CN110305884B - Gene NtAIS 1 for improving jasmonic acid content of tobacco leaves and cloning method and application thereof - Google Patents
Gene NtAIS 1 for improving jasmonic acid content of tobacco leaves and cloning method and application thereof Download PDFInfo
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Abstract
The invention discloses a gene NtAAS 1 for improving the jasmonic acid content of tobacco leaves, and a cloning method and application thereof. The product can increase jasmonic acid content in tobaccoNtAOS1The nucleotide sequence of (a) is shown as SEQ ID NO:1 is shown. The amino acid sequence of the encoded protein is shown as SEQ ID NO:2 is shown in the specification; designing 1 segment of interference fragment by adopting a nucleic acid interference (RNAi) technology, constructing a plant expression vector by adopting a gateway technology, transforming tobacco, and selecting by adopting a qPCR technologyNtAOS1The gene transcription level is obviously reduced in transgenic plants. The jasmonic acid content detection shows that:NtAOS1the reduced level of gene expression instead resulted in a significantly higher jasmonic acid content than the wild-type control. The invention is thatNtAOS1The gene and the encoding protein thereof are applied to tobacco stress-resistant breeding and the subsequent research of jasmonic acid anabolism approaches in tobacco, and a material basis is provided.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a gene NtAOS1 for improving jasmonic acid content in tobacco leaves, and a cloning method and application thereof.
Background
Jasmonic Acid (JA) and methyl jasmonate (methyl jasmate) as a methyl jasmonic acid derivative are important growth regulators of plants and play a role in regulating and controlling the growth of the plants. On one hand, jasmonic acid can regulate the growth and development of plants, such as the germination and growth of seeds, the growth and development of organs, the senescence and apoptosis of plants and the like, and on the other hand, jasmonic acid also participates in the defense reaction of the plants against biotic stress and abiotic stress. The biosynthetic pathway of JA is also known as the octadecane-like biosynthetic pathway: linolenic acid is catalyzed by Lipoxygenase (LOX) to synthesize 13 (S) -hydroperoxyl linolenic acid (13-hydroperoxylidenoic acid, 13-HPOT), and the 13-HPOT generates 10, 11-dihydro-12-oxo phytodienoic acid (OPDA) under the catalysis of enzymes such as Allene Oxide Synthase (AOS) and Allene Oxide Cyclase (AOC). OPDA is oxidized by beta for three times to generate JA, so that AOS is a key enzyme of jasmonic acid synthesis pathway in plants. AOS belongs to a member of the P450 supergene family, is ubiquitous in plant tissues, and is involved in regulating many physiological processes in plants by regulating JA biosynthesis. Research in rice shows that rice insect pests can induce the expression of the AOS gene, and the over-expression of the AOS2 gene in the rice can improve the content of endogenous JA of the rice, induce the expression of PR genes related to the course of disease and enhance the resistance to rice blast; the injury stress can induce the expression of AOS genes in tomato leaves. Therefore, the AOS gene expression plays an important role in improving the adaptability of the plant to the physical environment and the interspecific competitiveness and enhancing the defense response of the plant.
Several upstream genes involved in the JA synthetic pathway are localized in chloroplasts, whereas β -oxidase is localized in peroxisomes (peroxisomes) and jasmonate-modifying enzymes are present in the cytoplasm. It has also been reported that the JAs synthesis related to development and JAs synthesis related to wound induction in arabidopsis thaliana are mutually overlapped, but are not completely the same, i.e. 2 JA synthesis pathways or 2 different synthesis regulation pathways may exist in the plant, which brings about a great difficulty to the research of the JA synthesis pathways, but at the same time indicates that more ways for regulating the synthesis pathways exist.
The research on the anabolism of jasmonic acid in tobacco is less, the invention discovers that the concentration of jasmonic acid in tobacco leaves is increased relative to wild tobacco by interfering with the NtAAS 1 gene through nucleic acid, and a transgenic plant generated by interference of RNAi has great application value for researching the anabolism of jasmonic acid in tobacco.
Disclosure of Invention
The first purpose of the invention is to provide a gene NtAAS 1 for improving the jasmonic acid content of tobacco leaves; the second purpose is to provide a cloning method of the gene NtAAS 1 for improving the jasmonic acid content of the tobacco leaves; the third purpose is to provide the application of the gene NtAOS1 for improving the jasmonic acid content of the tobacco leaves.
