CN116144698A - Application of tobacco alcohol dehydrogenase related gene NtADH1 in regulation and control of tobacco waist leaf width - Google Patents

Abstract

The invention discloses an application of a tobacco alcohol dehydrogenase related gene NtADH1 in regulating and controlling tobacco waist leaf width. The nucleotide sequence of the NtADH1 gene is shown as SEQ ID NO.1, and the amino acid sequence of the NtADH1 coding protein is shown as SEQ ID NO. 3. The CRISPR/Cas9 editing vector for knocking out the NtADH1 gene is constructed by a CRISPR/Cas9 mediated gene editing technology, and the tobacco plant with the NtADH1 gene knocked out is obtained after editing material creation and molecular detection identification. Compared with a control tobacco plant, the average waist leaf width of the NtADH1 gene knocked-out edited tobacco plant obtained by the invention is obviously increased. Provides a theoretical basis for further elucidating the regulating mechanism of the waist-leaf width of the tobacco, and provides a new genetic material for cultivating tobacco varieties with obviously changed waist-leaf width.

Description

Application of tobacco alcohol dehydrogenase related gene NtADH1 in regulation and control of tobacco waist leaf width

Technical Field

The invention relates to the technical field of plant genetic engineering, in particular to application of a tobacco alcohol dehydrogenase related gene NtADH1 in regulating and controlling tobacco waist leaf width.

Background

Alcohol dehydrogenase (alcohol dehydrogenase, ADH), which is a member of the medium-long chain dehydrogenase/reductase protein family, is a zinc-binding enzyme that catalyzes the reversible reaction between alcohol and acetaldehyde with NAD as a coenzyme. ADH is widely distributed in plants, plays an important role in plant growth and stress resistance, and along with the deep research, the role of ADH in plant aroma synthesis is also gradually paid attention to. The plant aroma substances mainly comprise esters, alcohols, aldehydes, terpenes and the like, aliphatic, branched or aromatic aldehydes in the fatty acid metabolic pathway and the amino acid metabolic pathway generate corresponding alcohols under the action of ADH, and finally carbonyl compounds, acids and esters are synthesized. Previous studies of ADH1 have focused mainly on resistance function studies in biotic and abiotic stresses (salt stress, drought stress, cold stress and pathogenic bacterial infection) and on effects on aroma components in plant fruits, while less studies have been conducted on agronomic traits of plants.

The length and width of the tobacco waist leaf are important indexes for influencing the yield of tobacco leaves and factors for influencing the appearance quality and the baking property of the tobacco leaves, so that the method has important significance in researching related genes for influencing the area of the tobacco waist leaf.

Disclosure of Invention

The invention aims to solve the technical problem of providing an application of a tobacco alcohol dehydrogenase related gene NtADH1 in regulating and controlling tobacco waist leaf width, and providing materials and references for tobacco alcohol dehydrogenase protein research and tobacco quality regulation and control.

The technical problems to be solved by the invention are realized by the following technical scheme:

an application of a tobacco alcohol dehydrogenase related gene NtADH1 in regulating and controlling tobacco waist leaf width.

Preferably, the nucleotide sequence of the NtADH1 gene is shown as SEQ ID NO.1, and the amino acid sequence of the NtADH1 coding protein is shown as SEQ ID NO. 3.

Preferably, the NtADH1 gene editing is performed by using a CRISPR/Cas9 mediated gene editing technology, a CRISPR/Cas9 editing vector for knocking out the NtADH1 gene is constructed, and a homozygous T-DNA-free tobacco plant subjected to NtADH1 gene editing is obtained after genetic transformation.

Preferably, the method for creating the tobacco plant edited by the NtADH1 gene comprises the following steps:

(1) Selecting a specific 20nt nucleotide sequence in the NtADH1 gene as a CRISPR/Cas9 guide sequence, and connecting, converting and PCR amplification detecting the sequence fragment with a CRISPR/Cas9 vector to obtain PCR positive clone, thus obtaining the CRISPR/Cas9-NtADH1 editing vector;

(2) And (3) carrying out genetic transformation and tissue culture by using the constructed CRISPR/Cas9-NtADH1 editing vector, and obtaining a T2 generation tobacco plant subjected to knockout editing on the homozygous tobacco NtADH1 gene without T-DNA through selfing.

