after re-watering for 48 h (0 h, 48 h, and RW 48 h) respectively. These seedlings had three biological replicates, with 100 seedlings in each, and a total of 18 samples were collected (3 time points × 2 varieties × 3 biological replicates).

[0046] Table 1

[0047] Example 2: The samples obtained above were subjected to the following tests: Physiological determination: Chlorophyll was measured in the largest functional leaves of cotton plants under different treatments using a portable chlorophyll meter (SPAD-502Plus). Referring to previous studies, the thiobarbituric acid colorimetric method, nitroblue tetrazolium photoreduction method, and guaiacol-hydrogen peroxide method were used to measure the content of malondialdehyde, superoxide dismutase activity, and peroxidase activity respectively.

[0048] Morphological observation: Using the vertical vein cutting method, leaves under different treatments were placed into FAA fixative respectively. After good fixation, trimming, dehydration, embedding, sectioning, staining, and mounting were carried out, and finally qualified specimens were examined under a microscope, and the sections were stained with safranin-fast green staining solution. Images were collected and scanned for browsing using a panoramic slide scanner (models: PANNORAMIC DESK / MIDI / 250 / 1000 and CaseViewer2.2) produced by 3DHISTECH (Hungary), and analyzed using Image-Pro Plus 6.0 produced by Media Cybemetics (U.S.A). The leaf thickness, upper epidermis thickness, palisade tissue thickness, spongy tissue thickness, and lower epidermis thickness at 5 locations in each section were measured respectively, and the mean values were taken.

[0049] RNA extraction and cDNA library construction: Leaves of ST and SS plants were collected at 0 h, 48 h of salt treatment, and 48 h of re-watering, and immediately frozen in liquid nitrogen. Then, all samples were stored at -80°C for RNA-seq analysis. The total RNA was extracted according to the product manual of the polysaccharide polyphenol total RNA extraction kit from Tiangen.

[0050] RNA degradation and contamination were detected by 1% agarose gel electrophoresis. Concentration was detected using Nanodrop 2000 (Thermo Fisher Nanodrop 2000, Shanghai, China), and then RNA integrity was calibrated using Agilent 2100 (Platinum Elmer LabChip GX, Beijing, China). RNA concentration was calibrated according to qubit quantification (Life Technologies, Beijing, China). Before constructing the library with high-quality RNA samples, the samples were calibrated again and used for RNA-seq. Under the same volume and quantity, OD260 / 280 was between 1.8 and 2.2, OD260 / 230 was between 1.8 and 2.2, RIN value ≥7, and baseline judgment was combined with GX detection. After the sample passed the test, magnetic beads with Oligo(dT) were used to enrich eukaryotic mRNA; Fragmentation Buffer was added to randomly fragment the mRNA; using the mRNA as a template, the first strand of cDNA was synthesized with random hexamers, and then buffer, dNTPs, RNase H, and DNA polymerase I were added to synthesize the second strand of cDNA, and the cDNA was purified using AMPure XP beads; the purified double-stranded cDNA was then subjected to end repair, A-tailing, and ligation of sequencing adapters, and then fragment size selection was performed using AMPure XP beads; finally, the cDNA library was obtained by PCR enrichment. After the library construction was completed, Qubit 3.0 fluorescence quantifier was first used for preliminary quantification, and the concentration needed to reach more than 1 ng / μL. Subsequently, the Qsep400 high-throughput analysis system was used to detect the inserted fragments of the library. After the inserted fragments met the expectations, Q-PCR method was used to accurately quantify the effective concentration of the library (library effective concentration > 2 nM) to ensure the library quality. After the library passed the quality inspection, the Illumina NovaSeq6000 (Illumina, San Diego, CA, USA) sequencing platform was used for PE150 mode sequencing.

