CN114891813B - Tobacco vacuolar membrane ATPase A1 subunit related gene and application thereof - Google Patents

Tobacco vacuolar membrane ATPase A1 subunit related gene and application thereof Download PDF

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CN114891813B
CN114891813B CN202210405271.XA CN202210405271A CN114891813B CN 114891813 B CN114891813 B CN 114891813B CN 202210405271 A CN202210405271 A CN 202210405271A CN 114891813 B CN114891813 B CN 114891813B
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许力
许�永
李雪梅
张伟
邓乐乐
高茜
米其利
杨叶昆
刘欣
杨文武
蒋佳芮
向海英
曾婉俐
张建铎
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China Tobacco Yunnan Industrial Co Ltd
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Abstract

The invention discloses a tobacco vacuole membrane ATPase A1 subunit related gene and application thereof, wherein the gene is named NtVHa-A1, and the nucleotide sequence is shown as SEQ ID NO.1 and comprises 2457bp bases. The gene editing technology mediated by CRISPR/Cas9 is utilized to knock out the NtVHa-A1 gene to obtain the material with the amino acid content changed, so that genetic materials and theoretical basis are provided for the study of the tobacco vacuolar membrane ATPase A1 subunit and the study of improving tobacco quality.

Description

Tobacco vacuolar membrane ATPase A1 subunit related gene and application thereof
Technical Field
The invention relates to the technical field of plant genetic engineering, in particular to a tobacco vacuolar membrane ATPase A1 subunit related gene and application thereof.
Background
Biological membrane H is currently known + There are three main classes of ATPase: p-type H + ATPase, type F H + ATPase and form V H + ATPase (vacuolar membrane ATPase). Wherein, V-shaped H + ATPase has complex structure, various functions and rich varieties and becomes H + New hot spot for ATPase study. V-shaped H + ATPase plays a key role in the regulation of ion balance in plant cells, pumping protons from the cytosol into the vacuoles to acidify them, creating a proton electrochemical gradient, and thus the enzyme can power secondary transport of other various ions and metabolites.
Plant vacuolar membrane ATPase (V type H) + ATPase) is an enzyme commonly encoded by a multi-subunit gene that is widely present on eukaryotic vacuole membranes, the main functions including active transport of protons coupled with ATP hydrolysis and control of other solutes into and out of vacuoles and cytoplasm. Thus playing an important role in maintaining cell homeostasis.
Studies in rice indicate that the A subunit of the OsVHA-A1 gene responsible for encoding is the main catalytic center of the vacuolar membrane ATPase and is involved in binding ATP. In the rice mutation, the OsVHA-A1 gene is studied to cause rice premature senility, and in the rice, glycine, serine and threonine metabolism and lysine biosynthesis are carried out in the gene mutant. It was thus deduced that the VHA-A1 mutation leads to the accumulation of these amino acids in the roots, and therefore these amino acids are thought to play an important role in helping the plant to cope with premature senescence.
Research of plant vacuolar membrane ATPase in plantsArabidopsis thaliana is the most abundant. In Arabidopsis, at least 26 genes encode the 12 subunits of the holoenzyme. The research shows that the functions of the enzyme in different parts of arabidopsis thaliana are greatly different. Such as: v-shaped H + Expression of the ATPase C1 subunit in the expanded cotyledon, the hypocotyl of the etiolated seedling, the root elongation region, supporting type V H + -the role of ATPase in cell volume increase; in root cap, V-shaped H + ATPase affects root growth and tolerance to neutral salt stress. V-type H in tomato (Lycopersicon esculentum) + The expression level of the ATPase A subunit occurs only transiently under salt stress conditions and thereafter exhibits a non-responsive state.
Tobacco is used as an important economic crop and a mode crop in China, and research on molecular mechanisms of tobacco vacuole membrane ATPase in response to amino acid to regulate and control tobacco maturity has important significance on tobacco quality. In tobacco vacuole membrane ATPase, few research reports exist at present, and according to the research in other plants, the change of amino acid content of a vacuole membrane ATPase A1 subunit editing plant and a control plant in the maturity period is detected, and the influence of the change of chemical components of the editing plant on tobacco quality is researched.
