Application of tobacco isopropyl malate synthase (NtIPMS) gene expression in the detection of tobacco seed aging

Abstract

This application discloses the application of the expression of the tobacco isopropyl malate synthase (NtIPMS) gene in the detection of tobacco seed aging. The expression level of the NtIPMS nucleotide sequence (SEQ ID NO. 1) in tobacco seeds has a quantitative relationship with seed vigor. Using this relationship, the degree of seed aging can be rapidly detected and seed vigor evaluated by detecting the expression level of SEQ ID NO. 1 in tobacco seeds.

Description

Technical Field

[0001] This application relates to the field of tobacco seed vigor detection technology, and in particular to the application of tobacco isopropyl malate synthase NtIPMS gene expression in the detection of tobacco seed aging degree. Background Technology

[0002] Seed vigor is formed during seed development and peaks at physiological maturity. After maturity, seeds undergo an irreversible process of gradual decline in vigor during storage, which can cause significant losses to germplasm resource preservation and agricultural production. Seed aging or deterioration is essentially a series of complex changes in cellular structure and physiological function, accompanied by changes in plasma membrane damage, organelle damage, decreased respiration efficiency and ATP production, and reduced enzyme activity. The continuous accumulation of toxic substances during seed storage can also have serious toxic effects on seeds. In addition, excessive ROS accumulation can lead to protein damage, lipid peroxidation, and genetic material damage, which is also one of the main factors contributing to seed deterioration.

[0003] In production, commonly used methods for detecting seed deterioration and aging during storage include the standard germination method, controlled deterioration method, accelerated aging method, tetrazolium staining method, methylene blue method, red ink staining method, and bromothymol blue method. Among these, the standard germination method, controlled deterioration method, and accelerated aging method are currently the most commonly used methods for seed vigor testing, but they have drawbacks such as being time-consuming, labor-intensive, and the results being easily affected by environmental and operational conditions. Other methods can only characterize seed viability. How to quickly and accurately evaluate the degree of tobacco seed aging and seed vigor, thereby improving seed value, remains an urgent problem to be solved. Therefore, it is necessary to develop molecular marker methods that can rapidly monitor tobacco seed aging and evaluate seed vigor, thereby accurately grasping the seed aging process, determining the optimal storage time, and avoiding production losses.

[0004] In existing technologies, α-isopropylmalate synthase is encoded by the leuA gene and participates in the synthesis of leucine in organisms. He et al. (Influence of isopropylmalate synthase OsIPMS1 on seed vigor associated with amino acid and energy metabolis min rice[J]. Yongqi He, Jinping Cheng, Ying He, Bin Yang, Yanhao Cheng, Can Yang, Hongsheng Zhang, Zhoufei Wang. Plant Biotechnology Journal. 2019(2)) cloned the α-isopropylmalate synthase gene OsIMPS1 located on chromosome 11 based on proteomic information, and used T-DNA and CRISPR / Cas9 mutant materials to reveal its mechanism of action in regulating rice seed vigor. However, existing research has only focused on the mechanism of action of this enzyme-related gene in rice seed vigor. Because the gene sequence of tobacco seeds and the expression of the enzymes contained therein are different from those of rice seeds, the expression of the enzyme OsIMPS1 is not found in the expression of tobacco seeds, and the existing technology has not seen the use of this enzyme-related gene in tobacco seed aging monitoring and tobacco seed vigor evaluation. Summary of the Invention

[0005] The purpose of this invention is to provide a method for detecting the aging degree of tobacco seeds, thereby solving the technical problem in the prior art that it is impossible to accurately and quickly evaluate seed vigor.

[0006] This application provides an application of the tobacco isopropyl malate synthase NtIPMS gene in the detection of tobacco seed aging, characterized in that the tobacco isopropyl malate synthase NtIPMS gene is SEQ ID NO.1.

[0007] This application first discovered a quantitative relationship between the expression level of the NtIPMS gene of this enzyme in tobacco seeds and the vigor of tobacco seeds. This can be used to rapidly detect the aging degree of tobacco seeds, quantitatively evaluate the vigor of tobacco seeds, improve detection efficiency, and improve the accuracy of seed vigor detection.

