Application of tobacco single copper oxidase-like protein gene NtSKU5

Abstract

The invention discloses an application of a tobacco single copper oxidase-like protein gene NtSKU5, the nucleotide sequence of the tobacco single copper oxidase-like protein gene NtSKU5 is shown as SEQ ID NO.1, the tobacco single copper oxidase-like protein gene NtSKU5 comprises 1818 basic groups, and the gene is derived from tobacco. According to the invention, agronomic traits of an NtSKU5 gene edited plant and a control (untransformed) plant are investigated in a squaring stage and a mature stage under normal conditions, and the plant height, stem girth, waist leaf length, waist leaf width and leaf area of the NtSKU5 gene edited plant are all higher than those of the control (untransformed). In a word, the NtSKU5 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, so that a genetic material and a theoretical basis are provided for the research on the single copper oxidase-like protein of the tobacco and the research on the growth and development of the tobacco.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of plant genetic engineering, in particular to an application of a tobacco monooxygenase-like protein gene NtSKU5. BACKGROUND

[0002] The monooxygenase-like protein SKU5 belongs to the SKS protein of the plant multicopper oxidase subfamily, the SKS family protein is a special subfamily existing in the plant multicopper oxidase gene family, and all belong to glycosylated proteins located on the cytoplasmic membrane. They only contain the conserved T2Cu connecting site in the multicopper oxidase and lack the histidine residues required for connecting other types of copper ions, and they may have lost the normal multicopper oxidase function and act on plant growth and development in a new way, but the understanding in this field is almost blank, and at present the only certainty is that this variation is not accidental, and for the plant body, this new type of multicopper oxidase must have an irreplaceable physiological function, which needs to be further explored.

[0003] At present, 19 members of the SKS protein family monooxygenase-like protein SKU5 and SKS1-SKS18 have been found in Arabidopsis thaliana, and this type of protein has also been found in plants such as Brassica napus, tobacco, corn and primula. The proteins encoded by the SKS family genes have effects on biological processes such as root growth direction, leaf vein form, pollen development, pollen tube elongation and hormone regulation, but the research on the whole SKS gene family has only focused on a few genes. Arabidopsis SKU5 is expressed in all tissues of Arabidopsis thaliana, and the expression amount is the largest in root tip meristem and distal elongation tissue. When SKU5 overexpression plants grow on agarose, the roots and hypocotyls are 15% shorter than the wild type, the roots deviate from the normal downward growth, and the yellowing hypocotyls also show counterclockwise axial rotation, which disrupts the normal downward growth of the root system. At present, the expression of SKU5 gene in alfalfa is extremely significantly negatively regulated by day length and extremely significantly positively regulated by temperature; the regulation results of SKU5 gene expression by day length and temperature in the leaves of two types of autumn dormancy alfalfa are inconsistent. The relative expression amount of SKU5 gene mRNA in the leaves of autumn dormancy alfalfa is significantly negatively correlated with plant height and leaf area.

[0004] At present, there is no report on the application of tobacco monooxygenase-like protein gene NtSKU5. Tobacco is an economic crop with leaf as product, and it is of great significance to study the molecular mechanism of tobacco monooxygenase-like protein gene on growth and development. SUMMARY

[0005] The technical problem to be solved by the present application is to provide an application of a tobacco monooxygenase-like protein gene NtSKU5, to provide genetic materials and theoretical basis for studying the functions of tobacco growth and development related genes.

[0006] The technical problem to be solved by the present application is solved by the following technical solutions:

[0007] The application of a tobacco monocupro oxidase-like protein gene NtSKU5 in regulating tobacco growth and development, the nucleotide sequence of the tobacco monocupro oxidase-like protein gene NtSKU5 is shown in SEQ ID NO. 1, containing 1818 bases, and the gene is derived from tobacco.

[0008] Preferably, in the above technical solution, the amino acid sequence of the protein encoded by the tobacco monocupro oxidase-like protein gene NtSKU5 is shown in SEQ ID NO. 2, containing 605 amino acids.

[0009] Preferably, in the above technical solution, CRISPR / Cas9-mediated gene editing technology is used to edit the tobacco monocupro oxidase-like protein gene NtSKU5, a CRISPR / Cas9 editing vector for knocking out the gene is constructed, and a tobacco mutant plant with NtSKU5 gene editing is obtained after genetic transformation. The plant with gene editing is significantly higher than the control plant in the plant height of the topped plant, the stem circumference, the waist leaf length, the waist leaf width and the leaf area at the mature stage.

[0010] Preferably, in the above technical solution, when the CRISPR / Cas9 editing vector is constructed, a specific nucleotide sequence of 23 nt in the NtSKU5 gene is selected as the guide sequence of CRISPR / Cas9, the guide sequence fragment is connected with the CRISPR / Cas9 vector, and after PCR amplification detection and sequencing confirmation, the CRISPR / Cas9-NtSKU5 editing vector is obtained.

