CN115011716A - Biomarker group for detecting boron deficiency stress of tobacco and application thereof - Google Patents
Biomarker group for detecting boron deficiency stress of tobacco and application thereof Download PDFInfo
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Abstract
The invention discloses a biomarker group for detecting boron deficiency stress of tobacco and application thereof. The biomarker group consists of 3 genes of Nitab4.5_0000595g0010, Nitab4.5_0011519g0020, Nitab4.5_0016396g0010 and the like, and the expression of the biomarkers is specifically up-regulated in the process of tobacco responding to boron stress. Therefore, the 3 genes can be used as a biomarker group for diagnosing the boron deficiency stress of the tobacco. The invention lays a foundation for establishing a rapid diagnosis method for boron deficiency of tobacco based on biomarkers.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a biomarker group which is obtained by screening through a genomics technology and can be used for detecting the boron deficiency stress of tobacco and application thereof.
Background
Tobacco (A)Nicotiana tabacum L.) Is a crop with higher economic value. In China, higher economic income can be created by planting tobacco. The tobacco is a plant of the genus Nicotiana of the family Solanaceae, which is annual or limited, has large biomass and strong nutrient absorption capacity, and needs various nutrient elements in the growth and development process.
Boron is one of trace elements necessary for the growth and development of tobacco, participates in various physiological and biochemical activities such as protein metabolism, substance transportation, alkaloid synthesis and the like, and influences the normal development and stress resistance of the tobacco. Boron is involved in uracil and chlorophyll synthesis, carbohydrate transport, cell division and growth, ribonucleic acid metabolism, and the like during tobacco growth and development. Boron can also improve photosynthesis and transpiration of tobacco and promote accumulation of saccharide. The lack of boron can cause withering of tobacco terminal buds, growth is hindered and stopped, plants are in a cluster shape, transportation of carbohydrate and protein is affected, and the quality of tobacco leaves is reduced.
By utilizing a genomics strategy, molecular biomarkers of response of tobacco to boron deficiency stress are mined and screened, and a nutrition diagnosis method based on the biomarkers is established, so that the method has practical significance for constructing a green tobacco planting system.
Disclosure of Invention
The invention aims to provide a biomarker group for detecting boron deficiency stress of tobacco and application thereof.
In order to achieve the purpose, the invention provides the following scheme:
the invention firstly provides a biomarker group for diagnosing response of tobacco to boron deficiency stress, which consists of Nitab4.5_0000595g0010, Nitab4.5_0011519g0020 and Nitab4.5_0016396g 0010; 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;
the invention also provides application of the biomarker group in the boron deficiency nutrition diagnosis of tobacco.
Further, the expression level of the biomarker group is remarkably up-regulated when the tobacco is stressed by boron deficiency.
The invention also provides a kit for diagnosing the boron deficiency stress of the tobacco, which comprises a reagent for detecting the biomarker group by using an RT-qPCR technology.
Furthermore, the reagent comprises specific primers of Nitabb 4.5_0000595g0010, Nitabb 4.5_0011519g0020 and Nitabb 4.5_0016396g 0010.
Further, the nucleotide sequences of the specific primers of Nitab4.5_0000595g0010, Nitab4.5_0011519g0020 and Nitab4.5_0016396g0010 are as follows:
specific primers for Nitab4.5_0000595g 0010:
RT-0595-F: 5ʹ-AATCAATAACGCCACTTC-3ʹ，
RT-0595-R: 5ʹ-ATCACATACACTTACAACAC-3ʹ；
specific primers for Nitab4.5-0011519 g 0020:
RT-1519-F: 5ʹ-TTCGTTCTCAAGCCTATATGG-3ʹ，
RT-1519-R: 5ʹ-GTCTGTGGAGCAACAACTATA-3ʹ；
specific primers for Nitab4.5_0016396g 0010:
RT-6396-F: 5ʹ-TCTTTGCTTCCCTTTGTG-3ʹ，
RT-6396-R: 5ʹ-CCTTGTCATCATCAGTAGTG-3ʹ。
the invention also provides application of the kit in the boron deficiency nutrition diagnosis of tobacco.
The invention also provides a method for diagnosing the boron deficiency nutrition of the tobacco, which comprises the following steps:
1) collecting tobacco leaves to be detected, extracting total RNA, and performing reverse transcription to obtain cDNA;
2) using cDNA obtained by reverse transcription as a template, and performing RT-qPCR detection by using the specific primer; and judging whether the tobacco to be detected is stressed by boron deficiency according to whether the gene is up-regulated and expressed.
The invention has the following remarkable advantages:
the invention researches the mechanism of tobacco responding to boron stress through a transcriptome strategy, provides a biomarker group consisting of Nitab4.5_0000595g0010, Nitab4.5_0011519g0020 and Nitab4.5_0016396g0010, and can diagnose the nutrient deficiency condition of the boron element in the tobacco by utilizing the biomarker group, thereby adjusting the fertilization direction in time and having extremely high practical application value.
