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

Abstract

This invention discloses a set of gene biomarkers for the diagnosis of zinc deficiency in tobacco and its applications, belonging to the field of biotechnology. The gene biomarker set consists of Nitab4.5_0000143g0340, Nitab4.5_0000572g0110, Nitab4.5_0001724g0060, Nitab4.5_0003605g0060, and Nitab4.5_0006169g0020. This set of gene biomarkers shows significant upregulation in tobacco responses to zinc deficiency stress and can be used to diagnose this condition. This invention provides a valuable reference for establishing a rapid diagnostic method for zinc deficiency in tobacco based on gene biomarkers.

Description

Technical Field

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

[0002] tobacco( Nicotiana tabacum L. Tobacco (Nicotiana spp.) is an annual or short-lived perennial plant belonging to the Solanaceae family and the Nicotiana genus. It has high economic and medicinal value. Tobacco is widely cultivated worldwide. To ensure the normal growth and development of tobacco, a variety of nutrients are needed. Zinc (Zn) is one such nutrient. 2+ Zinc is one of the essential micronutrients for the normal growth and development of tobacco. It participates in the synthesis of various enzymes and chlorophyll in plants, and is also an important substance for the synthesis of tryptophan, a precursor to auxin. Zinc deficiency affects the activity of many enzymes and also impairs photosynthesis, leading to a decrease in the plant's ability to synthesize polysaccharides. Zinc deficiency can also affect plant development, resulting in symptoms such as stunted growth and slowed development.

[0003] Genomic strategies based on high-throughput sequencing technology can more accurately and efficiently discover and screen molecular biomarkers of tobacco response to zinc deficiency stress, establish biomarker-based nutritional diagnostic methods, and provide new methods and ideas for building a green tobacco planting system. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a set of gene markers for the diagnosis of zinc deficiency in tobacco and their applications.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention first provides a set of gene biomarkers for the diagnosis of zinc deficiency in tobacco. The set of gene biomarkers consists of Nitab4.5_0000143g0340, Nitab4.5_0000572g0110, Nitab4.5_0003605g0060, Nitab4.5_0001724g0060, and Nitab4.5_0006169g0020. The nucleotide sequence of Nitab4.5_0000143g0340 is shown in SEQ ID NO:1, the nucleotide sequence of Nitab4.5_0000572g0110 is shown in SEQ ID NO:2, the nucleotide sequence of Nitab4.5_0003605g0060 is shown in SEQ ID NO:3, and the nucleotide sequence of Nitab4.5_0001724g0060 is shown in SEQ ID NO:4. As shown in NO:4, the nucleotide sequence of Nitab4.5_0006169g0020 is shown in SEQ ID NO:5.

[0007] This invention also provides the application of the above-mentioned set of gene markers in the diagnosis of zinc deficiency in tobacco nutrition.

[0008] The present invention also provides a diagnostic kit for zinc deficiency in tobacco, the kit comprising reagents for detecting the set of gene markers described above using RT-qPCR technology.

[0009] Furthermore, the above kit includes primers specifically for detecting Nitab4.5_0000143g0340, Nitab4.5_0000572g0110, Nitab4.5_0003605g0060, Nitab4.5_0001724g0060, and Nitab4.5_0006169g0020.

[0010] Furthermore, the primer sequences for the specific detection of Nitab4.5_0000143g0340, Nitab4.5_0000572g0110, Nitab4.5_0003605g0060, Nitab4.5_0001724g0060, and Nitab4.5_0006169g0020 are as follows:

[0011] Specific primers for Nitab4.5_0000143g0340:

[0012] RT-0143-F: 5ʹ-CGGCTTGTTACACCTCATTACTC-3ʹ,

[0013] RT-0143-R: 5ʹ-TTCACCACTTGCTTCAACTCTTC-3ʹ;

[0014] Specific primers for Nitab4.5_0000572g0110:

[0015] RT-0572-F: 5ʹ-GATAGACCTCTTGCTTCC-3ʹ,

[0016] RT-0572-R: 5ʹ-GATTAGTTGCTTGTGATGG-3ʹ;

[0017] Specific primers for Nitab4.5_0003605g0060:

[0018] RT-3605-F: 5ʹ-TTCAGCAAGAGTAATTGTTG-3ʹ,

[0019] RT-3605-R: 5ʹ-ATTCGGAAGTCGTCAAG-3ʹ;

[0020] Specific primers for Nitab4.5_0001724g0060:

[0021] RT-1724-F: 5ʹ-GCTTCAGTCATTCCTTAG-3ʹ,

[0022] RT-1724-R: 5ʹ-TGCTGTCAATATCATCCT-3ʹ;

[0023] Specific primers for Nitab4.5_0006169g0020:

[0024] RT-6169-F: 5ʹ-TCTCACTTACAACTCCAG-3ʹ,

[0025] RT-6169-R: 5ʹ- AATACTCCTTCCACAATGA-3ʹ.

