A biomarker group for detecting boron deficiency stress of tobacco and application thereof

Abstract

The application discloses a biomarker group for detecting boron deficiency stress of tobacco and application thereof. The biomarker group is composed of three genes of Nitab4.5_0000595g0010, Nitab4.5_0011519g0020 and Nitab4.5_0016396g0010, and the three genes are specifically up-regulated in the process of tobacco responding to boron stress. Therefore, the three genes can be used as the biomarker group for diagnosing boron deficiency stress of tobacco. The application lays a foundation for establishing a rapid diagnosis method of boron deficiency of tobacco based on biomarkers.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a biomarker group for detecting boron deficiency stress of tobacco and application thereof screened by genomics technology. BACKGROUND

[0002] Tobacco ( Nicotiana tabacum L. ) is a crop with high economic value. In China, planting tobacco can create high economic income. Tobacco is an annual or limited perennial Solanaceae Nicotiana plant, has large biomass and strong nutrient absorption capacity, and needs multiple nutrients during its growth and development.

[0003] Boron is one of the trace elements necessary for the growth and development of tobacco, participates in various physiological and biochemical activities such as protein metabolism, material transport and alkaloid synthesis, and affects the normal development and stress resistance of tobacco. During the growth and development of tobacco, boron participates in the synthesis of uracil and chlorophyll, the transport of carbohydrates, cell division and growth, ribonucleic acid metabolism and the like. Boron can also improve photosynthesis and transpiration of tobacco and promote the accumulation of sugar substances. Boron deficiency will cause the top bud of tobacco to die, the growth to be hindered and stagnated, the plant to be in a tufted state, the transport of sugar substances and proteins to be affected, and the quality of tobacco leaves to be reduced.

[0004] Using genomics strategy, the molecular biomarkers of tobacco responding to boron deficiency stress are mined and screened, and a nutrient diagnosis method based on biomarkers is established, which has practical significance for constructing a green tobacco planting system. SUMMARY

[0005] The present application aims to provide a biomarker group for detecting boron deficiency stress of tobacco and application thereof.

[0006] To achieve the above-mentioned purpose, the present application provides the following solutions.

[0007] The present application first provides a biomarker group for diagnosing boron deficiency stress of tobacco, which consists of Nitab4.5_0000595g0010, Nitab4.5_0011519g0020 and Nitab4.5_0016396g0010; wherein the nucleotide sequence of Nitab4.5_0000595g0010 is shown in SEQ ID NO: 1, the nucleotide sequence of Nitab4.5_0011519g0020 is shown in SEQ ID NO: 2, and the nucleotide sequence of Nitab4.5_0016396g0010 is shown in SEQ ID NO: 3.

[0008] The present application also provides the application of the above-mentioned biomarker group in the diagnosis of boron deficiency nutrition of tobacco.

[0009] Further, the expression level of the above-mentioned biomarker group is significantly up-regulated when tobacco is under boron deficiency stress.

[0010] The present application also provides a kit for diagnosing boron deficiency stress of tobacco, which comprises reagents for detecting the above-mentioned biomarker group by RT-qPCR technology.

[0011] Further, the above-mentioned reagents comprise specific primers of Nitab4.5_0000595g0010, Nitab4.5_0011519g0020 and Nitab4.5_0016396g0010.

[0012] Further, the nucleotide sequences of the specific primers of Nitab4.5_0000595g0010, Nitab4.5_0011519g0020 and Nitab4.5_0016396g0010 are as follows:

[0013] Specific primers of Nitab4.5_0000595g0010:

[0014] RT-0595-F: 5ʹ-AATCAATAACGCCACTTC-3ʹ,

[0015] RT-0595-R: 5ʹ-ATCACATACACTTACAACAC-3ʹ;

[0016] Specific primers of Nitab4.5_0011519g0020:

[0017] RT-1519-F: 5ʹ-TTCGTTCTCAAGCCTATATGG-3ʹ,

[0018] RT-1519-R: 5ʹ-GTCTGTGGAGCAACAACTATA-3ʹ;

[0019] Specific primers of Nitab4.5_0016396g0010:

[0020] RT-6396-F: 5ʹ-TCTTTGCTTCCCTTTGTG-3ʹ,

[0021] RT-6396-R: 5ʹ-CCTTGTCATCATCAGTAGTG-3ʹ.

