CN116004877A - Gene marker set for tobacco zinc deficiency nutrition diagnosis and application thereof - Google Patents
Gene marker set for tobacco zinc deficiency nutrition diagnosis and application thereof Download PDFInfo
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Abstract
The invention discloses a gene marker set for tobacco zinc deficiency nutrition diagnosis and application thereof, and belongs to the technical field of biology. The gene marker set consists of Nitab4.5_0000143g0340, nitab4.5_0000572g0110, nitab4.5_0001724g0060, nitab4.5_0003605g0060 and Nitab4.5_0006169g0020. The gene marker set specifically and obviously up-regulates the expression in response to the zinc deficiency stress of the tobacco, and can diagnose the zinc deficiency stress of the tobacco. The invention has reference significance for establishing a rapid diagnosis method of the zinc deficiency of the tobacco based on the gene markers.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a gene marker set for tobacco zinc deficiency nutrition diagnosis and application thereof.
Background
Tobacco (tobacco)Nicotiana tabacum L.) Is a plant of the genus Nicotiana of the family Solanaceae, which has high economic and medicinal values. Tobacco is widely planted throughout the world. In order to ensure the normal growth and development of tobacco, various nutrient elements are required to be provided. Zinc (Zn) 2+ ) Is one of microelements necessary for the normal growth and development of tobacco. Zinc is an important substance for synthesizing tryptophan, a precursor of auxin, and is involved in synthesis of various enzymes in plants and synthesis of chlorophyll. Zinc deficiency affects the activity of numerous enzymesAt the same time, the photosynthesis of plants is affected, resulting in the decrease of the plant polysaccharide synthesis ability. Zinc deficiency can also affect plant development, resulting in symptoms of dwarfing, reduced growth, and the like.
The genomics strategy based on the high-throughput sequencing technology can more accurately and efficiently excavate and screen molecular biomarkers of tobacco responding to zinc deficiency stress, establish a nutrition diagnosis method based on the biomarkers, and provide a new method and a new idea for constructing a green planting system of tobacco.
Disclosure of Invention
In view of the above, the invention aims to provide a gene marker set for tobacco zinc deficiency nutrition diagnosis and application thereof.
In order to achieve the above object, the present invention provides the following solutions:
the invention firstly provides a gene marker set for nutrition diagnosis of zinc deficiency of tobacco, which 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 as SEQ ID NO. 1, the nucleotide sequence of Nitab4.5_0000572g0110 is shown as SEQ ID NO. 2, the nucleotide sequence of Nitab4.5_0003605g0060 is shown as SEQ ID NO. 3, the nucleotide sequence of Nitab4.5_0001724g0060 is shown as SEQ ID NO. 4, and the nucleotide sequence of Nitab4.5_0006169g0020 is shown as SEQ ID NO. 5.
The invention also provides application of the gene marker set in nutrition diagnosis of zinc deficiency of tobacco.
The invention also provides a tobacco zinc deficiency nutrition diagnosis kit, which comprises a reagent for detecting the gene marker set by using an RT-qPCR technology.
Further, the kit comprises primers for specifically detecting Nitab4.5_0000143g0340, nitab4.5_0000572g0110, nitab4.5_0003605g0060, nitab4.5_0001724g0060 and Nitab4.5_0006169g0020.
Further, 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 primer for Nitab4.5_0000143g 0340:
RT-0143-F: 5ʹ-CGGCTTGTTACACCTCATTACTC-3ʹ，
RT-0143-R: 5ʹ-TTCACCACTTGCTTCAACTCTTC-3ʹ；
specific primer of Nitab4.5_0000572g 0110:
RT-0572-F: 5ʹ-GATAGACCTCTTGCTTCC-3ʹ，
RT-0572-R: 5ʹ-GATTAGTTGCTTGTGATGG-3ʹ；
specific primer for Nitab4.5_0003605g 0060:
RT-3605-F: 5ʹ-TTCAGCAAGAGTAATTGTTG-3ʹ，
RT-3605-R: 5ʹ- ATTCGGAAGTTCGTCAAG-3ʹ；
specific primer for Nitab4.5_0001724g 0060:
RT-1724-F: 5ʹ-GCTTCAGTCATTCCTTAG-3ʹ，
RT-1724-R: 5ʹ-TGCTGTCAATATCATCCT-3ʹ；
specific primer for Nitab4.5_0006169g 0020:
RT-6169-F: 5ʹ-TCTCACTTACAACTCCAG-3ʹ，
RT-6169-R: 5ʹ- AATACTCCTTCCACAATGA-3ʹ。
the invention also provides application of the tobacco zinc deficiency nutrition diagnosis kit in tobacco zinc deficiency nutrition diagnosis.