[0026] This invention also provides the application of the above-mentioned tobacco zinc deficiency nutritional diagnostic kit in the diagnosis of tobacco zinc deficiency.

[0027] The present invention also provides a method for diagnosing zinc deficiency in tobacco, the method comprising the following steps:

[0028] 1) Collect tobacco leaves for testing, extract total RNA, and reverse transcribe it into cDNA;

[0029] 2) Using the cDNA obtained by reverse transcription as a template, RT-qPCR was performed using the specific primers mentioned above. The presence or absence of upregulated gene expression was used to determine whether the tobacco sample was subjected to zinc deficiency stress.

[0030] Furthermore, if the expression of Nitab4.5_0000143g0340, Nitab4.5_0000572g0110, Nitab4.5_0003605g0060, Nitab4.5_0001724g0060, and Nitab4.5_0006169g0020 in the tested tobacco is significantly upregulated compared to normal tobacco, then the tested tobacco is under zinc deficiency stress.

[0031] The significant advantages of this invention are:

[0032] This invention utilizes a transcriptome strategy to study the mechanism of tobacco response to zinc stress. It screened five genes—Nitab4.5_0000143g0340, Nitab4.5_0000572g0110, Nitab4.5_0003605g0060, Nitab4.5_0001724g0060, and Nitab4.5_0006169g0020—which can serve as a set of gene markers for diagnosing poor tobacco growth caused by zinc deficiency. This invention has practical significance. Attached Figure Description

[0033] Figure 1 The growth and development phenotypes of tobacco plants cultured in nutrient solutions with different zinc concentrations (white scale bar is 10cm).

[0034] Figure 2 The expression level of Nitab4.5_0000143g0340 in tobacco plants cultured in nutrient solutions with different zinc concentrations.

[0035] Figure 3 The expression level of Nitab4.5_0000572g0110 in tobacco plants cultured in nutrient solutions with different zinc concentrations.

[0036] Figure 4 The expression levels of Nitab4.5_0003605g0060 in tobacco plants cultured in nutrient solutions with different zinc concentrations.

[0037] Figure 5 The expression level of Nitab4.5_0001724g0060 in tobacco plants cultured in nutrient solutions with different zinc concentrations.

[0038] Figure 6 Results of Nitab4.5_0006169g0020 expression levels in tobacco plants cultured in nutrient solutions with different zinc concentrations. Detailed Implementation

[0039] The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0040] The formulations of the zinc-rich nutrient solution, zinc-deficient nutrient solution, zinc-insufficient nutrient solution, and zinc-excess nutrient solution used in the following examples are shown in Table 1.

[0041] Table 1. Nutrient solution formula (unit: mg / L)

[0042]

[0043] Example 1

[0044] I. Materials and Methods

[0045] 1. Plant cultivation and treatment

[0046] Using Cuibi No. 1 as the test variety, tobacco samples were cultured in soil. After a period of cultivation, tobacco seedlings that were at the 3-leaf stage, growing well, and of uniform size were selected. Soil residue was carefully removed from the roots of the seedlings, and they were transplanted into culture tanks containing a complete nutrient solution with an appropriate zinc concentration (0.00077 mM) for recovery. They were then transferred to an artificial climate chamber for cultivation. The cultivation conditions were: 20°C dark cultivation for 12 hours; 25°C light cultivation for 12 hours.

[0047] Five days after seedling recovery, a culture experiment with different zinc supply gradients was formally conducted. Poorly recovered tobacco plants were removed, and the well-recovered seedlings were randomly divided into four groups. Each group was then transferred to one of four nutrient solutions: zinc-deficient (0 mM), zinc-insufficient (0.00019 mM), zinc-adequate (0.00077 mM), or zinc-excessive (0.00308 mM). The culture conditions were: 12 hours of dark culture at 20°C followed by 12 hours of light culture at 25°C.