[0051] Data and differential gene expression analysis: Raw reads were obtained using Illumina. First, the fastp software was used to filter and perform quality control on the data, and clean reads were obtained while calculating the Q30 and GC content. Then, the HISAT2 software was used to map the clean reads to the cotton reference genome Gossypium_hirsutum.TM_1_v2.1.genome.fa, and quantification was performed using the StringTie software based on Fragments per kilobase of transcript per million Fragments mapped (FPKM) to estimate the gene expression level of each transcript. Using the Edge R package, genes with FoldChange≥2 and FDR<0.01 as the screening criteria were designated as DEGs. The false discovery rate (FDR) was calculated by adjusting the p-value by multiple corrections of the false discovery rate.

[0052] Construction of weighted gene co-expression network: Through the BMKCloud cloud platform (www.biocloud.net), a cotton salt tolerance co-expression network was established for the 32,390 DEGs generated by ST and SS after salt stress and rewatering. In the scale-free weighted gene network, nodes correspond to DEGs, and edges are determined by the similar expression profiles of paired genes calculated by Pearson correlation. Therefore, in the embodiment of the present invention, an optimal soft threshold β = 26 was selected to construct a network based on the adjacency matrix (R 2 > 0.8). The topological overlap matrix was calculated through the adjacency matrix, and the dynamic tree cutting algorithm was used to segment the modules according to the default parameter settings. The grouping matrix of 18 samples was used as the trait for specific module identification. The co-expression network was visualized using Cytoscape 3.9.1 software.

[0053] qRT-PCR verification of transcriptome: To verify the results of Illumina sequencing data, RNA was extracted from SS and ST at three time points of 0, 48 h, and RW48 h under salt stress. 10 DEGs were randomly selected, and the conserved regions of the sequences were found using BlastX and ORF finder in NCBI, and qRT-PCR primers were designed using the online website (https: / / www.primer3plus.com). The change in gene expression was detected using the two-step method program on the 7500 Fast System of the American ABI company, with GbUBQ7 as the internal reference. Each reaction was repeated 3 times. According to the Ct value, the relative expression level of each gene was calculated.

[0054] Data analysis: Data statistics and analysis were performed using Excel 2010 and SPSS 27, and the sequencing results were analyzed through the BMKCloud cloud platform (www.biocloud.net).

[0055] Example 3: The test results and analysis of the above Example 2 are as follows: 1. Phenotypic observation of cotton seedlings under salt stress and after re-watering: Cotton seedlings at the three-leaf stage were treated with 200 mM NaCl solution, and the growth conditions of cotton seedlings at different time periods under salt stress were observed (as Figure 1 shown). During 0 - 48 h of NaCl stress, the leaves and stems of ST and SS seedlings gradually softened, and the cotyledons wilted severely at 48 h. The changes in SS seedlings were more obvious than those in ST seedlings. At 48 h, the cotyledons withered and began to fall off, and the margins of the true leaves were slightly coked.

[0056] 2. Physiological responses of cotton seedlings under salt stress and after re-watering: The changes in the MDA content of cotton seedlings under salt stress and after re-watering are as Figure 2 shown. Figure 2 In, the same letters indicate no significant difference between treatments, and different letters indicate significant differences (P < 0.05, LSD method); Salt stress can disrupt the ion balance within plant cells, leading to the production of adverse factors such as ion toxins, osmotic pressure, and reactive oxygen species, thereby affecting the growth and development of cotton. Under 48 h of NaCl treatment, the leaves of cotton seedlings were severely damaged, and the malondialdehyde (MDA) concentrations in the leaves of ST and SS seedlings increased significantly, while the chlorophyll content and superoxide dismutase activity decreased significantly. After re-watering treatment, ST and SS recovered to a certain extent, with the MDA concentration decreasing, and the chlorophyll content, superoxide dismutase activity, and peroxidase activity all increasing. ST seedlings recovered better than SS seedlings, and their MDA concentration and superoxide dismutase activity basically returned to the CK level.