Drawings
FIG. 1 shows the phenotype of tobacco plants cultured under conditions of boron deficiency (0 mM), moderate boron content (0.046 mM), and excess boron content (0.184 mM) for 12 days and 19 days, with a white scale of 10 cm.
FIG. 2 shows the results of the gene expression level of Nitabb 4.5-0000595 g0010 detected by qRT-PCR, wherein the tobacco plants are cultured for 5 days, 12 days and 19 days under the conditions of boron deficiency (0 mM), proper amount of boron (0.046 mM) and excess amount of boron (0.184 mM).
FIG. 3 shows the results of the gene expression level of Nitabb 4.5-0011519 g0020 by qRT-PCR, wherein the tobacco plants are cultured for 5 days, 12 days and 19 days under the conditions of boron deficiency (0 mM), proper amount of boron (0.046 mM) and excess amount of boron (0.184 mM).
FIG. 4 shows the results of the gene expression level of Nitabb 4.5-0016396 g0010 detected by qRT-PCR after tobacco plants are cultured for 5 days, 12 days and 19 days under the conditions of boron deficiency (0 mM), proper boron amount (0.046 mM) and boron excess (0.184 mM).
Detailed Description
In order to make the present invention more comprehensible, the technical solutions of the present invention are further described below with reference to specific embodiments, but the present invention is not limited thereto.
Example 1
Materials and methods
1. Plant and experimental design
The formulas of the complete nutrient solution with proper amount of boron, the complete nutrient solution with boron deficiency and the complete nutrient solution with excessive boron are shown in table 1.
TABLE 1 complete nutrient solution formulation (unit: mg/L)
The seedlings of tobacco Jade No. 1 with 3 leaf stages, consistent growth and good condition are obtained by soil culture. Transplanting the tobacco green No. 1 seedling into a culture tank filled with complete nutrient solution with proper boron (the boron concentration is 0.046 mM), and transferring into an artificial climate box for culture; the conditions were set at 25 ℃ for 12 hours in light culture and 20 ℃ for 12 hours in dark culture. After 5 days of culture, different supply gradient culture tests of boron are carried out, and the tobacco plant seedlings are randomly divided into three groups and cultured in complete nutrient solution with boron deficiency (0 mM), proper boron (0.046 mM) and excess boron (0.184 mM).
2. Sample collection
As shown in figure 1, after the tobacco plants are cultured by complete nutrient solutions with different boron concentrations for 5 days, the growth conditions of the tobacco plants in each treatment group are good, and the phenotype difference is small; after tobacco plants are cultured by complete nutrient solutions with different boron concentrations for 12 days, leaves of the plants cultured by nutrient solutions lacking boron (0 mM) are curled and grow slowly, and the plants cultured by nutrient solutions with proper boron (0.046 mM) and excessive boron (0.184 mM) have no obvious abnormality; after tobacco plants are cultured by complete nutrient solutions with different boron concentrations for 19 days, leaves of the plants cultured by the nutrient solutions with boron deficiency (0 mM) are seriously curled, terminal buds die and are clustered, the plants become short and small due to growth stagnation, and the plants cultured by the nutrient solutions with proper boron (0.046 mM) and excessive boron (0.184 mM) have no obvious abnormality.
After 5 days, 12 days, 19 days of complete nutrient solution culture at different boron concentrations, the first new leaf of the completely unfolded tobacco plant is taken respectively and stored in liquid nitrogen, and 3 biological replicates are set.
3. Transcriptome sequencing and analysis
Extracting total RNA of tobacco sample leaves by adopting a Trizol method, carrying out quality detection on the extracted RNA, constructing a eukaryotic RNA-seq library after the quality detection is qualified, and sequencing by utilizing an IlluminaHiSeq platform.
And further performing quality control on the original data of the RNA-seq by using FASTP software, filtering and removing a linker sequence and a sequence with lower sequencing quality to obtain high-quality purified data. The purified data were aligned using the HISAT2 software with a tobacco reference genome from the solanaceous genomic database website (https:// solgenomics. net/organism/Nicotiana _ tabacum/genome) of 4.5 GB, co-annotated 69500 genes encoding proteins.
Using edgR software (http://bioconductor.org/packages/release/bioc/html/ edgeR.html) Carrying out differential expression analysis, calculating q-value, and screening significant difference genes according to the calculation result; genes with significantly up-regulated or down-regulated differential expression are screened using a threshold q-value of less than 0.05 and a fold difference log (base 2) of greater than 1. And performing GO enrichment analysis by using CluterProfiler according to the analysis result of differential expression.
