Gene marker set for diagnosis of sulfur deficiency nutrition of tobacco and application thereof

Abstract

The application discloses a gene marker set for diagnosing sulfur deficiency of tobacco and application thereof, and belongs to the technical field of biotechnology.The gene marker set is composed of Nitab4.5_0001735g0030, Nitab4.5_0005001g0030, Nitab4.5_0008753g0030 and Nitab4.5_0013359g0010, and the gene marker set is specifically up-regulated in the process of tobacco responding to sulfur deficiency stress.Through the gene marker set and a reagent for detecting the gene marker set, the sulfur deficiency stress of tobacco can be diagnosed.Therefore, the application lays a foundation for establishing a rapid diagnosis method of sulfur deficiency of tobacco based on gene markers.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a set of gene markers for the diagnosis of sulfur deficiency nutrition in tobacco and their applications. Background Technology

[0002] tobacco( Nicotiana tabacum Tobacco (L.) is a crop with high economic value and is widely cultivated worldwide. Especially in my country, tobacco can generate high economic income for farmers. Tobacco is an annual or short-lived perennial plant belonging to the genus Nicotiana in the Solanaceae family. During its growth and development, it needs to ingest a variety of nutrients, such as macroelements like nitrogen, phosphorus, and potassium, macroelements like magnesium, calcium, and sulfur, and microelements like copper, zinc, iron, boron, and manganese.

[0003] Sulfur is an essential macronutrient element for tobacco growth. It participates in the synthesis of various organic compounds, such as thiamine, vitamin H, and sulfur-containing amino acids. Sulfur is also a crucial medium for the synthesis of chlorophyll, glutathione, and coenzymes, thus playing a key role in plant growth and development. Studies have shown that sulfur deficiency affects tobacco plant growth and development, causing yellowing leaves, rough leaves, poor plant development, decreased leaf quality, poor combustibility, and reduced aroma.

[0004] With the rapid development of genomics technology, utilizing genomics strategies to discover and screen molecular biomarkers of tobacco's response to sulfur deficiency stress and to establish biomarker-based nutritional diagnostic methods is of great significance for building a green tobacco planting system.

[0005] Based on this, the present invention develops a set of gene biomarkers for the diagnosis of sulfur deficiency nutrition in tobacco and their applications. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide a set of gene markers for the diagnosis of sulfur deficiency nutrition in tobacco and their applications.

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

[0008] This invention first provides a set of gene biomarkers for the diagnosis of sulfur deficiency nutrition in tobacco, the set of gene biomarkers consisting of Nitab4.5_0001735g0030, Nitab4.5_0005001g0030, Nitab4.5_0008753g0030, and Nitab4.5_0013359g0010; wherein, the nucleotide sequence of Nitab4.5_0001735g0030 is shown in SEQ ID NO:1, the nucleotide sequence of Nitab4.5_0005001g0030 is shown in SEQ ID NO:2, the nucleotide sequence of Nitab4.5_0008753g0030 is shown in SEQ ID NO:3, and the nucleotide sequence of Nitab4.5_0013359g0010 is shown in SEQ ID NO:4.

[0009] This invention also provides the application of the above-mentioned set of gene markers in the diagnosis of sulfur deficiency nutrition in tobacco.

[0010] The present invention also provides a diagnostic kit for sulfur deficiency nutrition in tobacco, the kit comprising reagents for detecting the above-mentioned set of gene markers by means of RT-qPCR technology.

[0011] Furthermore, the above kit includes specific primers for Nitab4.5_0001735g0030, Nitab4.5_0005001g0030, Nitab4.5_0008753g0030, and Nitab4.5_0013359g0010.

[0012] Furthermore, the nucleotide sequences of the specific primers for Nitab4.5_0001735g0030, Nitab4.5_0005001g0030, Nitab4.5_0008753g0030, and Nitab4.5_0013359g0010 are as follows:

[0013] Specific primers for Nitab4.5_0001735g0030:

[0014] RT-01735-F: 5ʹ-AAATGGGAGAGAAATGGT-3ʹ,

[0015] RT-01735-R: 5ʹ-GAGCAATGAAGTGTAGTAG-3ʹ;

[0016] Specific primers for Nitab4.5_0005001g0030:

[0017] RT-05001-F: 5ʹ-GCTCCTTCACCATTCTACA-3ʹ,

[0018] RT-05001-R: 5ʹ-GCAGTATCCGACCTTCAA-3ʹ;

[0019] Specific primers for Nitab4.5_0008753g0030:

[0020] RT-08753-F: 5ʹ-GATGGAGAATGGATTAGAG-3ʹ,

[0021] RT-08753-R: 5ʹ-GTTGAGGAAGAATGGAAT-3ʹ;

[0022] Specific primers for Nitab4.5_0013359g0010:

[0023] RT-13359-F: 5ʹ-AAATGGGAGAGAAATGGT-3ʹ,

[0024] RT-13359-R: 5ʹ-GAGCAATGAAGTGTAGTAG-3ʹ.

