Application of tobacco salicylic acid carboxymethyl transferase-like gene NtSAMT in promotion of tobacco seed germination

Abstract

The invention discloses an application of a tobacco salicylic acid carboxymethyl transferase-like gene NtSAMT in promoting tobacco seed germination, and belongs to the technical field of tobacco gene engineering. The CDS sequence of the NtSAMT gene is shown as SEQ ID NO.1. The gene is knocked out in a targeted manner through a gene editing technology, and the germination rate of tobacco seeds can be remarkably increased. By knocking out the NtSAMT gene, a tobacco strain with a high germination rate can be created. By utilizing the NtSAMT gene knockout mutant material created by the invention, an ideal target is provided for molecular design breeding, a new tobacco variety which rapidly germinates and neatly emerges can be cultivated, and the NtSAMT gene knockout mutant material has important application values for improving the tobacco seedling raising efficiency, reducing the production cost and guaranteeing stable yield and high quality.

Description

Technical Field

[0001] This invention relates to the field of tobacco genetic engineering technology, specifically to a tobacco salicylate carboxymethyltransferase-like gene. NtSAMT Application in promoting tobacco seed germination. Background Technology

[0002] tobacco( Nicotiana tabacum As an important economic crop and model plant, the rapid and uniform germination of tobacco seeds is the foundation for seedling cultivation and high-yield cultivation. Tobacco seeds are small and have limited storage substances, and their germination process is extremely sensitive to environmental conditions (such as temperature and moisture). Especially under adverse conditions, the germination rate of seeds decreases and the uniformity deteriorates, directly affecting seedling efficiency and early plant growth.

[0003] Currently, conventional techniques for promoting seed germination mainly include physical methods (such as seed soaking and germination induction), chemical methods (such as treatment with exogenous hormones or agents like gibberellin and potassium nitrate), and agronomic regulation. These methods can improve germination to some extent, but they have limitations such as unstable effectiveness, cumbersome operation, high cost, and potential environmental and residue risks. In particular, exploring key genes that can significantly enhance seed vigor and germination potential by investigating the intrinsic genetic regulatory mechanisms of plants and developing their applications has become a more promising research direction in the field of crop genetic improvement. SAMT The gene encodes salicylate carboxyltransferase, which current research confirms is highly conserved in dicotyledonous plants and mainly participates in the regulation of salicylic acid metabolism, playing a role in stress resistance and floral aroma synthesis. Currently, tobacco seed germination regulation still relies on traditional methods such as PEG soaking and acid-base treatment, leaving a gap in gene-level targeted regulation technology. Given... SAMT The function of this gene in promoting tobacco seed germination has not yet been explored, and its related applications have not been reported. Therefore, it is urgent to clarify this novel function of the gene in order to fill the gap in existing technology.

[0004] It should be noted that the information disclosed in the background section above is only used to enhance the understanding of the background of this disclosure, and therefore may include information that does not constitute prior art known to those skilled in the art. Summary of the Invention

[0005] The purpose of this invention is to provide a method for improving the germination rate of tobacco seeds, thereby addressing the problem of low germination rates in tobacco seeds. To achieve the above objectives, the present invention provides a tobacco salicylate carboxymethyltransferase-like gene. NtSAMT Application in promoting tobacco seed germination, NtSAMT The CDS nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0006] Furthermore, the above applications utilize gene editing technology to knock out [elements] in tobacco. NtSAMT Genetic implementation.

[0007] Furthermore, the above applications utilize CRISPR / Cas9 technology for gene editing, with the target sequence selected from any of the target sites shown in SEQ ID NO.7-9.

[0008] The present invention also provides a method for knocking out NtSAMT The gene is a CRISPR / Cas9 vector containing an amplified fragment of any of the targets shown in SEQ ID NO.7-9.

[0009] Preferably, the CRISPR / Cas9 vector is selected from the pHSbdcas9i vector.

[0010] The present invention also provides an Agrobacterium strain containing the above-mentioned CRISPR / Cas9 vector.

[0011] This invention also provides a tobacco salicylate carboxymethyltransferase-like gene. NtSAMT Application in creating tobacco lines with high germination rates.

[0012] The present invention also provides a set of methods for detection NtSAMT The primer pair for gene expression has the upstream primer shown in SEQ ID NO.3 and the downstream primer shown in SEQ ID NO.4.