[0011] Preferably, in the above technical solution, the specific nucleotide sequence of 23 nt is shown in SEQ ID NO. 5.

[0012] A method for creating a tobacco mutant plant by knocking out a tobacco monocupro oxidase-like protein gene NtSKU5 using CRISPR / Cas9-mediated gene editing technology, which uses CRISPR / Cas9-mediated gene editing technology to edit the tobacco monocupro oxidase-like protein gene NtSKU5, constructs a CRISPR / Cas9 editing vector for knocking out the gene, and obtains a tobacco mutant plant with NtSKU5 gene editing after genetic transformation.

[0013] Preferably, the above technical solution specifically comprises the following steps:

[0014] (1) Constructing CRISPR / Cas9-NtSKU5 editing vector: selecting a specific nucleotide sequence of 23 nt in NtSKU5 gene as a guide sequence of CRISPR / Cas9, connecting the guide sequence fragment with a CRISPR / Cas9 vector, after PCR amplification detection, sequencing the positive PCR clone to obtain the CRISPR / Cas9-NtSKU5 editing vector;

[0015] (2) Genetic transformation: taking Honghuadajinyuan tobacco as the transformation object, using leaf disc method, transforming the CRISPR / Cas9-NtSKU5 editing vector obtained in step (1) into tobacco plants through Agrobacterium mediation to obtain regenerated tobacco plants after transformation;

[0016] (3) Molecular detection and plant screening: sampling leaves of the regenerated tobacco plants obtained in step (2), determining the plants in which NtSKU5 gene is knocked out through molecular detection, that is, tobacco mutant plants.

[0017] Preferably, in the technical solution, the tobacco mutant plants obtained by the method have a significantly higher topping plant height, stem circumference, waist leaf length, waist leaf width and leaf area than the control plants at the mature stage.

[0018] The above technical solution of the application has the following beneficial effects:

[0019] The application clones a tobacco monooxygenase-like protein gene NtSKU5 by homologous cloning method.

[0020] The application constructs a CRISPR / Cas9 editing vector for knocking out NtSKU5 gene by CRISPR / Cas9-mediated gene editing technology, and obtains Honghuadajinyuan mutant plant with edited NtSKU5 gene after genetic transformation.

[0021] The application investigates the agronomic traits of the NtSKU5 gene editing plant and the control (untransformed) plant under normal conditions, at the budding stage and the mature stage, and the plant height, stem circumference, waist leaf length, waist leaf width and leaf area of the NtSKU5 gene editing plant are higher than those of the control (untransformed) plant.

[0022] In summary, the NtSKU5 gene is knocked out by CRISPR / Cas9-mediated gene editing technology to obtain a gene editing plant affecting plant growth and development, which provides genetic materials and theoretical basis for the research of tobacco monooxygenase-like protein and the research of tobacco growth and development. BRIEF DESCRIPTION OF DRAWINGS

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

[0024] Figure 1 Figure 1 is a gel map of NtSKU5 gene cloning;

[0025] Figure 2 Figure 2 is a sequence alignment of NtSKU5 protein;

[0026] Figure 3 The plant height, stem girth, waist leaf length, waist leaf width and leaf area of the control (untransformed) plants and the gene edited plants were determined under normal conditions at the mature stage. DETAILED DESCRIPTION

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

[0028] The experimental methods used in the following examples are conventional methods unless otherwise specified, 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 unless otherwise specified.

[0029] Example 1

[0030] This example mainly describes the process of obtaining the tobacco monooxygenase-like protein gene NtSKU5, and is briefly introduced as follows:

[0031] The total RNA of tobacco leaves was extracted using an RNA extraction kit, and reverse transcribed into cDNA for standby use, with the leaves of the cultivated species tobacco Honghuadajinyuan as the sample.

[0032] The total RNA of tobacco was extracted according to the instructions of the plant RNA extraction kit.

[0033] 1 μg of total RNA extracted from the leaves was used for reverse transcription, and the transcription system was as follows:

[0034] Total RNA 1 μg

[0035] Oligo(dT) (10 μM) 1.5 μL

[0036] ddH2O up to 15 μL

[0037] After mixing the above system, it was placed in PCR for 5 min at 70°C, and immediately removed and placed on ice for 5 min. Then the following reagents were added to the system:

[0038]

[0039] The system is placed in a PCR instrument, 42℃ for 65 min, 65℃ for 10 min, 4℃ for 10 min, and then stored in a -20℃ refrigerator for use.

[0040] By homologous alignment method, referring to the sequence of Arabidopsis genes and known tobacco partial gene sequences, the primer sequence is designed as follows:

[0041] F: 5'-ATGATGGCATTTCTTAAGGG-3' (SEQ ID No. 3),

[0042] R: 5'-TTATGATGAAATCCGCCC-3' (SEQ ID No. 4).