[0048] 2. Sample Collection

[0049] After tobacco plants were treated in three different nutrient solutions for 5, 15, and 25 days, the first fully unfolded new leaf was taken and rapidly transferred to liquid nitrogen for preservation. Three biological replicates were set up for the experiment.

[0050] like Figure 1 As shown, after 5 days of treatment, the tobacco plants in all treatment groups grew normally with no significant phenotypic differences. On day 15, the leaves of plants cultured in zinc-deficient (0 mM) nutrient solution curled slightly and turned yellow, affecting plant growth and slowing its rate. No obvious abnormalities were found in plants cultured in zinc-deficient (0.00019 mM), zinc-appropriate (0.00077 mM), and zinc-excess (0.00308 mM) nutrient solutions. On day 25, the leaves of plants cultured in zinc-deficient (0 mM) nutrient solution curled severely, turned yellow, and partially withered. The plants were stunted and grew slowly. The symptoms of the leaves of plants cultured in zinc-deficient (0.00019 mM) nutrient solution were relatively mild, with yellowing leaves, stunted growth, and no withering. No obvious abnormalities were found in plants cultured in zinc-appropriate (0.00077 mM) and zinc-excess (0.00308 mM) nutrient solutions.

[0051] 3. Transcriptome sequencing and analysis

[0052] After obtaining the experimental samples, total RNA was extracted from tobacco leaves using the Trizol method. The extracted RNA was quality tested, and substandard samples were discarded. A eukaryotic RNA-seq library was then constructed, and transcriptome sequencing was performed using the Illumina HiSeq platform. The sequencing work was completed by Novogene Biotechnology Co., Ltd.

[0053] After transcriptome sequencing was completed, FASTP software was used to perform quality control on the raw RNA-seq data to obtain high-quality purified sequencing data. The next step was to use HISAT2 software to align the purified data with a tobacco reference genome, obtained from the Solanaceae Genome Database (https: / / solgenomics.net / organism / Nicotiana_tabacum / genome). This reference genome is 4.5 GB in size and contains 69,500 protein-coding genes annotated. Differential expression analysis was performed using edgR software (http: / / bioconductor.org / packages / release / bioc / html / edgeR.html), and q-values ​​were calculated. The threshold was set to a q-value less than 0.05 and a fold change logarithm (base 2) greater than 1. Genes that were significantly upregulated or significantly downregulated were selected based on the calculation results.

[0054] As shown in Table 2, after treating tobacco plants with different zinc concentrations of nutrient solution for 5 days, a total of 166 genes were significantly differentially expressed between the zinc-deficient (0 mM) group and the zinc-adequate (0.00077 mM) group (157 genes were upregulated and 9 genes were downregulated), 188 genes were significantly differentially expressed between the zinc-deficient (0.00019 mM) group and the zinc-adequate (0.00077 mM) group (179 genes were upregulated and 9 genes were downregulated), 789 genes were significantly differentially expressed between the zinc-deficient (0 mM) group and the zinc-excess (0.00308 mM) group (117 genes were upregulated and 672 genes were downregulated), and 675 genes were significantly differentially expressed between the zinc-deficient (0.00019 mM) group and the zinc-excess (0.00308 mM) group. Significant differential expression was detected between the zinc deficiency (0 mM) and zinc excess (0.00077 mM) groups (82 genes were upregulated and 593 genes were downregulated). After 15 days of treatment, a total of 802 genes were significantly differentially expressed between the zinc deficiency (0 mM) and zinc excess (0.00077 mM) groups, 120 genes were significantly differentially expressed between the zinc deficiency (0.00019 mM) and zinc excess (0.00077 mM) groups, 1711 genes were significantly differentially expressed between the zinc deficiency (0 mM) and zinc excess (0.00308 mM) groups, and 755 genes were significantly differentially expressed between the zinc deficiency (0.00019 mM) and zinc excess (0.00308 mM) groups. After 25 days of treatment, a total of 1328 genes were significantly differentially expressed between the zinc deficiency (0 mM) and zinc excess (0.00077 mM) groups. Significant differential expression was observed between the zinc deficiency (0 mM) and zinc excess (0 mM) groups. 1121 genes showed significant differential expression between the zinc deficiency (0 mM) and zinc excess (0 mM) groups. 3696 genes showed significant differential expression between the zinc deficiency (0 mM) and zinc excess (0 mM) groups. 1915 genes showed significant differential expression between the zinc deficiency (0 mM) and zinc excess (0 mM) groups.