The first purpose of the invention is realized by that the nucleotide sequence of the gene NtAAS 1 for improving the jasmonic acid content of the tobacco leaves is shown in a sequence table SEQ ID NO:1 is shown.
The second object of the present invention is achieved by comprising the steps of:
A. extracting tobacco root tissue RNA, and performing reverse transcription to obtain first-strand cDNA;
B. designing and synthesizing a specific primer according to the NtAAS 1 gene sequence, carrying out PCR amplification by taking the first strand cDNA obtained by reverse transcription as a template, and recovering and purifying a PCR product;
D. the purified product is connected with a carrier and is connected with a TOPO carrier through a kit reaction, and a connection system and the process are as follows: 4 μ L of purified product, 5X buffer 2 μ L, T4 ligase 1 μ L, H 2 O 2μL、 1μL PCR ® -Blunt II-TOPO (invitrogen) and water bath at 25 ℃ for 10min; and (3) carrying out heat shock transformation on the connected vector to enter escherichia coli DH5 alpha, adding a liquid culture medium for shaking culture, then coating the obtained product on an LB (lysogeny broth) plate containing 100mg/L kanamycin for overnight culture at 37 ℃, picking a bacterial colony for PCR (polymerase chain reaction) positive detection, picking a positive bacterial colony, shaking the bacterial colony to extract a plasmid, and sequencing a positive clone.
The third purpose of the invention is realized by the application of the gene NtAOS1 for improving the jasmonic acid content of tobacco leaves in obtaining transgenic tobacco plants with obviously improved jasmonic acid content of the tobacco leaves.
Drawings
FIG. 1 is an agarose gel electrophoresis image of NtAO 1 gene amplified using the following primers NtAO _1F/NtAO 1_ R, the amplification product size being approximately 1600bp, M: DL2000,1,3 is the NtAAS 1 gene band;
FIG. 2 shows agarose gel electrophoresis of RNAi fragments after LR reaction Recombinant Nucleic Acid Interference (RNAi) pHELLSGATE 12-NtAAS 1 vector, digested with Xba I and Xho I. M: DL2000;1, pHELLSGATE 12-NtAAS 1 vector plasmid; 2, cutting two bands of about 200bp and 3500bp after digesting the vector by the nucleic acid interference PCR fragment and 3 Xba I enzyme; 4, cutting two bands of about 200bp and 3500bp after Xho I enzyme digestion of the vector;
FIG. 3 is T 0 The gene expression level of the generation NtAAS 1 gene RNAi transgenic plant NtAAS 1 is analyzed, WT is a wild type control, and RNAi is a transgenic strain;
FIG. 4 is a standard curve prepared by measuring jasmonic acid concentration in tobacco leaves;
FIG. 5 shows the jasmonic acid content analysis of leaves of T0 generation NtAAS 1 gene transgenic plants, WT is a wild type control, and RNAi is a transgenic line.
Detailed Description
The present invention is further described with reference to the following examples and drawings, but the present invention is not limited thereto in any way, and any modifications or alterations based on the teaching of the present invention are within the scope of the present invention.
The nucleotide sequence of the gene NtAOS1 for improving the jasmonic acid content of the tobacco leaves is shown in a sequence table SEQ ID NO:1 is shown.
The amino acid sequence coded by the gene NtAAS 1 for improving the jasmonic acid content of the tobacco leaves is shown as SEQ ID NO:2, respectively.
The cloning method of the gene NtAAS 1 for improving the jasmonic acid content of the tobacco leaves comprises the following steps:
A. extracting tobacco root tissue RNA, and performing reverse transcription to obtain first-strand cDNA;
B. designing and synthesizing a specific primer according to the NtAAS 1 gene sequence, carrying out PCR amplification by taking the first strand cDNA obtained by reverse transcription as a template, and recovering and purifying a PCR product;
D. the purified product is connected with a carrier and is connected with a TOPO carrier through a kit reaction, and a connection system and the process are as follows: 4 μ L of purified product, 5X buffer 2 μ L, T4 ligase 1 μ L, H 2 O 2μL、 1μL PCR ® -Blunt II-TOPO (invitrogen) and water bath at 25 ℃ for 10min; and (3) carrying out heat shock transformation on the connected vector to enter escherichia coli DH5 alpha, adding a liquid culture medium for shaking culture, then coating the obtained product on an LB (lysogeny broth) plate containing 100mg/L kanamycin for overnight culture at 37 ℃, picking a bacterial colony for PCR (polymerase chain reaction) positive detection, picking a positive bacterial colony, shaking the bacterial colony to extract a plasmid, and sequencing a positive clone.
