CN115747227A - Tobacco NtGA3ox1 gene and application thereof in regulating and controlling epidermal glandular hair density - Google Patents

Abstract

The invention discloses a method for cultivating multi-glandular hair tobacco by using a gene editing technology. The invention belongs to the technical field of plant genetic engineering, and particularly relates to a tobacco gibberellin 3-beta-dioxygenase gene NtGA3ox1 gene and application thereof in regulating and controlling the glandular hair density of tobacco. The tobacco NtGA3ox1 gene comprises 1063bp, and the base sequence is shown as SEQ ID NO. 1. The amino acid sequence of the tobacco NtGA3ox1 gene is shown in SEQ ID NO. 2. Compared with the wild type, the glandular hair density of the tobacco NtGA3ox1 gene editing plant is obviously increased. The gene editing technology is utilized to target NtGA3ox1, and the gene editing technology has great potential in molecular regulation and control of the tobacco glandular hair density and targeted improvement of the glandular hair.

Description

Tobacco NtGA3ox1 gene and application thereof in regulating and controlling epidermal glandular hair density

Technical Field

The invention belongs to the technical field of plant genetic engineering, and particularly relates to a tobacco NtGA3ox1 gene and application thereof in regulating and controlling epidermal glandular hair density.

Background

Plant glandular hairs are used as specialized structures of plant epidermal cells and can be classified into non-secretory type and secretory type according to different secretory behaviors. The non-secretory epidermal hair is a single-cell or multi-cell epidermal hair without secretion capacity, secretory glandular hair has the capacity of synthesizing and secreting various compounds, and compounds synthesized and secreted by the secretory glandular hair represented by sweet wormwood herb and mint comprise polysaccharides, organic acids, proteins, terpenes, alkaloids, polyphenols and the like, and the compounds endow plants with special odor, application and economic value. Essentially, glandular hairs, whether secreted or not, their presence is an evolutionary consequence of the adaptation of the plant itself to the external environmental stimuli, a way of interacting with the external environment.

The tobacco glandular hair is a specialized structure of the epidermal cells of tobacco and has important influence on the resistance of tobacco plants and the quality of tobacco leaves. The tobacco gland is rich and various. Cembrane diterpene and sugar ester compounds are main components of glandular hair secretion of tobacco, and the components are not only important fragrance precursor substances, but also have obvious repelling effect on aphids. Therefore, increasing the glandular hair density of the tobacco is one of the important ways to improve the resistance of the tobacco and the quality of the tobacco leaves. The density of the tobacco glandular hairs is influenced by various conditions such as illumination, temperature, moisture, soil, fertilizer, ecological environment and the like, but genetic factors still play a decisive role in the density of the tobacco glandular hairs. Different tobacco types, varieties, leaf positions and development periods of leaves have great differences in glandular hair density and secretion content and components, and the quality and style of tobacco leaves are influenced to a certain extent. The cultivation of tobacco varieties with high glandular hair density and high secretory type is one of the important targets of the conventional tobacco breeding. Therefore, how to effectively regulate and control the factors influencing the development of glandular hairs and effectively utilize the factors has very important research theoretical significance and practical value for improving the quality of tobacco.

Disclosure of Invention

The application aims to provide a tobacco gibberellin 3-beta-dioxygenase gene tobacco NtGA3ox1 gene, a tobacco NtGA3ox1 protein coded by the gene and application of the protein in regulation and control of epidermal glandular hair density, and the gene has very important application value.

The technical scheme of the invention is as follows:

the first aspect of the invention discloses a tobacco NtGA3ox1 gene, which comprises 3141bp, and the base sequence is shown as SEQ ID NO. 1.

Preferably, the encoded NtGA3ox1 protein of the tobacco NtGA3ox1 gene.

Preferably, the amino acid sequence of the encoded NtGA3ox1 protein is as shown in SEQ ID No. 2.

The second aspect of the invention discloses the application of the tobacco NtGA3ox1 gene in tobacco.

Preferably, the plants in which the NtGA3ox1 gene is genetically edited have increased epidermal glandular hairs and increased glandular hair density compared to control plants.