[0055] Table 2. Number of differentially expressed genes under zinc deficiency stress in tobacco detected by RNA-Seq

[0056]

[0057] Note: Zn0 is a tobacco sample cultured in a culture medium with a zinc concentration of 0 mM; Zn0.00019 is a tobacco sample cultured in a culture medium with a zinc concentration of 0.00019 mM; Zn0.00077 is a tobacco sample cultured in a culture medium with a zinc concentration of 0.00077 mM; Zn0.00308 is a tobacco sample cultured in a culture medium with a zinc concentration of 0.00308 mM.

[0058] Further comparison and analysis of transcriptome data from zinc-deficient (0 mM), zinc-insufficient (0.00019 mM), zinc-adequate (0.00077 mM), and zinc-excess (0.00308 mM) groups revealed five differentially expressed genes that showed extremely low expression levels in the zinc-adequate (0.00077 mM) and zinc-excess (0.00308 mM) groups, but significantly upregulated expression in the zinc-deficient (0 mM) and zinc-insufficient (0.00019 mM) groups (Table 3). These five genes are Nitab4.5_0000143g0340, Nitab4.5_0000572g0110, Nitab4.5_0003605g0060, Nitab4.5_0001724g0060, and Nitab4.5_0006169g0020.

[0059] Table 3. Five differentially expressed genes identified by RNA-Seq under zinc deficiency stress in tobacco.

[0060]

[0061] Note: Zn0 is a tobacco sample cultured in a culture medium with a zinc concentration of 0 mM; Zn0.00019 is a tobacco sample cultured in a culture medium with a zinc concentration of 0.00019 mM; Zn0.00077 is a tobacco sample cultured in a culture medium with a zinc concentration of 0.00077 mM; Zn0.00308 is a tobacco sample cultured in a culture medium with a zinc concentration of 0.00308 mM.

[0062] 4. RT-qPCR to validate the expression levels of candidate differentially expressed genes.

[0063] Gene differential expression analysis identified a number of significantly expressed genes: Nitab4.5_0000143g0340, Nitab4.5_0000572g0110, Nitab4.5_0003605g0060, Nitab4.5_0001724g0060, and Nitab4.5_0006169g0020, which were then validated using qRT-PCR. cDNA was obtained through reverse transcription using a reverse transcription kit. After quality testing of the cDNA, qualified samples were selected for RT-qPCR validation. Both the reverse transcription kit and the RT-qPCR detection kit were provided by Novizan Biosciences Co., Ltd.

[0064] The sequences of primers specifically detecting Nitab4.5_0000143g0340, Nitab4.5_0000572g0110, Nitab4.5_0003605g0060, Nitab4.5_0001724g0060, and Nitab4.5_0006169g0020 are as follows:

[0065] Specific primers for Nitab4.5_0000143g0340:

[0066] RT-0143-F: 5ʹ-CGGCTTGTTACACCTCATTACTC-3ʹ,

[0067] RT-0143-R: 5ʹ-TTCACCACTTGCTTCAACTCTTC-3ʹ;

[0068] Specific primers for Nitab4.5_0000572g0110:

[0069] RT-0572-F: 5ʹ-GATAGACCTCTTGCTTCC-3ʹ,

[0070] RT-0572-R: 5ʹ-GATTAGTTGCTTGTGATGG-3ʹ;

[0071] Specific primers for Nitab4.5_0003605g0060:

[0072] RT-3605-F: 5ʹ-TTCAGCAAGAGTAATTGTTG-3ʹ,

[0073] RT-3605-R: 5ʹ-ATTCGGAAGTCGTCAAG-3ʹ;

[0074] Specific primers for Nitab4.5_0001724g0060:

[0075] RT-1724-F: 5ʹ-GCTTCAGTCATTCCTTAG-3ʹ,

[0076] RT-1724-R: 5ʹ-TGCTGTCAATATCATCCT-3ʹ;

[0077] Specific primers for Nitab4.5_0006169g0020:

[0078] RT-6169-F: 5ʹ-TCTCACTTACAACTCCAG-3ʹ,

[0079] RT-6169-R: 5ʹ- AATACTCCTTCCACAATGA-3ʹ.

[0080] The qRT-PCR reaction system is as follows: 2×SYBRMix 5 μL, Primer-F 0.2 μL, Primer-R 0.2 μL, cDNA 1 μL, RNase-free ddH2O 3.6 μL.

[0081] 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.

