CN103012571B - Gene capable of reducing nicotine content of tobacco and application thereof - Google Patents

Abstract

The invention discloses a gene capable of reducing nicotine content of tobacco and an application thereof. The gene is a tobacco JAZ protein gene which is named as NtJAZ1, and protein encoded by the gene disclosed by the invention contains a typical TIFY structural domain and a jas structural domain and is induced and expressed by jasmonate. An RNAi (Ribonucleic Acid Interfere) transgenic carrier of the NtJAZ1 gene is constructed, and the tobacco is converted, so that expression of the NtJAZ1 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 gene disclosed by the invention provides an important basis and a technical support for low-nicotine-content tobacco breeding.

Description

Reduce gene and the application of tobacco nicotine content

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, thus he how reduces that harm of smoking has become that national governments, medical expert, botanist go all out to pay close attention to, one of problem of research.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.To Nicotine, biosynthetic research mainly concentrates on its metabolism involved enzyme of clone and analyzes its physiology and chemistry characteristic aspect 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 ofanatabine at the expense of nicotine.Plant Molecular Biology 53:87-105.).

The Nicotine biosynthesizing is subject 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 is 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 subject to the plant wound induce inducing of hormone-Jasmonate and express, thereby form Nicotine.Found jasmonic so far from 1962, 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.Nature 448: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.Nature 448:U661-U662.; Yan et al., 2007.A DownstreamMediator in the Growth Repression Limb ofthe Jasmonate Pathway.Plant Cell 19,2470-2483.).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; While having Jasmonate to exist in plant materials, 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.Phytochemistry 58:729-738.), 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 Academyof Sciences of the United States of America 95, 8113-8118.), this concentration can make most of leaf-eating insects dead immediately, thereby protection is subject 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, find altogether 12 members in Arabidopis thaliana, this is not difficult to make us to think deeply, and have in tobacco 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 subject to inducing of Jasmonate and expresses.Disturb its expression separately by the RNAi technology 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 NtJAZ1, 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 derivative protein has the function identical with (i) described protein.

SEQ ID No:1 sequence in sequence table is comprised of 239 amino-acid residues, wherein the 102nd to the 107th amino acids residue be the TIFY structural domain, the 187th to the 205th 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, lacked or added, and can be realized by the ordinary skill in the art the detection of correlation function.

Tobacco JAZ protein gene NtJAZ1 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.

Carry out de novo splicing by tobacco being transcribed to the group sequencing result, 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 NtJAZ1, 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-time PCR 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 NtJAZ1 gene, successfully obtained and suppressed the transgenic cell line that NtJAZ1 expresses in tobacco cell, 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 NtJAZ1 gene and can obtain the transgene tobacco that nicotine content reduces, this makes low Nicotine content tobacco breeding become possibility, for reducing the tobacco nicotine content, provides a kind of effective technique means.

In example of the present invention, at first, for building the RNAi binary expression vector of NtJAZ1, selecting one section nucleic acid fragment (the 1-538bp sequence of sequence table SEQ ID NO:2) more special in this gene is the homing sequence of RNAi, and the design primer obtains this sequence.Then, this nucleic acid fragment is inserted into to the RNAi binary expression vector that is configured to NtJAZ1 in plant expression vector pHZPRi-Hyg with positive and negative both direction.As shown in Figure 2, two NtJAZ1 specific fragments in this carrier are separated by one section GUS sequence, by the guiding of CaMV 35S promoter, are expressed.Afterwards, this plasmid is proceeded to Agrobacterium LBA4404, and via Agrobacterium, this plasmid is proceeded in tobacco BY-2 suspension cell.And then the efficiency by the Real-time PCR method to RNAi detects.Result shows, effectively inhibition (Fig. 3) of the expression of NtJAZ1 quilt in the transgenic cell line obtained.

For NtJAZ1-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 NtJAZ1 to change, and wherein: left figure has shown that the NtJAZ1 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; Take Actin as internal reference.

Fig. 2, NtJAZ1-RNAi plant expression vector construction schematic diagram, in this carrier, the RNAi homing sequence of NtJAZ1 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 NtJAZ1-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 NtJAZ1 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 NtJAZ1, WT is 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 Yellow 2)

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 24 ° of C, 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 be processed, collected sample at 0,0.5,2,6,12,24 hour 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 at MeJA, processes and collects sample in 72 hours, in-80 ℃ of cryogenic refrigerators, preserves, to detect for nicotine content.

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, by the 0.1%DEPC aqueous solution soaking, spends the night, and autoclaving is placed in 80 ℃ of loft drier dry.Be used for the electrophoresis chamber of RNA electrophoresis after cleaning, with soaking 30min in dehydrated alcohol, then at 30%H ₂O ₂Middle immersion 30min, finally process water flushing 5 times with the DEPC of sterilizing.