Preferably, the plant after gene editing is a tobacco plant with an edited NtGA3ox1 gene obtained by genetic transformation through constructing a CRISPR/Cas9 editing vector for knocking out the NtGA3ox1 gene by a CRISPR/Cas9 mediated gene editing technology.

Preferably, the recombinant CRISPR/Cas9 expression vector for knocking out the tobacco NtGA3ox1 gene enables the expression level of the tobacco NtGA3ox1 in a transformed plant to be obviously reduced or not expressed.

Preferably, the genetically edited tobacco plant has a significantly delayed flowering time and a significantly reduced plant height in tobacco compared to control tobacco.

The application of the tobacco NtGA3ox1 gene in regulating and controlling the density of the epidermal glandular hairs comprises the following steps: cloning a tobacco NtGA3ox1 gene to obtain a tobacco epidermal glandular hair density regulating gene; the gene editing technology is utilized to obtain a gene editing carrier of the tobacco NtGA3ox1 gene with function loss, so that the epidermal glandular hairs are increased, and the glandular hair density is increased; meanwhile, the plant height of the gene editing plant is obviously shortened, and the flowering time is obviously prolonged.

Compared with the prior art, the method has the beneficial effect that the epidermal glandular hair density of the tobacco is effectively regulated and controlled by utilizing the NtGA3ox1 gene of the tobacco. The gene editing technology is used for targeting NtGA3ox1, has great potential in molecular regulation and control of the glandular hair density of tobacco and directional improvement of the glandular hair, and has very important research theoretical significance and practical value for cultivating high glandular hair density and improving tobacco quality.

Drawings

FIG. 1 is an electrophoretogram of a clone of the NtGA3ox1 gene provided in the examples of the present invention.

FIG. 2 is a comparison of glandular hair density of control sample (left) and gene-edited tobacco plants (right) of non-gene-edited tobacco plants

FIG. 3 is a photomicrograph of the glandular hairs of the edge of a control (left) and a gene-edited tobacco plant (right) of a tobacco plant not subjected to gene editing

FIG. 4 is an overall phenotypic observation of a control of non-genetically-edited tobacco plants (left) and genetically-edited tobacco plants (right).

Detailed Description

The present application is further illustrated by the following examples.

Before describing specific embodiments, a brief description will be given of some of the biological materials, test reagents, test devices, and the like in the following embodiments.

Biological material:

tobacco variety: the seeds adopted in the embodiment are provided by the preservation of the national tobacco gene research center.

Carrier: pEASY-T1 Simple vector, purchased from Beijing Quanyujin Biotechnology, inc.

The CRISPR/Cas9 vector is provided by key laboratory affection of China in Bombyx mori genome biology at southwest university.

The strain is as follows:

trans1-T1 chemically competent cells, purchased from Beijing Quanji Biotechnology, inc.;

LBA4404 Agrobacterium strains, common strains in biological experiments, can be obtained publicly;

the synthesis of the primers and the DNA sequencing are completed by the Beijing Liu-He Hua Dagen science and technology Co., ltd;

experimental reagent:

RNA extraction Kit, superpure Plant polyRNA Kit;

fluorescent quantitative PCR enzyme (SYBR qPCR kit) purchased from Zhengzhou Ansai Biotech Co., ltd;

a reverse transcription kit and T4 ligase purchased from Takara Bio engineering (Dalian) Co., ltd;

restriction enzyme BsaI, available from NEB;

DNA amplification enzyme, purchased from Beijing Quanji Biotech, inc.;

the plant genome extraction kit and the DNA purification kit were purchased from QIAGEN.

Experimental equipment:

PCR synthesizer TProfessional Thermocycler, biometra;

quantitative PCR instrument CFX96, bio-Rad;

uv gel imaging system biopspectrum, UVP corporation.

Example 1

This example is briefly described below mainly with respect to the process of obtaining a clone of the NtGA3ox1 gene.