The invention also provides a method for diagnosing zinc deficiency nutrition of tobacco, which comprises the following steps:
1) Collecting tobacco leaves to be detected, extracting total RNA, and reversely transcribing the total RNA into cDNA;
2) And (3) using the cDNA obtained by reverse transcription as a template, carrying out RT-qPCR detection by using the specific primer, and judging whether the tobacco to be detected is stressed by zinc deficiency according to whether the gene is up-regulated.
Further, if the expression of Nitab4.5_0000143g0340, nitab4.5_0000572g0110, nitab4.5_0003605g0060, nitab4.5_0001724g0060 and Nitab4.5_0006169g0020 in the tobacco to be tested is significantly up-regulated compared with that of normal tobacco, the tobacco to be tested is in a zinc deficiency stress state.
The invention has the remarkable advantages that:
the invention utilizes transcriptome strategy to research the mechanism of responding zinc stress of tobacco, and screens five genes of Nitab4.5_0000143g0340, nitab4.5_0000572g0110, nitab4.5_0003605g0060, nitab4.5_0001724g0060 and Nitab4.5_0006169g0020, which can be used as gene marker sets for diagnosing bad growth of tobacco caused by zinc deficiency.
Drawings
FIG. 1 shows the growth phenotype (white scale: 10 cm) of tobacco plants grown in nutrient solutions of different zinc concentrations.
FIG. 2 shows Nitab4.5_0000143g0340 expression levels of tobacco plants cultured in different zinc concentration nutrient solutions.
FIG. 3 shows the expression levels of Nitab4.5_0000572g0110 in tobacco plants cultured in different zinc concentration nutrient solutions.
FIG. 4 shows Nitab4.5_0003605g0060 expression levels of tobacco plants cultured in different zinc concentration nutrient solutions.
FIG. 5 shows Nitab4.5_0001724g0060 expression levels of tobacco plants cultured in different zinc concentration nutrient solutions.
FIG. 6 shows the results of Nitab4.5-0006169 g0020 expression levels of tobacco plants cultured in different zinc concentration nutrient solutions.
Detailed Description
The following embodiments are illustrative of the present invention, but are not intended to limit the scope of the invention.
The formulations of the zinc-appropriate nutrient solution, the zinc-deficient nutrient solution and the zinc-excessive nutrient solution used in the following examples are shown in Table 1.
Table 1 formulation of nutrient solution (Unit: mg/L)
Example 1
1. Materials and methods
1. Plant cultivation and treatment
Culturing tobacco samples by using No. 1 green as a test variety through soil, and selecting tobacco seedlings with good growth conditions and consistent sizes in the 3-leaf period after culturing for a period of time. Carefully removing soil residues at root parts of seedlings, transplanting the seedlings into a culture tank filled with a proper amount of complete nutrient solution (the zinc concentration is 0.00077 mM) for resuscitation, and transferring the seedlings into an artificial climate box for culture; culturing at 20deg.C for 12 hr; light culture at 25℃for 12 hours.
After the seedlings are revived for 5 days, the gradient culture tests of different zinc element supplies are formally developed. Removing the tobacco plants with poor recovery, randomly dividing the tobacco plant seedlings with good recovery into four groups, and transferring each group into four nutrient solutions with zinc deficiency (zinc element concentration 0 mM), zinc deficiency (zinc element concentration 0.00019 mM), zinc proper amount (zinc element concentration 0.00077 mM) and zinc excess (zinc element concentration 0.00308 mM), and continuously culturing at 20 ℃ for 12 hours in a dark condition; light culture at 25℃for 12 hours.