[0025] This invention also provides the application of the above-mentioned tobacco sulfur deficiency nutrition diagnostic kit in the diagnosis of tobacco sulfur deficiency nutrition.

[0026] The present invention also provides a method for diagnosing sulfur deficiency in tobacco, the method comprising the following steps:

[0027] 1) Collect tobacco leaves for testing, extract total RNA, and reverse transcribe it into cDNA;

[0028] 2) Using the cDNA obtained by reverse transcription as a template, RT-qPCR was performed using specific primers Nitab4.5_0001735g0030, Nitab4.5_0005001g0030, Nitab4.5_0008753g0030, and Nitab4.5_0013359g0010. The presence or absence of upregulated gene expression was used to determine whether the tobacco samples were subjected to sulfur deficiency stress.

[0029] Furthermore, if the expression of Nitab4.5_0001735g0030, Nitab4.5_0005001g0030, Nitab4.5_0008753g0030, and Nitab4.5_0013359g0010 in the tested tobacco is significantly upregulated compared to normal tobacco, then the tested tobacco is under sulfur deficiency stress.

[0030] The significant advantages of this invention are:

[0031] This invention investigates the mechanism of tobacco response to sulfur stress using a transcriptome strategy. A gene biomarker set consisting of Nitab4.5_0001735g0030, Nitab4.5_0005001g0030, Nitab4.5_0008753g0030, and Nitab4.5_0013359g0010 was developed. This gene biomarker set is significantly upregulated in sulfur-deficient tobacco, thus it can be used to diagnose adverse growth reactions in tobacco caused by sulfur deficiency. Furthermore, the PCR method is simple to operate and the results are easy to interpret. Attached Figure Description

[0032] Figure 1 Phenotypes of tobacco plants cultured in nutrient solutions with different sulfur concentrations are shown. The white scale bar is 10 cm.

[0033] Figure 2 The expression level of Nitab4.5_0001735g0030 in tobacco plants cultured in nutrient solutions with different sulfur concentrations.

[0034] Figure 3 The expression levels of Nitab4.5_0005001g0030 in tobacco plants cultured in nutrient solutions with different sulfur concentrations.

[0035] Figure 4 The expression level of Nitab4.5_0008753g0030 in tobacco plants cultured in nutrient solutions with different sulfur concentrations.

[0036] Figure 5 The expression level of Nitab4.5_0013359g0010 in tobacco plants cultured in nutrient solutions with different sulfur concentrations. Detailed Implementation

[0037] To make the content of this invention easier to understand, the technical solution of this invention will be further described below with reference to specific embodiments, but this invention is not limited thereto.

[0038] The formulations of the sulfur-containing nutrient solution, sulfur-deficient nutrient solution, and sulfur-excess nutrient solution used in the following examples are shown in Table 1.

[0039] Table 1. Nutrient solution formula (unit: mg / L)

[0040]

[0041] Example 1

[0042] 1. Plants and Experimental Design

[0043] Cuibi No. 1 was selected as the test variety. First, tobacco seedlings with uniform growth at the 3-leaf stage were obtained through soil cultivation. Then, the seedlings were transplanted into culture tanks containing complete nutrient solution with appropriate sulfur content and placed in an artificial climate chamber for uniform cultivation. The artificial climate chamber was set with the following parameters: 12 hours of light cultivation at 25℃; 12 hours of dark cultivation at 20℃.

[0044] On the 5th day of hydroponic cultivation in the artificial climate chamber, a cultivation experiment was conducted with different sulfur supply gradients. The tobacco seedlings were randomly divided into three groups and transferred to three different nutrient solutions: sulfur-deficient (0 mM), sulfur-appropriate (1.89 mM), and sulfur-excessive (7.56 mM), respectively, for further cultivation.