[0057] 3. Microscopic structure of cotton leaves under salt stress and after re-watering: The salt tolerance mechanism of plants is very complex. Salt stress not only affects the physiological and metabolic processes of plants but also causes various changes in plant morphology, anatomical structure, etc. Observation of the microscopic structure of cotton leaves under salt stress and after re-watering shows that (as Figure 3 shown. Figure 3 In, Up: upper epidermis; Pt: palisade tissue; St: spongy tissue; Le: lower epidermis), under normal conditions, the sections of ST and SS seedlings were stained darker, the microscopic structure was arranged more closely, the bulliform cells were in the shape of a single layer of irregular quadrilaterals and were closely arranged; under salt stress, the bulliform cells were oval-shaped, the palisade tissue and spongy tissue cells were arranged more loosely, and the leaves became softened. After re-watering, the bulliform cells were in the shape of long ovals, the leaves were flatter, and gradually resumed normal growth. In addition, the data on the structural characteristics of the leaf epidermis of cotton seedlings under salt stress and after re-watering are shown in Table 2.

[0058] Table 2

[0059] The data in Table 2 show that under normal conditions, there was no significant difference in the leaf epidermal structure between ST and SS cotton seedlings; under NaCl stress, the leaf thickness, palisade tissue thickness and spongy tissue thickness of ST and SS decreased, and recovered after rehydration. After NaCl treatment, the leaf thickness, palisade tissue thickness and spongy tissue thickness of ST cotton seedlings decreased by 16.17%, 13.92% and 18.46% respectively compared with the control, which was not significantly different from the control; the leaf thickness, palisade tissue thickness and spongy tissue thickness of SS decreased by 32.43%, 30.13% and 41.10% respectively compared with the control, which was significantly different from the control.

[0060] 4. Transcriptome sequencing analysis of cotton seedlings after salt stress and rehydration: In order to explore important genes related to cotton salt tolerance, the present invention samples two cotton materials with huge differences in salt tolerance at three growth time periods of 0 and 48h under 200 mM NaCl stress and then rehydrated for 48h (0 hour, 48 hours and RW 48h). Each treatment has three biological replicates. For the 18 samples collected (3 time points × 2 varieties × 3 biological replicates), 18 RNA-seq libraries were constructed and sequenced and analyzed by Illumina HiSeq 4000, and a total of 127.58Gb of Clean Data was obtained, and the Q30 base percentage of each sample was not less than 91.70%. The Total Reads of each sample were sequenced with the upland cotton reference genome (TM-1). According to the comparison results, the comparison efficiency of the Reads of each sample with the reference genome was between 94.06% and 96.60%, and the GC content of each sample exceeded 43.47%. These results all prove that the RNA-seq provided in the embodiment of the present invention is of high quality and can be used for subsequent analysis (the RNA-seq results of salt stress and rehydration are shown in Table 3).

[0061] Based on the comparison results, 11,125 new genes were discovered, of which 6,974 were functionally annotated. The normalized FPKM value was used to measure the expression level of each gene, and the Pearson correlation coefficient (PCC) was used to detect the correlation between all samples. It was found that the overall correlation between the three biological replicates of the same growth stage of the two varieties was high, and the sample clustering also showed a good correlation between the biological replicates of the same growth environment (such as Figure 4 To further confirm the correlation between ST and SS at different stages of salt stress and rehydration, principal component analysis was performed on the above-mentioned expressed genes (as shown in A). Figure 4As shown in B), it shows that two cotton varieties with a huge difference in salt tolerance have significantly different expression patterns in different environments. SS and ST are consistent in three biological replicates in the same environment, indirectly proving the reliability of the transcriptome data.

[0062] To verify the reliability of RNA-seq, in the examples of the present invention, 10 genes were randomly selected for qRT-PCR experiments, and the results were basically consistent with the expression trends of the transcriptome expression profiles, proving that the transcriptome data is reliable for the next step of analysis (as Figure 5 and 6 shown). In the salt-sensitive SS, 43233, 40970, and 42529 genes were expressed in sequence, among which 36890 genes were co-expressed; in ST, 43593, 41028, and 43943 genes were expressed in sequence, among which 37256 genes were co-expressed (as Figure 7 shown).