2. Sample collection
After the tobacco is treated in three nutrient solutions for 5 days, 15 days and 25 days, a first new leaf of the tobacco plant which is fully developed is taken and quickly transferred into liquid nitrogen for preservation. The test was set up with 3 biological replicates.
As shown in fig. 1, after tobacco samples were treated for 5 days, each treatment group had normal tobacco plants grown with no obvious difference in phenotype; on day 15 of treatment, the leaves of the plants cultivated by the zinc-deficient (0 mM) nutrient solution are slightly curled and yellow, the growth of the plants is affected, the growth speed is slowed down, and the plants cultivated by the zinc-deficient (0.00019 mM), zinc-moderate (0.00077 mM) and zinc-excessive (0.00308 mM) nutrient solution are not obviously abnormal; on day 25, the sample leaves of the plants cultivated with the zinc-deficient (0 mM) nutrient solution had severe curling, yellow leaves and partial withering, short plants and slow growth, while the sample leaves cultivated with the zinc-deficient (0.00019 mM) nutrient solution had relatively light symptoms, yellow leaves, short plants, no withering, and the plants cultivated with the zinc-moderate (0.00077 mM) and zinc-excessive (0.00308 mM) nutrient solutions had no obvious abnormalities.
3. Transcriptome sequencing and analysis
And after a test sample is obtained, extracting total RNA of tobacco leaves by using a Trizol method, detecting the quality of the extracted RNA, constructing a eukaryotic RNA-seq library after rejecting unqualified samples, and performing transcriptome sequencing by using an IlluminaHiseq platform. Sequencing work was done by norelgen biotechnology limited.
After the transcriptome sequencing work is completed, the quality control of the raw data of the RNA-seq is further performed by using FASTP software, in order to obtain high quality purified sequencing data. The next step was to use HISAT2 software to compare the purified data with a tobacco reference genome from the Solanaceae genome database website (https:// solgenemics. Net/organization/nicotiana_tabacum/genome), a reference genome size of 4.5 GB, annotated 69500 encoded protein genes. Differential expression analysis is performed on the comparative data by using edgR software (http:// bioconductor. Org/packages/release/bioc/html/edgel. Html), q-value is calculated, the threshold is set to be that q-value is less than 0.05, the logarithm (based on 2) of the fold difference (fold) is greater than 1, and gene genes which are significantly up-regulated or significantly down-regulated are screened according to the calculation result.
As shown in table 2, after tobacco plants were treated with different zinc concentration nutrient solutions for 5 days, a total of 166 genes were detected as significantly differentially expressed between the zinc deficient (0 mM) group and the zinc moderate (0.00077 mM) group (wherein 157 genes were up-regulated in expression, 9 genes were down-regulated in expression), 188 genes were significantly differentially expressed between the zinc deficient (0.00019 mM) group and the zinc moderate (0.00077 mM) group (wherein 179 genes were up-regulated in expression, 9 genes were down-regulated in expression), 789 genes were significantly differentially expressed between the zinc deficient (0 mM) group and the zinc excessive (0.00308 mM) group (wherein 117 genes were up-regulated in expression, 672 genes were down-regulated in expression), 675 genes were significantly differentially expressed between the zinc deficient (0.00019 mM) group and the zinc excessive (0.00308 mM) group (wherein 82 genes were up-regulated in expression, 593 genes were down-regulated in expression); after 15 days of treatment, a total of 802 genes were detected as significantly differentially expressed between the zinc deficient (0 mM) and zinc moderate (0.00077 mM) groups, 120 genes as significantly differentially expressed between the zinc deficient (0.00019 mM) and zinc moderate (0.00077 mM) groups, 1711 genes as significantly differentially expressed between the zinc deficient (0 mM) and zinc excessive (0.00308 mM) groups, 755 genes as significantly differentially expressed between the zinc deficient (0.00019 mM) and zinc excessive (0.00308 mM) groups; after 25 days of treatment, a total of 1328 genes were detected as significantly differentially expressed between the zinc deficient (0 mM) group and the zinc moderate (0.00077 mM) group, 1121 genes were significantly differentially expressed between the zinc deficient (0.00019 mM) group and the zinc moderate (0.00077 mM) group, 3696 genes were significantly differentially expressed between the zinc deficient (0 mM) group and the zinc excessive (0.00308 mM) group, and 1915 genes were significantly differentially expressed between the zinc deficient (0.00019 mM) group and the zinc excessive (0.00308 mM) group.