[0022] The present application also provides the use of the above-mentioned kit in diagnosing boron deficiency stress of tobacco.

[0023] The present application also provides a method for diagnosing boron deficiency stress of tobacco, which comprises the following steps:

[0024] 1) Collecting the tobacco leaves to be tested, extracting total RNA, and reverse transcribing into cDNA;

[0025] 2) Using the cDNA obtained by reverse transcription as a template, and using the specific primers described above to perform RT-qPCR detection; according to whether the gene is up-regulated, it is judged whether the tobacco to be tested is subjected to boron stress.

[0026] The present application has the following advantages:

[0027] The present application provides a biomarker group composed of Nitab4.5_0000595g0010, Nitab4.5_0011519g0020 and Nitab4.5_0016396g0010 by studying the mechanism of tobacco responding to boron stress through transcriptome strategy, and by using the biomarker group involved in the present application, the nutritional deficiency of boron elements in tobacco can be diagnosed, so that the fertilization direction can be adjusted in time, and the present application has very high practical application value. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 The phenotypes of tobacco plants under the conditions of boron deficiency (0 mM), appropriate boron (0.046 mM) and boron excess (0.184 mM) for 12 days and 19 days, and the white scale is 10 cm.

[0029] Figure 2 The results of qRT-PCR detection of the expression level of Nitab4.5_0000595g0010 gene of tobacco plants under the conditions of boron deficiency (0 mM), appropriate boron (0.046 mM) and boron excess (0.184 mM) for 5 days, 12 days and 19 days.

[0030] Figure 3 The results of qRT-PCR detection of the expression level of Nitab4.5_0011519g0020 gene of tobacco plants under the conditions of boron deficiency (0 mM), appropriate boron (0.046 mM) and boron excess (0.184 mM) for 5 days, 12 days and 19 days.

[0031] Figure 4 The results of qRT-PCR detection of the expression level of Nitab4.5_0016396g0010 gene of tobacco plants under the conditions of boron deficiency (0 mM), appropriate boron (0.046 mM) and boron excess (0.184 mM) for 5 days, 12 days and 19 days. DETAILED DESCRIPTION

[0032] 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.

[0033] Example 1

[0034] I. Materials and Methods

[0035] 1. Plants and Experimental Design

[0036] The formulations of boron-rich complete nutrient solutions, boron-deficient complete nutrient solutions, and boron-excess complete nutrient solutions are shown in Table 1.

[0037] Table 1. Formula for complete nutrient solution (unit: mg / L)

[0038]

[0039] Tobacco seedlings of the 'Cui Bi No. 1' variety, exhibiting uniform growth and good condition at the 3-leaf stage, were obtained through soil cultivation. These seedlings were then transplanted into culture tanks containing a complete nutrient solution with an appropriate boron concentration (0.046 mM) and placed in an artificial climate chamber for further cultivation. The conditions were set as follows: 12 hours of light cultivation at 25°C, followed by 12 hours of dark cultivation at 20°C. After 5 days of cultivation, a boron supply gradient culture experiment was conducted. The seedlings were randomly divided into three groups and cultured in complete nutrient solutions with boron deficiency (0 mM), appropriate boron concentration (0.046 mM), and boron excess (0.184 mM), respectively.

