Application of premna microphylla turcz diene oxygenase gene NtPO

Abstract

The invention discloses application of a premna microphylla turcz diene oxygenase gene NtPO, the nucleotide sequence of the premna microphylla turcz diene oxygenase gene NtPO is shown as SEQ ID NO.1, the premna microphylla turcz diene oxygenase gene NtPO contains 450 basic groups, the gene is derived from tobacco, and the premna microphylla turcz diene oxygenase gene NtPO is obtained by editing the NtPO gene. The topped plant height, stem girth, waist leaf length, waist leaf width and leaf area of the gene editing plant in the mature period are all higher than those of a control plant. In a word, according to the application, the NtPO gene is knocked out by utilizing a CRISPR / Cas9 mediated gene editing technology to obtain a gene editing plant which affects the growth and development of the plant, and a genetic material and a theoretical basis are provided for tobacco premna microphylla diene oxygenase gene research and tobacco growth and development research.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and in particular to the application of the tobacco tofu spirodiene oxygenase gene NtPO. Background Technology

[0002] Premnaspirodiene oxygenase is a key enzyme in the synthesis of the phytosalicylate spirovelocity. Premnaspirodiene synthase catalyzes the cyclization of fenofosyl diphosphate (FPP) to form premnaspirodiene. Under the catalysis of premnaspirodiene oxygenase, premnaspirodiene undergoes hydroxylation and oxidation to form spirovelocity. TAKAHASHI et al. cloned the HPO gene from *Hypericum hemlock*, whose encoded protein belongs to the P450 superfamily. Cytochrome P450 (CYP) enzymes are a class of heme protein enzymes with various catalytic activities, including oxidation, hydroxylation, and demethylation. If the amino acid sequence similarity is higher than 40%, they belong to the same family; if it is higher than 55%, they belong to the same subfamily.

[0003] The gene DoPO, an oxygenase for zebulidiene in Dendrobium officinale, was found to belong to the P450 superfamily. Its nucleotide sequence shows 70% amino acid similarity to HPO (A6YIH8) from Hesperidinium henryi, placing them in the same subfamily. It is speculated that DoPO catalyzes the formation of spirophyllin from zebulidiene. Furthermore, it is hypothesized that DoPO plays a crucial role in the biosynthesis of phytoalexins and disease resistance mechanisms in Dendrobium officinale.

[0004] Tobacco (Nicotiana tabacum L.) belongs to the Solanaceae family and is an important economic crop. However, research reports on the *Cypripedium tumefaciens* gene in tobacco are currently scarce. We hope to discover the function of this gene in tobacco through molecular biology and genetic engineering research, which may be of great significance for research on tobacco growth, development, disease resistance, and stress tolerance. Summary of the Invention

[0005] The technical problem to be solved by this invention is to provide an application of the tobacco tofu snail diene oxygenase gene NtPO, which provides genetic materials and theoretical basis for studying the function of genes related to tobacco growth and development.

[0006] The technical problem to be solved by the present invention is achieved through the following technical solution:

[0007] The application of the tobacco tofu snail diene oxygenase gene NtPO in regulating tobacco growth and development. The nucleotide sequence of the tobacco monocopper oxidase-like protein gene NtPO is shown in SEQ ID NO.1, containing 450 bases. This gene is derived from tobacco. By editing the NtPO gene, the plant with this gene editing has higher plant height, stem circumference, middle leaf length, middle leaf width and leaf area at maturity than the control plant.

[0008] The amino acid sequence of the protein encoded by the NtPO gene of the tobacco tofu snail diene oxygenase is shown in SEQ ID NO.2, and contains 149 amino acids.

[0009] The NtPO gene of *Synostemma pentaphyllum* was edited using CRISPR / Cas9-mediated gene editing technology. A CRISPR / Cas9 editing vector for knocking out this gene was constructed, and NtPO gene-edited tobacco mutant plants were obtained after genetic transformation.

[0010] When constructing the CRISPR / Cas9 editing vector, a specific nucleotide sequence of 23 nt in the NtPO gene was selected as the guide sequence for CRISPR / Cas9. This guide sequence fragment was ligated to the CRISPR / Cas9 vector, and after PCR amplification and sequencing confirmation, the CRISPR / Cas9-NtPO editing vector was obtained.

[0011] The specific nucleotide sequence of the 23nt is shown in SEQ ID NO.3.

[0012] A method for creating tobacco mutant plants by knocking out the NtPO gene of *Synthia suspensa* using CRISPR / Cas9-mediated gene editing technology is disclosed. The method involves editing the NtPO gene of *Synthia suspensa* using CRISPR / Cas9-mediated gene editing technology, constructing a CRISPR / Cas9 editing vector for knocking out this gene, and obtaining NtPO gene-edited tobacco mutant plants after genetic transformation.