Preferably, the more specific 20nt nucleotide sequence of the NtADH1 gene in step (1) is shown in SEQ ID No. 6.

Preferably, the agronomic and phytological property surveys of the edited plant and the control plant are carried out in the bud stage and the topping later stage, and the result shows that the waist leaf width of the mature stage of the NtADH1 gene homozygous edited tobacco plant is remarkably widened.

The technical scheme of the invention has the following beneficial effects:

(1) The CRISPR/Cas9 editing vector for knocking out the NtADH1 gene is constructed by a CRISPR/Cas9 mediated gene editing technology, and the tobacco plant with the NtADH1 gene knocked out is obtained after creation of edited materials and molecular detection and identification. Compared with a control tobacco plant, the average waist leaf width of the NtADH1 gene knocked-out edited tobacco plant obtained by the invention is obviously increased.

(2) The invention uses CRISPR/Cas9 mediated gene editing technology to knock out the NtADH1 gene to obtain edited tobacco material with obviously widened waist leaf width, which provides theoretical basis for further elucidating tobacco waist leaf width regulating mechanism and provides new genetic material for cultivating tobacco variety with obviously changed waist leaf width.

Drawings

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

FIG. 1 shows the waist length and waist width (p < 0.01) of a mature tobacco plant.

Detailed Description

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

All experimental methods used in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the following examples were commercially available unless otherwise specified.

Unless otherwise indicated, the percentages are by volume and the proportions are by volume.

The tobacco variety used in the present application is safflower Dajinyuan, a commercial tobacco variety.

Example 1

Taking leaf of Honghua Dajinyuan of cultivar tobacco as sample, using Vazyme

Plant DNA Isolation Mini Kit extracting genomic DNA of tobacco leaves; use of Shangai Promega +.>

The Super total RNA extraction kit extracts tobacco leaf total RNA, and then uses Vazyme +.>

1st Strand cDNA Synthesis Kit (+gDNA wind) reverse transcription kit reverse transcribes RNA into cDNA for later use.

By a homology comparison method, referring to the sequence of the Arabidopsis gene and the sequence of the known tobacco part gene, the amplification primer sequence is designed as follows:

NtADH1F：5'-ATGTCAAGCAATACTGCTGGG-3'(SEQ ID No.4)；

NtADH1R：5'-TCAATGCTCCATAGTGATCATG-3'(SEQ ID No.5)；

PCR amplification was performed using the DNA and cDNA prepared as described above as templates and the above primers:

amplification system (50 μl):

and (3) carrying out PCR amplification after uniformly mixing and centrifuging, wherein the PCR reaction conditions are as follows: 95 ℃ for 1min; 15sec at 95 ℃, 15sec at 56 ℃ and 3min/1min at 72 ℃ for 35 cycles; 72 ℃ for 10min; hold at 12 ℃.

And (3) purifying the amplified product, and sequencing to obtain the sequence of the tobacco alcohol dehydrogenase related gene NtADH1, wherein the DNA sequence is shown as SEQ ID No.1 and comprises 3355bp of bases, the CDS sequence is shown as SEQ ID No.2 and comprises 1146bp of bases. After the gene sequence is translated, the coded protein sequence is shown as SEQ ID No.3, which totally comprises 381 amino acids, and further comparative analysis shows that the protein contains a sequence with high homology and is highly conserved.

Example 2

The invention further constructs a CRISPR/Cas9 vector by utilizing the gene NtADH1 sequence of the tobacco alcohol dehydrogenase obtained in the example 1, and obtains a gene editing plant by utilizing leaf disc method transformation.