Disclosure of Invention
The invention aims to solve the technical problem of providing a gene related to a tobacco vacuolar membrane ATPase A1 subunit (VHa-A1) and application thereof, and provides genetic materials and theoretical basis for researching tobacco quality and gene functions.
The technical problems to be solved by the invention are realized by the following technical scheme:
a gene related to a tobacco vacuolar membrane ATPase A1 subunit is named NtVHa-A1, and the nucleotide sequence of the gene is shown as SEQ ID NO.1 and comprises 2457bp bases.
Preferably, in the above technical scheme, the NtVHA-A1 gene encodes a protein, and the amino acid sequence is shown as SEQ ID NO.2 and comprises 818 amino acids.
Use of a gene related to the atpase A1 subunit of the vacuolar membrane of tobacco for improving tobacco quality, said gene being the NtVHa-A1 gene.
Preferably, in the technical scheme, the NtVHA-A1 gene editing plant is constructed by using a CRISPR/Cas9 mediated gene editing technology to knock out a CRISPR/Cas9 editing vector of the NtVHA-A1 gene, and the plant subjected to the NtVHA-A1 gene editing is obtained after genetic transformation.
Preferably, in the above technical scheme, the method for creating the tobacco plant edited by the NtVHA-A1 gene specifically includes:
(1) Selecting a 23nt nucleotide sequence which is more specific in the NtVHA-A1 gene as a CRISPR/Cas9 guide sequence, connecting the sequence fragment with a CRISPR/Cas9 vector, converting and performing PCR amplification detection to obtain PCR positive clone, and obtaining the CRISPR/Cas9-NtVHA-A1 editing vector;
(2) And (3) utilizing the constructed CRISPR/Cas9-NtVHA-A1 editing vector plasmid to carry out genetic transformation so as to knock out the NtVHA-A1 gene in the plant body and obtain the NtVHA-A1 gene editing plant.
Preferably, in the above technical scheme, the specific 23nt nucleotide sequence of the NtVHA-A1 gene in the step (1) is shown as SEQ ID No. 5.
Preferably, in the above technical scheme, the contents of serine, arginine, aspartic acid, leucine and methionine in the NtVHA-A1 gene editing plant are lower than those of the control material, the content of lysine is higher than that of the control material, and glycine and proline have no obvious difference from those of amino acids in the control.
The technical scheme of the invention has the following beneficial effects:
the invention constructs a CRISPR/Cas9 editing vector for knocking out the NtVHA-A1 gene by a CRISPR/Cas9 mediated gene editing technology, and obtains a safflower Dajinyuan plant edited for the NtVHA-A1 gene after genetic transformation. According to the invention, under normal conditions, amino acid content of fresh tobacco leaves of mature editing materials and red-big control materials is detected, and the change of the amino acid content is found.
In conclusion, the gene editing technology mediated by CRISPR/Cas9 is utilized to knock out the NtVHa-A1 gene to obtain the material with the amino acid content changed, so that genetic materials and theoretical basis are provided for the study of the tobacco vacuolar membrane ATPase A1 subunit and the study of improving tobacco quality.
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 is a graph comparing glycine changes in fresh tobacco leaves at maturity for control (untransformed) plants and gene-edited plants.
FIG. 2 is a graph comparing proline changes in fresh tobacco leaves at maturity for control (untransformed) plants and gene-edited plants.
FIG. 3 is a graph comparing serine changes in fresh tobacco leaves at maturity for control (untransformed) plants and gene-edited plants.
FIG. 4 is a graph comparing arginine changes in fresh tobacco leaves at maturity for control (untransformed) plants and gene-edited plants.
FIG. 5 is a graph comparing changes in aspartic acid in fresh tobacco leaves at maturity for control (untransformed) plants and gene-edited plants.
FIG. 6 is a graph comparing leucine changes in fresh tobacco leaves at maturity for control (untransformed) plants and gene-edited plants.
