CN103012573B - Tobacco JAZ protein as well as gene and application thereof - Google Patents

Abstract

The invention discloses a tobacco JAZ protein as well as a gene and an application thereof. The tobacco JAZ protein contains a typical TIFY structural domain and a jas structural domain, and the gene of the tobacco JAZ protein is named as NtJAZ10 and is induced and expressed by jasmonate. An RNAi (Ribonucleic Acid Interfere) transgenic carrier of the NtJAZ10 gene is constructed, and the tobacco is converted, so that expression of the NtJAZ10 in tobacco cells is successfully inhibited, and the nicotine content of the obtained transgenic tobacco cell line is obviously reduced compared with that of a wild type tobacco cell line. The tobacco JAZ protein disclosed by the invention provides an important basis and a technical support for low-nicotine-content tobacco breeding.

Description

One grow tobacco JAZ albumen and gene and application

Technical field

The present invention relates to the biosynthesis and regulation genes involved of tobacco Nicotine, the particularly synthetic application of gene aspect reduction tobacco nicotine content of regulation and control tobacco Nicotine.

Background technology

Smoking serious harm people's is healthy, thereby how to reduce harm of smoking and become national governments, medical expert, botanist one of the problem paying close attention to, study of going all out.The top tobacco company in the much world drops into huge fund pathways metabolism, the regulatory mechanism of tobacco secondary metabolite is studied.Nicotine is a kind of pyridine alkaloid, mainly is present in Solanaceae Nicotiana (Nicotiana) plant, is a kind of important secondary metabolite in the tobacco body.Biosynthetic research mainly concentrates on its metabolism involved enzyme of clone and analyzes its physiology and chemistry characteristic aspect to Nicotine at present.Nicotine is that the root plant forms and passes through xylem and is transported under the blade deposition.The synthetic precursor of Nicotine is basic amino acid: arginine and ornithine and aspartic acid.Their key enzyme PMT in a series of metabolism route of synthesis, the catalysis such as QPT and MPO finally generates Nicotine (Chintapakorn and Hamill, 2003.Antisense-mediated down-regulation of putrescineN-methyltransferase activity in transgenic Nicotiana tabacum L.can lead to elevated levels o fanatabine at the expense of nicotine.Plant Molecular Biology53:87-105.).

The Nicotine biosynthesizing is subjected to the regulation and control of many factors, and the hormone signal of developmental regulation is exactly a very important class.Jasmonate (JAs) is the general designation of jasmonic and derivative thereof, is to play the plant growth regulating substance that global regulation acts in plant materials, is distributed widely in the reproductive organ of tender tissue, flower and growth of plant.And when plant was subject to biology and coerces with abiotic stress, JAs can also change the defensive raction of genetic expression and mediated plant in cell.Studies have shown that the key enzyme on the nicotine metabolite approach, as MPT, QPT and MPO all are subjected to the plant wound induce inducing of hormone-Jasmonate and express, thereby form Nicotine.From 1962, find jasmonic so far, people understand the route of synthesis of jasmonic very much, but growing and the molecular mechanism of stress reaction unclear to the signal transduction path of Jasmonate and regulating plant thereof.

Three seminars in 2007 in succession be reported in Arabidopis thaliana found a class new transcribe the inhibition regulon, be named as Jasmonate ZIM domain protein family (being called for short JAZ albumen).It brings into play its physiological function (Chini et al., 2007.The JAZ family of repressors is the missing link in jasmonate signalling.Nature448:U664-U664 by the expression that suppresses the Jasmonate responsive genes; Thines et al., 2007.JAZ repressor proteins are targets of the SCFCO11complex during jasmonate signalling.Nature448:U661-U662.; Yan et al., 2007.A Downstream Mediator in the Growth Repression Limb of the Jasmonate Pathway.Plant Cell19,2470-2483.).When there is no Jasmonate, the C-of JAZ albumen end is combined in the N-end of MYC2 transcription factor, suppresses the expression of Jasmonate induced gene; When in plant materials, having Jasmonate to exist, the same SCF of JAZ albumen ^COI1Mixture interacts, thereby causes the poly-ubiquitin of JAZ albumen and degraded by the 26S proteasome, discharges the expression that the MYC2 transcription factor activates the Jasmonate induced gene.

