Application of zeatin glucose transferase gene in regulating tobacco seed germination time

Abstract

The application discloses application of a tobacco zeatin glucosyltransferase gene in regulating tobacco seed germination time. The gene is NtZOG, the nucleotide sequence of which is shown as SEQ ID NO. 1, and the amino acid sequence of which is shown as SEQ ID NO. 2. Research finds that by knocking out the tobacco zeatin glucosyltransferase gene NtZOG from wild tobacco K326, a tobacco mutant with the NtZOG gene knocked out is constructed. By comparing the germination of the wild tobacco K326 and the mutant, it is found that the germination rate of the mutant is significantly lower than that of the wild tobacco at 4.5 days of development, and is as low as about 50%, so it can be seen that the tobacco zeatin glucosyltransferase gene NtZOG has an important regulating effect on tobacco seed germination, and the expression of the gene can be regulated to regulate the germination time of tobacco, and the application has a potential application prospect in the tobacco planting field.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the application of a tobacco zeatin glucosyltransferase gene in regulating the germination time of tobacco seeds. Background Technology

[0002] Seed vigor is a key factor in agricultural production, affecting crop germination and growth. Zeatin is an important plant growth regulator, and its O-glycosylation modification is widely present in plant tissues, playing a crucial role in regulating the level of active zeatin. Zeatin glucosyltransferase (ZOG) can convert zeatin into its O-glycosylated form, thereby affecting its activity. Reports on zeatin glucosyltransferases in other crops mainly focus on their role in regulating plant growth, development, and stress resistance. Research on the molecular mechanisms by which zeatin glucosyltransferase (ZOG) regulates seed germination is limited, especially regarding the impact of zeatin glucosyltransferase genes in tobacco on tobacco germination. Summary of the Invention

[0003] The purpose of this invention is to provide the use of the tobacco zeatin glucosyltransferase gene NtZOG in regulating tobacco seed germination, providing a reference for regulating tobacco germination time, and showing promising application prospects in the field of tobacco cultivation.

[0004] To achieve the above objectives, the present invention provides a novel use for the tobacco zeatin glucosyltransferase gene NtZOG, which can delay the germination time of tobacco by reducing the expression of the NtZOG gene; wherein the nucleotide sequence of the NtZOG gene is shown in SEQ ID NO.1.

[0005] The present invention also provides an application of the tobacco zeatin glucosyltransferase gene NtZOG in the field of tobacco cultivation. By reducing the expression of the NtZOG gene, the germination time of tobacco can be delayed; wherein, the nucleotide sequence of the NtZOG gene is shown in SEQ ID NO.1.

[0006] Preferably, the reduction of NtZOG gene expression is achieved by constructing an NtZOG gene knockout vector, and then transferring the obtained knockout vector into tobacco to obtain an NtZOG gene knockout tobacco mutant.

[0007] Preferably, the knockout vector is selected from the pHSbdcas9i vector.

[0008] Preferably, the target sequence targeted by the above knockout vector has a nucleotide sequence as shown in SEQ ID NO.3 or SEQ ID NO.4.

[0009] Preferably, the tobacco mutant described above is obtained by constructing a knockout vector using Agrobacterium-mediated transformation.

[0010] The present invention also provides an application of the tobacco zeatin glucosyltransferase gene NtZOG in promoting early germination of tobacco seeds. By constructing a tobacco line overexpressing this gene, it can be used to advance the germination time of the tobacco line. The nucleotide sequence of the NtZOG gene is shown in SEQ ID NO.1.

[0011] This invention also provides an application of the tobacco zeatin glucosyltransferase gene NtZOG in the preparation of early-germinating tobacco lines. By constructing an overexpression vector of the gene NtZOG and transferring the overexpression vector into tobacco, early-germinating tobacco lines can be obtained, resulting in tobacco lines with fast germination.

[0012] The present invention has the following advantages:

[0013] This invention isolates and clones the NtZOG gene from tobacco. By constructing a CRISPR / Cas9 mutant, it is the first time that this gene is involved in the regulation of tobacco seed germination. Reducing the expression of this gene leads to delayed germination of tobacco seeds, which in turn shows that increasing the expression of this gene has the potential to promote early germination of tobacco. This invention has reference and guiding significance in the study of the mechanism of tobacco germination.

[0014] This invention provides a foundation for screening tobacco varieties with fast germination and also provides important genetic resources for improving the germination speed of tobacco. It is of great significance to production and has potential application value in the field of tobacco production and planting. Attached Figure Description

[0015] Figure 1 This shows the expression of the tobacco NtZOG gene at different seed imbibition times in this invention.