[0040] 2. Sample Collection

[0041] like Figure 1 As shown, after culturing tobacco plants in complete nutrient solutions with different boron concentrations for 5 days, the plants in each treatment group showed good growth and little phenotypic difference. After culturing tobacco plants in complete nutrient solutions with different boron concentrations for 12 days, the leaves of plants cultured in boron-deficient (0 mM) nutrient solution curled and the growth was slow, while the plants cultured in nutrient solutions with adequate boron (0.046 mM) and excess boron (0.184 mM) showed no significant abnormalities. After culturing tobacco plants in complete nutrient solutions with different boron concentrations for 19 days, the leaves of plants cultured in boron-deficient (0 mM) nutrient solution curled severely, the terminal buds withered, and the plants grew in clumps. The plants became stunted due to growth stagnation, while the plants cultured in nutrient solutions with adequate boron (0.046 mM) and excess boron (0.184 mM) showed no significant abnormalities.

[0042] After culturing in complete nutrient solutions with different boron concentrations for 5, 12, and 19 days, the first fully unfolded new leaf of each tobacco plant was taken and stored in liquid nitrogen, with three biological replicates.

[0043] 3. Transcriptome sequencing and analysis

[0044] Total RNA was extracted from tobacco leaf samples using the Trizol method. The extracted RNA was subjected to quality testing. After passing the quality test, a eukaryotic RNA-seq library was constructed and sequenced using the Illumina HiSeq platform.

[0045] The raw RNA-seq data were further quality-controlled using FASTP software, filtering and removing adapter sequences and sequences with low sequencing quality to obtain high-quality purified data. The purified data were then compared with the tobacco reference genome using HISAT2 software. This reference genome, sourced from the Solanaceae Genome Database (https: / / solgenomics.net / organism / Nicotiana_tabacum / genome), is 4.5 GB in size and contains annotations for 69,500 protein-coding genes.

[0046] Using edgR software ( http: / / bioconductor.org / packages / release / bioc / html / edgeR.html Differential expression analysis was performed, and q-values ​​were calculated. Genes with significant differential expression were screened based on the calculation results. A threshold of q-value less than 0.05 and a fold change logarithm (base 2) greater than 1 was used to screen genes with significantly upregulated or downregulated expression. GO enrichment analysis was performed using CluterProfiler based on the differential expression analysis results.

[0047] After quality assessment and biological repeatability analysis, transcriptome data are used for screening differentially expressed genes. After culturing tobacco plants in complete nutrient solutions with different boron concentrations for 5 days, a total of 1042 genes showed significant differential expression between the boron-deficient (0 mM) group and the boron-adequate (0.046 mM) group, with 883 genes upregulated and 159 genes downregulated. 389 genes showed significant differential expression between the boron-deficient (0 mM) group and the boron-excess (0.184 mM) group, with 49 genes upregulated and 340 genes downregulated. After culturing tobacco plants in complete nutrient solutions with different boron concentrations for 12 days, a total of 5901 genes showed significant differential expression between the boron-deficient (0 mM) group and the boron-adequate (0.046 mM) group, and 6169 genes showed significant differential expression between the boron-deficient (0 mM) group and the boron-excess (0.184 mM) group. After culturing tobacco plants in complete nutrient solutions with different boron concentrations for 19 days, a total of 2278 genes showed significant differential expression between the boron-deficient (0 mM) and boron-adequate (0.046 mM) groups. Significantly different expression of 7734 genes was observed between the boron-deficient (0 mM) group and the boron-appropriate (0.046 mM) group, and between the boron-excess (0.184 mM) group (Table 2).

[0048] Table 2. Number of differentially expressed genes in tobacco under boron deficiency stress detected by RNA-Seq

[0049]

[0050]

[0051] Note: B0 is a tobacco sample cultured in a medium with a boron concentration of 0 mM; B0.046 is a tobacco sample cultured in a medium with a boron concentration of 0.046 mM; B0.184 is a tobacco sample cultured in a medium with a boron concentration of 0.184 mM.