[0045] 2. Sample Collection

[0046] After tobacco plants were treated in three nutrient solutions for 5, 15, and 25 days, the first fully unfolded new leaf of the tobacco plant was taken and stored in liquid nitrogen at low temperature, with 3 biological replicates set up.

[0047] like Figure 1 As shown, after 5 days of treatment with nutrient solutions of different sulfur concentrations, the tobacco plants in each treatment group showed good growth with no significant differences. On day 15, the leaves of plants cultured in sulfur-deficient (0 mM) nutrient solution turned slightly yellow, the leaves of plants cultured in sulfur-excessive (7.56 mM) nutrient solution curled slightly, and the plants cultured in nutrient solution with appropriate sulfur (1.89 mM) nutrient solution showed no significant abnormalities. On day 25, the leaves of plants cultured in sulfur-deficient (0 mM) nutrient solution turned significantly yellow and the plants were stunted, the leaves of plants cultured in sulfur-excessive (7.56 mM) nutrient solution curled significantly, while the plants cultured in nutrient solution with appropriate sulfur (1.89 mM) nutrient solution showed no significant abnormalities.

[0048] 3. Transcriptome sequencing and analysis

[0049] Transcriptome sequencing was performed by Novogene Biotechnology Co., Ltd. After obtaining the experimental samples, total RNA was extracted from tobacco leaves, and the extracted RNA underwent quality testing, with substandard samples being discarded. The RNA from the quality-tested samples was used to construct eukaryotic RNA-seq libraries, and sequencing was performed using the Illumina HiSeq platform.

[0050] After sequencing, FASTP software was used to perform quality control on the raw RNA-seq data, filtering and removing low-quality adapter sequences and other sequences to obtain high-quality sequences. The tobacco genome in the Solanaceae Genome Database (https: / / solgenomics.net / organism / Nicotiana_tabacum / genome) was used as the reference genome. This reference genome is 4.5 GB in size and contains annotations for 69,500 protein-coding genes.

[0051] The purified RNA-seq sequences were aligned with a reference genome using HISAT2 software. The edgeR software was also used. http: / / bioconductor.org / packages / release / bioc / html / edgeR.html Differential expression analysis was performed, and the FDR-adjusted p-value (i.e., q-value) was calculated. Genes with significant differential expression were screened based on the calculation results. The thresholds used were a q-value less than 0.05 and a fold change logarithm (base 2) greater than or less than 1, to screen genes with significantly upregulated or downregulated expression. Based on the differential expression analysis results, CluterProfiler software was used (…). http: / / bioconductor.org / GO and KEGG enrichment analysis were performed.

[0052] As shown in Table 2, transcriptome data analysis revealed that after 5 days of treatment with different sulfur concentrations of nutrient solution, 74 genes showed significant differential expression between the sulfur-deficient (0 mM) group and the sulfur-appropriate (1.89 mM) group (55 genes were upregulated and 19 genes were downregulated), and 87 genes showed significant differential expression between the sulfur-deficient (0 mM) group and the sulfur-excess (7.56 mM) group (29 genes were upregulated and 58 genes were downregulated). After 15 days of treatment with different sulfur concentrations of nutrient solution, 403 genes showed significant differential expression between the sulfur-deficient (0 mM) group and the sulfur-appropriate (1.89 mM) group, and 328 genes showed significant differential expression between the sulfur-deficient (0 mM) group and the sulfur-excess (7.56 mM) group. After 25 days of treatment with different sulfur concentrations of nutrient solution, significant differential expression was found between the sulfur-deficient (0 mM) group and the sulfur-appropriate (1.89 mM) group. A total of 831 genes were significantly expressed differently between the sulfur-deficient (0 mM) group and the sulfur-excess (7.56 mM) group, and 1072 genes were significantly expressed differently between the sulfur-deficient (0 mM) group and the sulfur-excess (7.56 mM) group.

[0053] Table 2. Number of differentially expressed genes under sulfur deficiency stress in tobacco detected by RNA-Seq

[0054]

[0055] Note: S0 is a tobacco sample cultured in a culture medium with a sulfur concentration of 0 mM; S1.89 is a tobacco sample cultured in a culture medium with a sulfur concentration of 1.89 mM; S7.56 is a tobacco sample cultured in a culture medium with a sulfur concentration of 7.56 mM.