[0016] Figure 2 This shows the germination phenotype of the tobacco NtZOG mutant material in this invention. Detailed Implementation

[0017] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0018] Note: Unless otherwise specified, the experimental methods in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0019] Experimental Example 1: Analysis of NtZOG Gene Expression in Tobacco

[0020] Seeds of wild-type tobacco variety K326 were selected and subjected to imbibition treatment for 0, 12, 24, 36, and 48 hours, respectively. After the treatment, the seeds were frozen in liquid nitrogen and then quickly ground into powder. The powder samples were stored at -80℃. The experiment was repeated three times.

[0021] RNA was extracted from each sample using the TransZol Plant kit (Transgen, www.transgen.com); II. The Reverse Transcriptase System (Vazyme Biotech Co., Ltd.) kit was used to reverse transcribe cDNA. Using the cDNA as a template, the expression of the tobacco zeatin glucosyltransferase gene NtZOG was analyzed by quantitative real-time PCR. The nucleotide sequence of the NtZOG gene is shown in SEQ ID NO.1, and its amino acid sequence is shown in SEQ ID NO.2. Primer sequences for quantitative real-time PCR detection of NtZOG were designed. The upstream primer nucleotide sequence is shown in SEQ ID NO.9, and the downstream primer nucleotide sequence is shown in SEQ ID NO.10. The tobacco internal reference gene EF1A primer was used. The upstream primer nucleotide sequence is shown in SEQ ID NO.11, and the downstream primer nucleotide sequence is shown in SEQ ID NO.12. The expression of the tobacco NtZOG gene at different seed imbibition times was obtained as follows: Figure 1 As shown, the results indicate that the expression level of the NtZOG gene gradually decreased during seed development and imbibition. This suggests that the expression level of the NtZOG gene gradually decreased as the seed developed.

[0022] Nucleotide sequence of the NtZOG gene (SEQ ID NO.1):

[0023]

[0024] The amino acid sequence encoded by the NtZOG gene (SEQ ID NO.2):

[0025] MAKTSNLENHDQEKVAVVMVPLPAQGHLNQLLHLSRLISAYHLPLHYVGTTTHTRQAKIRVQGWDPHSISNIHFHEFSTPSCEIPPPNPQSSTKFPSQLMPSFHATCHLRDPITSLLRE LARCNKRVIVIYDSLIAWVLQDAPFIANVECYSFRSISALNIHSLALEFAGKSTEPNLPSIEGCFTQEFSEFSRVQTEFRELVNSGDLYNSCYEIEGLYLDLLAKEKSASHKQWAVGPLN PVIPYDKKDSNKRHKCLEWLDKQEPNSVIFVSFGTTTSLSDNEIEELAIGLEQRQQKFIWVLRDADKGDIFTGDVRKVELPKGYEERVIGRGLIVRDWAPQLEILGHLSTGGFMSHCGW NSCMESISMGVPIIAWPMHSDQPRNAFLVTNVLKIGVVLKDWALREELVTSVMVEECVKRLMDSVEGDKMRERAVELRQSVVDGGGSHKEMDSFITHITR*; NtZOG gene fluorescence quantification primer F (SEQ ID NO.9):

[0026] TTCGGATCAGCCAAGAAATGCG;

[0027] NtZOG gene fluorescence quantitative primer R (SEQ ID NO.10):

[0028] TCTGAGTGCCCAATCCTTCAGC;

[0029] Tobacco internal reference gene EF1A primer F (SEQ ID NO.11):

[0030] AGCTTCACCACCCAGGTCATC;

[0031] Primer R for the tobacco internal reference gene EF1A (SEQ ID NO.12):

[0032] AGAACGCCTGTCAATTCTTGG.

[0033] Example 2: Construction of tobacco NtZOG gene mutant

[0034] Based on the target gene sequence, gDNA targets were screened using conventional methods. Two gDNA targets were selected, designated as target 1 and target 2, with nucleotide sequences shown in SEQ ID NO.3 and SEQ ID NO.4, respectively. Amplification primers F and R were designed based on these target sequences. The nucleotide sequence of primer F is shown in SEQ ID NO.5, and the nucleotide sequence of primer R is shown in SEQ ID NO.6. These primers are universal for both targets; target 1 is located on primer F, and target 2 is located on primer R. The specific sequences are as follows. PCR products containing the target targets were amplified using a Vazyme P520 (Vazyme Biotech Co., Ltd.). The PCR products were purified and recovered, and then constructed into the pHSbdcas9i vector using T4 ligase.