[0052] Further comparison and analysis of transcriptome data from the boron-deficient (0 mM), boron-adequate (0.046 mM), and boron-excess (0.184 mM) groups revealed three differentially expressed genes (Table 3) that showed extremely low expression levels in the boron-adequate (0.046 mM) and boron-excess (0.184 mM) groups, but significantly upregulated expression in the boron-deficient (0 mM) group. These genes were Nitab4.5_0000595g0010, Nitab4.5_0011519g0020, and Nitab4.5_0016396g0010. The gene sequences of Nitab4.5_0000595g0010 are shown in SEQ ID NO:1, Nitab4.5_0011519g0020 in SEQ ID NO:2, and Nitab4.5_0016396g0010 in SEQ ID NO:3.

[0053] Table 3. RNA-Seq identification of expression values ​​of three significantly differentially expressed genes under boron deficiency stress in tobacco.

[0054]

[0055] Note: B0 is a tobacco sample cultured in a medium with a boron concentration of 0 mM; B0.046 is a tobacco sample cultured in a medium with a boron concentration of 0.046 mM; B0.184 is a tobacco sample cultured in a medium with a boron concentration of 0.184 mM.

[0056] 4. qRT-PCR to verify the expression levels of candidate differentially expressed genes

[0057] Transcriptome data analysis identified a group of differentially expressed genes (Nitab4.5_0000595g0010, Nitab4.5_0011519g0020, Nitab4.5_0016396g0010), which were further validated by quantitative real-time qRT-PCR. Total RNA was extracted from tobacco leaf samples using the Trizol method, and reverse transcription experiments were performed using specific primers and a reverse transcription kit provided by Novizan Biosciences Co., Ltd. cDNA was then obtained and detected by qRT-PCR.

[0058] The specific primers for Nitab4.5_0000595g0010 are as follows:

[0059] RT-0595-F: 5ʹ-AATCAATAACGCCACTTC-3ʹ,

[0060] RT-0595-R: 5ʹ-ATCACATACACTTACAACAC-3ʹ;

[0061] The specific primers for Nitab4.5_0011519g0020 are:

[0062] RT-1519-F: 5ʹ-TTCGTTCTCAAGCCTATATGG-3ʹ,

[0063] RT-1519-R: 5ʹ-GTCTGTGGAGCAACAACTATA-3ʹ;

[0064] The specific primers for Nitab4.5_0016396g0010 are:

[0065] RT-6396-F: 5ʹ-TCTTTGCTTCCCTTTGTG-3ʹ,

[0066] RT-6396-R: 5ʹ-CCTTGTCATCATCAGTAGTG-3ʹ.

[0067] The qRT-PCR experimental 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.

[0068] The qRT-PCR experimental reaction program consists of three stages: Stage 1: 95℃, 30 s; Stage 2: (95℃,

[0069] Phase 1: 40 cycles (10 s, 60 ℃, 30 s, 95 ℃, 15 s); Phase 2: 60 ℃, 60 s, 95 ℃, 15 s.

[0070] qRT-PCR validation results showed that after 5 days of treatment with complete nutrient solutions of different boron concentrations, the Nitab4.5_0000595g0010 gene was almost undetectable in tobacco plants in the boron-deficient (0 mM), boron-adequate (0.046 mM), and boron-excess (0.184 mM) groups. After 12 or 19 days of treatment, the Nitab4.5_0000595g0010 gene was also only detected at very weak expression levels in the boron-adequate (0.046 mM) and boron-excess (0.184 mM) groups, while it was detected at a highly significant level in the boron-deficient (0 mM) group. Figure 2 ).

[0071] qRT-PCR validation results showed that after 5 days of treatment with complete nutrient solutions of different boron concentrations, the Nitab4.5_0011519g0020 gene was almost not expressed in tobacco plants in the boron-deficient (0 mM), boron-adequate (0.046 mM), and boron-excess (0.184 mM) groups. After 12 or 19 days of treatment, the expression level of the Nitab4.5_0000595g0010 gene in the boron-deficient (0 mM) group was significantly higher than that in the boron-adequate (0.046 mM) or boron-excess (0.184 mM) groups. Figure 3 ).