[0056] Further comparison and analysis of transcriptome data from the sulfur-deficient (0 mM), sulfur-appropriate (1.89 mM), and sulfur-excess (7.56 mM) groups revealed four differentially expressed genes that were significantly upregulated in the sulfur-deficient (0 mM) group but extremely low in the sulfur-appropriate (1.89 mM) and sulfur-excess (7.56 mM) groups (Table 3). These four genes are Nitab4.5_0001735g0030, Nitab4.5_0005001g0030, Nitab4.5_0008753g0030, and Nitab4.5_0013359g0010. The nucleotide sequences of Nitab4.5_0001735g0030 are shown in SEQ ID NO:1, Nitab4.5_0005001g0030 are shown in SEQ ID NO:2, Nitab4.5_0008753g0030 are shown in SEQ ID NO:3, and Nitab4.5_0013359g0010 are shown in SEQ ID NO:4.

[0057] Table 3. RNA-Seq identification of four significantly differentially expressed genes under tobacco sulfur deficiency stress.

[0058]

[0059] Note: S0 is a tobacco sample cultured in a culture medium with a sulfur concentration of 0 mM; S1.89 is a tobacco sample cultured in a culture medium with a sulfur concentration of 1.89 mM; S7.56 is a tobacco sample cultured in a culture medium with a sulfur concentration of 7.56 mM.

[0060] 4. qRT-PCR to verify the expression levels of candidate differentially expressed genes

[0061] Transcriptome data analysis initially identified differentially expressed genes Nitab4.5_0001735g0030, Nitab4.5_0005001g0030, Nitab4.5_0008753g0030, and Nitab4.5_0013359g0010 in tobacco response to sulfur deficiency stress. The reliability of the transcriptome data was further validated using quantitative real-time PCR. RNA reverse transcription was performed using a reverse transcription kit to obtain cDNA. Subsequently, qRT-PCR detection was performed using specific primers and a qRT-PCR detection kit. Both the reverse transcription kit and the qRT-qPCR detection kit were purchased from Novizan Biosciences Co., Ltd.

[0062] The nucleotide sequences of the specific primers for Nitab4.5_0001735g0030, Nitab4.5_0005001g0030, Nitab4.5_0008753g0030, and Nitab4.5_0013359g0010 are as follows:

[0063] Specific primers for Nitab4.5_0001735g0030:

[0064] RT-01735-F: 5ʹ-AAATGGGAGAGAAATGGT-3ʹ,

[0065] RT-01735-R: 5ʹ-GAGCAATGAAGTGTAGTAG-3ʹ;

[0066] Specific primers for Nitab4.5_0005001g0030:

[0067] RT-05001-F: 5ʹ-GCTCCTTCACCATTCTACA-3ʹ,

[0068] RT-05001-R: 5ʹ-GCAGTATCCGACCTTCAA-3ʹ;

[0069] Specific primers for Nitab4.5_0008753g0030:

[0070] RT-08753-F: 5ʹ-GATGGAGAATGGATTAGAG-3ʹ,

[0071] RT-08753-R: 5ʹ-GTTGAGGAAGAATGGAAT-3ʹ;

[0072] Specific primers for Nitab4.5_0013359g0010:

[0073] RT-13359-F: 5ʹ-AAATGGGAGAGAAATGGT-3ʹ,

[0074] RT-13359-R: 5ʹ-GAGCAATGAAGTGTAGTAG-3ʹ.

[0075] The qRT-PCR experimental reaction system was as follows: 2 × SYBRMix 5 μL, Primer-F 0.2 μL, Primer-R 0.2 μL, cDNA 1 μL, and RNase-free ddH2O 3.6 μL.

[0076] The qRT-PCR experimental reaction program consists of three stages: Stage 1: 95℃, 30 s; Stage 2: (95℃, 10 s, 60℃, 30 s, 95℃, 15 s) 40 cycles; Stage 3: 60℃, 60 s, 95℃, 15 s.

[0077] qRT-PCR validation results showed that after 5 days of treatment with different sulfur concentrations of nutrient solution, the Nitab4.5_0001735g0030 gene was almost undetectable in tobacco plants in the sulfur-deficient (0 mM), sulfur-appropriate (1.89 mM), and sulfur-excess (7.56 mM) groups. After 15 or 25 days of treatment, the Nitab4.5_0001735g0030 gene showed extremely high expression levels in the sulfur-deficient (0 mM) group, while expression remained almost undetectable in the sulfur-appropriate (1.89 mM) and sulfur-excess (7.56 mM) groups. Figure 2 ).