The specific primers are as follows:
a forward primer: ntAOS1_ F: ATGGCAGTAGCAACAGCAACAG
Reverse primer: ntAOS1_ R: TCAAATTTCCGTTCCAACTCCGATC.
And C, selecting a Phusion high-fidelity amplification enzyme reaction system as a PCR amplification reaction system in the step C, wherein the total volume of the system is 50 mu L, and the method comprises the following steps: 200ng cDNA,5 XPHUSION HF reaction buffer 10. Mu.L, 10mM dNTP 1. Mu.L, 2U Phusion ® High-Fidelity DNA Polymerase, 1. Mu.L each of 10. Mu.M forward and reverse primers, and water was added to 50. Mu.L.
The reaction condition for PCR amplification in step C is in Mastercycler ® The pro amplification instrument is used for carrying out the following reaction procedures: 30 seconds at 98 ℃; 35 cycles of 98 ℃,7 seconds, 58 ℃,30 seconds, 72 ℃,30 seconds; extension at 72 ℃ for 7 minutes.
The application of the gene NtAO 1 for improving the jasmonic acid content of the tobacco leaves is the application of the gene NtAO 1 for improving the jasmonic acid content of the tobacco leaves in obtaining transgenic tobacco plants with obviously improved jasmonic acid content of the leaves.
The invention is further illustrated by the following specific examples:
example 1-isolated cloning of the NtAIS 1 Gene, comprising the following steps:
A. verifying the NtAAS 1 gene sequence;
the NtAOS1 gene was cloned using the following primers:
a forward primer: ntAOS1_ F: ATGGCAGTAGCAACAGCAACAG
Reverse primer: ntAOS1_ R: TCAAATTTCCGTTCCAACTCCGATC
B. Extracting tobacco leaf tissue RNA, and performing reverse transcription to obtain first-strand cDNA;
C. performing PCR amplification by using a primer NtAOS 1F/NtAOS 1R by using a first-strand cDNA obtained by reverse transcription as a template, and recovering and purifying a PCR product;
D. the purified product is connected with a carrier, and the connection system and the process are as follows:
a connection system: 10 μ L
mu.L of purified product, 1. Mu.L of PCR-Blunt II-TOPO (Invitrogen) mixed well, 5 Xbuffer 2. Mu. L, T4 ligase 1. Mu. L, H 2 O 2μL。
The reaction steps are as follows:
the purified product reacts for 10min at 25 ℃,
vector transformation Escherichia coli DH5a was transformed by heat shock. After culture, 100 mu L of bacterial liquid is coated on an LB plate containing 100mg/L kanamycin for overnight culture at 37 ℃, colonies are picked for colony PCR positive detection, the positive colonies are picked for bacteria shaking to extract plasmids, and the positive clones are sequenced.
Example 2
A. Constructing an RNAi vector:
taking the positive clone after NtAAS 1 gene verification as a template, carrying out PCR amplification by using a primer containing a Gateway joint sequence, purifying an amplification product by using a PCR product, and inserting the amplification product into a pDONR Zeo vector of Invitrogen company through BP reaction. The constructed BP reaction vector is used for replacing the NtAO 1-RNAi fragment into the pHELLSGATE12 interference expression vector through LR reaction. The method comprises the following specific steps:
(1) Designing a primer according to a tobacco gene NtAOS1 gene sequence:
RNAi-F: 5’-GCTGGAAAGGATTTTGTGGTGC-3’
RNAi-R: 5’-TCAAATTTCCGTTCCAACTCCGATC-3’
according to the requirement of BP reaction in the Gateway system, a 5'-GGGACAAGTTTGTACAAAAAAGCAGGCTGC-3' sequence is added in front of a forward primer, and a 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTC-3' sequence is added in front of a reverse primer. The Gateway reaction primer sequences were obtained as follows:
attB-L-RNAi-F:
5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTGCGCTGGAAAGGATTTTGTGGTGC-3’
attB-R-RNAi-R:
5’-GGGGACCACTTTGTACAAGAAAGCTGGGTCTCAAATTTCCGTTCCAACTCCGATC-3’
(2) PCR cloning is carried out by Phusion high-fidelity polymerase in all PCR reactions.