[0035] The obtained pHSbdcas9i vector plasmid containing the target gene NtZOG was transformed into Agrobacterium. The Agrobacterium carrying the transformation plasmid was then transformed into wild-type tobacco variety K326. PCR amplification and sequencing were performed using homozygous mutant screening primers, and homozygous mutants were screened by comparison with the wild type. The upstream primer sequence is shown in SEQ ID NO.7, and the downstream primer sequence is shown in SEQ ID NO.8. The specific sequences are as follows. After identification of the transfected tobacco mutants, two tobacco gene NtZOG mutants, Ntzog-1 and Ntzog-2, were obtained.

[0036] gDNA target sequence 1 (SEQ ID NO.3):

[0037] AGGTGGAATTTCACAAGAT;

[0038] gDNA target sequence 2 (SEQ ID NO.4):

[0039] TGCTTGACGGGTGTGAGTGG;

[0040] Amplification primer F (SEQ ID NO.5):

[0041] CAGTGGTCTCATGCAAGGTGGAATTTCACAAGATGGTTTTAGAGCT AG;

[0042] Amplification primer R (SEQ ID NO.6):

[0043] CAGTGGTCTCAAAACCCACTCACACCCGTCAAGCATG;

[0044] Homozygous mutant screening primer F (SEQ ID NO.7):

[0045] GCAACGCTCTGTCATCGTTACAAT;

[0046] Primer R (SEQ ID NO.8) for screening homozygous mutants:

[0047] GCGATTAAGTTGGGTAACGCCAGGG.

[0048] Phenotypic analysis of tobacco NtZOG gene mutant materials in Experiment Example 3

[0049] Germination experiments were conducted using the constructed NtZOG CRISPR / Cas9 mutants Ntzog-1 and Ntzog-2, and the wild-type tobacco variety K326. The specific method is as follows: Healthy, plump seeds were selected for each iteration, placed in petri dishes, and 8 mL of distilled water was added. The dishes were then incubated at 25°C under light and darkness for 12 hours each. Germination occurred in the water for 4.5 days, and the germination rate at different time points was recorded. The experiment was repeated three times. The germination phenotypes of various seeds are shown below. Figure 2 As shown in A, the germination rate was quantitatively statistically analyzed, and the results are shown in [Figure A]. Figure 2 As shown in B and C, the germination rate of the Ntzog mutant was significantly reduced at 4.5 days. The germination rate of wild-type K326 (WT) was 85%, while that of the mutant Ntzog-1 was 31% and that of the mutant Ntzog-2 was 35%. Compared with wild-type seeds, the seedling survival rate of seeds mutated with the Ntzog gene was reduced by 50%. This indicates that the Ntzog gene plays an important regulatory role in tobacco seed germination, and the reduction in its expression can significantly delay tobacco seed germination.

[0050] Based on conventional knowledge in this field and the above conclusions, it can be concluded that reducing the expression of this gene can decrease the germination rate of tobacco seeds at 4.5 days, thus delaying germination time. Therefore, the expression level of this gene is inversely proportional to the germination rate of tobacco seeds. Consequently, increasing the expression of this gene can promote earlier seed germination. Based on this, a mutant overexpressing the tobacco NtZOG gene can be constructed to promote earlier germination in this tobacco mutant.

[0051] In summary, this invention discloses the application of the tobacco zeatin glucosyltransferase gene in regulating tobacco seed germination time. By knocking out the NtZOG gene of the tobacco zeatin glucosyltransferase gene in wild-type tobacco K326, an NtZOG gene knockout tobacco mutant was constructed. The germination rate of this mutant was significantly lower than that of wild-type tobacco at 4.5 days of development, reaching as low as about 50%. This demonstrates that the tobacco zeatin glucosyltransferase gene NtZOG plays a crucial regulatory role in tobacco seed germination time. Controlling the expression of this gene can significantly delay tobacco germination time, showing potential application prospects in the tobacco cultivation field.

[0052] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

[0053]

[0054]

[0055]

[0056]

[0057]

Claims

1. A tobacco zeatin glucosyltransferase gene NtZOG Its application in regulating tobacco seed germination time is characterized by... By knocking out the above NtZOG Gene expression delays the germination time of tobacco seeds; wherein, the aforementioned NtZOG The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The knockout NtZOG Gene expression, enabling the construction of NtZOG Gene knockout vector, and then the knockout vector is transferred into tobacco to obtain... NtZOG Gene knockout tobacco mutant.

3. The application according to claim 2, characterized in that, The knockout vector is selected from the pHSbdcas9i vector.

4. The application according to claim 2, characterized in that, The target sequence targeted by the knockout vector has a nucleotide sequence as shown in SEQ ID NO.3 or SEQ ID NO.

4.

5. The application according to claim 2, characterized in that, The tobacco mutant is constructed by transforming the knockout vector using Agrobacterium-mediated transformation.

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