[0078] qRT-PCR validation results showed that after 5 days of treatment with different sulfur concentrations of nutrient solution, the Nitab4.5_0005001g0030 gene expression was almost undetectable in tobacco plants in the sulfur-deficient (0 mM), sulfur-appropriate (1.89 mM), and sulfur-excess (7.56 mM) groups. After 15 or 25 days of treatment, the Nitab4.5_0005001g0030 gene expression was significantly high in the sulfur-deficient (0 mM) group, while only very weak expression levels were detected in the sulfur-appropriate (1.89 mM) and sulfur-excess (7.56 mM) groups. Figure 3 ).

[0079] qRT-PCR validation results showed that after 5 days of treatment with different sulfur concentration nutrient solutions, the expression level of the Nitab4.5_0008753g0030 gene was almost undetectable in tobacco plants in the sulfur-deficient (0 mM), sulfur-appropriate (1.89 mM), and sulfur-excess (7.56 mM) groups. After 15 or 25 days of treatment, the expression level of the Nitab4.5_0005001g0030 gene in the sulfur-deficient (0 mM) group was significantly higher than that in the sulfur-appropriate (1.89 mM) or sulfur-excess (7.56 mM) groups. Figure 4 ).

[0080] qRT-PCR validation results showed that after 5 days of treatment with different sulfur concentrations of nutrient solution, the Nitab4.5_0013359g0010 gene was almost undetectable in tobacco plants in the sulfur-deficient (0 mM), sulfur-appropriate (1.89 mM), and sulfur-excess (7.56 mM) groups. After 15 or 25 days of treatment, the Nitab4.5_0013359g0010 gene was also almost undetectable in tobacco plants in the sulfur-appropriate (1.89 mM) and sulfur-excess (7.56 mM) groups, while highly significant high-level expression was detected in the sulfur-deficient (0 mM) group. Figure 5 ).

[0081] The RT-qPCR results of the four genes were consistent with their RNA-seq analysis results, indicating that the expression levels of all four genes were significantly upregulated after tobacco plants were in a sulfur-deficient state for a certain period. Under conditions of adequate or excessive sulfur, the expression levels of the four genes were basically at very low levels. In conclusion, the four genes Nitab4.5_0001735g0030, Nitab4.5_0005001g0030, Nitab4.5_0008753g0030, and Nitab4.5_0013359g0010 can be used as a set of gene biomarkers for diagnosing tobacco responses to sulfur deficiency stress.