The transcriptome data is applied to screening of differential expression genes after quality evaluation and biological repeated analysis. After culturing tobacco plants in complete nutrient solution with different boron concentrations for 5 days, 1042 genes are detected to be significantly and differentially expressed between a boron deficiency (0 mM) group and a boron proper (0.046 mM) group, wherein 883 genes are up-regulated and 159 genes are down-regulated, and 389 genes are significantly and differentially expressed between the boron deficiency (0 mM) group and the boron excess (0.184 mM) group, wherein 49 genes are up-regulated and 340 genes are down-regulated; after the tobacco plants are cultured by complete nutrient solutions with different boron concentrations for 12 days, 5901 genes are detected to be significantly and differentially expressed between a boron deficiency (0 mM) group and a boron proper amount (0.046 mM) group, and 6169 genes are detected to be significantly and differentially expressed between the boron deficiency (0 mM) group and the boron excess (0.184 mM) group; after culturing tobacco plants in complete nutrient solution at different boron concentrations for 19 days, 2278 genes were detected to be significantly differentially expressed between the boron deficiency (0 mM) group and the boron proper (0.046 mM) group, and 7734 genes were significantly differentially expressed between the boron deficiency (0 mM) group and the boron excess (0.184 mM) group (Table 2).
TABLE 2 RNA-Seq detection of the number of differentially expressed genes under the condition of boron deficiency stress in tobacco
Note: b0 is a tobacco sample cultured by a culture solution with the boron element concentration of 0 mM; b0.046 is a tobacco sample cultured by a culture solution with the concentration of boron element of 0.046 mM; b0.184 is a tobacco sample cultured in a culture solution with a boron element concentration of 0.184 mM.
Further comparing and mining transcriptome data of samples of boron deficiency (0 mM) group, boron adequate (0.046 mM) group and boron excess (0.184 mM) group, 3 differential genes (Table 3) expressing extremely low values in boron adequate (0.046 mM) group and boron excess (0.184 mM) group and significantly up-regulated in boron deficiency (0 mM) group were screened, namely Nitab4.5_0000595g0010, Nitab4.5_0011519g0020 and Nitab4.5_0016396g0010, respectively. Wherein, the gene sequence of Nitabb 4.5_0000595g0010 is shown in SEQ ID NO. 1, the gene sequence of Nitabb 4.5_0011519g0020 is shown in SEQ ID NO. 2, and the gene sequence of Nitabb 4.5_0016396g0010 is shown in SEQ ID NO. 3.
TABLE 3 RNA-Seq identification of expression values of 3 significant difference genes under the condition of boron deficiency stress of tobacco
Note: b0 is a tobacco sample cultured by a culture solution with the boron element concentration of 0 mM; b0.046 is a tobacco sample cultured by a culture solution with the boron element concentration of 0.046 mM; b0.184 is a tobacco sample cultured in a culture solution with a boron element concentration of 0.184 mM.
4. qRT-PCR verification of candidate differential gene expression level
By analyzing transcriptome data, a batch of genes (Nitab4.5 _0000595g0010, Nitab4.5_0011519g0020 and Nitab4.5_0016396g 0010) with obvious differential expression are screened out, and further verified by fluorescence quantitative qRT-PCR. Extracting total RNA of tobacco sample leaves by adopting a Trizol method, and performing RNA reverse transcription experiment by utilizing a specific primer and a reverse transcription kit provided by Novozan biotechnology, Inc.; after obtaining cDNA, qRT-PCR detection is carried out.
Specific primers of Nitab4.5_0000595g0010 are as follows:
RT-0595-F: 5ʹ-AATCAATAACGCCACTTC-3ʹ，
RT-0595-R: 5ʹ-ATCACATACACTTACAACAC-3ʹ；
specific primers for Nitab4.5_0011519g0020 are:
RT-1519-F: 5ʹ-TTCGTTCTCAAGCCTATATGG-3ʹ，
RT-1519-R: 5ʹ-GTCTGTGGAGCAACAACTATA-3ʹ；
specific primers of Nitab4.5_0016396g0010 are as follows:
RT-6396-F: 5ʹ-TCTTTGCTTCCCTTTGTG-3ʹ，
RT-6396-R: 5ʹ-CCTTGTCATCATCAGTAGTG-3ʹ。
the qRT-PCR experiment reaction system is as follows: 2 × SYBRMix 5 ul, Primer-F0.2 ul, Primer-R0.2 ul, cDNA 1 ul, RNase-free ddH 2 O 3.6 ul。
The qRT-PCR experimental reaction program is divided into three stages, namely: at 95 ℃ for 30 s; and a second stage: (95 ℃ C.),
10 s, 60 ℃, 30 s, 95 ℃, 15 s) 40 cycles; and a third stage: 60 ℃, 60 s, 95 ℃ and 15 s.