[0072] qRT-PCR validation results showed that after 5 days of treatment with complete nutrient solutions of different boron concentrations, the Nitab4.5_0016396g0010 gene was almost undetectable in tobacco plants in the boron-deficient (0 mM), boron-adequate (0.046 mM), and boron-excess (0.184 mM) groups. After 12 or 19 days of treatment, the Nitab4.5_0016396g0010 gene was also almost undetectable in the boron-adequate (0.046 mM) and boron-excess (0.184 mM) groups, while highly significant high-level expression was detected in the boron-deficient (0 mM) group. Figure 4 ).

[0073] As can be seen, the qRT-PCR results are consistent with the RNA-seq analysis results, indicating that when tobacco plants are in a boron-deficient state for a certain period, the expression levels of the three genes Nitab4.5_0000595g0010, Nitab4.5_0011519g0020, and Nitab4.5_0016396g0010 are significantly upregulated; under conditions of adequate or excessive boron, the expression of the three genes is basically at the background level. In conclusion, the three genes Nitab4.5_0000595g0010, Nitab4.5_0011519g0020, and Nitab4.5_0016396g0010 can be used as biomarkers for diagnosing the response of tobacco to boron deficiency stress.

[0074] 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> Fujian Provincial Tobacco Monopoly Bureau Tobacco Science Research Institute, Minjiang University <120> A biomarker set for detecting boron deficiency stress in tobacco and its applications <130> <160> 9 <170> PatentIn version 3.3 <210> 1 <211> 690 <212> DNA <213> Artificial sequence <400> 1 gagccaacta aaaagatata aagagcatca tatcatacat agcaataaaa gaaagatgca 60 aggacagtgg atagcggcaa gagaccttac aattacatgg gtgaacaatc ctcagttctg 120 gacatggaaa actgttgatc ctaatattga agtggcggag cttcgtagcg taaattggct 180 tgacatttat ggaaagatag agacaaaaaa tcttattcaa acgactagtt atgcagtata 240 tttagtgttc aagttaacag ataaccctcg tggacttgaa cgatccattg catcgctaag 300 atttgtgaag gaggtggcaa aggacgcagg cattgagggt accactgttt tcatctcgaa 360 gaaaaaggaa ttaccaggag aacttggccg gtttccacat cttcgaagcg atggctggtt 420 agaaatcaag cttggtgagt ttttcaacaa cttaggagag gatggtgaag tcgaaatgag 480 gttgatggaa atcaataacg ccacttcgaa atctggcatc attgttaagg gcttcgacat 540 tcgtccaaat taattggata gatctctagc ctttctattc tgcatgttgt tgtgtttgtc 600 ttttcagatt ttctaataaa atctctctct atgataagag tgagcgtgtt gtaagtgtat 660 gtgatcttat atatgatcta tcctttttac 690 <210> 2 <211> 642 <212> DNA <213> Artificial sequence <400> 2 atggaaaagg ttcatgagaa accgctatct ctttataatg gatcattatc tgtttttaga 60 ttaggagatt tggctgtgaa aataacaaag gtgaaaaagg gatcattaaa taatgataat 120 ctttcacccc caacttcatt gttagttgtt tcaccaatca taccaggaac ttatccagtt 180 ttactctttt ttcatggctt cgttctcaag cctatatggt acaagtctct ccttcaacat 240 atttcttccc acggctatat agttgttgct ccacagacgg tccctgtggc ggtgatcggc 300 gctggcttgt caaaccaaag tgcgaattgt atctttccac ccttcgcacc aaacggtgtc 360 aaccattcgg agttttttaa cgagtccaaa ccaccttgct gttattttct ggctaaaaat 420 tatggacata ctgatatgtt agatgacaga attgctgcaa ttgcgagttg gatttcaaag 480 agtgggaagg gacccaagga ccttatgaga aaggctgttg gagggattgt tgtggctttt 540 cttgaggcta aattgggaga gaaagtggat aatctaaatg ccattgttca agaaccttct 600 cttgctccca tcatccttga cccagtcata tctgtcaaat aa 642 <210> 3 <211> 729 <212> DNA <213> Artificial Sequence <400> 3 atgaaatcct ttatttttgg cttcctcttg ctttcaacta ctctctcttt gcttcccttt 60 gtggtttttt cttcatcttt cacttccacc aatcacattg tccttcccac cactactgat 120 gatgacaagg ttttccctat ccctggattc tccgaagtgc tagacataaa cggcgaaccc 180 ctccatgtcg gcgaagagta ccacattatc tccgctatcc gtggagccgg tggcggcggc 240 gtatacttaa cctatgttgg aaataccaaa tgtccaaacg gcgtactcca gcatgggagg 300 gacacccgtc ccggcatgcc cgtgaaattc gttaccactc actcacgacc ttttaatgtc 360 gtacgtgaaa atactgacat taatattatg ttctctgttt caacgtcacg actctgtgtt 420 aatgaaactg tttggaaagt tggtgatccc gacttaacta cacgagggac taggtttgtg 480 gtaaccggtg gaaccctagg aaattcaaga cccgaaacaa caaacaactg gtttaagatt 540 gagaaagtaa caaaacaagc acctttctac aagttaaggt attgtcctga taaatttttg 600 tgtccagagt gccaccccgt tgattgttta gatgtcggtg tgactgacca tttcggatat 660 aggcgtctgg ctctcaccaa gcagcctttt atggttttct tcaggaaatt ccagaagact 720 gatggataa 729 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <400> 4 aatcaataac gccacttc 18 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <400> 5 atcacataca cttacaacac 20 <210> 6 <211> twenty one <212> DNA <213> Artificial sequence <400> 6 ttcgttctca agcctatatg g 21 <210> 7 <211> twenty one <212> DNA <213> Artificial sequence <400> 7 gtctgtggag caacaactat a 21 <210> 8 <211> 18 <212> DNA <213> Artificial sequence <400> 8 tctttgcttc cctttgtg 18 <210> 9 <211> 20 <212> DNA <213> Artificial sequence <400> 9 ccttgtcatc atcagtagtg 20