[0013] The present invention has the following advantages: This invention discloses for the first time the application of the tobacco SMAT gene. By knocking out the expression of this gene, the germination rate of the knocked-out seeds can be significantly improved, which helps to increase the seed germination rate and can be used to create tobacco lines with rapid germination. It has the effect of shortening the seed germination time, allowing the crop to enter the growth cycle earlier, while reducing the cost of using high-quality seeds, and has significant application and economic value. Attached Figure Description

[0014] Figure 1 This shows the expression of the tobacco SMAT gene at different germination times in this invention.

[0015] Figure 2 These are the sequencing identification results of wild-type tobacco and mutants in this invention.

[0016] Figure 3 This is a statistical result of the germination of seeds from wild tobacco and gene mutant plants 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] Example 1 Tobacco NtSAMT Gene expression analysis Seeds of wild-type tobacco variety K326 were selected, and samples were taken at different germination times of 0, 1, 3, 5, 7, and 9 days. After germination, the seeds of each treatment were frozen in liquid nitrogen and then quickly ground into powder. The powder samples were stored at -80 ℃. The experiment was repeated 3 times.

[0020] RNA was extracted from seed samples from each treatment and reverse transcribed into cDNA using the HiScript® II Reverse Transcriptase system (Vazyme Biotech Co., Ltd). Using the cDNA as a template, the tobacco salicylate carboxymethyltransferase-like gene was analyzed by quantitative real-time PCR. NtSAMT We will analyze the expression of [the information].

[0021] Among them, the NtSAMT The CDS nucleotide sequence of the gene is shown in SEQ ID NO.1, and its encoded amino acid sequence is shown in SEQ ID NO.2. The specific sequences are as follows: A quantitative real-time PCR detection method was designed. NtSAMT The primer sequences are as follows: the upstream primer nucleotide sequence is shown in SEQ ID NO.3, and the downstream primer nucleotide sequence is shown in SEQ ID NO.4. The tobacco internal reference gene was used. EF1A The primers have the following nucleotide sequences: the upstream primer is shown in SEQ ID NO.5, and the downstream primer is shown in SEQ ID NO.6. All nucleic acid sequences are in the 5'-3' orientation.

[0022] NtSAMT Gene CDS sequence (SEQ ID NO.1):

[0023] NtSAMT The amino acid sequence encoded by the gene (SEQ ID NO.2): MKVVEVLHMNGGIGDISYAKNSLVQQKVILMTKPITEQAITDLYCSLFPQNLCIADLGCSSGANTFIVVSELIKIVEKERKKHGFQSPEFHFNFNDLPGNDFNTIFQSLDIFQQDLRKQIGEEFGPCFFSGVSGSFYTRLFPSNSLHFVHSSYSLMWLSQVPDAVENNKGNIYMASTSP PSVIKAYYKQYEKDFSNFLKYRSEELMKGGKMVLTFLGRESEDPTSKECCYIWELLAMALNELVVEGLIEEEKVDSFNIPQYTPSPADVKYVVEKEGSFTINQLEATRVHWNACNDKYKNVGYSVSRCMRAVAEPLLVSQFGEELMDLVFHKYEQIISECMSKAQTEFTNVIVSLTKTN.

[0024] NtSAMT Upstream primer for quantitative gene sequencing (SEQ ID NO.3): TCCTTGGTTCAGCAAAAGGT; NtSAMT Gene fluorescence quantitative downstream primer (SEQ ID NO.4): TGAAAGTGTTCGCTCCAGAGG; Fluorescent quantitative internal control primer F (SEQ ID NO.5): AGCTTCACCACCCAGGTCATC; Fluorescent quantitative internal control primer R (SEQ ID NO.6): AGAACGCCTGTCAATTCTTGG.

[0025] Obtain tobacco NtSAMT Gene expression at different times during seed germination, such as Figure 1 As shown, the results indicate that during seed germination... NtSAMT The gene expression level showed a gradually upward trend, indicating that as the seed germinates normally, NtSAMT Gene expression levels gradually increase.

[0026] Example 2 Tobacco NtSAMT Construction of gene mutants Based on the target gene sequence, gDNA targets were screened using conventional methods. Three gDNA targets were selected, and their primer nucleotide sequences are shown in SEQ ID NO.7-9. Amplification primers were designed based on the sequences corresponding to the targets. 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.

[0027] Knockout target 1 (SEQ ID NO.7): CCTCTGGAGCGAACACTTTCATA; Knockout target 2 (SEQ ID NO.8): TGGAGCGAACACTTTCATAGTGG; Knockout target 3 (SEQ ID NO.9): TGCTTTTTCAGTGGAGTGTCTGG.