(3) BP reaction:
(a) Prepare 8 μ L of the reaction system in a 200 μ L centrifuge tube, comprising: 1-7 mu L of attB-PCR product (about 15-150 ng, mass concentration is more than or equal to 10 ng/mu L), 1 mu L of 150 ng/mu L pdonr-zeo carrier and a proper amount of TE buffer solution (pH 8.0), and mixing uniformly at room temperature;
(b) Standing BP Clonase II enzyme mixture on ice for 2 min to melt, shaking gently for 2 times, and mixing well for use;
(c) Adding 2. Mu.L of a BP clone II enzyme mixture to the sample prepared in (1), gently mixing the system;
(d) (II) storing the BP clone ™ II enzyme mixture back at-20 ℃ or-80 ℃;
(e) Placing the reaction system in a 25 ℃ warm bath of 1 h;
(f) Adding 1 mu L of proteinase K solution into the reaction system, shaking gently, and then placing the sample in a warm bath at 37 ℃ for 10min so as to terminate the BP reaction;
(g) And (3) after the mixed solution is transformed into escherichia coli, coating the transformed bacterium solution on an LB plate containing Zeacin resistance, picking a bacterial colony to a culture medium solution containing corresponding antibiotics for shake culture, and extracting pDONR-Zeocin plasmid of positive clone for later use after confirmation.
(4) LR reaction:
(a) Prepare 8 μ L of the reaction in a 200 μ L centrifuge tube, including: 1-7. Mu.L of the obtained pDONR-Zeocin plasmid (50-150 ng), 1. Mu.L of 150 ng/. Mu.L of pHELLSGATE12 vector and an appropriate amount of TE buffer (pH 8.0), and mixing them at room temperature;
(b) Standing LR clone ™ II enzyme mixture on ice for 2 min to melt, shaking gently for 2 times to mix well;
(c) Adding 2 μ L LR clone II enzyme mixture, and mixing the system by gentle shaking;
(d) (II) storing the LR clone ™ II enzyme mixture at-20 ℃ or-80 ℃ in a freezer;
(e) Placing the reaction system at 25 ℃ for a warm bath reaction of 1 h;
(f) Adding 1 mu L of proteinase K solution into the reaction system to stop LR reaction, after gently shaking, placing the sample at 37 ℃ and standing for 10min; the pHELLSGATE12 recombinant vector is obtained.
The vector is transformed into escherichia coli through heat shock, added with a culture medium, subjected to shake culture, coated on an LB plate containing 100mg/L spectinomycin for overnight culture, and subjected to strain selection and plasmid extraction, and then XbaI/XhoI double enzyme digestion is used for identifying a target plasmid.
B. And (3) agrobacterium transformation:
taking out the agrobacterium-infected cells from a refrigerator at the temperature of-80 ℃, placing the agrobacterium-infected cells on ice for dissolving, and adding 4 mu L of a recombinant expression vector pHellsgate12-NtAOS 1; quickly freezing for 1min with liquid nitrogen, transferring into 37 deg.C water bath for 5 min, ice-bathing for 2 min, adding 1mL LB liquid culture medium into the mixture, culturing at 28 deg.C and 220rpm for 3~4 hr; the culture is coated on LB solid culture medium containing 100mg/L spectinomycin and 25mg/L rifampicin, inverted culture is carried out for 2~3 days at 28 ℃, and agrobacterium clone containing a target vector can be seen;
C. introducing the RNAi vector into tobacco, culturing a transgenic plant:
(a) Selecting agrobacterium clone containing a target vector, streaking on an LB (Langmycin) plate containing spectinomycin and rifampicin, and culturing for 2~3 days at 28 ℃; scraping streak plaque to inoculate bacteria in an MS culture medium containing spectinomycin and rifampicin, carrying out shake culture at 28 ℃ and 220rpm, and infecting when the concentration of the bacteria liquid reaches OD =0.5 to 0.8;
(b) Putting the tobacco leaves into a 500mL wide-mouth bottle, adding a proper amount of 75% ethanol, and rinsing for 1min; removing the ethanol, adding 0.1% HgCl2 solution, and placing on a shaking table to oscillate for 15-30 minutes at room temperature; discarding the solution, and washing with sterile water for 6 times;