[0082] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention. SEQUENCE LISTING <110> Minjiang University, Fujian Provincial Tobacco Monopoly Bureau Tobacco Science Research Institute <120> Gene biomarkers for the diagnosis of sulfur deficiency in tobacco and their applications <130> <160> 12 <170> PatentIn version 3.3 <210> 1 <211> 912 <212> DNA <213> Nitab4.5_0001735g0030 <400> 1 atgaggttta agagggtagc agcggcatttt gatgaggtgg CAAggcgag attttgcgaa 60 agcagtgta gcgagcactc gtcggttgag agcgtgacag atttgtacgg tcttgtaaat 120 tcgtttattg aagaggaaa tggttttaga ggaggagaa atgagaga gattattgat 180 aatgatagag aatttagt agaaatga ttaagcaata attgttgtga tgatttagag 240 attaggatta agttaggaa attgttaggtt tatgagaaa gtgattagt gagagaaat 300 attcattccg tcgtggaaaa tgcatggctt gaatcggtg atcggagtac ggtggagttc 360 aaacggcggt tgatgactcg gttgcgtaat agaggattg atgctgatct gtgcaaatca 420 aaatgggaga gaatggtca acttccatct ggaaattacg agtacattga cattacatg 480 aacgaaaatc gctatataat cgaagtttat ctgccagag aattcgaaat agctagacca 540 acaccctact acacctcatt gctcgagatt ttcctca tatttgttgg aaagtggaa 600 gattgaagc aagtggtgaa actaatgagt agagctaca agaatcaat gagaaaatg 660 gatattcatg ttccaccttg gagacaactt agttatatgc aagctaagtg gttttggttct 720 tacaaagaa caatcaatga gttcttgtta gatgatgata acagaatct tgatttttct 780 tacaagtgtt tggctaagaa aagagcagta ggttttgtgt ccatcccaac aatttctttc 840 tattgcagag aaaactttgc ttctacaat ggtataaaag ttggaactt ggctgcagct 900 912 <210> 2 <211> 2571 <212> DNA <213> Nitab4.5_0005001g0030 <400> 2 atgacaactt tgaaaatcac CAaaagcat cacaacatt tcaacaatcc tttccctgcc 60 aacccaaaga ctctcccatt gatatatgga actgatctt tgaatttca gaaactgcct 120 tcttcacatc agatctacac cattggaaaa gatttccagt tgagttgtc ttcaaagat 180 ggaggatttc ttcaatctc cacaagtct gagccagcaa ggtccctgtg gtccacaatc 240 cctagagaag ctttcatttc tgcagctatt gctgaaactg aagtcgaaga aagtagaggt 300 tcattcctga tcaaagataa acatgttcat ttcgtatgta atcaccaaac cattgaagag 360 ataaacatca taaaccaatc tgatatcact acgtcctcac acgatcaaga tcaatttttg 420 ccaaagaatt cccagtttcc tgttttgatg atcacaggga aagtttatgg tgtcaataaa 480 acaaagaagg ttagatttcc aagacgaaaa gagttgatgg aatcttcaga gaaagaaact 540 tccacttctg caagatattg gcttctgttt gatcaaaaaa actgtaatca acttggtttc 600 caagtgagaa ttggaaaacc agatttacaa cttccacaga gagtttctcc aagaagttac 660 agaagctttt ctctaaaatt tggtcgaatt cggagacgta gagctgggtg gtttggattt 720 ctgtcaagaa aaaaaactgt cgctgaggag aacgtggtga tgaaaagtgc cggagttagt 780 gatttcaata ggatttgttt aacatatgca agtgaaagaa atgagagatt ctttggtttc 840 ggcgagcaat tttctcattt agactttaaa ggaaagaggg tccccatttt tgttcaagaa 900 caagggattg gaagaggcga tcaacctatt acctttgcag ctaatttagt tagctacagg 960 gcaggggggg actggagcac aacttatgct ccttcaccat tctacatgac atcaaagatg 1020 aggtcacttt acttggaggg ttatgactat tcagtgtttg atctaacaag agatgataga 1080 attcaaatac agttacatgg gaactcattt gaaggtcgga tactgcacgg caactcacct 1140 tgtgagctca ttgaacatct cacagaaagc attggaaggc ccccacctct tccagaatgg 1200 attatttctg gtgcagtggt gggaatgcaa ggtggcacgg acaccgtccg cagtatttgg 1260 aatgaaatgc aaagatatga tgtcccagta tcagcattct ggttgcagga ttgggtaggg 1320 cagagagaga cagtcattgg gtcacaactt tggtggaatt gggaagcaga tgaaactaga 1380 tactcaggat ggcaacaact aattcgagac cttaatatga aacatatcaa agtgatgaca 1440 tattgcaatc cttgtctggc tccgatggat aaaaagccaa atataaggag acaccatttt 1500 gaggaggcaa agaagttgga tatcttagtg aaagacaaga atggggaacc atatatggtg 1560 cccaatacag catttgatgt agggatgctg gatttgacac atccacatac cgcgaattgg 1620 tttaagcaga ttctgcaaga aatggtagat gatggagtga gaggatggat ggcagatttc 1680 ggcgaaggcc tgccagtgga tgcctgcttg tattcaggcg aagatccaat tgcagcacat 1740 aatagatatc ctgaattatg ggcaaaactc aacagggaat ttgtggatga atggcgaagc 1800 acacatgtag gccaagaaag agaagatcca gaggagactt tggttttctt catgagggct 