[0082] qRT-PCR results using Nitab4.5_0000143g0340 showed that after 5 days of treatment with different zinc concentrations of nutrient solution, the gene was expressed at low levels in tobacco samples with insufficient zinc (0.00019 mM), but almost not expressed in tobacco samples with zinc deficiency (0 mM), adequate zinc (0.00077 mM), and excess zinc (0.00308 mM). After 15 and 25 days of treatment, the gene was significantly expressed in tobacco samples with zinc deficiency (0 mM) and insufficient zinc (0.00019 mM), especially with extremely significant high-level expression in the zinc deficiency (0 mM) group, while it was expressed very weakly in tobacco samples with adequate zinc (0.00077 mM) and excess zinc (0.00308 mM). Figure 2 ).

[0083] qRT-PCR validation results using Nitab4.5_0000572g0110 showed that after 5 days of treatment with different zinc concentrations of nutrient solution, this gene was almost not expressed in tobacco samples from the zinc-deficient (0 mM), zinc-insufficient (0.00019 mM), zinc-adequate (0.00077 mM), and zinc-excessive (0.00308 mM) groups. After 15 days of treatment, this gene was significantly highly expressed in the zinc-deficient (0 mM) group of tobacco samples. Figure 3 ).

[0084] qRT-PCR validation results of Nitab4.5_0003605g0060 showed that after 5 days of treatment with different zinc concentrations of nutrient solution, the gene was expressed at low levels in tobacco samples with insufficient zinc (0.00019 mM), and extremely weakly in tobacco samples with zinc deficiency (0 mM), adequate zinc (0.00077 mM), and excess zinc (0.00308 mM). After 15 days of treatment, the Nitab4.5_0003605g0060 gene was significantly expressed in tobacco samples with insufficient zinc (0.00019 mM), and extremely significantly expressed in tobacco samples with zinc deficiency (0 mM). However, it was weakly expressed in tobacco samples with adequate zinc (0.00077 mM) and excess zinc (0.00308 mM). Figure 4 ).

[0085] qRT-PCR validation results of Nitab4.5_0001724g0060 showed that after 5 days of treatment with different zinc concentrations of nutrient solution, the Nitab4.5_0001724g0060 gene was almost not expressed in tobacco samples from the zinc-deficient (0 mM), zinc-insufficient (0.00019 mM), zinc-adequate (0.00077 mM), and zinc-excessive (0.00308 mM) groups. On day 15, the Nitab4.5_0001724g0060 gene was highly significantly expressed in the zinc-deficient (0 mM) and zinc-insufficient (0.00019 mM) groups, while its expression was extremely weak in the zinc-adequate (0.00077 mM) and zinc-excessive (0.00308 mM) groups. Figure 5 ).

[0086] qRT-PCR validation results of Nitab4.5_0006169g0020 showed that after 5 days of treatment with different zinc concentrations of culture medium, the Nitab4.5_0001724g0060 gene was almost not expressed in tobacco samples from the zinc-deficient (0 mM), zinc-insufficient (0.00019 mM), zinc-adequate (0.00077 mM), and zinc-excessive (0.00308 mM) groups. After 15 days of treatment, the Nitab4.5_0001724g0060 gene was highly significantly expressed in the zinc-deficient (0 mM) and zinc-insufficient (0.00019 mM) groups, while expression was not significant in the zinc-adequate (0.00077 mM) and zinc-excessive (0.00308 mM) groups. Figure 6 ).

[0087] Comparative analysis showed that the qRT-PCR results of the five genes were consistent with their corresponding RNA-seq results, indicating that the expression levels of the five genes were significantly upregulated after tobacco plants were in a zinc deficiency or insufficiency state for a period of time; under adequate or excessive zinc conditions, the expression of the other three genes remained at baseline levels. In conclusion, the five genes Nitab4.5_0000143g0340, Nitab4.5_0000572g0110, Nitab4.5_0003605g0060, Nitab4.5_0001724g0060, and Nitab4.5_0006169g0020 can be used as biomarkers for diagnosing the response of tobacco plants to zinc deficiency stress.