Claims

1. A biomarker set for diagnosing boron deficiency in tobacco, characterized in that: The biomarker group consists of Nitab4.5_0000595g0010, Nitab4.5_0011519g0020, and Nitab4.5_0016396g0010; wherein the nucleotide sequence of Nitab4.5_0000595g0010 is shown in SEQ ID NO:1, the nucleotide sequence of Nitab4.5_0011519g0020 is shown in SEQ ID NO:2, and the nucleotide sequence of Nitab4.5_0016396g0010 is shown in SEQ ID NO:

3.

2. The application of the biomarker group as described in claim 1 in analyzing the response of tobacco to boron deficiency stress.

3. A reagent kit, characterized in that: The kit includes reagents for detecting the biomarker group of claim 1 using RT-qPCR technology.

4. The reagent kit according to claim 3, characterized in that: The reagents include specific primers for Nitab4.5_0000595g0010, Nitab4.5_0011519g0020, and Nitab4.5_0016396g0010.

5. The reagent kit according to claim 4, characterized in that: The nucleotide sequences of the specific primers for Nitab4.5_0000595g0010, Nitab4.5_0011519g0020, and Nitab4.5_0016396g0010 are as follows: Specific primers for Nitab4.5_0000595g0010: RT-0595-F: 5'-AATCAATAACGCCACTTC-3', RT-0595-R: 5'-ATCACATACACTTACAACAC-3'; Specific primers for Nitab4.5_0011519g0020: RT-1519-F: 5'-TTCGTTCTCAAGCCTATATGG-3', RT-1519-R: 5'-GTCTGTGGAGCAACAACTATA-3'; Specific primers for Nitab4.5_0016396g0010: RT-6396-F: 5'-TCTTTGCTTCCCTTTGTG-3', RT-6396-R: 5'-CCTTGTCATCATCAGTAGTG-3'.

6. The application of the kit as described in claim 3 in analyzing the response of tobacco to boron deficiency stress.

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