(1) Preparation of cDNA as template for cloning

Taking 100mg of stems of Wangwang tobacco (Honghuadajinyuan) as samples, fully grinding in liquid nitrogen, extracting total RNA according to the instruction of an RNA extraction kit, and then carrying out reverse transcription to obtain cDNA for later use;

(2) Designing primers and carrying out PCR amplification

The gene primer sequences designed for amplifying tobacco NtGA3ox1 were as follows:

NtGA3ox1-F:5'-ATGCCTTCAAGAATCTCAGA-3'(SEQ ID No.3)，

NtGA3ox1-R:5'-TTAGCCAACTTGGACTCTATTA-3'(SEQ ID No.4)；

and (2) performing PCR amplification by using the cDNA prepared in the step (1) as a template and the primers, wherein the PCR amplification conditions are as follows: pre-denaturation at 94 ℃ for 3min, denaturation at 94 ℃ for 20s, annealing at 58 ℃ for 20s, extension at 72 ℃ for 30s, and complete extension at 72 ℃ for 10min after the circulation is finished; and storing the PCR amplification product at 4 ℃ for later use, or directly carrying out electrophoresis detection analysis.

And (3) purifying the product after PCR amplification according to the gel recovery kit specification, and connecting the purified product to a pEASY-T1 vector by using a connection system as follows:

DNA amplification product, 6. Mu.L;

pEASY-T1 vector, 1. Mu.L;

after mixing, the mixture was connected at 25 ℃ for 25min.

The ligation products were transformed into E.coli competent cells, and the specific transformation procedure is briefly described as follows:

taking out the competent cells from a refrigerator at-80 ℃, putting the competent cells on ice to dissolve the competent cells, adding the ligation product into 50 mu L of Trans1-T1 competent cells, flicking and uniformly mixing, and carrying out ice bath for 30min;

heat shock is carried out in water bath at 42 ℃ for 30s, and the mixture is immediately placed on ice for 2min; adding 250 μ L LB (without antibiotic) balanced to room temperature, and shaking at 37 deg.C for 1h;

mu.L of 500mM IPTG and 40. Mu.L of 20mg/mL X-gal were mixed and applied evenly to LB solid plate (containing 60. Mu.g/. Mu.L ampicillin) and the plate was inverted and incubated overnight at 37 ℃.

Extracting each plasmid DNA after picking out the white spot amplification culture, identifying the recombinant plasmid through plasmid PCR amplification, and sending a sample for sequencing the corresponding positive clone to obtain the NtGA3ox1 gene sequence.

Sequencing analysis results show that the length of the coding region of the NtGA3ox1 gene is 1643bp nucleotides, and is specifically shown as SEQ ID NO. 1; after the gene is analyzed, the amino acid sequence of the encoded NtGA3ox1 protein is shown as SEQ ID NO. 2. FIG. 1 is an electrophoretogram of the NtGA3ox1 gene clone provided in this example. As can be seen from FIG. 1, the PCR product was detected by agarose gel electrophoresis to obtain a PCR band of about 1000bp in size, and the sequencing result showed that the total length of the protein coding region (CDS) of the tobacco NtGA3ox1 gene was 1092bp.

Example 2

A CRISPR/Cas9 vector is further constructed by utilizing the tobacco NtGA3ox1 gene obtained in the embodiment 1, and a gene editing plant is obtained by utilizing the leaf disc method for transformation.

A specific 23nt nucleotide sequence (SEQ ID No. 5) in the NtGA3ox1 gene is selected as a guide sequence of CRISPR/Cas9, and knockout primer sequences GA3ox1-K-F and GA3ox1-K-R are designed as follows:

GA3OX1-K-F:5’-GATTGAATTACCCGAATCCCATGCA-3’(SEQ ID No.6)，

GA3OX1-K-R:5’-AAATGCATGGGATTCGGGTAATT-3’(SEQ ID No.7)；

obtaining a DNA double strand of a target site (annealing) by using the primer sequence; and connecting the obtained DNA double strand (annealing product) of the target site with the CRISPR/Cas9 vector after BsaI enzyme digestion, converting, screening, detecting by PCR amplification, and sending the PCR positive clone to a sequencing company for sequencing confirmation to finally obtain the CRISPR/Cas9-NtGA3ox1 editing vector.

The CRISPR/Cas9-NtGA3ox1 editing vector plasmid constructed in the previous step is utilized, and genetic transformation and tissue culture are carried out by taking a safflower macrogol as an example to obtain a plant with a gene NtGA3ox1 related to the tobacco flowering time subjected to knockout editing, and related experimental processes are briefly introduced as follows:

inoculating sterilized tobacco seed onto MS culture medium, culturing to 4 cotyledons (15-20 d), transferring into culture bottle containing MS solid culture medium, and culturing at 25 + -1 deg.C under illumination intensity of 30-50 μmol/(m) ² S) and culturing for 35-40d under the condition of 16h/d illumination time for later use.