[0063] Table 3

[0064] 5. Expression analysis of differentially expressed genes (DEGs) between two cotton materials: To further analyze the differential mechanism between two cotton materials, with Fold Change≥2 and FDR<0.01 as the screening criteria, the differentially expressed genes (DEGs) of the same treatment of the two materials were obtained (as Figure 8 shown). A total of 10589 genes were screened, among which 5960 genes were up-regulated, including 136 genes under normal conditions, 2151 genes under salt stress, and 3673 genes under re-watering; 4629 genes were down-regulated, including 228 genes under normal conditions, 2271 genes under salt stress, and 2130 genes under re-watering. As the growth period increased, the number of differentially expressed genes between the two cotton materials increased, indicating that the mechanisms of these two cotton materials in response to salt stress and re-watering are different.

[0065] The DEGs between the two materials were compared to the GO database (as Figure 9 shown). In terms of biological processes, the DEGs were mainly enriched in metabolic processes, cellular processes, single-organism processes, etc. In terms of cellular components, they were mainly involved in cells and cellular components. In terms of molecular functions, they were mainly enriched in binding and catalytic activities. Under normal conditions, the number of down-regulated genes was relatively large, while after re-watering, the number of up-regulated genes was relatively large. This fully illustrates the adaptability of cotton to the re-watering treatment. Subsequently, KEGG enrichment analysis was performed on the differentially expressed genes. As Figure 10 shown, the differentially expressed genes of the salt-tolerant cotton ST were significantly enriched in photosynthesis - antenna proteins (ko00196), carbon fixation in photosynthetic organisms (ko00710), and vitamin B6 metabolism (ko00750).

[0066] 6. Expression analysis of differentially expressed genes (DEGs) under salt stress and re-watering: To further analyze the differential mechanisms under salt stress and re-watering, with Fold Change ≥ 2 and FDR < 0.01 as the screening criteria, the differential gene expressions (DEGs) under salt stress and re-watering were obtained (as Figure 11 shown), and a total of 32,390 genes were screened. For highly salt-tolerant cotton ST, 15,780 genes were screened under salt stress, among which 7,996 genes were up-regulated and 7,784 genes were down-regulated. Under re-watering treatment, 2,454 genes were screened, among which 659 genes were up-regulated and 1,795 genes were down-regulated. For salt-sensitive cotton SS, a total of 14,156 genes were screened under salt stress, among which 6,925 genes were up-regulated and 7,231 genes were down-regulated. Under re-watering treatment, 9,390 genes were screened, among which 4,666 genes were up-regulated and 4,724 genes were down-regulated. Under salt stress, the number of DEGs screened in ST was more than that in SS, but the number of DEGs generated after re-watering treatment was much less than that in SS, which may be related to the better recovery of ST after re-watering.

[0067] The DEGs of the two materials after salt stress and re-watering were aligned to the GO database (as Figure 12 shown). In terms of biological processes, the DEGs were mainly enriched in metabolic processes, cellular processes, single-organism processes, etc. In terms of cellular components, they were mainly involved in cells, cell components, and membranes. In terms of molecular functions, they were mainly enriched in binding and catalytic activities.

[0068] The DEGs annotated by KEGG were divided into different categories (as Figure 13As shown in the figure, the response of cotton to salt stress and its adaptation after rewatering may be related to the interactions of photosynthesis (ko 00195), photosynthesis - antenna proteins (ko 00196), plant hormone signal transduction (ko 04075), starch and sucrose metabolism (ko 00500), and porphyrin and chlorophyll metabolism (ko 00860), etc. In addition, salt - tolerant cotton ST responds to salt stress through pathways such as circadian rhythm - plant (ko 04712), carbon fixation in photosynthetic organisms (ko 00710), and carbon metabolism (ko 01200), and regulates its adaptation under rewatering treatment through pathways such as glycerolipid metabolism (ko 00561), pentose and glucuronate interconversions (ko 00040), circadian rhythm - plant (ko 04712), and phenylpropanoid biosynthesis (ko 00940). Sensitive cotton SS responds to or resists salt stress through pathways such as fatty acid degradation (ko 00071), degradation of valine, leucine, and isoleucine (ko 00280), brassinosteroid biosynthesis (ko 00905), and sphingolipid metabolism (ko 00600). The damage caused by salt stress is alleviated through pathways such as plant - pathogen interaction (ko 04626), MAPK signaling pathway - plant (ko 04016), and α - linolenic acid metabolism (ko 00592).