[0008] Preferably, the tobacco seeds include: naked tobacco seeds and coated tobacco seeds.

[0009] Preferably, it includes the following steps:

[0010] The expression levels of SEQ ID NO.1 in the tobacco seeds to be tested and fresh tobacco seeds were obtained, respectively;

[0011] The gene expression level of the seed to be tested relative to that of the fresh seed is obtained by the following formula: Gene expression level of the seed to be tested / Gene expression level of the fresh seed = Gene expression level fold;

[0012] The viability of the tobacco seeds to be tested was evaluated based on the fold change in gene expression levels.

[0013] Preferably, when the expression level in the naked tobacco seeds to be tested is increased by more than or equal to 4 times compared with the expression level in fresh naked tobacco seeds, the germination index decreases by more than or equal to 50%, and the seedling rate after 12 days decreases by less than or equal to 78.0%; when the expression level in the coated tobacco seeds to be tested is increased by more than or equal to 2.2 times compared with the expression level in fresh coated tobacco seeds, the germination index decreases by more than or equal to 26%, and the seedling rate after 12 days decreases by less than or equal to 81.3%.

[0014] The fresh seeds described in this application include: newly produced and processed seeds stored for less than one month at 17°C and a relative moisture content of 40%.

[0015] This application first discovered that the primer pair can be used to achieve real-time quantitative PCR analysis of NtIPMS gene expression in tobacco seeds.

[0016] Preferably, in tobacco seeds, the upstream primer of the primer pair used for NtIPMS detection of the tobacco isopropyl malate synthase gene is shown in SEQ ID NO. 2, and the downstream primer is shown in SEQ ID NO. 3. The tobacco gene NtEF-1α is used as an internal reference gene, and the upstream primer of this gene is shown in SEQ ID NO. 4, and the downstream primer is shown in SEQ ID NO. 5.

[0017] Experimental studies have revealed a quantitative relationship between the expression of this gene sequence in tobacco seeds and seed vigor. Based on this quantitative relationship, the aging degree of tobacco seeds can be detected, and seed vigor can be quantitatively evaluated.

[0018] Another aspect of this application provides a method for detecting the aging degree of tobacco seeds based on the expression of the tobacco isopropyl malate synthase NTIPMS gene, comprising the following steps:

[0019] Step S1: After freezing the tobacco seeds to be tested and fresh tobacco seeds with liquid nitrogen, quickly grind them into powder to obtain the test sample, which is stored at -80℃. Coated tobacco seeds need to be washed with water in an 80-mesh bag to obtain the naked tobacco seeds, and the surface moisture is quickly dried before preparing the test sample.

[0020] Step S2: Detect the expression level of the tobacco isopropyl malate synthase NtIPMS gene SEQ ID NO.1 as described in any one of claims 1 to 4 in the tested tobacco seeds and fresh tobacco seeds;

[0021] Step S3: Obtain the fold change in NtIPMS gene expression level between the tobacco seeds to be tested and fresh seeds to evaluate the viability of the tobacco seeds.

[0022] This method can detect the aging degree of tobacco seeds, quantitatively evaluate seed vigor, improve seed detection efficiency, and avoid production losses.

[0023] Preferably, when the expression level in the unprocessed tobacco seeds to be tested is increased by more than or equal to 4 times compared with the expression level in fresh unprocessed tobacco seeds, the germination index decreases by more than or equal to 50%, and the seedling rate after 12 days decreases by less than or equal to 78.0%; when the expression level in the coated tobacco seeds to be tested is increased by more than or equal to 2.2 times compared with the expression level in fresh coated tobacco seeds, the germination index decreases by more than or equal to 26%, and the seedling rate after 12 days decreases by less than or equal to 81.3%.

[0024] Preferably, step S2 includes the following steps: extracting RNA from the sample to be tested and reverse transcribing it to obtain cDNA, and using the cDNA as a template to detect the expression level of the SEQ ID NO.1 gene in tobacco seeds by real-time PCR.