FIG. 7 is a graph comparing methionine change in fresh tobacco leaves at maturity for control (untransformed) plants and gene-edited plants.
FIG. 8 is a graph comparing lysine changes in fresh tobacco leaves at maturity for control (untransformed) plants and gene-edited plants.
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.
Example 1
This example is mainly described below with respect to the process of obtaining the gene NtVHa-A1 related to the ATPase A1 subunit of the tobacco vacuolar membrane.
The method comprises the steps of taking tobacco safflower Dajinyuan leaves as a sample, extracting total RNA of the tobacco leaves by using an RNA extraction kit, and carrying out reverse transcription to obtain cDNA for later use:
extracting total RNA of tobacco according to the instruction of the plant RNA extraction kit.
1 μg total RNA extracted from leaf for reverse transcription was as follows:
Total RNA 1μg；
Oligo(dT)(10μM) 1.5μL；
ddH 2 O up to 15μL；
mixing the above systems, placing in PCR, maintaining at 70deg.C for 5min, removing, immediately placing on ice for 5min, and adding the following reagents:
placing the above system into a PCR instrument, keeping temperature at 42deg.C for 65min, 65deg.C for 10min, and 4deg.C, and storing in a refrigerator at-20deg.C.
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:
F：5’-ATGGAGTACATAGACAACATGCCGCC-3’(SEQ ID No.3)；
R：5’-CTAATCGTCATCATCTGCTAACAAGGC-3’(SEQ IDNo.4)；
PCR amplification was performed using the cDNA prepared as described above as a template 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℃10sec,52℃30sec,72℃2.5min for 30 cycles; 72 ℃ for 10min; hold at 12 ℃.
And (3) purifying and sequencing the amplified product to obtain a gene NtVHA-A1 sequence related to the tobacco ethylene response transcription factor, wherein the base sequence is shown as SEQ ID No.1 and comprises 2457bp bases. After the gene sequence is translated, the coded protein sequence is shown as SEQ ID No.2, and contains 818 amino acids, and further, the comparison 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 NtVHa-A1 related to the ATPase A1 subunit of the tobacco vacuolar membrane obtained in the example 1, and obtains a gene editing plant by utilizing a leaf disk method for transformation.
The 23nt nucleotide sequence (SEQ ID No. 5) which is more specific in the NtVHA-A1 gene is selected as a CRISPR/Cas9 guiding sequence, the sequence fragment is connected with a CRISPR/Cas9 carrier (provided by southwest university) to obtain transformed clones, PCR amplification detection is carried out, and then the PCR positive clones are sent to a sequencing company for sequencing confirmation, so that the CRISPR/Cas9-NtVHA-A1 editing carrier is finally obtained.
And (3) editing a vector plasmid by using the CRISPR/Cas9-NtVHA-A1 constructed in the last step, taking safflower Dajinyuan as an example, and carrying out a genetic transformation test to knock out a gene NtVHA-A1 related to a tobacco ethylene response transcription factor in a plant body, wherein the related experimental process is briefly described below.
Obtaining aseptic seedlings: the tobacco seeds are planted in a culture dish, after the tobacco seeds grow to 4 cotyledons (15-20 d), the tobacco seeds can be transferred into culture bottles (containing 80mL MS liquid culture medium), 2 plants are planted in each bottle, and the illumination intensity is 30-50 mu mol/(m) at 25+/-1 DEG C 2 S) the culture was continued for 40d at 16h/d for further use.
Transformation of Agrobacterium: 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. Mu.L of plasmid containing the edited NtVHA-A1 gene was added, gently flicked, mixed and placed on ice. Then, transformation culture was performed.
Transformation and culture of tobacco leaf discs: making tobacco leaf disc into square leaf disc with side length of 1cm in ultra clean workbench, preparing agrobacterium containing CRISPR/Cas9-NtVHA-A1 editing vector with MS liquidFalling into suspension (OD) 600 =0.6-0.8). And soaking and infecting tobacco leaf discs for 10min by using suspension agrobacterium liquid. Leaf disc subcultures were then performed and it was determined that NtVHA-A1 gene-edited plants were obtained.