And (3) selecting a specific 20nt nucleotide sequence (SEQ ID No. 6) in the NtADH1 gene as a CRISPR/Cas9 guide sequence by using a CRISPR-P2.0 website, connecting the sequence fragment with a CRISPR/Cas9 vector (provided by southwest university), converting and detecting by PCR amplification, and sending a sequencing company to carry out sequencing confirmation on PCR positive clone to obtain the CRISPR/Cas9-NtADH1 editing vector.

The CRISPR/Cas9-NtADH1 editing vector plasmid constructed in the previous step is used for carrying out genetic transformation and tissue culture by taking safflower Dajinyuan as an example to obtain a plant with the knockout editing of the gene NtADH1 related to the amino acid transport of tobacco, and the related experimental process is briefly described as follows.

And (3) after the surfaces of the tobacco seeds are disinfected, dibbling the tobacco seeds on an MS culture medium, growing until 4 cotyledons (15-20 d) are grown, transferring the cotyledons into a culture bottle containing an MS solid culture medium, and continuously culturing for 35-40d at the temperature of 25+/-1 ℃ under the condition that the illumination intensity is 30-50 mu mol/(m 2 s) and the illumination time is 16h/d for standby.

LBA4404 stored at-80℃was removed and competent Agrobacterium cells were electrotransformed and frozen and thawed on ice. When the competence was just thawed, 2 μl of CRISPR/Cas9-NtADH1 editing vector plasmid was added, mixed well and placed on ice. Transferring the uniformly mixed competence into a precooled electric rotating cup, placing the electric rotating cup into an electric rotating instrument for conversion, adding 0.5mL of YEB liquid culture medium to be mixed with the conversion liquid after the conversion is finished, and placing the mixture into a shaking table for culturing at 28 ℃ and 200rpm for 1.5-2 hours. The cells were centrifuged at 8000rpm to discard the supernatant, and then the cells were suspended in 200. Mu.L of YEB liquid medium and plated on YEB solid medium containing 50mg/L rifampicin, 50mg/L streptomycin and 50mg/L kanamycin for 2-3d in an inverted dark culture at 28 ℃.

Square leaf discs with side length of 1cm were made in an ultra clean bench and agrobacterium colony suspension containing CRISPR/Cas9-NtADH1 editing vector (od600=0.6-0.8) was prepared with MS liquid. And soaking and infecting tobacco leaf discs for 10min by using suspension agrobacterium liquid. Then, the leaf discs were placed on MS solid medium containing 2.0mg/L NAA+0.5 mg/L6-BA, at 28℃in the dark, and co-cultured for 3d. Then carrying out secondary culture, and placing on an MS solid culture medium containing 2.0mg/L NAA+0.5 mg/L6-BA+250 mg/LCb+50mg/L Kan, wherein the culture conditions are as follows: culturing at 28deg.C for 16h/d with light intensity of 30-50 μmol/(m2.s), culturing at 25deg.C in dark for 8h/d, culturing for 45-60d until differentiation bud forms, and changing differentiation culture medium for 3-4 times every 7-10 d; culturing until differentiation buds are formed; cutting off the callus formed by the existing differentiation buds, placing the callus on an MS culture medium containing 500mg/L carbenicillin and 50mg/L kanamycin for culture, and culturing for 8-14 days when the differentiation buds on the callus grow to 2-4cm high under the condition consistent with the differentiation culture condition; rooting and culturing regenerated plants, cutting off differentiated buds, inserting the cut off differentiated buds into an MS culture medium containing 500mg/L of carbenicillin and 50mg/L of kanamycin for rooting and culturing, wherein the culture conditions are consistent with the differentiation culture conditions, culturing for 20-30d, regenerating and transplanting the cut off buds to a flowerpot for culturing, sampling leaves of the transformed plants, sending the samples to a sequencing company for molecular detection by using NtADH1-1F/NtADH1-1R primers, determining that the NtADH1 gene homozygous edited plants are obtained, and then carrying out selfing. T to be homozygously edited ₀ And planting the generation seeds, sampling the leaves of the single plant when the plants grow to 5-6 leaves, carrying out molecular detection by using a Cas9F/Cas9R primer, determining to obtain a T-DNA-free plant subjected to homozygous editing of the NtADH1 gene, and then carrying out self-copulation to obtain the T1 generation seeds (T2 generation).