(c) Taking out the tobacco leaves, washing off surface liquid by using sterile absorbent paper, cutting the sterile leaves into small pieces of 1cm multiplied by 1cm by using scissors, putting the cut tobacco leaves into sterile MS liquid culture medium suspension bacterial liquid containing a target carrier, and standing for 15-20min; taking out tobacco leaf, removing excess bacterial liquid with sterile filter paper, and performing dark culture in MS culture medium containing 6-BA (0.02 mg/L) and NAA (2 mg/L) at 25 deg.C for two days; transferring tobacco leaf into differentiation culture medium, contacting the cut with the culture medium, wherein the differentiation culture medium is MS culture medium containing 6-BA (0.5 mg/L), NAA (0.1 mg/L), kanamycin (100 mg/L) and cefamycin (500 mg/L), subculturing once every 2~3 weeks, gradually forming callus at the cut, and finally differentiating to obtain buds;
(d) Cutting off buds growing to 3 to 5cm, transferring the buds into an MS culture medium to induce rooting, taking out the rooted transgenic plants from the rooting culture medium, washing the culture medium with tap water, and transplanting the transgenic plants into sterilized nutrient soil;
(e) And carrying out PCR verification on the transgenic plant by using a Kana gene specific primer to identify a transgenic positive plant.
D. Transgenic plant screening
Extracting total RNA of wild plants and 25T 0 generation plants transformed into NtAAS 1, performing real-time fluorescent quantitative PCR (qRT-PCR) analysis, and analyzing the expression conditions of different strains because an internal reference gene is 26S. Selecting a tobacco strain with the lowest expression quantity of NtAAS 1.
NtAOS1 qRT primer:
NtAOS1_qRT_F：5’- CGATCCTTCTGAACCGAACCATG -3’；
NtAOS1_qRT_R：5’- GATTTAGCCAAGAAATTAAACGCGGC-3’。
26s reference gene primer:
26s_F：5’-GAAGAAGGTCCCAAGGGTTC-3’；
26s_R：5’-TCTCCCTTTAACACCAACGG-3’。
jasmonic acid content determination
A. The instrument comprises the following steps: RIGOL L3000 high performance liquid chromatograph, kromasil C18 reversed phase chromatographic column (250mm x 4.6mm,5 μm), KQ2200DE type numerical control ultrasonic cleaner, saimefei Thermo Micro CL 17R high speed refrigerated centrifuge, vortex mixer, NDK200-2 numerical control temperature nitrogen blower (Hangzhou Mi Europe instruments Co., ltd.).
B. Reagent: methyl jasmonate standard (sigma, purity is more than or equal to 99.9%), HPLC grade acetonitrile (J & K CHEMICAL LTD, purity is more than or equal to 99.9%), HPLC grade methanol (J & K CHEMICAL LTD, purity is more than or equal to 99.9%), ethyl acetate (national drug group chemical reagent limited, analytical purity is more than or equal to 99.5%), trimethylsilylated diazomethane, petroleum ether (national drug group chemical reagent limited, analytical purity is more than or equal to 99.5%), hydrochloric acid (national drug group chemical reagent limited, premium grade purity, 36.0 to 38.0%), n-hexane (national drug group chemical reagent limited, premium grade purity, purity is more than or equal to 97.0%), glacial acetic acid (crude, group purity is more than or equal to 98%), phosphoric acid (national drug chemical reagent limited, premium grade purity, purity is more than or equal to 85.0%).
C. Pretreatment operation: about 1g of the sample was weighed, ground in a mortar, added with 6 mL pre-cooled 80% aqueous methanol and leached overnight at 4 ℃. Centrifuging at 8000g for 10min, collecting supernatant, leaching residue with 2mL of 80% methanol aqueous solution for 2 h, centrifuging, taking out supernatant, combining supernatants twice, evaporating at 40 deg.C under reduced pressure until no organic phase is contained, adding 3mL of petroleum ether for extraction and decolorization for three times, adding 2mol/L of hydrochloric acid aqueous solution with appropriate amount to adjust pH to 2-3, adding 2mL of ethyl acetate for extraction twice, transferring upper organic phase to a new EP tube, blowing dry with nitrogen, and derivatizing.