The LBA4404 stored at-80 ℃ was taken out and electrically transformed into competent Agrobacterium cells, and frozen and thawed on ice. When competence is just thawed, 2 mu L of CRISPR/Cas9-NtGA3ox1 editing vector plasmid is added, mixed evenly and placed on ice. Then transferring the mixed competence to a precooled electric rotor, placing the electric rotor in an electric rotor for transformation, adding 1mL of YEB liquid culture medium after the transformation is finished, mixing with the transformation liquid, and then placing the mixture on a shaking table at 28 ℃ for culturing for 1.5-2h at 200 rpm. The cells were centrifuged at 8000rpm to discard the supernatant medium, and then suspended in 200. Mu.L of YEB liquid medium, spread on YEB solid medium containing 50mg/L rifampicin, 50mg/L streptomycin and 50mg/L kanamycin, and cultured in 28 ℃ inversion dark for 2-3d.

Tobacco leaf disks are made into square leaf disks with the side length of 1cm in a clean bench, and agrobacterium colonies containing CRISPR/Cas9-NtGA3ox1 editing vectors are prepared into suspension bacteria liquid by MS liquid (OD 600= 0.6-0.8). And soaking and infecting the tobacco leaf discs for 10min by using the suspension agrobacterium liquid. Then, the leaf discs were placed on MS solid medium containing 2.0mg/L NAA +0.5 mg/L6-BA, and were co-cultured at 28 ℃ in the dark for 3 days. Then, subculture is carried out, and the subcultured cells are placed on an MS solid culture medium containing 2.0mg/L NAA, 0.5 mg/L6-BA, 250mg/L Cb and 50mg/L Kan under the culture conditions that: culturing at 28 deg.C under illumination for 16h/d with illumination intensity of 30-50 μmol/(m 2 s), culturing at 25 deg.C in dark for 8h/d, culturing for 45-60d until differentiated bud is formed, and changing differentiation culture medium every 7-10d for 3-4 times; culturing until a differentiated bud is formed; cutting off callus formed by existing differentiated bud, culturing on MS culture medium containing carbenicillin 500mg/L and kanamycin 50mg/L, culturing for 8-14d when the differentiated bud on callus grows to 2-4cm high and the culture condition is the same as that of differentiated culture; and (2) performing rooting culture on the regenerated plant, cutting a differentiated bud, inserting the cut differentiated bud into an MS culture medium containing 500mg/L carbenicillin and 50mg/L kanamycin to perform rooting culture, wherein the culture condition is consistent with the differentiation culture condition, culturing for 20-30d, performing regeneration and transplantation to a flowerpot, then performing transformation plant leaf sampling, delivering a Huamao gene to perform molecular detection, determining to obtain an NtGA3ox1 gene editing plant, and then performing seed harvesting to obtain T0 generation editing plant seeds. Carrying out selfing homozygous propagation on the T0 generation seeds by 23 times, when the plants grow to 5-6 leaves, sampling leaves of single plants, delivering Huada genes to carry out molecular detection, determining to obtain plants subjected to homozygous editing of the NtGA3ox1 gene, and then harvesting to obtain T1 generation seeds subjected to homozygous editing of the NtGA3ox1 gene.

The application of the tobacco NtGA3ox1 gene is that the expression of the NtGA3ox1 gene is reduced in a tobacco plant body, compared with a control epidermal glandular hair, the gene editing plant is increased, and the glandular hair density is increased; meanwhile, the plant height of the gene editing plant is obviously shortened, and the flowering time is obviously prolonged. Methods of reducing gene expression or gene silencing commonly used in the art are suitable for use in the present invention.