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, acutely sway centrifuge tube 15s with hand, 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 1ml 75% 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

Survey the absorbance value of RNA sample at 260nm and 280nm on GBC Cintra 10e ultraviolet spectrophotometer, by absorption value, calculate the concentration of RNA sample and the purity of judgement RNA sample.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 detect the integrity of RNA by 1% agarose gel electrophoresis.

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-freeDNase Buffer, RNase inhibitor 20unit, 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 finishes, with phenol/chloroform extracting, the ethanol precipitation reclaims the RNA sample.

2, the clone of NtJAZ1 full-length cDNA

Extract the BY-2 cell total rna that Jasmonate is processed 2 hours, reverse transcription is cDNA, and take this cDNA as template pcr amplification NtJAZ1 full length sequence.Idiographic flow is as follows:

Utilize the RevertAid of Fermentas company ^TMFirst Strand cDNA Synthesis Kit is reversed, 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 L 5 * 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 NtJAZ1 encoding gene: according to the Blast analytical sequence, design total length primer, used high-fidelity DNA polymerase pfu amplification NtJAZ1 encoding gene.Reaction system is as follows: 2 * PCR pfu Mix, 25 μ L, upstream primer 1 μ M, downstream primer 1 μ M, cDNA 1.0 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 '-CACCATGGGGTCATCGGAGATTGTAGA-3 ' (SEQ ID No:3);

Downstream primer: 5 '-CTAAAAGAACTGCTCAGTTTTCAC-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 use pENTR/D-TOPO cloning kit test kit that clone's gene is connected with the pENTR/D-TOPO carrier, the use linked system is as follows: PCR product 0.5-4 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-NtJAZ1.

The a small amount of of plasmid pENTY-NtJAZ1 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 and make the abundant cracking of thalline for 6-8 time, 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, carefully supernatant is transferred to (adsorption column adds in collection tube) in adsorption column with pipettor, room temperature is 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, the H to 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-NtJAZ1 identifies: by PCR, the total length of NtJAZ1 is carried out to the electrophoresis evaluation, will obtain the positive colony of total length Insert Fragment, send company to be checked order, the final full length sequence that obtains NtJAZ1 gene ORF of identifying.

3, Jasmonate is induced the expression of NtJAZ1 gene

The sequential analysis of ■ NtJAZ1 conserved regions

The sequential analysis of NtJAZ1 conserved regions: the NtJAZ1 albumen that the clone is obtained carries out sequence alignment and domain analyses, find that its N end contains conservative tify structural domain (the 102nd to the 107th amino acids residue) and C-terminal contains Jas structural domain (the 187th to the 205th 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 NtJAZ1 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 '-CGAACTCACGTGCCAATATC-3 ' (SEQ IDNo:5);

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

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

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

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

Take Actin as internal reference, and the primer that it is carried out to Real time-PCR is as follows:

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

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

The detection of expression of NtJAZ1 under ■ BY-2 cell different time

Draw materials: tobacco BY-2 suspension cell

Time: 50 μ M MeJA process 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.Add 10 μ L 2 * ABI powerSYBR green PCR master mix in 8 quantitative PCR pipes, 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 ABI 7500 real-time fluorescence quantitative PCR instrument, by normal process, carries out the PCR reaction.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 finishes, application software ABI 7500 Software v2.0 analyze 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, process tobacco BY-2 cells with 50 μ M MeJA, respectively at 0h, 0.5h, 2h, 6h, 12h and 24h collecting cell material, extract RNA, find that by Real-timePCR the NtJAZ1 gene is induced by Jasmonate and expressed, when the stimulation that is subject to Jasmonate, NtJAZ1 gene expression amount in 30min has significant rising, and along with the JA prolongation of action time, the expression amount of gene continues in very high level, reach maximum value at 24h, the expression amount of positive control PMT is significantly induced after processed by Jasmonate.

4, the acquisition of the transgenic tobacco cells of NtJAZ1 gene silencing system

The Clone and sequence of ■ gene silencing trigger sequence

According to sequence alignment, choose the fragment that specificity is higher (design of primers is as follows) in the ORF of NtJAZ1 frame, the specimen material that the Jasmonate of take is 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 NtJAZ1-RNAi in binary vector pHZPRi-Hyg.Primer sequence is as follows:

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

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

Application Agrobacterium infestation method proceeds to the NtJAZ1 gene silencing transgene carrier (NtJAZ1-RNAi) built 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 by after screening, and carry out the Jasmonate processing, collecting 0hour and 12hour sample extraction RNA reverse transcription is cDNA, detects the expression (Fig. 3) of NtJAZ1 by Real-time PCR, 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 cell that approximately 0.1 ~ 0.15g grinds 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 NtJAZ1, nicotine content has substantial degradation (as shown in Figure 4), and WT is tobacco wild-type BY-2 cell.

Claims (6)

1. a 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 1-538 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 contrary 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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