[0013] Specifically, the following steps are included:

[0014] (1) Construction of CRISPR / Cas9-NtPO editing vector: The specific nucleotide sequence of 23nt in the NtPO gene was selected as the guide sequence of CRISPR / Cas9. The guide sequence fragment was ligated to the CRISPR / Cas9 vector. After PCR amplification and detection, the PCR positive clones were sequenced to confirm and obtain the CRISPR / Cas9-NtPO editing vector.

[0015] (2) Genetic transformation: Using Tobacco Dajinyuan as the transformation target, the leaf disc method was adopted, and the CRISPR / Cas9-NtPO editing vector obtained in step (1) was transformed into tobacco plants through Agrobacterium-mediated transformation to obtain transformed tobacco regenerated plants.

[0016] (3) Molecular detection and plant screening: Leaf samples were taken from the tobacco regenerated plants obtained in step (2), and the plants with the NtPO gene knocked out were identified by molecular detection, which are the tobacco mutant plants.

[0017] The tobacco mutant plants obtained by the method had higher plant height, stem circumference, leaf length, leaf width, and leaf area at maturity than the control plants.

[0018] The above-described technical solution of the present invention has the following beneficial effects:

[0019] This invention obtains the NtPO gene of *Cypripedium tofu* spathidiene oxygenase by comparing it with the China Tobacco Database and NCBI sequence.

[0020] This invention utilizes CRISPR / Cas9-mediated gene editing technology to construct a CRISPR / Cas9 editing vector for knocking out the NtPO gene. After genetic transformation, mutant plants of *Safflower 'Da Jin Yuan'* with NtPO gene editing were obtained.

[0021] This invention investigates the agronomic traits of NtPO gene-edited plants and control (untransformed) plants under normal conditions at maturity. The gene-edited plants showed higher plant height, stem circumference, leaf length, leaf width, and leaf area than the control (untransformed) plants.

[0022] In summary, the use of CRISPR / Cas9-mediated gene editing technology to knock out the NtPO gene and obtain gene-edited plants that affect plant growth and development provides genetic materials and theoretical basis for the study of the Tobacco Tofu Cyrusadiene Oxygenase gene and the study of tobacco growth and development. Attached Figure Description

[0023] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments of the invention and, together with their description, serve to explain the principles of the invention.

[0024] Figure 1 Phylogenetic tree of tobacco tofu snail diene oxygenase protein;

[0025] Figure 2 To measure plant height, stem circumference, leaf length, leaf width, and leaf area of ​​control (untransformed) plants and tobacco tofu spathidiene oxygenase gene-edited plants under normal conditions at maturity.

[0026] Figure 3The phenotypes of control (untransformed) plants and tobacco tofu spathidiene oxygenase gene-edited plants under normal conditions at maturity were compared. Detailed Implementation

[0027] Various exemplary embodiments of the present invention will now be described in detail with reference to the accompanying drawings. It should be noted that, unless otherwise specifically stated, the relative arrangement, numerical expressions, and values ​​of the components and steps set forth in these embodiments do not limit the scope of the invention.

[0028] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available. Unless otherwise specified, the equipment used in the experiments is well known to those skilled in the art.

[0029] Unless otherwise stated, in this invention, the percentage sign refers to volume percentage, and the ratio refers to volume ratio.

[0030] The tobacco variety used in this application is Honghua Dajinyuan, a commercially available tobacco variety.

[0031] Example 1

[0032] This embodiment mainly describes the process of obtaining the NtPO gene from tobacco, tobacco, tofu, and chabiosis diene oxygenase, which is briefly introduced below.

[0033] Using leaves of cultivated tobacco cultivar 'Honghua Dajinyuan' as samples, the gene for tobacco tofu snail diene oxygenase was obtained by homologous cloning. The gene sequence was confirmed by sequence comparison with the China Tobacco Database, and the gene was identified as NtPO.

[0034] Then, by confirming the gene sequence, the protein sequence was obtained, and compared with NCBI. By constructing a phylogenetic tree, the phylogenetic relationship between tobacco tofu seviperene oxygenase and different species was determined.

[0035] The NtPO sequence of the *Tobacco tofu* spirodiene oxygenase gene was obtained, as shown in SEQ ID No. 1, comprising 450 bp. Translation of this gene sequence yielded the protein sequence shown in SEQ ID No. 2, comprising 149 amino acids. Further comparative analysis indicated that the protein contains highly homologous and conserved sequences (results are shown in...). Figure 1 (As shown).

[0036] Example 2

[0037] Using the tobacco tofu razorberide oxygenase gene NtPO obtained from the China Tobacco Database in Example 1, this invention further constructed a CRISPR / Cas9 vector and obtained gene-edited plants by leaf disc transformation.