[0025] Preferably, the upstream primer used for the quantitative real-time PCR detection of the NtIPMS gene is shown in SEQ ID NO. 2, and the downstream primer used for the quantitative real-time PCR detection of the NtIPMS gene is shown in SEQ ID NO. 3. The tobacco gene NtEF-1α is used as an internal reference gene, and the upstream primer for this gene is shown in SEQ ID NO. 4, and the downstream primer is shown in SEQ ID NO. 5.

[0026] By using the tobacco gene NtEF-1α as an internal reference gene and in conjunction with the primer pair used, the expression level of this gene in tobacco seeds can be accurately detected.

[0027] The beneficial effects that this application can produce include:

[0028] 1) The application of the expression of tobacco isopropyl malate synthase NtIPMS gene provided in this application in the detection of the aging degree of tobacco seeds. By detecting the expression of isopropyl malate synthase NtIPMS gene in seeds with different aging degrees, the relationship between gene expression and aging degree during seed aging is established. The gene is used as a molecular marker for seed aging detection, thereby effectively monitoring the aging degree of seeds during storage.

[0029] 2) The application of the tobacco isopropyl malate synthase (NtIPMS) gene expression method provided in this application in the detection of tobacco seed aging involves detecting the NtIPMS expression levels of both tested and fresh tobacco seeds, and obtaining the rate of change in the gene expression levels of both, thereby rapidly and accurately evaluating the seed vigor. This method has high detection efficiency and can significantly shorten the seed vigor evaluation time, making it particularly suitable for tobacco seed vigor evaluation in emergency situations. Attached Figure Description

[0030] Figure 1 is a schematic diagram of the seed experiment in Example 1 of this application, where A is a photo of each experimental group, B is a bar chart of the relationship between germination index and aging time after aging treatment for each experimental group, and C is a bar chart of the relationship between seedling rate after aging treatment and aging time for each experimental group.

[0031] Figure 2 shows the relationship between the relative expression level of NTIPMS in seeds aged at different aging times in Example 2 of this application and aging time;

[0032] Figure 3 is a schematic diagram of the coated seed experiment in Example 3 of this application, where A is a photo of each experimental group; B is a bar chart showing the relationship between the germination index and aging time of each experimental group after aging treatment; and C is a bar chart showing the relationship between the seedling rate after aging treatment and aging time of each experimental group.

[0033] Figure 4 shows the relationship between the relative expression level of NTIPMS in seeds coated at different aging times and aging time in Example 4 of this application; Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. The components of the embodiments of the present invention described and shown in the accompanying drawings can generally be arranged and designed in various different configurations.

[0035] Therefore, the following detailed description of the embodiments of the invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the invention without inventive effort are within the scope of protection of the invention.

[0036] Technical features not intended to solve the technical problems of this application are all set or installed according to common methods in the prior art, and will not be described in detail here. Example

[0037] Unless otherwise specified, all instruments and materials used in the following examples were purchased through commercial channels.

[0038] Unless otherwise specified, all methods used in the examples are conventional methods. Primers and sequencing were performed by Tianyi Huiyuan Biotechnology Co., Ltd. All enzymes and reverse transcription kits used in the experiments were purchased from Novizan Biotechnology Co., Ltd., and the methods were performed in accordance with the instructions.

[0039] Example 1: Comparison of viability of naked tobacco seeds at different aging times

[0040] The seeds of MS Yunyan 87 were placed in an artificial aging chamber and then placed on a layer of moist filter paper.

[0041] The seeds were aged for 0 days, 3 days, 6 days, and 8 days under conditions of 45℃ and 100% relative humidity to obtain a control group, a second aged seed group, a third aged seed group, and a fourth aged seed group.

[0042] Germination tests were conducted using a control group, a second aged seed group, a third aged seed group, and a fourth aged seed group. Seeds from these groups were first soaked in 75% alcohol for 30 seconds, then in 30% sodium hypochlorite for 10 minutes, washed 4-5 times with sterile water, and then soaked in sterile water for 10 minutes. The seeds were then wiped clean and placed in a 9cm diameter petri dish lined with two layers of filter paper. 5mL of distilled water was added, and the dish was incubated at 25℃ (12 hours in light / 12 hours in darkness).

[0043] During cultivation in a light-incubator, the number of germinations and seedlings was recorded daily. Photos of each group after the experimental treatments are shown below. Figure 1 As shown in A. The above experiment was repeated 3 times for the same group of seeds.