Claims

1. A set of gene markers for the diagnosis of zinc deficiency in tobacco, characterized in that: The set of gene markers consists of Nitab4.5_0000143g0340, Nitab4.5_0000572g0110, Nitab4.5_0003605g0060, Nitab4.5_0001724g0060, and Nitab4.5_0006169g0020. The nucleotide sequence of Nitab4.5_0000143g0340 is shown in SEQ ID NO:1, the nucleotide sequence of Nitab4.5_0000572g0110 is shown in SEQ ID NO:2, the nucleotide sequence of Nitab4.5_0003605g0060 is shown in SEQ ID NO:3, the nucleotide sequence of Nitab4.5_0001724g0060 is shown in SEQ ID NO:4, and the nucleotide sequence of Nitab4.5_0006169g0020 is shown in SEQ ID NO:

4. As shown in NO:5; the diagnosis of zinc deficiency in tobacco is based on whether the expression of gene markers is upregulated to determine whether the tobacco being tested is under zinc deficiency stress.

2. A reagent kit for diagnosing zinc deficiency 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 primers for specifically detecting Nitab4.5_0000143g0340, Nitab4.5_0000572g0110, Nitab4.5_0003605g0060, Nitab4.5_0001724g0060, and Nitab4.5_0006169g0020; the sequences of the primers for specifically detecting Nitab4.5_0000143g0340, Nitab4.5_0000572g0110, Nitab4.5_0003605g0060, Nitab4.5_0001724g0060, and Nitab4.5_0006169g0020 are as follows: Specific primers for Nitab4.5_0000143g0340: RT-0143-F: 5'-CGGCTTGTTACACCTCATTACTC-3', RT-0143-R: 5'-TTCACCACTTGCTTCAACTCTTC-3'; Specific primers for Nitab4.5_0000572g0110: RT-0572-F: 5'-GATAGACCTCTTGCTTCC-3', RT-0572-R: 5'-GATTAGTTGCTTGTGATGG-3'; Specific primers for Nitab4.5_0003605g0060: RT-3605-F: 5'-TTCAGCAAGAGTAATTGTTG-3', RT-3605-R: 5'-ATTCGGAAGTTCGTCAAG-3'; Specific primers for Nitab4.5_0001724g0060: RT-1724-F: 5'-GCTTCAGTCATTCCTTAG-3', RT-1724-R: 5'-TGCTGTCAATATCATCCT-3'; Specific primers for Nitab4.5_0006169g0020: RT-6169-F: 5'-TCTCACTTACAACTCCAG-3', RT-6169-R: 5'-AATACTCCTTCCACAATGA-3'; The diagnosis of zinc deficiency in tobacco is based on whether the expression of a set of gene markers is upregulated to determine whether the tobacco being tested is under zinc deficiency stress.

3. A method for diagnosing zinc deficiency in tobacco, characterized in that: The method for determining whether the tested tobacco is under zinc deficiency stress is achieved by detecting the expression level of the gene biomarker set described in claim 1 in tobacco; the method includes the following steps: 1) Collect tobacco leaves for testing, extract total RNA, and reverse transcribe it into cDNA; 2) Using the cDNA obtained from reverse transcription as a template, RT-qPCR detection was performed using specific primers; the sequences of the primers specifically detecting Nitab4.5_0000143g0340, Nitab4.5_0000572g0110, Nitab4.5_0003605g0060, Nitab4.5_0001724g0060, and Nitab4.5_0006169g0020 are as follows: Specific primers for Nitab4.5_0000143g0340: RT-0143-F: 5'-CGGCTTGTTACACCTCATTACTC-3', RT-0143-R: 5'-TTCACCACTTGCTTCAACTCTTC-3'; Specific primers for Nitab4.5_0000572g0110: RT-0572-F: 5'-GATAGACCTCTTGCTTCC-3', RT-0572-R: 5'-GATTAGTTGCTTGTGATGG-3'; Specific primers for Nitab4.5_0003605g0060: RT-3605-F: 5'-TTCAGCAAGAGTAATTGTTG-3', RT-3605-R: 5'-ATTCGGAAGTTCGTCAAG-3'; Specific primers for Nitab4.5_0001724g0060: RT-1724-F: 5'-GCTTCAGTCATTCCTTAG-3', RT-1724-R: 5'-TGCTGTCAATATCATCCT-3'; Specific primers for Nitab4.5_0006169g0020: RT-6169-F: 5'-TCTCACTTACAACTCCAG-3', RT-6169-R: 5'-AATACTCCTTCCACAATGA-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; If the expression of Nitab4.5_0000143g0340, Nitab4.5_0000572g0110, Nitab4.5_0003605g0060, Nitab4.5_0001724g0060, and Nitab4.5_0006169g0020 in the tested tobacco is significantly upregulated compared to normal tobacco, then the tested tobacco is under zinc deficiency stress.

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