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 PDFInfo
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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
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
<110> Yunnan Zhongyan industry Limited liability company
<120> a gene related to ATPase A1 subunit of tobacco vacuolar membrane and application thereof
<130> WPC221147
<141> 2022-04-18
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ggacttctcc aattccgtga tctaaatgcc gaaaagagcc ctttccagag aacatttgta 180
aaccaggtaa aacgatgcgc ggagatgtca agaaaactaa gatatttcaa agatcagata 240
cacaaagccg gtctattgcc tcctcctctt cctgcttccc aacctgatat tgaattagaa 300
gaattggaga tacaactggc agagcatgaa catgagttga ttgaaatgaa tgctaatagt 360
gaaaaattgc ggcaatcata taatgagctg cttgagttta agatggtatt gcaaaaggct 420
agtggcttcc ttgtttcaag tagtcatacc actgatcagg aaacagaatt ggatgaaaat 480
gtgtactcca atgataacca tgcggataca gcatcattac ttgagcagga gatgcgttca 540
gaactgtcaa atcagtctgg agttagattt attagtggca ttatctgcaa gtccaaggtt 600
gttcaatttg aaagaatgct gtttcgtgct acaaggggta atatgctttt caatcaggca 660
gttgctgatg atgaaatact ggatccttcc tcaaacgaaa tggttgagaa agtagtcttt 720
gtagtattct tttcaggtga gcaggcaaga acaaaaatac tgaaaatatg tgaggcattt 780
ggtgcaaatt gctatcctgt tcctgaagac acgacaaaga gaaggcagat aactcaagaa 840
gttttgtctc ggctatctga attagagacc actctggatg ctggactgcg ccatagagat 900
aaggctttga cctccatagg ttatcacctt acaaaatgga taaatatggc taaaacacaa 960
aaggcagtgt atgacacatt aaatatgcta aatttcgatg ttacaaagaa gtgccttgtg 1020
ggtgagggct ggtgtccaat atttgctaaa accaagatac aagaggcttt gcagcgtgca 1080
acatttgata gcagttcaca agtggggatt atatttcatg tgatggatgc tgtggagtca 1140
cctccaacat actttaggac aaacagtttc acaaatgcat ttcaagaaat tgttgatgca 1200
tatggtgttg ctaaatacca ggaggcaaat ccagctgttt ataccattgt tacatttcct 1260
ttcctttttg ctgtgatgtt tggggactgg ggtcatggaa tctgcttgct gttgggagca 1320
ttagttctta tcgcaaggga aagcaaactt agctctcaga aactaggcag cttcatggag 1380
atgctctttg ggggtcgcta tgtactcgtg ttgatgtcaa tattttcaat ttactgtggc 1440
ttgctatata atgaattctt ctcagttccc tttcacatat ttggtgaatc agcgtacaaa 1500
tgccgagatg ctacatgcag tgatgcacgg acagttggtt tagtaaaata caaggatcca 1560
tatccatttg gcgtggaccc aagctggaga ggcagccgtt cagaacttcc tttcttgaat 1620
tctcttaaga tgaagatgtc tattttgttg ggtgtggccc agatgaacct cggaattatt 1680
ttaagttatt tcaatgcacg tttcttcagc agctcaattg atattaagta tcagtttatt 1740
ccacaagtga tctttctcaa cagcctcttt ggataccttt cgcttctcat tcttgtcaaa 1800
tggtgcactg gttctcaagc agatctatat catgttatga tttatatgtt cttaagtcct 1860
tttgaggctc ttggtgaaaa taggttgttc tggggccaga gtgtgcttca ggtaatattg 1920
ctgcttttgg cacttgttgc tgttccatgg atgctcttcc caaaaccttt tattttgaaa 1980
agacttcata tggagagatt tcaaggtcgt acatacggga tacttggaac ctccgagatg 2040
ggtattgacg aagaaccagg ctctgccagg cagcatgctg aggagtttaa ctttagtgag 2100
gtttttgtgc accaaatgat acactctatt gaatttgtac ttggtgctgt ttctaacact 2160
gcatcgtatc ttcggctttg ggctttgagt ttggctcact ctgaattatc tactgtgttc 2220
tatgagaaag ttctcctcct tgcctggggg tatgataaca tcgtcatacg gctagtgggg 2280
ctggcagttt ttgcctttgc gacaactttt atactactta tgatggagac tcttagtgca 2340
ttccttcatg cattgcgtct tcattgggtg gaattccaga acaagtttta tcatggggat 2400
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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.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
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| 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)
| Title |
|---|
| PREDICTED: Nicotiana tabacum V-type proton ATPase subunit a1-like (LOC107818994), mRNA.Genbank:XM_016645073.1.2016,全文. * |
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