The metabolic regulation of research Nicotine is a very significant job.Nicotine has very strong physiological stimulation effect to human body, is the basic substance that the tobacco commerciality is used.For tobacco, Nicotine is a kind of defensive material of resisting insect pest infestation.In the tobacco plant do not come to harm, nicotine content is approximately 0.1%-1.0%.After being subject to plant-feed insect infringement or physical abuse, in tobacco plant, Jasmonate content can be increased to 9 times of original (Ziegler et al., 2001.Herbivore-inducedallene oxide synthase transcripts and jasmonic acid in Nicotiana attenuata.Phytochemistry58:729-738.), and nicotine content can reach doubly (Baldwin of 4-10, 1998.Jasmonate-induced responses arecostly but benefit plants under attack in native populations.Proceedings of the National Academy of Sciences of the United States of America95, 8113-8118.), this concentration can make most of leaf-eating insects dead immediately, thereby protection is subjected to the tissue of insect infringement, reach pest-resistant purpose.The results show Jasmonate can regulate and control the generation of Nicotine, but its molecular mechanism unclear.

At present, for the research of JAZ albumen, in Arabidopis thaliana, find altogether 12 members, this is not difficult to make us to think deeply, and in tobacco, have and there is no such JAZ albumen, and whether these albumen has participated in the synthetic of tobacco Nicotine that Jasmonate induces.

Summary of the invention

The object of the invention is to find the functional gene relevant to the tobacco nicotine content, and then a kind of method that reduces the tobacco nicotine content is provided, for low Nicotine content tobacco breeding provides technique means.

Research of the present invention finds, some NtJAZs in tobacco is subjected to inducing of Jasmonate and expresses.By the RNAi technology, disturb its expression separately in tobacco, obtain transgenic cell line, detect the relative wild-type of content of finding Nicotine in these transgenic cell lines much lower.Infer thus, utilize transgenic technology, disturb, reticent or knock out some NtJAZs, can be and obtain low Nicotine content tobacco breeding important scientific basis is provided.In view of the using value of this genoid and utilize the application prospect of potentiality, be necessary to be protected by patent.

Gene for reducing the tobacco nicotine content provided by the invention belongs to JAZ family protein gene, derives from tobacco (Nicotiana tabacum), called after NtJAZ10NtJAZ10, and coding following proteins (i) or (ii):

(i) protein of aminoacid sequence shown in the SEQ ID No:1 in sequence table;

(ii) aminoacid sequence shown in the SEQ ID No:1 in sequence table is through replacement, disappearance or the interpolation of one to ten amino-acid residue and derivative protein, and the protein derived has the function identical with (i) described protein.

SEQ ID No:1 sequence in sequence table is comprised of 231 amino-acid residues, wherein the 24th to the 29th amino acids residue be the TIFY structural domain, the 180th to the 198th amino acids residue be the jas structural domain.One to ten amino-acid residue of described replacement, disappearance or interpolation can be the amino-acid residue in non-said structure territory, and its change can not exert an influence to the function of this albumen.Amino-acid residue is replaced, lacks or adds, and can realize by the ordinary skill in the art the detection of correlation function.

Tobacco JAZ protein gene NtJAZ10 of the present invention can be its cDNA sequence, can be also genomic dna sequence, or has 90% above homology and the DNA sequence dna of the identical function albumen of encoding with these sequences.The DNA sequence dna shown in SEQ ID NO:2 for example.

By tobacco being transcribed to the group sequencing result, carry out de novo splicing, utilize the tobacco obtained to transcribe the tobacco gene group sequence fragment of group data and NCBI announcement and the Blast analysis that the EST fragment is carried out Arabidopis thaliana homology JAZ, correlative study of the present invention has obtained a series of tobacco JAZ albumen (NtJAZs) member.Wherein, the ORF full length sequence (SEQ ID NO:2) that the design primer has been cloned NtJAZ10, the protein sequence of its coding is as shown in the SEQ ID No:1 in sequence table.The sequencing results shows, this albumen contains conservative TIFY structural domain and jas structural domain.Secondly, utilize by RT-PCR and Real-timePCR and find that this gene is (Fig. 1) that induced by Jasmonate to express.