The qRT-PCR verification result shows that: after the tobacco plants are treated by complete nutrient solution with different boron concentrations for 5 days, Nitab4.5_0000595g0010 gene can hardly be expressed in the tobacco plants of a boron deficiency group (0 mM), a proper boron amount group (0.046 mM) and a boron excess group (0.184 mM); after 12 days or 19 days of treatment, the Nitab4.5_0000595g0010 gene was also detected only at a very weak expression level in tobacco plants of the boron-adequate (0.046 mM) and boron-excess (0.184 mM) groups, and at a very significantly high level in the boron-deficient (0 mM) group (FIG. 2).
The qRT-PCR verification result shows that: after the tobacco plants are treated by complete nutrient solution with different boron concentrations for 5 days, Nitab4.5_0011519g0020 gene is basically not expressed in the tobacco plants of a boron deficiency group (0 mM), a proper boron amount group (0.046 mM) and a boron excess group (0.184 mM); after 12 or 19 days of treatment, the expression level of the Nitab4.5_0000595g0010 gene in tobacco plants in the boron deficiency (0 mM) group was significantly higher than that in tobacco plants in the boron adequate (0.046 mM) group or the boron excess (0.184 mM) group (FIG. 3).
The qRT-PCR verification result shows that: after the tobacco plants are treated by complete nutrient solution with different boron concentrations for 5 days, the Nitabb 4.5_0016396g0010 gene is hardly expressed in the tobacco plants of a boron deficiency group (0 mM), a boron proper amount group (0.046 mM) and a boron excess group (0.184 mM); after 12 days or 19 days of treatment, the Nitab4.5-0016396 g0010 gene was also barely detectable in tobacco plants of the boron-adequate (0.046 mM) group and the boron-excess (0.184 mM) group, while it could be detected at a very significantly high level in tobacco plants of the boron-deficient (0 mM) group (FIG. 4).
As can be seen, the qRT-PCR detection result is consistent with the RNA-seq analysis result, and shows that the expression levels of three genes, namely Nitab4.5_0000595g0010, Nitab4.5_0011519g0020 and Nitab4.5_0016396g0010 are obviously up-regulated after the tobacco plant is in a boron-deficient state for a certain period of time; under the condition of proper amount of boron or excessive boron, the expression of the three genes is basically at the background level. In conclusion, the three genes of 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.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
SEQUENCE LISTING
<110> Nicotiana tabacum scientific research institute of tobacco monopoly, Minjiang academy
<120> biomarker group for detecting boron deficiency stress of tobacco and application thereof
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<170> PatentIn version 3.3
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gaaaaaggaa ttaccaggag aacttggccg gtttccacat cttcgaagcg atggctggtt 420
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Claims (7)
1. A biomarker panel for detecting boron deficiency stress in tobacco, comprising: the biomarker group consists of Nitab4.5_0000595g0010, Nitab4.5_0011519g0020 and Nitab4.5_0016396g 0010; 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. Use of the biomarker panel of claim 1 in the diagnosis of boron deficiency in tobacco.
3. A kit for diagnosing the boron deficiency stress of tobacco is characterized in that: the kit comprises reagents for detecting the biomarker panel of claim 1 by RT-qPCR techniques.
4. The kit of claim 3, wherein: the reagent comprises specific primers of Nitabb 4.5_0000595g0010, Nitabb 4.5_0011519g0020 and Nitabb 4.5_0016396g 0010.
5. The kit of claim 4, wherein: the nucleotide sequences of the specific primers of the Nitab4.5_0000595g0010, the Nitab4.5_0011519g0020 and the Nitab4.5_0016396g0010 are as follows:
specific primers for Nitab4.5_0000595g 0010:
RT-0595-F: 5ʹ-AATCAATAACGCCACTTC-3ʹ，
RT-0595-R: 5ʹ-ATCACATACACTTACAACAC-3ʹ；
specific primers for Nitab4.5_0011519g 0020:
RT-1519-F: 5ʹ-TTCGTTCTCAAGCCTATATGG-3ʹ，
RT-1519-R: 5ʹ-GTCTGTGGAGCAACAACTATA-3ʹ；
specific primers for Nitab4.5_0016396g 0010:
RT-6396-F: 5ʹ-TCTTTGCTTCCCTTTGTG-3ʹ，
RT-6396-R: 5ʹ-CCTTGTCATCATCAGTAGTG-3ʹ。
6. use of the kit according to claim 3 in the diagnosis of boron deficiency in tobacco.
7. A method for diagnosing boron deficiency nutrition of tobacco is characterized by comprising the following steps: the method comprises the following steps:
1) collecting tobacco leaves to be detected, extracting total RNA, and performing reverse transcription to obtain cDNA;
2) performing RT-qPCR detection by using cDNA obtained by reverse transcription as a template and the specific primer in claim 5; and judging whether the tobacco to be detected is stressed by boron deficiency according to whether the gene is up-regulated and expressed.
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