Derivatization of jasmonic acid: adding 200uL of reagent III for redissolution, adding 20uL of reagent IV, mixing uniformly, and standing at 25 ℃ for 30min. Adding 20uL of reagent five, uniformly mixing, and standing at the normal temperature of 25 ℃ for 30min. Blowing the mixture by nitrogen in ice water bath. Adding 0.5mL of mobile phase for redissolution, taking a proper amount of solution, filtering the solution by using a needle head type filter, and measuring the solution in a sample bottle with a lining tube.
And (3) reagent III: weighing 9mL of diethyl ether and 1mL of methanol, and uniformly mixing:
and (4) reagent IV: 2mmol/L trimethylsilylated diazomethane (dissolved in n-hexane);
and a fifth reagent: 2mol/L glacial acetic acid n-hexane solution.
D. Liquid chromatography conditions:
chromatograph: RIGOL L3000 high performance liquid chromatograph with wavelength of 210nm.
And (3) chromatographic column: kromasil C18 reverse phase chromatography column (250mm 4.6mm,5 μm)
Column temperature: 30. c
Flow rate: 0.8mL/min, injection volume: 10 μ L
Mobile phase: 0.1% phosphoric acid aqueous solution: acetonitrile = 45: 55 (V/V)
E. And (3) standard curve determination:
accurately weighing methyl jasmonate standard substance, dissolving with methanol, preparing into 5-6 standard solutions with different mass concentrations, sequentially detecting peak area of each standard solution according to the chromatographic conditions, and calculating to obtain standard curve, linear range and correlation coefficient of methyl jasmonate by taking peak area as ordinate and concentration as abscissa
| X (concentration. Mu.g/mL) | 0.1 | 0.5 | 1 | 5 | 10 |
| y (Peak area) | 0.304 | 1.326 | 2.743 | 14.288 | 27.556 |
The results show that the jasmonic acid content is improved by more than 50 percent compared with the control.
SEQUENCE LISTING
<110> research institute of tobacco agricultural science in Yunnan province
<120> gene NtAOS1 for improving jasmonic acid content of tobacco leaves, and cloning method and application thereof
<130> 2019
<160> 11
<170> PatentIn version 3.3
<210> 1
<211> 1578
<212> DNA
<213> nucleotide sequence of NtAOS1 Gene
<400> 1
atggcagtag caacagcaac agcaacatta tcttcgtctt ctgtttttcc tctccattct 60
cttcaccaac agtttccatc aaaatacttc actgttcgtc ccattacagt ctctttatcc 120
gaaaaaatac cagcagtgac acaatcgtct gagctcacaa aattaccgat ccgtaaaatt 180
cccggcgatt atggtcttcc tttaattggt ccatggaaag atagacaaga ttatttttac 240
aatcaaggta aagaagaatt cttcagatca agaattcaaa aatacaaatc tactgtattt 300
aaaaccaata tgccacctgg aaatttcatt tcttccaatc caaacgttgt cgttttgctc 360
gacggcaaga gttttccgac cctcttcgac atttctaaag tcgagaaaaa ggatctcttc 420
accggaactt tcatgccgtc gactgaactc accggcggtt atcgtgttct ttcttatctc 480
gatccttctg aaccgaacca tgaaaaatta aaaaagctcc tcttttttct tcttacttct 540
cgtcgtgatt acataatacc ccagtttcac gaaagctata cggagctgtt taaaacccta 600
gaaaaggaaa tggagaaaaa tggtaaagct gatttaaact cagctaatga tcaagccgcg 660