Based on above-mentioned research, the present invention by the RNAi technique construction RNAi transgene carrier of NtJAZ10 gene, successfully obtained and suppressed NtJAZ10 expresses in tobacco cell transgenic cell line, the transgenic tobacco cells obtained system has the special phenotype that the nicotine content relative comparison obviously reduces.Visible, utilize transgenic technology, disturb, reticent or knock out the NtJAZ10 gene and can obtain the transgene tobacco that nicotine content reduces, this makes low Nicotine content tobacco breeding become possibility, provides a kind of effective technique means for reducing the tobacco nicotine content.

In example of the present invention, at first, for building the RNAi binary expression vector of NtJAZ10, select one section nucleic acid fragment (the 1-513bp sequence of sequence table SEQ ID NO:2) more special in this gene to be the homing sequence of RNAi, the design primer obtains this sequence.Then, this nucleic acid fragment is inserted into to the RNAi binary expression vector that is configured to NtJAZ10 in plant expression vector pHZPRi-Hyg with positive and negative both direction.As shown in Figure 2, two NtJAZ10 specific fragments in this carrier are separated by one section GUS sequence, are expressed by the guiding of CaMV35S promotor.Afterwards, this plasmid is proceeded to Agrobacterium LBA4404, and via Agrobacterium, this plasmid is proceeded in tobacco BY-2 suspension cell.And then, by the efficiency of Real-timePCR method to RNAi, detect.Result shows, effectively inhibition (Fig. 3) of the expression of NtJAZ10 quilt in the transgenic cell line obtained.

For NtJAZ10-RNAi transgenic tobacco cells system, utilize the gas chromatography mass spectrometry analytical technique of mass spectrum to measure Jasmonate and process after 72 hours the content of Nicotine in cell.Compare according to the wild-type tobacco cell, the nicotine content of transformation cell lines has obvious reduction (Fig. 4).Therefore, the present invention provides important scientific basis and technical support for obtaining low Nicotine content tobacco breeding.

The accompanying drawing explanation

Fig. 1, Real-time PCR detect Jasmonate and induce the expression of lower NtJAZ10 to change, and wherein: left figure has shown that the NtJAZ10 gene is induced by Jasmonate (JA), and the expression increased gradually along with the prolongation expression amount of JA effect; Right figure is positive control, and the key enzyme PMT in the Nicotine route of synthesis induces the expression under processing at JA; The Actin of take is internal reference.

Fig. 2, NtJAZ10-RNAi plant expression vector construction schematic diagram, in this carrier, the RNAi homing sequence of NtJAZ10 inserts the both sides of plant expression vector GUS sequence with positive and negative both direction; In figure, RB, LB show the border, left and right of sequence; 2 * 35S shows promotor, and Hyg shows hygromycin gene.

The Molecular Identification of Fig. 3, Transgenic Tobacco clone: be depicted as the Real-time PCR qualification result of the tobacco suspension cell transcriptional level that transforms the NtJAZ10-RNAi expression vector, wherein WT is tobacco wild-type BY-2 cell.

Fig. 4, Transgenic Tobacco clone nicotine content detect: utilize the expression of RNAi technology Antisense Suppression NtJAZ10 in tobacco BY-2 cells, collection processes without MeJA and MeJA processes 72 hours samples, utilize gas chromatography mass spectrometry analytical technique of mass spectrum (GC-MS) to carry out the nicotine content detection, nicotine content decrease after Antisense Suppression NtJAZ10, WT are the contrast of tobacco wild-type BY-2 cell.

Embodiment

Following experimental technique, if no special instructions, be ordinary method.The reagent that following experimental technique is used, if no special instructions, be from routine biochemistry reagent company and buy and obtain.

Vegetable material: tobacco suspension cell is BY-2(Nicotiana tabacum L.cv. Bright Yellow2)

1, the extraction of the acquisition of experiment material and RNA

The method of drawing material of ■ different time MeJA treatments B Y-2 cell

Tobacco suspension cell BY-2 is at MS(4.3g/lMS, 100mg/l inositol, 30g/l sucrose, 200mg/l potassium primary phosphate, 1mg/lVB1,0.2mg/l2,4-D, pH=5.8) liquid nutrient medium in 24C, suspension culture in the 120rpm shaking table, 7 days one-period.