Example 3

The difference between the gene-edited material and the wild type was obtained by analyzing the phenotype of leaves and the whole observation using the plants determined to be homozygous knockout of the NtGA3ox1 gene by molecular detection in example 2. FIG. 2 is a comparison of leaf glandular hair density of control (left) and right) gene-edited tobacco plants in which no gene editing occurred; FIG. 3 is a photomicrograph of the border glandular hairs of a control (left) and a gene-edited tobacco plant (right) of an unorganized tobacco plant; FIG. 4 is an overall phenotypic observation of a control of non-genetically-edited tobacco plants (left) and genetically-edited tobacco plants (right). As can be seen from FIGS. 2, 3 and 4, the tobacco plants edited by the NtGA3ox1 gene have increased epidermal glandular hairs and increased glandular hair density; meanwhile, the plant height of the gene editing plant is obviously shortened, and the flowering time is obviously prolonged.

The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Sequence table information:

DTD version V1_3

The file name is tobacco NtGA3ox1 gene and the application in regulating and controlling the density of epidermal glandular hairs

Software name WIPO Sequence

Software version 2.1.2

Production date 2022-09-25

Basic information:

filing name of the present application/applicant RIB220618

Applicant name or name-tobacco industry Limited liability company in Yunnan

Zh name or name/language of applicant

Applicant's name or name/Latin name CHINA TOBACCO YUNNAN INDUSTRIAL CO., LTD

Inventor name Deng Lele

Zh inventor name/language

Inventor name/Latin name DENG Lele

The invention discloses a tobacco NtGA3ox1 gene and application thereof in regulating and controlling epidermal glandular hair density (zh)

Total amount of sequences 7

The sequence is as follows:

serial number (ID): 1

Length of 1643

Molecular type DNA

Feature location/qualifier:

-source,1..1643

>mol_type,other DNA

>organism,synthetic construct

residue:

serial number (ID): 2

Length: 363

Molecular type AA

Feature location/qualifier:

-source,1..363

>mol_type,protein

>organism,synthetic construct

residue:

serial number (ID): 3

Length of 20

Molecular type DNA

Feature location/qualifier:

-source,1..20

>mol_type,other DNA

>organism,synthetic construct

residue:

atgccttcaa gaatctcaga 20

serial number (ID): 4

Length: 22

Molecular type DNA

Feature location/qualifier:

-source,1..22

>mol_type,other DNA

>organism,synthetic construct

residue (b):

ttagccaact tggactctat ta 22

serial number (ID): 5

Length: 23

Molecular type DNA

Feature location/qualifier:

-source,1..23

>mol_type,other DNA

>organism,synthetic construct

residue:

aattacccga atcccatgca tgg 23

serial number (ID): 6

Length 25: 25

Molecular type DNA

Feature location/qualifier:

-source,1..25

>mol_type,other DNA

>organism,synthetic construct

residue:

gattgaatta cccgaatccc atgca 25

serial number (ID): 7

Length: 23

Molecular type DNA

Feature location/qualifier:

-source,1..23

>mol_type,other DNA

>organism,synthetic construct

residue:

aaatgcatgg gattcgggta att 23

END。

Claims (8)

1. the tobacco NtGA3ox1 gene is characterized by comprising 1063bp, and the base sequence is shown as SEQ ID NO. 1.

2. The tobacco NtGA3ox1 gene according to claim 1, wherein the encoded NtGA3ox1 protein of the tobacco NtGA3ox1 gene.

3. The tobacco NtGA3ox1 gene according to claim 2, wherein the amino acid sequence of the encoded NtGA3ox1 protein is as shown in SEQ ID No. 2.

4. Use of the tobacco NtGA3ox1 gene according to any one of claims 1-3 in tobacco.

5. Use according to claim 4, characterized in that the epidermal glandular hair density of the plant is increased after the genetic editing of the NtGA3ox1 gene compared to the control plant.

6. The application of claim 5, wherein the plant after gene editing is a tobacco plant with an NtGA3ox1 gene edited by genetic transformation, wherein a CRISPR/Cas9 editing vector for knocking out the NtGA3ox1 gene is constructed by a CRISPR/Cas9 mediated gene editing technology.

7. The use as claimed in claim 6, wherein the recombinant CRISPR/Cas9 expression vector for knocking out the tobacco NtGA3ox1 gene can obviously reduce or not express the tobacco NtGA3ox1 expression level in the transformed plant.

8. The use according to claim 5, wherein the genetically edited tobacco plant has a significantly delayed flowering time and a significantly reduced plant height compared to control tobacco.

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