tttaatttct tggctaaatc attgtacgga gcgaacccag ttgagactaa gctcggaact 720
gatggtccca cattgatcgg aaaatgggtg ttgtttcagc ttcatccttt gctcactctt 780
ggtcttccga aggttctaga cgactttctc ctccataatt tccggttacc gccagctcta 840
gtgaagaaag attaccagag actctacgat ttcttctatg agagctccac tactgtgcta 900
aacgaagctg aaaattttgg tatttcgcgc gacgaagctt gtcataacct tctcttcgct 960
acgtgtttca attcctttgg cgggatgaag attttctttc ctaatatgct gaaatggata 1020
gctagagcag gagtagagct ccatatccgg ttagcgaacg agatccgatc cgccgtaaaa 1080
tccgccggcg ggaagatcac gatgtcggcg atggagaaaa tgcctgtgat gaaatcggtt 1140
gtatacgagg ctttacgaat tgatccacca gttgcttcgc aatacgggag agccaaacgt 1200
gaccttatga tcgaatcaca cgacgccgtt tttgaggtga agaaaggtga attgttattc 1260
gggtaccaac cgtttgcgac gaaggatccg aagagttttg accgacccga tgagttcgta 1320
cccgatcggt tcgtaggtga agaaggggaa aagctattga aacatgtatt atggtctaac 1380
ggacctgaaa cggagagtcc gacggtggag aataaacaat gtgctggaaa ggattttgtg 1440
gtgctggttt cgagattgtt agtaacggag ttttttctcc gttatgacac gttggatatc 1500
gacgtcggca cgtcgccgtt aggagctaag attactataa cttctttgaa gatgatcgga 1560
gttggaacgg aaatttga 1578
<210> 2
<211> 525
<212> PRT
<213> amino acid sequence encoded by NtAAS 1 gene
<400> 2
Met Ala Val Ala Thr Ala Thr Ala Thr Leu Ser Ser Ser Ser Val Phe
1 5 10 15
Pro Leu His Ser Leu His Gln Gln Phe Pro Ser Lys Tyr Phe Thr Val
20 25 30
Arg Pro Ile Thr Val Ser Leu Ser Glu Lys Ile Pro Ala Val Thr Gln
35 40 45
Ser Ser Glu Leu Thr Lys Leu Pro Ile Arg Lys Ile Pro Gly Asp Tyr
50 55 60
Gly Leu Pro Leu Ile Gly Pro Trp Lys Asp Arg Gln Asp Tyr Phe Tyr
65 70 75 80
Asn Gln Gly Lys Glu Glu Phe Phe Arg Ser Arg Ile Gln Lys Tyr Lys
85 90 95
Ser Thr Val Phe Lys Thr Asn Met Pro Pro Gly Asn Phe Ile Ser Ser
100 105 110
Asn Pro Asn Val Val Val Leu Leu Asp Gly Lys Ser Phe Pro Thr Leu
115 120 125
Phe Asp Ile Ser Lys Val Glu Lys Lys Asp Leu Phe Thr Gly Thr Phe
130 135 140
Met Pro Ser Thr Glu Leu Thr Gly Gly Tyr Arg Val Leu Ser Tyr Leu
145 150 155 160
Asp Pro Ser Glu Pro Asn His Glu Lys Leu Lys Lys Leu Leu Phe Phe
165 170 175
Leu Leu Thr Ser Arg Arg Asp Tyr Ile Ile Pro Gln Phe His Glu Ser
180 185 190
Tyr Thr Glu Leu Phe Lys Thr Leu Glu Lys Glu Met Glu Lys Asn Gly
195 200 205
Lys Ala Asp Leu Asn Ser Ala Asn Asp Gln Ala Ala Phe Asn Phe Leu
210 215 220
Ala Lys Ser Leu Tyr Gly Ala Asn Pro Val Glu Thr Lys Leu Gly Thr
225 230 235 240
Asp Gly Pro Thr Leu Ile Gly Lys Trp Val Leu Phe Gln Leu His Pro
245 250 255
Leu Leu Thr Leu Gly Leu Pro Lys Val Leu Asp Asp Phe Leu Leu His
260 265 270
Asn Phe Arg Leu Pro Pro Ala Leu Val Lys Lys Asp Tyr Gln Arg Leu
275 280 285
Tyr Asp Phe Phe Tyr Glu Ser Ser Thr Thr Val Leu Asn Glu Ala Glu
290 295 300
Asn Phe Gly Ile Ser Arg Asp Glu Ala Cys His Asn Leu Leu Phe Ala
305 310 315 320
Thr Cys Phe Asn Ser Phe Gly Gly Met Lys Ile Phe Phe Pro Asn Met
325 330 335
Leu Lys Trp Ile Ala Arg Ala Gly Val Glu Leu His Ile Arg Leu Ala
340 345 350
Asn Glu Ile Arg Ser Ala Val Lys Ser Ala Gly Gly Lys Ile Thr Met
355 360 365
Ser Ala Met Glu Lys Met Pro Val Met Lys Ser Val Val Tyr Glu Ala
370 375 380