Real-time PCR tests material therefor: when carrying out Jasmonate (MeJA) processing, the growth suspension cell of 4 days is reached without 2, in the MS substratum of 4-D, after 24 hours, add 50 μ M Jasmonates (final concentration) to process, at 0,0.5,2,6,12,24 hour, collect sample respectively, in the centrifuge tube of the precooling of packing into.After liquid nitrogen flash freezer, transfer in-80 ℃ of cryogenic refrigerators and preserve, with for the Real-time pcr analysis.

Nicotine content test experience material therefor: the same Real-time PCR experiment material therefor is processed, and processes and collects sample in 72 hours at MeJA, in-80 ℃ of cryogenic refrigerators, preserves, for nicotine content, to detect.

The RNA enzyme that goes of ■ glasswork, plastics and electrophoresis chamber is processed

Glasswork used in RNA related experiment process is before use in 180 ℃ of baking 8h.Plastics, comprise various types of rifle heads and centrifuge tube, spends the night by the 0.1%DEPC aqueous solution soaking, and autoclaving is placed in 80 ℃ of loft drier dry.Be used for the electrophoresis chamber of RNA electrophoresis after cleaning, with in dehydrated alcohol, soaking 30min, then at 30%H ₂O ₂Middle immersion 30min, finally process water with sterilizing DEPC and rinse 5 times.

The extraction of ■ different time MeJA treatments B Y-2 cell total rna

The Trizol method is extracted cell total rna, at first get the vegetable material (about 0.1g) that is sub-packed in the 1.5ml centrifuge tube, liquid nitrogen grinding, every pipe adds 1ml Trizol liquid, mix rapidly, under room temperature, standing 5-10 minute is beneficial to dissociating of nucleic acid protein complex body, centrifugal 12000g, 10min, get the chloroform that new centrifuge tube adds 1ml, the supernatant liquor that adds previous step, carefully be not drawn onto lower sediment, cover tightly centrifuge tube, with hand, acutely sway centrifuge tube 15s, the standing 5min of room temperature, centrifugal 10 minutes of 12000g, get supernatant liquor (water) and proceed to a new centrifuge tube, add the equal-volume Virahol, place 2h for-20 ℃, 4 ℃, the centrifugal 10min of 12000g, abandoning supernatant, add 1ml75% ethanol, slowly put upside down centrifuge tube, the centrifugal supernatant of abandoning, repeat once.Drying at room temperature.Add appropriate DEPC and process water dissolution RNA.

■ RNA quality examination

On GBC Cintra 10e ultraviolet spectrophotometer, survey the absorbance value of RNA sample at 260nm and 280nm, calculate the concentration of RNA sample and the purity of judgement RNA sample by absorption value.RNA absorbance value and concentration conversion formula: 1OD ₂₆₀=40 μ g/mL.Purity determination methods: pure RNA, its OD ₂₆₀/ OD ₂₈₀Be 2.0, if polluted protein or phenol, OD ₂₆₀/ OD ₂₈₀Ratio is starkly lower than this value.Then by 1% agarose gel electrophoresis, detect the integrity of RNA.

The removal of a small amount of DNA in ■ RNA sample

By residual DNA in the digestion of the DNase without RNase RNA sample, reaction system comprises: 1x RQ1 RNase-free DNase Buffer, RNase inhibitor 20 unit, RQ1 RNase-free DNase 1 μ L, RNA sample 50 μ g, use DEPC-H ₂O supplies system to 50 μ L.Above-mentioned system is in 37 ℃ of incubation 30min.After the DNA enzyme digestion reaction finished, with phenol/chloroform extracting, the ethanol precipitation reclaimed the RNA sample.