[0044] The results showed that, compared with the fresh seeds in the control group, seed vigor gradually decreased with increasing aging time (see...). Figure 1 (B, C) After aging naked seeds for 6 days, the germination index decreased to 9.79, a reduction of 50%, and the seedling survival rate decreased to 78.0% after 12 days. After aging naked seeds for 8 days, the germination index decreased to 4.65, a reduction of 76%, and the seedling survival rate decreased to 37.3% after 12 days.

[0045] Example 2: Gene expression analysis of naked tobacco seed at different aging times

[0046] The control group, the second aged seed group, and the third aged seed group obtained in Example 1 were taken respectively.

[0047] Seed samples from the fourth aged seed group. Seeds were rapidly ground into powder after being frozen in liquid nitrogen, and the samples were stored at -80℃. The experiment was repeated three times.

[0048] RNA was extracted from each sample using the EZNA® HPPlantRNA Kit (Omega, Bio-tek, Inc.); cDNA was generated by reverse transcription using the HiScript® II Reverse Transcriptase System (Vazyme Biotech Co., Ltd.) kit, and used as a template; analysis was performed by quantitative real-time PCR. The primer sequences were detected by quantitative real-time PCR. The upstream primer sequence is shown in SEQ ID NO. 2, and the downstream primer sequence is shown in SEQ ID NO. 3. Primers for the tobacco internal reference gene NtEF-1α were used. The upstream primer sequence is shown in SEQ ID NO. 4, and the downstream primer sequence is shown in [the sequence listing text is missing].

[0049] As shown in SEQ ID NO.5.

[0050] The results showed that the transcription level of the NtIPMS gene in seeds gradually increased with the increase of aging time in naked seeds. (See attached results). Figure 2 Compared with fresh seeds, the expression of the NtIPMS gene was upregulated to 14 after 6 days of aging in naked seeds, an increase of 9.7 times. After 8 days of aging in naked seeds, the expression of the NtIPMS gene was upregulated to 28.

[0051] Based on Example 1, it was found that in the third aged seed group after 6 days of aging treatment, when the NtIPMS gene expression was upregulated to 9.7-fold, the seed germination index dropped to below 10, which was 50% lower than the control. In the fourth aged seed group after 8 days of aging treatment, when the NtIPMS gene expression was upregulated to 19.5-fold, the seed germination index dropped to 4.65, which was 76% lower than the control.

[0052] It is evident that the expression of the NtIPMS gene can serve as a molecular marker for the aging degree of naked tobacco seeds. By detecting the expression of this gene during the aging process of naked seeds and comparing it with the expression of fresh seeds, a quantitative relationship between the two was obtained: when the gene is upregulated by ≥9.7 times, the germination index decreases significantly by ≥50%, and the seedling rate after 12 days decreases significantly by ≤78.0%.

[0053] Example 3: Comparison of vigor of tobacco-coated seeds at different aging times

[0054] Pre-germinated and coated seeds of MS Yunyan 87 were placed in an artificial aging chamber on two layers of moist filter paper and aged for 0 days, 1 day, 2 days, and 3 days at a temperature of 47℃ and a relative humidity of 100%. This resulted in a control group, the fifth aged seed group, the sixth aged seed group, and the seventh aged seed group.

[0055] Germination tests were conducted using a control group, a fifth-aged seed group, a sixth-aged seed group, and a seventh-aged seed group. Seeds were placed in petri dishes (9 cm in diameter) lined with two layers of filter paper, 10 mL of distilled water was added, and the dishes were placed in a 25°C incubator (12 hours of light / 12 hours of darkness). The number of germinated seeds and seedlings was recorded daily. Photos of each group after the experimental treatments are shown below. Figure 3 As shown in A. The above experiment was repeated 3 times for the same group of seeds.

[0056] The results showed that, compared with the control group of coated seeds, seed vigor gradually decreased with increasing aging time (see...). Figure 3 (B, C) After one day of aging, the germination index of the coated seeds decreased to 13.90.