Example 3
The molecular detection in example 2 was used to determine that NtVHA-A1 gene knockout plants were used for seed collection to obtain gene editing materials.
Planting an edited plant and a control plant, sampling fresh tobacco leaves in a maturity stage, detecting 8 amino acids, and measuring 8 free amino acids in the fresh tobacco leaves, wherein the detection comprises glycine, proline, serine, arginine, aspartic acid, leucine, methionine and lysine, the content of serine, arginine, aspartic acid, leucine and methionine in the edited material is obviously lower than that in the control material, the content of lysine is obviously higher than that in the control material, and the glycine, proline and the amino acids in the control are not obviously different (the results are shown in figures 1-8).
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.
Sequence listing
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Gly Ile Ile Phe His Val Met Asp Ala Val Glu Ser Pro Pro Thr Tyr
370 375 380
Phe Arg Thr Asn Ser Phe Thr Asn Ala Phe Gln Glu Ile Val Asp Ala
385 390 395 400
Tyr Gly Val Ala Lys Tyr Gln Glu Ala Asn Pro Ala Val Tyr Thr Ile
405 410 415
Val Thr Phe Pro Phe Leu Phe Ala Val Met Phe Gly Asp Trp Gly His
420 425 430
Gly Ile Cys Leu Leu Leu Gly Ala Leu Val Leu Ile Ala Arg Glu Ser
435 440 445
Lys Leu Ser Ser Gln Lys Leu Gly Ser Phe Met Glu Met Leu Phe Gly
450 455 460
Gly Arg Tyr Val Leu Val Leu Met Ser Ile Phe Ser Ile Tyr Cys Gly
465 470 475 480
Leu Leu Tyr Asn Glu Phe Phe Ser Val Pro Phe His Ile Phe Gly Glu
485 490 495
Ser Ala Tyr Lys Cys Arg Asp Ala Thr Cys Ser Asp Ala Arg Thr Val
500 505 510
Gly Leu Val Lys Tyr Lys Asp Pro Tyr Pro Phe Gly Val Asp Pro Ser
515 520 525
Trp Arg Gly Ser Arg Ser Glu Leu Pro Phe Leu Asn Ser Leu Lys Met
530 535 540
Lys Met Ser Ile Leu Leu Gly Val Ala Gln Met Asn Leu Gly Ile Ile
545 550 555 560
Leu Ser Tyr Phe Asn Ala Arg Phe Phe Ser Ser Ser Ile Asp Ile Lys
565 570 575
Tyr Gln Phe Ile Pro Gln Val Ile Phe Leu Asn Ser Leu Phe Gly Tyr
580 585 590
Leu Ser Leu Leu Ile Leu Val Lys Trp Cys Thr Gly Ser Gln Ala Asp
595 600 605
Leu Tyr His Val Met Ile Tyr Met Phe Leu Ser Pro Phe Glu Ala Leu
610 615 620
Gly Glu Asn Arg Leu Phe Trp Gly Gln Ser Val Leu Gln Val Ile Leu
625 630 635 640
Leu Leu Leu Ala Leu Val Ala Val Pro Trp Met Leu Phe Pro Lys Pro
645 650 655
Phe Ile Leu Lys Arg Leu His Met Glu Arg Phe Gln Gly Arg Thr Tyr
660 665 670
Gly Ile Leu Gly Thr Ser Glu Met Gly Ile Asp Glu Glu Pro Gly Ser
675 680 685
Ala Arg Gln His Ala Glu Glu Phe Asn Phe Ser Glu Val Phe Val His
690 695 700
Gln Met Ile His Ser Ile Glu Phe Val Leu Gly Ala Val Ser Asn Thr
705 710 715 720
Ala Ser Tyr Leu Arg Leu Trp Ala Leu Ser Leu Ala His Ser Glu Leu
725 730 735
Ser Thr Val Phe Tyr Glu Lys Val Leu Leu Leu Ala Trp Gly Tyr Asp
740 745 750
Asn Ile Val Ile Arg Leu Val Gly Leu Ala Val Phe Ala Phe Ala Thr
755 760 765
Thr Phe Ile Leu Leu Met Met Glu Thr Leu Ser Ala Phe Leu His Ala
770 775 780
Leu Arg Leu His Trp Val Glu Phe Gln Asn Lys Phe Tyr His Gly Asp
785 790 795 800
Gly Tyr Lys Phe Lys Pro Phe Ser Phe Ala Leu Leu Ala Asp Asp Asp
805 810 815
Asp
<210> 3
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
atggagtaca tagacaacat gccgcc 26
<210> 4
<211> 27
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