2, the clone of NtJAZ10 full-length cDNA

Extract the BY-2 cell total rna that Jasmonate was processed 2 hours, reverse transcription is cDNA, and take this cDNA and be template pcr amplification NtJAZ10 full length sequence.Idiographic flow is as follows:

Utilize the RevertAid of Fermentas company ^TMFirst Strand cDNA Synthesis Kit reverses, and obtains cDNA, and I is as follows for the preparation reaction system: the total RNA of 2 μ g, 1 μ L Oligo dT and add DEPC and process water polishing to 12 μ L are placed on ice rapidly after 65 ℃ of incubation 5min.Then add reaction system II:4 μ L5 * reaction buffer, 1 μ L RNase inhibitor, 2 μ L 10mM dNTP Mix and 1 μ L M-MuLV reverse transcriptase.Mix reaction system I and II, 42 ℃ of reverse transcription reaction 60min, process 5min for 70 ℃, is placed in 2min on ice, and packing also is stored in-20 ℃.

Pcr amplification NtJAZ10 encoding gene: according to the Blast analytical sequence, design total length primer, used high-fidelity DNA polymerase pfu amplification NtJAZ10 encoding gene.Reaction system is as follows: 2 * PCR pfu Mix, 25 μ L, upstream primer 1 μ M, downstream primer 1 μ M, cDNA1.0 μ L, use dd H ₂O postreaction system to 50.0 μ L.PCR reaction conditions: 95 ℃ of denaturation 5min; 95 ℃ of sex change 45sec, 55 ℃ of annealing 45sec, 72 ℃ are extended 2min, 30 circulations; Last 72 ℃ are extended 7min.Wherein primer sequence is as follows:

Upstream primer: 5 '-CACCATGGAGGTGGCCGTAAATCAGCC-3 ' (SEQ ID No:3);

Downstream primer: 5 '-CTATAACTTGAAATTGAGATCGAGT-3 ' (SEQ ID No:4).

The PCR product is connected into the ENTY carrier cloning: utilize the multifunctional dna purifying of Bo Maide biology to reclaim test kit, the PCR product is reclaimed, and the gene that uses pENTR/D-TOPO cloning kit test kit to clone is connected with the pENTR/D-TOPO carrier, the use linked system is as follows: PCR product0.5-4 μ l, Salt solution 1 μ l, TOPO vetor 1 μ l, Sterilewater mends to 6 μ l systems.Mix gently, 25 ℃ of connections are spent the night, and after this carry out DH5 α conversion, obtain plasmid pENTY-NtJAZ10.

The a small amount of of plasmid pENTY-NtJAZ10 is extracted: use the little extraction reagent kit of Beijing Suo Laibao company plasmid, connect bacterium to overnight incubation in the 1-5mlLB/Kan substratum, next day, centrifugal 13000rpm 1min collected thalline.Add 250 μ L solution I (please first check and whether added RNaseA), after vibration mixes, add 250 μ l solution II, leniently spin upside down 6-8 time and make the abundant cracking of thalline, add 350 μ l solution III, leniently spin upside down 6-8 time immediately, fully mix, now there will be white flocks.The centrifugal 10min of 12000rpm, with pipettor, carefully supernatant is transferred to (adsorption column adds in collection tube) in adsorption column, room temperature was placed 2 minutes, the centrifugal 1min of 12000rpm, outwell the waste liquid in collection tube, adsorption column is relay and reclaims in collector, add 500 μ l rinsing liquids (please first check before use and whether added dehydrated alcohol), centrifugal 1 minute of 12000rpm, abandon waste liquid, adsorption column is put into to collection tube, repeat once, then the 12000rpm sky is from 2min.Drying at room temperature, to the H of the unsettled dropping 50 μ l of adsorption film central authorities through 65 ℃ of water-bath preheatings ₂O, room temperature is placed 5min, and the centrifugal 2min of 12000rpm, obtain plasmid.

The PCR of plasmid pENTY-NtJAZ10 identifies: by PCR, the total length of NtJAZ10 is carried out to the electrophoresis evaluation, will obtain the positive colony of total length Insert Fragment, send company to check order, the final full length sequence that obtains NtJAZ10 gene ORF of identifying.