[0057] The germination rate decreased by 26%, and the seedling survival rate decreased to 81.3% after 12 days. After 2 days of aging, the germination index of coated seeds decreased to 11.68, a decrease of 38%, and the seedling survival rate decreased to 70.0% after 12 days. After 3 days of aging, the germination index of coated seeds decreased to 7.81, a decrease of 58%, and the seedling survival rate decreased to 53.0% after 12 days.

[0058] Example 4: Gene expression analysis of tobacco-coated seeds at different aging times

[0059] The control group, the fifth aged seed group, and the sixth aged seed group obtained in Example 3 were taken respectively.

[0060] Coated seed samples from the seventh aging seed group. The coated seeds were washed with water in an 80-mesh bag to obtain the naked tobacco seeds. After quickly drying the surface moisture, the seeds were frozen in liquid nitrogen and then rapidly ground into powder. The samples were stored at -80℃. The experiment was repeated three times.

[0061] RNA was extracted from each sample using the EZNA® HPPlantRNA Kit (Omega, Bio-tek, Inc.); cDNA was reverse transcribed using the HiScript® II Reverse Transcriptase System (Vazyme Biotech Co., Ltd.) kit, and used as a template; analysis was performed using quantitative real-time PCR. The primer sequences were detected by quantitative real-time PCR. The upstream primer sequence is shown in SEQ ID NO. 2, and the downstream primer sequence is shown in SEQ ID NO. 3. Primers for the tobacco internal reference gene NtEF-1α were used. The upstream primer sequence is shown in SEQ ID NO. 4, and the downstream primer sequence is shown in SEQ ID NO. 5.

[0062] The results showed that the transcription level of the NtIPMS gene in the seeds gradually increased with the increase of the aging time of the coated seeds (see [link to relevant documentation]). Figure 4Compared with the control, the NtIPMS gene expression in the seventh aged seed group after 1 day of aging treatment was upregulated to 2.21, an increase of 2.2-fold. The NtIPMS gene expression in the seventh aged seed group after 2 days of aging treatment was upregulated to 2.44, an increase of 2.4-fold. The NtIPMS gene expression in the seventh aged seed group after 2 days of aging treatment was upregulated to 3.94, an increase of 3.9-fold.

[0063] Based on the findings in Example 3, the expression of the NtIPMS gene can serve as a molecular marker for the aging degree of tobacco coated seeds. By detecting the expression of this gene during the aging process of coated seeds and comparing it with the expression in fresh seeds, a quantitative relationship was obtained: when the expression of the NtIPMS gene in aged seeds was upregulated by ≥2.2 times, the germination index decreased significantly by ≥26%, and the seedling rate after 12 days decreased significantly by ≤81.3%.

[0064] Example 5: Method for Detecting the Aging Degree of Naked Tobacco Seeds

[0065] Naked tobacco seeds and fresh seeds were collected as test seeds. The test seeds and fresh seeds were frozen in liquid nitrogen and then rapidly ground into powder to obtain the test samples, which were stored at -80℃. The experiment was repeated three times.

[0066] RNA was extracted from the test samples using the EZNA® HPPlantRNA Kit (Omega, Bio-tek, Inc.); cDNA was generated by reverse transcription using the HiScript® II Reverse Transcriptase System (Vazyme Biotech Co., Ltd.) kit, and used as a template; analysis was performed using quantitative real-time PCR. The primer sequences for quantitative real-time PCR detection are shown in SEQ ID NO. 2 and SEQ ID NO. 3 of the sequence listing. Primers for the tobacco internal reference gene NtEF-1α were used; the upstream primer sequence is shown in SEQ ID NO. 4 and the downstream primer sequence is shown in the sequence listing.

[0067] As shown in SEQ ID NO. 5;

[0068] The NtIPMS gene expression levels in naked tobacco seeds and fresh seeds were obtained, and the fold increase in NtIPMS gene expression level between the tested tobacco seeds and fresh seeds was calculated using the following formula:

[0069] Gene expression level fold increase = gene expression level of seed to be tested / gene expression level of fresh seed.

[0070] Following the above method, after the seeds of MS Yunyan 87 were harvested and stored, they were stored at 17℃ and 40% relative moisture content. The NtIPMS gene expression level was detected within one month of storage. The NtIPMS gene expression level of the MS Yunyan 87 seeds to be tested was detected, and the gene expression level of the seeds to be tested relative to fresh seeds was obtained.