[0069] 7. Weighted co - expression network analysis of differentially expressed genes (DEGs) under salt stress and after re - watering: Sample clustering analysis: To further understand gene expression under salt stress and after re - watering and to identify salt - tolerance candidate genes, the differentially expressed genes under salt stress and after re - watering were used to construct a co - expression network by WGCNA. The expression matrix was imported, the Euclidean distance matrix was calculated using the dist function, and the hclust clustering function (hierarchical clustering function) was used. The "average" clustering algorithm was selected to cluster the samples. As Figure 14 shown, it can be seen from the figure that there are no outlier samples among the 18 samples. It can also be seen from the figure that there are significant differences between different treatments of ST and SS, and the re - watering treatment of ST and the re - watering of SS can be clearly distinguished, which further indicates that the specificity of the expression matrix is good and can well distinguish different treatments of ST and SS with higher salt tolerance. In the embodiment of the present invention, the optimal soft threshold 26 is selected to construct a gene co - expression that is infinitely close to a scale - free topological distribution. In the embodiment of the present invention, the genes are divided into 6 modules, and the grey module has the highest correlation with the re - watering treatment of SS (R = 0.86, p < 0.0001).

[0070] Module hub gene mining: By screening using the cytohubba plugin in the cytoscape software, the top five genes with the highest correlation are used as the hub genes of the module. As Figure 15As shown in Table 4, five key hub genes were screened out from the MEgrey module, namely: GH_A01G1528, GH_A08G2688, GH_D08G2683, GH_D01G1620, and GH_A10G0617.

[0071] In summary, salt causes osmotic damage by restricting the water absorption of plants and leads to ionic stress due to the accumulation of excessive ion content in plant cells. Like other plants, cotton has evolved a series of responses to salinity and other environmental stresses. These strategies include morphological, physiological, biochemical, and molecular processes to cope with environmental stresses. These processes can avoid, tolerate, escape, or recover after stress, enabling plants to survive under abiotic stresses. The difference between salt-tolerant materials and salt-sensitive materials may lie in that salt-tolerant materials have a faster adaptation, physiological adjustment, and tolerance compared to salt-sensitive materials when encountering external high-salt stress. Under 48 h of NaCl treatment, the leaves of cotton seedlings were severely damaged, and the malondialdehyde concentration in the leaves of ST and SS seedlings increased significantly, while the chlorophyll content and superoxide dismutase activity decreased significantly. After rehydration treatment, ST and SS were somewhat restored, with the malondialdehyde concentration decreasing, and the chlorophyll content, superoxide dismutase activity, and peroxidase activity increasing. The salt-tolerant materials showed greater salt tolerance and faster recovery after rehydration than the sensitive materials.