[0028] The obtained containing the target gene NtSAMT The pHSbdcas9i vector plasmid targeting the target was transformed into Agrobacterium. The Agrobacterium carrying the transformation plasmid was then transformed into wild-type tobacco variety K326. Homozygous mutants were screened by PCR amplification using homozygous mutant screening primers and sequenced. Homozygous mutants were screened by comparison with the wild-type. The upstream primer F sequence is shown in SEQ ID NO. 10, and the downstream primer R sequence is shown in SEQ ID NO. 11. After identification of the transfected tobacco mutants, two tobacco genes were obtained. NtSAMT homozygous mutant Ntsamt-1 , Ntsamt-2 The sequencing identification results for wild-type and mutant strains are shown below. Figure 2 As shown, where, Figure 2 In the diagram, A represents the target gene editing site. Figure 2 B in the sequence comparison result is the result of the sequence comparison.

[0029] Screening primer F (SEQ ID NO.10): TGAACAGCAAAAGGTAATTCTCA; Screening primer R (SEQ ID NO.11): GAATGGACCGTACCTCTTTAG.

[0030] Example 3 Tobacco NtSAMT Phenotypic analysis of gene mutant materials Using the constructed NtSAMTCRISPR / Cas9 mutant gene Ntsamt-1 , Ntsamt-2 Germination and seedling emergence tests were conducted on the wild-type tobacco variety K326 (WT). Specifically, healthy, plump seeds were repeatedly selected and placed in petri dishes. 8 mL of distilled water was added to each dish, and the dishes were incubated at 25°C under light and darkness for 12 hours each. Both treatments germinated for 7 days, and the seedling emergence rate was recorded at least twice daily. The results showed that the greatest phenotypic difference occurred at 4.5 days. Figure 3 As shown, where, Figure 3 In the diagram, A represents the phenotypic representation of seed germination. Figure 3 B in the figure represents the statistical results of germination and seedling formation.

[0031] In summary, this invention utilizes tobacco... NtSAMT After gene knockout, the seed germination rate significantly increased, suggesting that this gene can negatively regulate the seed germination process. Tobacco was created using gene editing technology. NtSAMT The discovery of a new gene loss-of-function germplasm can be used to cultivate crop varieties with rapid seedling emergence and high seedling establishment rates. This achievement has significant application value in agricultural production. The gene can serve as a key target for molecular design breeding, accelerating the selection and breeding of new high-yield, high-efficiency, and stress-resistant crop lines, and providing genetic resources and technical support for ensuring food security and sustainable agricultural development.

[0032] 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.

Claims

1. A tobacco salicylate carboxymethyltransferase-like gene NtSAMT In promoting germination of tobacco seeds, said NtSAMT The CDS nucleotide sequence of the gene is shown as SEQ ID NO.

1.

2. Use according to claim 1, characterized in that, Knocking out genes in tobacco by gene editing technology NtSAMT is achieved.

3. Use according to claim 2, characterized in that, Gene editing employs CRISPR / Cas9 technology.

4. Use according to claim 3, characterized in that, The targeting sequence of the CRISPR / Cas9 is selected from any one of the target points shown in SEQ ID NO. 7-9.

5. A CRISPR / Cas9 vector for knocking out a gene, characterized in that, NtSAMT a gene, characterized in that, An amplified fragment comprising any one of the target sites shown in SEQ ID NO. 7-9, wherein the amplified fragment is NtSAMT The CDS nucleotide sequence of the gene is shown in SEQ ID NO.

1.

6. The CRISPR / Cas9 vector of claim 5, wherein, The vector is selected from a pHSbdcas9i vector.

7. An Agrobacterium strain containing the vector of any one of claims 5 or 6.

8. A tobacco gene for salicylic acid carboxymethyltransferase-like NtSAMT In the creation of high germination tobacco lines, the gene NtSAMT The CDS nucleotide sequence of the gene is shown as SEQ ID NO.

1.

9. A primer pair for detecting NtSAMT gene expression, characterized in that The upstream primer is shown as SEQ ID NO. 3, and the downstream primer is shown as SEQ ID NO. 4; wherein the CDS nucleotide sequence of the gene is shown as SEQ ID NO.

1. NtSAMT The CDS nucleotide sequence of the gene is shown as SEQ ID NO. 1.

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