[0071] In this embodiment, when the NtIPMS gene expression level of the seed to be tested was 19.5 times that of the gene expression level of the fresh seed, the germination index of the seed to be tested decreased to 4.65, and the seedling rate after 12 days decreased to 37.3%.

[0072] The method provided in this application enables rapid evaluation of the viability of naked tobacco seedlings.

[0073] Example 6: Method for Detecting the Aging Degree of Tobacco-Coated Seeds

[0074] Repeat Example 5, except that the tobacco coated seeds need to be washed with water in an 80-mesh bag to obtain the naked tobacco seeds, and the surface moisture is quickly dried.

[0075] In this embodiment, when the NtIPMS gene expression level of the MS Yunyan 87 coated seeds was 2.44 times that of the fresh coated seeds, the germination index of the coated seeds decreased to 11.68 and the seedling rate after 12 days decreased to 70.0%.

[0076] The method provided in this application enables rapid evaluation of the vigor of tobacco-coated seeds.

[0077] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A tobacco isopropyl malate synthase NtIPMS The application of genes in the detection of tobacco seed aging is characterized by, The tobacco isopropyl malate synthase NtIPMS The gene is SEQ ID NO.1; the application is based on isopropyl malate synthase in tobacco seeds. NtIPMS Gene expression levels are used to detect the aging degree of tobacco seeds.

2. The application according to claim 1, characterized in that, Tobacco seeds include naked tobacco seeds and coated tobacco seeds.

3. The application according to claim 1, characterized in that, The upstream primer of the primer pair used for detecting the tobacco isopropyl malate synthase NtIPMS gene in tobacco seeds is shown in SEQ ID NO.2, and the downstream primer is shown in SEQ ID NO.

3. The tobacco gene NtEF-1α is used as an internal reference gene, and the upstream primer of the internal reference gene is shown in SEQ ID NO.4, and the downstream primer is shown in SEQ ID NO.

5.

4. A tobacco isopropyl malate synthase-based enzyme NtIPMS A method for detecting the aging degree of tobacco seeds by gene expression, characterized in that, Includes the following steps: Step S1: After freezing the tobacco seeds to be tested and fresh tobacco seeds with liquid nitrogen, they are quickly ground into powder to obtain the test sample. The test sample is stored at -80℃. Tobacco-coated seeds need to be washed with clean water in an 80-mesh bag to obtain naked tobacco seeds. After quickly drying the surface moisture, test samples are prepared. Step S2: Detect tobacco isopropyl malate synthase in the tobacco seeds to be tested and fresh tobacco seeds as described in any one of claims 1 to 3. NtIPMS Expression level of gene SEQ ID NO.1; Step S3: Obtain the tobacco seeds to be tested relative to fresh seeds. NtIPMS Gene expression level fold change is used to evaluate tobacco seed vigor.

5. The detection method according to claim 4, characterized in that, Using the tobacco seeds to be tested relative to fresh tobacco seeds NtIPMS Fold-up of gene expression levels to evaluate tobacco seed vigor; The gene expression level in the seed to be tested / the gene expression level in the fresh seed = the multiple of the gene expression level.

6. The detection method according to claim 4, characterized in that, Step S2 includes the following steps: extracting RNA from the sample to be tested and reverse transcribing it to obtain cDNA, and using the cDNA as a template to detect the expression level of the SEQ ID NO.1 gene in tobacco seeds by real-time PCR.

7. The detection method according to claim 6, characterized in that, The upstream primer used for quantitative real-time PCR detection of the NtIPMS gene is shown in SEQ ID NO.2 of the sequence listing, and the downstream primer used for quantitative real-time PCR detection of the NtIPMS gene is shown in SEQ ID NO.3 of the sequence listing.

8. The detection method according to claim 6, characterized in that, The tobacco gene NtEF-1α was used as an internal reference gene. The upstream primer of the internal reference gene is shown in SEQ ID NO.4 of the sequence listing, and the downstream primer is shown in SEQ ID NO.5 of the sequence listing.

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