[0072] Salt not only changes the metabolic mechanism of plants but also affects the normal growth of plants, especially their morphology and anatomy. Leaves are the center of photosynthesis in higher plants, and when suffering from adversity stress, the phenotypes, physiology, anatomical structures, and other traits of plant leaves can respond in a timely manner. The salt secretion by glandular hairs on the leaves of upland cotton is one of the specific manifestations of cotton's salt tolerance. The microscopic structural characteristics of leaves are an important aspect of plant adaptation to saline-alkali stress. Under NaCl stress, the component cells of cotton leaves lose water and shrink, and the thickness of the leaf cross-section becomes thinner, and the cell morphology of the palisade tissue and others is affected. In the examples of the present invention, under normal conditions, the microscopic structure of cotton leaves is arranged more closely, and the bulliform cells are in the shape of a single layer of irregular quadrilaterals and are closely arranged; under salt stress, the bulliform cells are oval-shaped, and the cell arrangement of the palisade tissue and spongy tissue is looser, and the leaves become softer. After rehydration, the bulliform cells are in the shape of long ovals, the leaves are flatter, and gradually return to normal growth. Under normal conditions, there is no significant difference in the leaf epidermal structure between ST and SS cotton seedlings; under NaCl stress conditions, the leaf thickness, palisade tissue thickness, and spongy tissue thickness of ST and SS all decrease and recover somewhat after rehydration.

[0073] Plant rehydration is one of the important means to restore the growth and physiological levels of plants under stress. In the embodiments of the present invention, the transcriptome changes of ST and SS after salt stress and rehydration were compared. Under salt stress, a large number of differentially expressed genes (15,780 and 14,156) were generated in both ST and SS. ST mainly responded to salt stress through pathways such as circadian rhythm-plant (ko 04712), carbon fixation in photosynthetic organisms (ko 00710), and carbon metabolism (ko 01200). While SS resisted salt stress through pathways such as fatty acid degradation (Ko 00071), degradation of valine, leucine and isoleucine (ko 00280), brassinosteroid biosynthesis (ko 00905), and sphingolipid metabolism (ko 00600). There are a large number of salt stress-induced transcription factor binding sites in the promoters of many circadian rhythm genes (Habte et al. 2014), and the circadian rhythm gene GIGANTEA (GI) affects plant salt tolerance by controlling the SOS (salt overlysensitive) pathway, and the GI mutant enhances salt stress tolerance.

[0074] After rehydration, SS still produced a large number of DEGs (9,390), while the number of DEGs produced by ST decreased significantly (2,454), which further indicates that ST has better salt stress adaptability than SS. ST regulated its adaptability under rehydration treatment through pathways such as glycerolipid metabolism (ko 00561), interconversion of pentose and glucuronate (ko 00040), circadian rhythm-plant (ko 04712), and phenylpropanoid biosynthesis (ko 00940). While SS alleviated the damage caused by salt stress through pathways such as plant-pathogen interaction (ko 04626), MAPK signaling pathway-plant (ko 04016), and α-linolenic acid metabolism (ko 00592).

[0075] Weighted gene co-expression network analysis (WGCNA) is a bioinformatics analysis method based on large-sample transcriptome data. It first assumes that the gene network follows a scale-free distribution, clusters genes with similar expression patterns to form different modules, analyzes the association between modules and specific traits or phenotypes, and constructs a co-expression regulatory network through gene clustering and association analysis results. Genes located at the center of the regulatory network are called hub genes, and such genes are usually key regulatory genes worthy of in-depth exploration and analysis.

[0076] In the embodiment of the present invention, one co-expression module grey associated with the recovery after cotton salt stress was enriched by WGCNA. By screening the grey module, several key genes were determined: GH_D01G1620 and GH_A01G1528 (encoding LTP1 and LTP4 lipid transfer proteins respectively. LTP1 and LTP4 lipid transfer proteins are important active proteins in plant life activities, accounting for about 4% of the cell soluble proteins. LTPs can respond to environmental changes such as low temperature, drought, salt stress, infection by bacterial and fungal pathogens, and some signal molecules such as abscisic acid (ABA), salicylic acid (SA), ethylene, etc. LTP4 may be used as a direct target gene of TDR1 to mediate the regulation of plant salt tolerance by TDR1.