[0038] The relatively specific 23nt nucleotide sequence (SEQ ID No. 3) in the NtPO gene was selected as the guide sequence for CRISPR / Cas9. This sequence fragment was ligated with the CRISPR / Cas9 vector (provided by Southwest University) to obtain transformed clones. PCR amplification was performed, and PCR-positive clones were sent to a sequencing company for sequencing confirmation. Finally, the CRISPR / Cas9-NtPO editing vector was obtained.

[0039] Using the CRISPR / Cas9-NtPO editing vector plasmid constructed in the previous step, a genetic transformation experiment was conducted with safflower 'Da Jin Yuan' as an example to knock out the NtPO gene of tobacco tofu 'Shizidiene oxygenase' and obtain edited plants with the knockout gene.

[0040] The regenerated plants that were transformed with the NtPO gene by Agrobacterium-mediated transformation were then sampled from the leaves of the transformed plants and sent to BGI Genomics for molecular detection to obtain NtPO gene-edited plants.

[0041] Example 3

[0042] Plants identified as NtPO gene knockout plants using molecular detection in Example 2 were harvested to obtain gene-edited material. Edited plants and control plants were planted, and tobacco agronomic traits were measured at maturity. The NtPO-edited material showed higher plant height, stem circumference, leaf length, leaf width, and leaf area than the control material (results are shown in Figure 2). Figure 2 ,like Figure 3 (As shown).

[0043] Although the present invention has been disclosed above with reference to embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various different choices and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention is defined by the claims and their equivalents.

Claims

1. The application of the tobacco tofu snail diene oxygenase gene NtPO in regulating tobacco growth and development, characterized in that, The nucleotide sequence of the tobacco single copper oxidase-like protein gene NtPO is shown in SEQ ID NO.1, containing 450 bases. This gene is derived from tobacco. By editing the NtPO gene, the plant with this gene-edited gene has higher plant height, stem circumference, waist leaf length, waist leaf width and leaf area at maturity than the control plant.

2. The application according to claim 1, characterized in that, The amino acid sequence of the protein encoded by the NtPO gene of the tobacco tofu snail diene oxygenase is shown in SEQ ID NO.2, and contains 149 amino acids.

3. The application according to claim 1, characterized in that, The NtPO gene of *Synostemma pentaphyllum* was edited using CRISPR / Cas9-mediated gene editing technology. A CRISPR / Cas9 editing vector for knocking out this gene was constructed, and NtPO gene-edited tobacco mutant plants were obtained after genetic transformation.

4. The application according to claim 3, characterized in that, When constructing the CRISPR / Cas9 editing vector, a specific nucleotide sequence of 23 nt in the NtPO gene was selected as the guide sequence for CRISPR / Cas9. This guide sequence fragment was ligated to the CRISPR / Cas9 vector, and after PCR amplification and sequencing confirmation, the CRISPR / Cas9-NtPO editing vector was obtained.

5. The application according to claim 4, characterized in that, The specific nucleotide sequence of the 23nt is shown in SEQ ID NO.

3.

6. A method for creating tobacco mutant plants by knocking out the NtPO gene, a type of spirodiene oxygenase, as described in claim 1, using CRISPR / Cas9-mediated gene editing technology, characterized in that... The tobacco single copper oxidase-like protein gene NtPO was edited using CRISPR / Cas9-mediated gene editing technology. A CRISPR / Cas9 editing vector for knocking out this gene was constructed, and NtPO gene-edited tobacco mutant plants were obtained after genetic transformation.

7. The method according to claim 6, characterized in that, Specifically, the following steps are included: (1) Construction of CRISPR / Cas9-NtPO editing vector: The specific nucleotide sequence of 23nt in the NtPO gene was selected as the guide sequence of CRISPR / Cas9. The guide sequence fragment was ligated to the CRISPR / Cas9 vector. After PCR amplification and detection, the PCR positive clones were sequenced to confirm and obtain the CRISPR / Cas9-NtPO editing vector. (2) Genetic transformation: Using Tobacco Dajinyuan as the transformation target, the leaf disc method was adopted, and the CRISPR / Cas9-NtPO editing vector obtained in step (1) was transformed into tobacco plants through Agrobacterium-mediated transformation to obtain transformed tobacco regenerated plants. (3) Molecular detection and plant screening: Leaf samples were taken from the tobacco regenerated plants obtained in step (2), and the plants with the NtPO gene knocked out were identified by molecular detection, which are the tobacco mutant plants.

8. The method according to any one of claims 6-7, characterized in that, The tobacco mutant plants obtained by the method had higher plant height, stem circumference, leaf length, leaf width, and leaf area at maturity than the control plants.

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