TABLE 2 RNA-Seq detection of the number of genes differentially expressed under tobacco Zinc deficiency stress conditions
Note that: zn0 is a tobacco sample cultured in a culture solution with zinc element concentration of 0 mM; zn0.00019 is a tobacco sample cultivated in a culture solution with the zinc element concentration of 0.00019 and mM; zn0.00077 is a tobacco sample cultivated in a culture medium with a zinc element concentration of 0.00077 mM; zn0.00308 is a tobacco sample cultivated in a medium with a zinc element concentration of 0.00308 mM.
Transcriptome data of zinc deficiency ((0. 0 mM), zinc deficiency (0.00019 mM), zinc moderate (0.00077 mM) and zinc excess (0.00308 mM) group samples were further compared and mined, five differential genes (table 3) expressing significant upregulation in zinc deficiency (0 mM) and zinc deficiency (0.00019 mM) were selected for extremely low expression values in zinc moderate (0.00077 mM) and zinc excess (0.00308 mM) groups, and nitab4.5_0000143g0340, nitab4.5_0000572g0110, nitab4.5_0003605g0060, nitab4.5_0001724g0060 and nitab4.5_0006169g0020, respectively.
TABLE 3 RNA-Seq identification of 5 significant differentially expressed genes under tobacco Zinc deficiency stress conditions
Note that: zn0 is a tobacco sample cultured in a culture solution with zinc element concentration of 0 mM; zn0.00019 is a tobacco sample cultivated in a culture solution with the zinc element concentration of 0.00019 and mM; zn0.00077 is a tobacco sample cultivated in a culture medium with a zinc element concentration of 0.00077 mM; zn0.00308 is a tobacco sample cultivated in a medium with a zinc element concentration of 0.00308 mM.
4. RT-qPCR (reverse transcription-quantitative polymerase chain reaction) verification of candidate differential gene expression quantity
A batch of significantly expressed genes, nitab4.5_0000143g0340, nitab4.5_0000572g0110, nitab4.5_0003605g0060, nitab4.5_0001724g0060 and nitab4.5_0006169g0020, were screened by gene differential expression analysis and verified using qRT-PCR. And (3) performing RNA reverse transcription experiment by using a reverse transcription kit to obtain cDNA, and selecting a qualified sample for RT-qPCR verification after quality detection of the cDNA. Both the reverse transcription kit and the RT-qPCR detection kit were supplied by Northenan Biotechnology Co., ltd.
The sequences of primers for specific detection of Nitab4.5_0000143g0340, nitab4.5_0000572g0110, nitab4.5_0003605g0060, nitab4.5_0001724g0060, nitab4.5_0006169g0020 were as follows:
specific primer for Nitab4.5_0000143g 0340:
RT-0143-F: 5ʹ-CGGCTTGTTACACCTCATTACTC-3ʹ，
RT-0143-R: 5ʹ-TTCACCACTTGCTTCAACTCTTC-3ʹ；
specific primer of Nitab4.5_0000572g 0110:
RT-0572-F: 5ʹ-GATAGACCTCTTGCTTCC-3ʹ，
RT-0572-R: 5ʹ-GATTAGTTGCTTGTGATGG-3ʹ；
specific primer for Nitab4.5_0003605g 0060:
RT-3605-F: 5ʹ-TTCAGCAAGAGTAATTGTTG-3ʹ，
RT-3605-R: 5ʹ- ATTCGGAAGTTCGTCAAG-3ʹ；
specific primer for Nitab4.5_0001724g 0060:
RT-1724-F: 5ʹ-GCTTCAGTCATTCCTTAG-3ʹ，
RT-1724-R: 5ʹ-TGCTGTCAATATCATCCT-3ʹ；
specific primer for Nitab4.5_0006169g 0020:
RT-6169-F: 5ʹ-TCTCACTTACAACTCCAG-3ʹ，
RT-6169-R: 5ʹ- AATACTCCTTCCACAATGA-3ʹ。
the qRT-PCR test reaction system is as follows: 2 XSBRMix 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 procedure is divided into three phases, namely, a phase I: 95 ℃,30 s; stage two: (95 ℃,10 s,60 ℃,30 s,95 ℃,15 s) for 40 cycles; stage three: 60 ℃,60 s,95 ℃,15 s.