ctaatcgtca tcatctgcta acaaggc 27
<210> 5
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 5
gcgcagtcca gcatccagag tgg 23

Claims (3)

1. The application of a gene related to a tobacco vacuolar membrane ATPase A1 subunit in improving tobacco quality is characterized in that the gene is named as NtVHa-A1, the nucleotide sequence is shown as SEQ ID NO.1, the amino acid sequence of the protein encoded by the NtVHa-A1 gene is shown as SEQ ID NO.2, wherein the contents of serine, arginine, aspartic acid, leucine and methionine in plants with the NtVHa-A1 knocked out are lower than those in control materials, the content of lysine is higher than those in the control materials, and the amino acids in glycine and proline are not obviously different from those in the control materials.
2. The use of the gene related to the ATPase A1 subunit of the vacuolar membrane of tobacco in improving the quality of tobacco according to claim 1, wherein the NtVHa-A1 gene knockout plant is obtained by constructing a CRISPR/Cas9 editing vector for knocking out the NtVHa-A1 gene through a CRISPR/Cas9 mediated gene editing technology and obtaining the plant from which the NtVHa-A1 gene is knocked out after genetic transformation.
3. The use of the gene related to the atpase A1 subunit of the vacuolar membrane of tobacco according to claim 2 for improving the quality of tobacco, characterized in that the method for creating plants from which the NtVHa-A1 gene is knocked out comprises:
(1) A 23nt nucleotide sequence which is more specific in the NtVHA-A1 gene is shown as SEQ ID No.5 and is a CRISPR/Cas9 guide sequence, and the sequence fragment is connected with a CRISPR/Cas9 vector, converted and subjected to PCR amplification detection to obtain PCR positive clone, so as to obtain the CRISPR/Cas9-NtVHA-A1 editing vector;
(2) And editing a vector plasmid by using the constructed CRISPR/Cas9-NtVHA-A1, and carrying out genetic transformation to knock out the NtVHA-A1 gene in the plant body to obtain a NtVHA-A1 gene knocked-out plant.
CN202210405271.XA 2022-04-18 2022-04-18 Tobacco vacuolar membrane ATPase A1 subunit related gene and application thereof Active CN114891813B (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111004808A (en) * 2019-12-19 2020-04-14 中国烟草总公司郑州烟草研究院 Tobacco protein NtVHA-a1 and application thereof
CN113151307A (en) * 2021-06-11 2021-07-23 云南中烟工业有限责任公司 Gene related to tobacco ethylene response transcription factor and application thereof
CN113999857A (en) * 2021-11-22 2022-02-01 云南中烟工业有限责任公司 Gene related to tobacco nicotine synthesis regulation and control and application thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111004808A (en) * 2019-12-19 2020-04-14 中国烟草总公司郑州烟草研究院 Tobacco protein NtVHA-a1 and application thereof
CN113151307A (en) * 2021-06-11 2021-07-23 云南中烟工业有限责任公司 Gene related to tobacco ethylene response transcription factor and application thereof
CN113999857A (en) * 2021-11-22 2022-02-01 云南中烟工业有限责任公司 Gene related to tobacco nicotine synthesis regulation and control and application thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
PREDICTED: Nicotiana tabacum V-type proton ATPase subunit a1-like (LOC107818994), mRNA.Genbank:XM_016645073.1.2016,全文. *

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