3, Jasmonate is induced the expression of NtJAZ10 gene

The sequential analysis of ■ NtJAZ10 conserved regions

The sequential analysis of NtJAZ10 conserved regions: the NtJAZ10 albumen that the clone is obtained carries out sequence alignment and domain analyses, find that its N end contains conservative tify structural domain (the 24th to the 29th amino acids residue) and C-terminal contains Jas structural domain (the 180th to the 198th amino acids residue), identical with the conserved domain of Arabidopis thaliana JAZs albumen.

■ Real time-PCR design of primers

Same family gene order similarity is higher, and therefore according to the sequence alignment situation of NtJAZ10 and other NtJAZs, picking distinguished sequence fragment is carried out Real time-PCR design of primers, and concrete primer sequence is as follows:

Upstream primer: 5 '-GCACCACAACAACAAAAGCA-3 ' (SEQ ID No:5);

Downstream primer: 5 '-GCGACGCAGAAGAAGTTGAT-3 ' (SEQ ID No:6).

Simultaneously, the key enzyme PMT of take in the Nicotine route of synthesis induces the expression under processing to be positive control at JA, as follows to its primer that carries out Real time-PCR:

Upstream primer: 5 '-TATGCACACAGGCTGAAAGC-3 ' (SEQ ID No:7);

Downstream primer: 5 '-AGTCAACTTCTGGCCCTTCA-3 ' (SEQ ID No:8).

The Actin of take is internal reference, as follows to its primer that carries out Real time-PCR:

Upstream primer: 5 '-AGCACCTCTTAACCCGAAGG-3 ' (SEQ ID No:9);

Downstream primer: 5 '-GGACAGTGTGGCTAACACCA-3 ' (SEQ ID No:10).

The detection of expression of NtJAZ10 under ■ BY-2 cell different time

Draw materials: tobacco BY-2 suspension cell

Time: 50 μ M MeJA processed 0,0.5,2,6,12,24 hour

Real-time PCR: use Trizol Plant test kit (Invitrogen) to extract total RNA, removing reverse transcription after DNA is cDNA, by 5 times of cDNA dilutions and be stored in-20 ℃ stand-by.In 8 quantitative PCR pipes, add 10 μ L2 * ABI powerSYBR greenPCR master mix, each 1 μ L of upstream and downstream primer (10 μ M) and cDNA, add dd H ₂O supplies reaction system to 20 μ L, mixes also of short duration centrifugal.Above-mentioned reaction system is placed in to ABI7500 real-time fluorescence quantitative PCR instrument, carries out the PCR reaction by normal process.Cycling condition is: 50 ℃ of 2min, 94 ℃ of denaturation 10min; 95 ℃ of sex change 15sec, anneal and extend 1min, 40 circulations for 60 ℃; Finally add a solubility curve and measure circulation.After reaction finished, application software ABI7500Software v2.0 analyzed experimental result.The key enzyme PMT of take in the Nicotine route of synthesis is positive control, with ACTIN(GenBank:ACH69153.1) be expressed as internal reference.Result as shown in Figure 1, with 50 μ M MeJA, process tobacco BY-2 cells, respectively at 0h, 0.5h, 2h, 6h, 12h and 24h collecting cell material, extract RNA, by Real-timePCR, find that the NtJAZ10 gene is induced by Jasmonate and expressed, and along with the JA prolongation of action time, the expression amount of gene increases gradually, at 2h, reach maximum value, the expression amount of positive control PMT is significantly induced after processed by Jasmonate.

4, the acquisition of the transgenic tobacco cells of NtJAZ10 gene silencing system

The Clone and sequence of ■ gene silencing trigger sequence

According to sequence alignment, in the ORF of NtJAZ10 frame, choose the fragment that specificity is higher (design of primers is as follows), the specimen material that the Jasmonate of take was processed 2 hours is template, carries out the PCR clone, obtains the DNA fragmentation as RNAi, connects ENTY carrier order-checking.Utilize Gateway LR Clonase II Enzyme Mix test kit to carry out the LR recombining reaction the correct gene silencing trigger sequence of order-checking and be connected to formation RNAi carrier NtJAZ10-RNAi in binary vector pHZPRi-Hyg.Primer sequence is as follows:

Upstream primer: 5 '-CACCATGGAGGTGGCCGTAAATCAGCC-3 ' (SEQ ID No:11);

Downstream primer: 5 '-AGAGCTGCTAGTTCCAGGTGTC-3 ' (SEQ ID No:12).