1860 ggttataggg atactcctaa gtgggcaatg ctattttggg agggagacca aatggtgagt 1920 tggcaaaaaa atgatggtat caagagtgca gtggttggct tgcttagcgg aggactttca 1980 ggatatgctc ttaatcacag tgatattgga ggctattgtg cagtaaactt accatttttc 2040 2100 gtgttccgga cacatgaagg aaacaagcca tcttgcaaca gccagttcta ctccaataat 2160 agaacactgt cacatttcgc acgtcttgca aagatctaca aagcatggaa gttttacagg 2220 attcaactag ttaaggaagc ctgtcagaaa gggctaccaa tttgtcgaca tctcttcctt 2280 cactatccag aagatgaaca tgtacatagc ttaacacatg agcagttcct agttggcaca 2340 gagatacttg tggtacccgt gctagaacaaa ggcaaagaat atgttaaggt ctattttccg 2400 ataggaaa gctcttcatg gaagcatatt tggagagaa aactgtattc aacacaaggt 2460 tccgaagctt gggtggaagc atcaatagga tatcctgcca ttttgttaa agttggttct 2520 cctgttggag aaaccttcct gggaaaactt aaaaataca atgtcttata a 2571 <210> 3 <211> 972 <212> DNA <213> Nitab4.5_0008753g0030 <400> 3 atgggagaag ttgatccagc ttcattcaa gatgtcgaac acaggcctaa gctcaccata 60 actgaagctg aaggaattcc agttatagat ctatcaatac tgaattatcc agatttctcc 120 tctgagaaat actccaagga attggaact ctagtggctg agataggcga tgcatcaag 180 aaatgggat tcttcact gataatcat ggggtaccct taaagcacag ggaaatatt 240 gagttggcat caaaaatt ctttgctttg tctaaggagg atagagaaa agtggggaca 300 gatgagttta accctttagg atattagat actgagcaca ccagaatgt aagggactgg 360 aaagaaaatc caacgttat accattttct cctgatcctg atgacaaca actgaagcag 420 ttgagcagcc aatggcctga ctaccaaca gagttcagct taggcttgcc tgcaaatagg 480 ttgaatggtt tctttaatga agaccaaact agttttgtta ggctgaatga ctacccacct 540 tgtcctatcc ctcatctggc tttaggatt ggtagacata aggatgctgg ggctcaca 600 atacttgctc aagataatgt tggactt gagtaaga gtaagcaga tggagaatgg 660 attagagtca aacccactcc tcatgcttat atataaata tggagacct tatccaggtt 720 tggagcaatg aagaatatga aagtgtgaa tattggggtga tggtgactc tgaaagagaa 780 agattttcaa ttccattctt cctcaaccca gcacactata catgggtcga gcccttggag 840 gagctggtga ctgagttaaa tcctgcaaaa tataaggcat atactgggg gaaatttttc 900 aaggcgga aggacagcaa ttcagaag catgatgttg aaaacattca atttatcat 960 call 972 <210> 4 <211> 909 <212> DNA <213> Nitab4.5_0013359g0010 <400> 4 atgaggttta agagggtagc cgcggcatttt gatgacgtgg aaggcgag attttgcgaa 60 agcagtgta gcgagcattc gtcggttgag agcgtgacgg atttgtacgg tcttgtgaat 120 tcgtttattg aagaggaaaaaaagaaaaaagaaaaaagaaaaaagaaaaaaaaaaaaaaaaaaggeah haved 180 gatgataatg agaagaga aaatgatta agcaatatt atttgatga tttagagatt 240 Agggaagt attaggttat gagaagtg attagtgaa gagaatatt 300 cattccgtcg tggaaaatgc atggcttgaa attggcgatt ggagtacggc ggagttcaa 360 cggcggttga tcactcggtt gcgtaataaa ggatttgatg ctggtctgtg caaatcaaa 420 tgggagagaa atggtcact tccatcagga attacgagt atttgacat taacatgaac 480 gaaaatcgct atataatcga agtttatctt gctagagaat tcgaatagc tcgaccaca 540 ccctactaca cttcattgct cgagattttc ccttcaat tgttggaaa agtggaagaa 600 ttgaagcaag tggtgaact atgactga gctatgaaa atcaatgaa gaaatggat 660 atttatgttc caccttggag gcaacttagt tatatgcaag ctaagtggtt tggttcttac 720 aaaagaaaaa tcaatgagta ttgttagat gatgataga agaatcttga ttttcttat 780 aagtgttttgg ctaagaaag agcagttggt ttgtgtcca tgccaacaat ttcttttac 840 tgcagagaaa actttgcttc tataatggt ataaaagttg gaaacttggc tgcagcttta 900 paragraph 909 <210> 5 <211> 18 <212> DNA <213> RT‐01735‐F <400> 5 aaatggggaga gaaatggt 18 <210> 6 <211> 19 <212> DNA <213> RT‐01735‐R <400> 6 gagcaatgaa gtgtagtag 19 <210> 7 <211> 19 <212> DNA <213> RT‐05001‐F <400> 7 gctccttcac cattctaca 19 <210> 8 <211> 18 <212> DNA <213> RT‐05001‐R <400> 8 gcagtatccg accttcaa 18 <210> 9 <211> 19 <212> DNA <213> RT‐08753‐F <400> 9 gatggagaat ggattagaat 19 <210> 10 <211> 18 <212> DNA <213> RT‐08753‐R <400> 10 gttgaggaag aatggaat 18 <210> 11 <211> 18 <212> DNA <213> RT‐13359‐F <400> 11 aaatggggaga gaaatggt 18 <210> 12 <211> 19 <212> DNA <213> RT‐13359‐R <400> 12 gagcaatgaa gtgtagtag 19