NtADH1-1F：5'-GCATGGTCTGTTTGGTGTGTG-3'(SEQ ID No.7)；

NtADH1-1R：5'-TTTCTGTGGTGGTGCAACCT-3'(SEQ ID No.8)；

Cas9F：5'-GATCTCCCAGTCACGACGTT-3'(SEQ ID No.9)；

Cas9R：5'-TGTAACGGCGTCTGGCGGTGCGCTTC-3'(SEQ ID No.10)；

The application of the tobacco alcohol dehydrogenase gene NtADH1 in the invention is that the expression of the NtADH1 gene is reduced in tobacco plants, and the waist leaf width of the tobacco in the mature period can be regulated and controlled. Methods of reducing gene expression or gene silencing commonly used in the art are suitable for use in the present invention.

Example 3

T determined as homozygous knockout of the NtADH1 Gene without T-DNA by molecular assay in example 2 ₁ The plants of the generation are subjected to selfing to obtain the seeds. And (3) planting the control safflower Dajinyuan and editing material T2 generation in a greenhouse, sampling single plant leaves when the plants grow to 5-6 leaves, and carrying out molecular detection to determine the T2 generation plants with homozygous editing of the NtADH1 gene without the T-DNA. The main agronomic traits and the phytology traits of the editing materials and the control are investigated in the bud stage and the topping later stage by referring to the industry standard YCT 142-2010 tobacco agronomic trait investigation and measurement method. Including systematic measurement of main agronomic traits (plant height, pitch, stem circumference, effective leaf number, waist leaf length, waist leaf width, etc.) and plant traits (plant type, leaf shape, leaf color, etc.) of the propagated population.

Control (unedited) and NtADH1 gene homozygous editing tobacco plants were compared for length and width of mature waist leaves (results shown in figure 1). The waist leaf width of the homozygous editing tobacco plants at maturity was significantly increased compared to the control, the NtADH1 gene.

Although the present invention has been described with reference to the above embodiments, it should be understood that the present invention is not limited thereto, and that various changes and modifications may be made by those skilled in the art without departing from the spirit and scope of the present invention, and the scope of the present invention is defined by the appended claims and their equivalents.

Claims (6)

1. An application of a tobacco alcohol dehydrogenase related gene NtADH1 in regulating and controlling tobacco waist leaf width.

2. The use according to claim 1, characterized in that the nucleotide sequence of the NtADH1 gene is shown in SEQ ID No.1 and the amino acid sequence of the NtADH 1-encoding protein is shown in SEQ ID No. 3.

3. The use according to claim 2, characterized in that the NtADH1 gene editing is by CRISPR/Cas9 mediated gene editing technology, a CRISPR/Cas9 editing vector for knocking out the NtADH1 gene is constructed, and a homozygous T-DNA free tobacco plant with the NtADH1 gene edited is obtained after genetic transformation.

4. The use according to claim 3, wherein the method for creating tobacco plants edited by the NtADH1 gene comprises:

(1) Selecting a specific 20nt nucleotide sequence in the NtADH1 gene as a CRISPR/Cas9 guide sequence, and connecting, converting and PCR amplification detecting the sequence fragment with a CRISPR/Cas9 vector to obtain PCR positive clone, thus obtaining the CRISPR/Cas9-NtADH1 editing vector;

(2) And (3) carrying out genetic transformation and tissue culture by using the constructed CRISPR/Cas9-NtADH1 editing vector, and obtaining a T2 generation tobacco plant subjected to knockout editing on the homozygous tobacco NtADH1 gene without T-DNA through selfing.

5. The use according to claim 4, wherein the more specific 20nt nucleotide sequence of the NtADH1 gene in step (1) is shown in SEQ ID No. 6.

6. The use according to claim 5, wherein agronomic and phytological investigation of the edited plants and control plants during the bud phase and the topping phase revealed a significant broadening of the waist leaf width in the maturity phase of the homozygous edited tobacco plant of the NtADH1 gene.

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