[0043] The above prepared cDNA is used as a template, and the above primer is used for PCR amplification:

[0044] Amplification system (50 μL):

[0045]

[0046] After mixing and centrifuging, PCR amplification is performed, and the PCR reaction conditions are as follows: 95℃ for 10 sec, 52℃ for 30 sec, 72℃ for 2.5 min, a total of 30 cycles; 72℃ for 10 min; 12℃ Hold.

[0047] After purification of the amplification product, sequencing is performed, and the tobacco mononuclear copper oxidase-like protein gene NtSKU5 sequence is obtained, the base sequence is shown as SEQ ID No. 1, and a total of 1818 bases are included (results are shown as Figure 1 After translation of the gene sequence, the encoded protein sequence is shown as SEQ ID No. 2, and a total of 605 amino acids are included, and further comparative analysis shows that the protein contains a sequence with high homology, which is highly conserved (results are shown as Figure 2

[0048] Example 2

[0049] Using the tobacco mononuclear copper oxidase-like protein gene NtSKU5 obtained in Example 1, the present application further constructs a CRISPR / Cas9 vector, and a gene editing plant is obtained by using leaf disc method.

[0050] ​The 23 nt nucleotide sequence (SEQ ID No. 5) which is more specific in the NtSKU5 gene is selected as the guide sequence of CRISPR / Cas9, and the sequence fragment is connected with the CRISPR / Cas9 carrier (provided by Southwest University) to obtain a transformed clone, PCR amplification detection is performed, and then the PCR positive clone is sent to a sequencing company for sequencing confirmation, and finally the CRISPR / Cas9-NtSKU5 editing vector is obtained.

[0051] The CRISPR / Cas9-NtSKU5 editing vector plasmid constructed in the above step is used to perform genetic transformation test by taking Honghuadajinyuan as an example to knock out the tobacco monooxygenase-like protein gene NtSKU5 in the plant body.

[0052] The regenerated plant transformed by the agrobacterium-mediated NtSKU5 gene is obtained, and then the leaf of the transformed plant is sampled and sent to Huada Gene for molecular detection to determine the NtSKU5 gene editing plant.

[0053] Example 3

[0054] The NtSKU5 gene knockout plant determined by the molecular detection in Example 2 is used to perform seed collection to obtain gene editing materials. The edited plant and the control plant are planted, and tobacco agronomic trait determination is performed at the mature stage, and the plant height, stem circumference, waist leaf length, waist leaf width and leaf area of the edited material are all higher than those of the control material (results are shown in Figure 3

[0055] Although the present application has been disclosed as above with examples, it is not intended to limit the present application, and any person skilled in the art can make various selections and modifications without departing from the spirit and scope of the present application, and therefore the protection scope of the present application is defined by the claims and equivalent forms thereof.​

Claims

1. The application of the tobacco monocopper oxidase-like protein gene NtSKU5 in regulating tobacco growth and development, characterized in that, The nucleotide sequence of the tobacco single copper oxidase-like protein gene NtSKU5 is shown in SEQ ID NO.1, containing 1818 bases, and this gene is derived from tobacco.

2. The application according to claim 1, characterized in that, The amino acid sequence of the protein encoded by the tobacco single copper oxidase-like protein gene NtSKU5 is shown in SEQ ID NO.2, containing 605 amino acids.

3. The application according to claim 1, characterized in that, The tobacco single copper oxidase-like protein gene NtSKU5 was edited using CRISPR / Cas9-mediated gene editing technology. A CRISPR / Cas9 editing vector for knocking out this gene was constructed. After genetic transformation, tobacco mutant plants with NtSKU5 gene editing were obtained. The plant height, stem circumference, middle leaf length, middle leaf width and leaf area of ​​the gene-edited plants at maturity were significantly higher than those of the control plants.

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 NtSKU5 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-NtSKU5 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.

5.

6. A method for creating tobacco mutant plants by knocking out the tobacco single copper oxidase-like protein gene NtSKU5 as described in claim 1 using CRISPR / Cas9-mediated gene editing technology, characterized in that, The tobacco single copper oxidase-like protein gene NtSKU5 was edited using CRISPR / Cas9-mediated gene editing technology. A CRISPR / Cas9 editing vector for knocking out this gene was constructed, and tobacco mutant plants with NtSKU5 gene editing 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-NtSKU5 editing vector: The specific nucleotide sequence of 23nt in the NtSKU5 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-NtSKU5 editing vector. (2) Genetic transformation: Using Tobacco Dajinyuan as the transformation target, the leaf disc method was adopted, and the CRISPR / Cas9-NtSKU5 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 NtSKU5 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 showed significantly higher plant height, stem circumference, leaf length, leaf width, and leaf area at maturity compared to the control plants.

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