Claims

1. A set of gene markers for the diagnosis of sulfur deficiency nutrition in tobacco, characterized in that: The set of gene markers consists of Nitab4.5_0001735g0030, Nitab4.5_0005001g0030, Nitab4.5_0008753g0030, and Nitab4.5_0013359g0010; wherein, the nucleotide sequence of Nitab4.5_0001735g0030 is shown in SEQ ID NO:1, the nucleotide sequence of Nitab4.5_0005001g0030 is shown in SEQ ID NO:2, the nucleotide sequence of Nitab4.5_0008753g0030 is shown in SEQ ID NO:3, and the nucleotide sequence of Nitab4.5_0013359g0010 is shown in SEQ ID NO:

4.

2. A reagent kit for diagnosing sulfur deficiency nutrition in tobacco, characterized in that: The kit includes reagents for detecting the gene biomarker set of claim 1 using RT-qPCR technology; the reagents include specific primers for Nitab4.5_0001735g0030, Nitab4.5_0005001g0030, Nitab4.5_0008753g0030, and Nitab4.5_0013359g0010; the nucleotide sequences of the specific primers for Nitab4.5_0001735g0030, Nitab4.5_0005001g0030, Nitab4.5_0008753g0030, and Nitab4.5_0013359g0010 are as follows: Specific primers for Nitab4.5_0001735g0030: RT-01735-F: 5'-AAATGGGAGAGAAATGGT-3', RT-01735-R: 5'-GAGCAATGAAGTGTAGTAG-3'; Specific primers for Nitab4.5_0005001g0030: RT-05001-F: 5'-GCTCCTTCACCATTCTACA-3', RT-05001-R: 5'-GCAGTATCCGACCTTCAA-3'; Specific primers for Nitab4.5_0008753g0030: RT-08753-F: 5'-GATGGAGAATGGATTAGAG-3', RT-08753-R: 5'-GTTGAGGAAGAATGGAAT-3'; Specific primers for Nitab4.5_0013359g0010: RT-13359-F: 5'-AAATGGGAGAGAAATGGT-3', RT-13359-R: 5'-GAGCAATGAAGTGTAGTAG-3'.

3. A method for diagnosing sulfur deficiency nutrition in tobacco, characterized in that: The method for determining whether the tobacco sample is subjected to sulfur deficiency stress is based on whether the gene marker set described in claim 1 is upregulated in expression; the method includes the following steps: 1) Collect tobacco leaves for testing, extract total RNA, and reverse transcribe it into cDNA; 2) Using cDNA obtained from reverse transcription as a template, RT-qPCR detection was performed using specific primers; The nucleotide sequences of the specific primers for Nitab4.5_0001735g0030, Nitab4.5_0005001g0030, Nitab4.5_0008753g0030, and Nitab4.5_0013359g0010 are as follows: Specific primers for Nitab4.5_0001735g0030: RT-01735-F: 5'-AAATGGGAGAGAAATGGT-3', RT-01735-R: 5'-GAGCAATGAAGTGTAGTAG-3'; Specific primers for Nitab4.5_0005001g0030: RT-05001-F: 5'-GCTCCTTCACCATTCTACA-3', RT-05001-R: 5'-GCAGTATCCGACCTTCAA-3'; Specific primers for Nitab4.5_0008753g0030: RT-08753-F: 5'-GATGGAGAATGGATTAGAG-3', RT-08753-R: 5'-GTTGAGGAAGAATGGAAT-3'; Specific primers for Nitab4.5_0013359g0010: RT-13359-F: 5'-AAATGGGAGAGAAATGGT-3', RT-13359-R: 5'-GAGCAATGAAGTGTAGTAG-3'; The RT-qPCR reaction system is as follows: 2 × SYBRMix 5ul, upstream primer 0.2ul, downstream primer 0.2ul, cDNA template 1ul, RNase-free ddH2O 3.6ul; The RT-qPCR reaction program is as follows: 95℃ for 30s; 95℃ for 10s, 60℃ for 30s, 95℃ for 15s, 40 cycles; 60℃ for 60s, 95℃ for 15s.

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