The NtJAZ10 gene silencing transgene carrier (NtJAZ10-RNAi) that application Agrobacterium infestation method will build proceeds in tobacco BY-2 suspension cell.

5, the evaluation of transgenic tobacco cells system and nicotine content are measured

The Molecular Identification of ■ Transgenic Tobacco clone

BY-2 suspension cell after Agrobacterium is infected screening and culturing on the hygromycin resistance substratum, until 2-4 after week, the picking resistant calli screens again, carry out afterwards suspension culture and succeeding transfer culture and by the method for RT-PCR and Real-time PCR, the transgenosis callus is carried out to Screening and Identification, the positive callus of acquisition.Concrete operations are carried out suspension cell for the positive callus after screening, and carry out the Jasmonate processing, collecting 0hour and 12hour sample extraction RNA reverse transcription is cDNA, by Real-time PCR, detects the expression (Fig. 3) of NtJAZ10, and screening obtains transgenic positive clone.

■ Transgenic Tobacco clone nicotine content detects

Positive callus after screening is carried out to suspension cell, and carry out the Jasmonate processing, collect the 72h sample and carry out the detection (Fig. 4) of nicotine content, concrete operations are as follows :-80 ℃ of refrigerators take out the 72h processing material of Collection and conservation, put into liquid nitrogen standby, the Liquid nitrogen precooler drill bit, grind cell with electric drill.Get the 1.5ml centrifuge tube, the weighing approximately cell of 0.1 ~ 0.15g grinding is put into, and adds the sodium hydroxide 1.25mL of 1.5mol/L, the quinoline inner mark solution 20 μ L that add again 0.0002 μ g/ml, ultrasonication 1min (total ultrasonic time 1min, ultrasonic 2s, interval 8s, intensity 36%).Hatch 4h for 37 ℃, take out and add the methylene dichloride of 5ml and the mixed solvent of methyl alcohol (volume ratio 3:1), jolt 5min.Standing, treat layering.Take off layer organic phase 2.5ml in new centrifuge tube, add glacial acetic acid 20 μ l, mix.With nitrogen, organic phase is dried up, add the 0.2ml methylene dichloride to dissolve determinand, whirlpool mixes, 2000rpm, and 5min is centrifugal, and sucking-off solution is put into chromatotube and is treated machine testing.

Utilize gas chromatography mass spectrometry analytical technique of mass spectrum (GC-MS) to detect nicotine content in sample, result shows, after Antisense Suppression NtJAZ10, nicotine content has substantial degradation (as shown in Figure 4), and WT is tobacco wild-type BY-2 cell.

Claims (6)

1. method that reduces the tobacco nicotine content, by transgenic technology disturb, reticent or knock out a tobacco JAZ protein gene, obtain the Transformation of tobacco plant that nicotine content reduces, the protein of aminoacid sequence shown in the SEQ ID No:1 in the sequence table of wherein said tobacco JAZ protein gene coding.

2. the method for claim 1, is characterized in that, the sequence of described tobacco JAZ protein gene is as shown in SEQ ID No:2 in sequence table.

3. the method for claim 1, is characterized in that, builds RNAi carrier the transformation of tobacco of described tobacco JAZ protein gene, obtains the transgene tobacco that nicotine content reduces.

4. method as claimed in claim 3, it is characterized in that, the RNAi carrier of described tobacco JAZ protein gene builds by following method: using the specific nucleic acid fragment of described tobacco JAZ protein gene as homing sequence, this specific nucleic acid fragment is inserted in plant expression vector with positive and negative both direction.

5. method as claimed in claim 4, is characterized in that, the specific nucleic acid fragment of described tobacco JAZ protein gene is the 1-513 position nucleotide sequence of SEQ ID No:2 in sequence table.

6. method as claimed in claim 4, it is characterized in that, in the RNAi carrier of described tobacco JAZ protein gene, two opposite described tobacco JAZ protein gene specific nucleic acid fragments of direction of insertion are separated by one section gus gene sequence, and are expressed by the guiding of CaMV35S promotor.

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