[0077] NtLTP4 participates in the abiotic stress response of plants as a positive regulator. Under stress conditions such as high salt and drought, compared with the wild-type control group, the overexpression lines of NtLTP4 showed extremely strong tolerance, with faster seed germination, longer seedling root length, and slower leaf water loss, etc. GH_A08G2688 (CER3) is one of the genes that play an important role in the biosynthesis of long-chain alkanes. GH_A10G0617 (PEPC1) is an important enzyme in plants. It regulates the carbon flow through the TCA cycle and controls the biosynthesis of proteins and oils. Cotton PEPC1 (1A and 1D) is induced by cold stress and salt stress.

[0078] The above embodiments only express several implementation manners of the present invention, and the description thereof is relatively specific and detailed, but it should not be construed as a limitation on the scope of the patent of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention should be subject to the appended claims.

Claims

1. A salt-alkali tolerance hub gene, characterized in that: It includes at least one of the following genes: GH_A01G1528, GH_A08G2688, GH_D08G2683, GH_D01G1620, and GH_A10G0617; the nucleotide sequences of the GH_A01G1528, GH_A08G2688, GH_D08G2683, GH_D01G1620, and GH_A10G0617 are shown in the sequence table SEQ ID NO.1-5, respectively.

2. A use of the salt-alkali tolerance hub gene as claimed in claim 1 to improve cotton's resistance to salt-alkali stress.

3. A use of the salt-alkali tolerance hub gene as claimed in claim 1 to improve the adaptability of cotton after rehydration under salt-alkali stress.

4. The use according to claim 3, characterized in that After the cotton containing the salt-alkali tolerance hub gene is rehydrated under salt-alkali stress, the malondialdehyde concentration is reduced, and the chlorophyll content, superoxide dismutase activity and peroxidase activity are all increased.

5. The use according to claim 3, characterized in that: Compared with cotton that does not contain the salt-alkali tolerance hub gene, cotton that contains the salt-alkali tolerance hub gene has fewer differentially expressed genes after rehydration under salt-alkali stress.

6. Use of the salt-alkali tolerance hub gene as claimed in claim 1 in cotton breeding.

7. A method for obtaining the salt-alkali tolerance hub gene as claimed in claim 1, characterized in that: The following steps are involved: Different types of cotton varieties were selected, and samples were collected under salinity stress and after rehydration treatment; RNA was extracted from the samples and transcriptome sequencing analysis was performed to obtain expression data; The differentially expressed genes were screened from the expression data to obtain the differentially expressed genes of different types of cotton varieties under salinity-alkali stress and after rehydration treatment; According to the differentially expressed genes of different types of cotton varieties under salinity-alkali stress and after rehydration treatment, a weighted gene co-expression network was constructed to obtain the cotton salinity-alkali tolerance co-expression network; The cotton salt-alkali tolerance co-expression network was divided into multiple co-expression modules; The salt-alkali tolerance phenotype of the sample was used as the trait input, and multiple co-expression modules were specifically identified to determine the modules related to the ability to recover from rehydration under salt-alkali stress, and the grey module was obtained; The connectivity of genes in the grey module is calculated, and highly connected genes are screened to obtain the salt-alkali tolerance hub gene.

8. A method for obtaining a salt-alkali tolerance hub gene according to claim 7, characterized in that: The cotton varieties include salt-tolerant cotton and salt-sensitive cotton.

9. A method for obtaining a salt-alkali tolerance hub gene according to claim 7, characterized in that: According to the differentially expressed genes of different types of cotton varieties under saline-alkali stress and after rehydration treatment, a weighted gene co-expression network is constructed to obtain the steps of the cotton saline-alkali tolerance co-expression network, which specifically includes: According to the differentially expressed genes of different types of cotton varieties under saline-alkali stress and after rehydration treatment, the soft threshold was determined through scale-free topological analysis, and a weighted gene co-expression network was constructed to obtain the cotton salt-alkali tolerance co-expression network.

10. A method for obtaining a salt-alkali tolerance hub gene according to claim 7, characterized in that: The method for dividing the cotton salt-alkali tolerance co-expression network into multiple co-expression modules is the dynamic tree cutting algorithm.

Images