The qRT-PCR results of Nitab4.5_0000143g0340 showed that the gene was expressed in small amounts in the zinc deficient (0.00019 mM) group tobacco samples and hardly in the zinc deficient ((0 mM), zinc moderate (0.00077 mM) and zinc excessive (0.00308 mM) group tobacco samples after 5 days of treatment, the gene was significantly expressed in the zinc deficient (0 mM) and zinc deficient (0.00019 mM) group tobacco samples, especially in the zinc deficient (0 mM) group tobacco samples, at very significantly high levels, and in the zinc moderate (0.00077 mM) and zinc excessive (0.00308 mM) group tobacco samples, after 15 days and 25 days of treatment (FIG. 2).
The results of qRT-PCR verification of Nitab4.5_0000572g0110 show that: after tobacco plants were treated with nutrient solutions of different zinc concentrations for 5 days, the gene was hardly expressed in all of the zinc deficiency (0. 0 mM), zinc deficiency (0.00019 mM), zinc moderate (0.00077 mM) and zinc excess (0.00308 mM) group tobacco samples. After 15 days of treatment, the gene was expressed at very significant high levels in zinc deficient (0 mM) group tobacco samples (fig. 3).
The qRT-PCR verification result of Nitab4.5_0003605g0060 shows that: after 5 days of treatment of tobacco plants with nutrient solutions of different zinc concentrations, the gene was expressed in small amounts in the tobacco samples of the group with insufficient zinc (0.00019, mM), while it was expressed very slightly in the tobacco samples of the group with insufficient zinc (0, mM), proper amount of zinc (0.00077 mM) and excessive zinc (0.00308 mM). After 15 days of treatment, the Nitab4.5_0003605g0060 gene was significantly expressed in the zinc deficient (0.00019 mM) group tobacco samples and very significantly expressed in the zinc deficient (0 mM) group tobacco samples. Whereas in the zinc-appropriate (0.00077 mM) and zinc-excess (0.00308 mM) groups of tobacco samples were weakly expressed (fig. 4).
The qRT-PCR verification result of Nitab4.5_0001724g0060 shows that: after tobacco plants were treated with nutrient solutions of different zinc concentrations for 5 days, the gene was hardly expressed in all of the zinc deficiency (0. 0 mM), zinc deficiency (0.00019 mM), zinc moderate (0.00077 mM) and zinc excess (0.00308 mM) group tobacco samples. On day 15, the Nitab4.5_0001724g0060 gene was very significantly expressed in zinc deficient (0 mM) and zinc deficient (0.00019 mM) group tobacco samples. While very slightly weakly expressed in the zinc-appropriate (0.00077 mM) and zinc-excess (0.00308 mM) group of tobacco samples (fig. 5).
The results of qRT-PCR verification of Nitab4.5_0006169g0020 show that: after 5 days of treatment of tobacco plants with different zinc concentration culture solutions, the gene is hardly expressed in the tobacco samples of the groups of zinc deficiency (0 mM), zinc deficiency (0.00019 mM), zinc right amount (0.00077 mM) and zinc excess (0.00308 mM). After 15 days of treatment, the Nitab4.5_0001724g0060 gene was very significantly expressed in zinc deficient (0 mM) and zinc deficient (0.00019 mM) group tobacco samples. Whereas expression was not evident in the zinc-appropriate (0.00077 mM) and zinc-excess (0.00308 mM) group of tobacco samples (fig. 6).