Leu Arg Ile Asp Pro Pro Val Ala Ser Gln Tyr Gly Arg Ala Lys Arg
385 390 395 400
Asp Leu Met Ile Glu Ser His Asp Ala Val Phe Glu Val Lys Lys Gly
405 410 415
Glu Leu Leu Phe Gly Tyr Gln Pro Phe Ala Thr Lys Asp Pro Lys Ser
420 425 430
Phe Asp Arg Pro Asp Glu Phe Val Pro Asp Arg Phe Val Gly Glu Glu
435 440 445
Gly Glu Lys Leu Leu Lys His Val Leu Trp Ser Asn Gly Pro Glu Thr
450 455 460
Glu Ser Pro Thr Val Glu Asn Lys Gln Cys Ala Gly Lys Asp Phe Val
465 470 475 480
Val Leu Val Ser Arg Leu Leu Val Thr Glu Phe Phe Leu Arg Tyr Asp
485 490 495
Thr Leu Asp Ile Asp Val Gly Thr Ser Pro Leu Gly Ala Lys Ile Thr
500 505 510
Ile Thr Ser Leu Lys Met Ile Gly Val Gly Thr Glu Ile
515 520 525
<210> 3
<211> 156
<212> DNA
<213> interfering fragment
<400> 3
gctggaaagg attttgtggt gctggtttcg agattgttag taacggagtt ttttctccgt 60
tatgacacgt tggatatcga cgtcggcacg tcgccgttag gagctaagat tactataact 120
tctttgaaga tgatcggagt tggaacggaa atttga 156
<210> 4
<211> 22
<212> DNA
<213> RNAi-F
<400> 4
gctggaaagg attttgtggt gc 22
<210> 5
<211> 25
<212> DNA
<213> RNAi-R
<400> 5
tcaaatttcc gttccaactc cgatc 25
<210> 6
<211> 53
<212> DNA
<213> attB-L-RNAi-F
<400> 6
ggggacaagt ttgtacaaaa aagcaggctg cgctggaaag gattttgtgg tgc 53
<210> 7
<211> 55
<212> DNA
<213> attB-R-RNAi-R
<400> 7
ggggaccact ttgtacaaga aagctgggtc tcaaatttcc gttccaactc cgatc 55
<210> 8
<211> 23
<212> DNA
<213> NtAOS1_qRT_F
<400> 8
cgatccttct gaaccgaacc atg 23
<210> 9
<211> 26
<212> DNA
<213> NtAOS1_qRT_R
<400> 9
gatttagcca agaaattaaa cgcggc 26
<210> 10
<211> 20
<212> DNA
<213> 26s_F
<400> 10
gaagaaggtc ccaagggttc 20
<210> 11
<211> 20
<212> DNA
<213> 26s_R
<400> 11
Claims (1)
1. The application of a gene NtAAS 1 in regulating the jasmonic acid content in tobacco leaves is disclosed, wherein the nucleotide sequence of the gene NtAAS 1 is shown in a sequence table SEQ ID NO:1, and the coded amino acid sequence is shown as SEQ ID NO:2, the method is characterized in that the expression of the tobacco gene NtAAS 1 is interfered by RNAi to obtain the transgenic tobacco with the NtAAS 1 expression quantity reduced, and the jasmonic acid concentration in the transgenic tobacco leaves is increased relative to that of wild tobacco.
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| CN111575305B (en) * | 2020-05-14 | 2022-03-15 | 安徽农业大学 | Allene oxide synthetase, coding gene CsAOS and application thereof |
| CN112094855A (en) * | 2020-09-27 | 2020-12-18 | 云南省烟草农业科学研究院 | Tobacco cytochrome C gene NtCYP94B3s and application thereof in improving content of tobacco jasmonic acid |
| CN112143738B (en) * | 2020-09-30 | 2023-04-11 | 云南省烟草农业科学研究院 | Tobacco receptor protein gene and cloning method and application thereof |
| CN115612691B (en) * | 2021-07-15 | 2024-04-23 | 安徽农业大学 | Method for synthesizing L-theanine in non-tea plant |
| CN117535316B (en) * | 2024-01-04 | 2024-03-29 | 湖南工程学院 | Ginseng PgJOX4 gene and application thereof in regulating ginsenoside biosynthesis |
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