Through comparison analysis, the qRT-PCR detection results of the five genes are consistent with the corresponding RNA-seq results, which shows that when the tobacco plant is in zinc deficiency or insufficient for a period of time, the expression quantity of the five genes is obviously up-regulated; in the proper amount of zinc or in the excessive zinc state, the expression of the three genes is basically at the background level. In summary, 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 tobacco response to zinc deficiency stress.
Claims (8)
1. A set of genetic markers for use in the diagnosis of zinc deficiency in tobacco, characterized in that: the gene marker set 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 as SEQ ID NO. 1, the nucleotide sequence of Nitab4.5_0000572g0110 is shown as SEQ ID NO. 2, the nucleotide sequence of Nitab4.5_0003605g0060 is shown as SEQ ID NO. 3, the nucleotide sequence of Nitab4.5_0001724g0060 is shown as SEQ ID NO. 4, and the nucleotide sequence of Nitab4.5_0006169g0020 is shown as SEQ ID NO. 5.
2. Use of the set of gene markers of claim 1 in the diagnosis of zinc deficiency in tobacco.
3. A tobacco zinc deficiency nutrition diagnosis kit is characterized in that: the kit comprises reagents for detecting the gene marker set of claim 1 by RT-qPCR technology.
4. A tobacco zinc deficiency nutritional diagnostic kit according to claim 3, characterized in that: the kit comprises primers for specifically detecting Nitab4.5_0000143g0340, nitab4.5_0000572g0110, nitab4.5_0003605g0060, nitab4.5_0001724g0060 and Nitab4.5_0006169g0020.
5. The tobacco zinc deficiency nutritional diagnostic kit of claim 4, wherein: 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 primer for Nitab4.5_0000143g 0340:
RT-0143-F: 5ʹ-CGGCTTGTTACACCTCATTACTC-3ʹ，
RT-0143-R: 5ʹ-TTCACCACTTGCTTCAACTCTTC-3ʹ；
specific primer of Nitab4.5_0000572g 0110:
RT-0572-F: 5ʹ-GATAGACCTCTTGCTTCC-3ʹ，
RT-0572-R: 5ʹ-GATTAGTTGCTTGTGATGG-3ʹ；
specific primer for Nitab4.5_0003605g 0060:
RT-3605-F: 5ʹ-TTCAGCAAGAGTAATTGTTG-3ʹ，
RT-3605-R: 5ʹ- ATTCGGAAGTTCGTCAAG-3ʹ；
specific primer for Nitab4.5_0001724g 0060:
RT-1724-F: 5ʹ-GCTTCAGTCATTCCTTAG-3ʹ，
RT-1724-R: 5ʹ-TGCTGTCAATATCATCCT-3ʹ；
specific primer for Nitab4.5_0006169g 0020:
RT-6169-F: 5ʹ-TCTCACTTACAACTCCAG-3ʹ，
RT-6169-R: 5ʹ- AATACTCCTTCCACAATGA-3ʹ。
6. use of the tobacco zinc deficiency nutritional diagnostic kit of claim 3 in tobacco zinc deficiency nutritional diagnosis.
7. A method for diagnosing zinc deficiency nutrition of tobacco, which is characterized by comprising the following steps: the method comprises the following steps:
1) Collecting tobacco leaves to be detected, extracting total RNA, and reversely transcribing the total RNA into cDNA;
2) RT-qPCR detection is carried out by using cDNA obtained by reverse transcription as a template and using the specific primer in claim 5, and whether the tobacco to be detected is under zinc deficiency stress is judged according to whether the gene is up-regulated for expression.
8. The method for the nutritional diagnosis of zinc deficiency in tobacco according to claim 7, characterized in that: if the expression of Nitab4.5_0000143g0340, nitab4.5_0000572g0110, nitab4.5_0003605g0060, nitab4.5_0001724g0060 and Nitab4.5_0006169g0020 in the tobacco to be tested is obviously up-regulated compared with that of normal tobacco, the tobacco to be tested is in a zinc deficiency stress state.
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