CN103012573A - 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 many worlds 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 to be transported under the blade deposition in the root formation of plant and through xylem.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 the 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 the cell.Studies have shown that the key enzyme on the nicotine metabolite approach, such as MPT, QPT and MPO are hindered by plant all to 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 and unclear to the signal transduction path of Jasmonate and regulating plant thereof.

Three seminars in 2007 in succession be reported in found in the Arabidopis thaliana 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 not having 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 having Jasmonate to exist in the 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 that does not come to harm, nicotine content is approximately 0.1%-1.0%.After being subject to plant-feed insect infringement or physical abuse; Jasmonate content can be increased to 9 times of original (Ziegler et al. in the tobacco plant; 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, reaches pest-resistant purpose.The results show Jasmonate can be regulated and control the generation of Nicotine, but its molecular mechanism and 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, having in the tobacco does not have such JAZ albumen, and whether these albumen have participated in the synthetic of tobacco Nicotine that Jasmonate induces.

Summary of the invention

The object of the invention is to seek the functional gene relevant with 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 the tobacco is subjected to inducing of Jasmonate and expresses.In tobacco, disturb its expression separately by the RNAi technology, 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 the sequence table;

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

SEQ ID No:1 sequence in the 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 the 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, also can be 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 the SEQ ID NO:2 for example.

Carry out de novo splicing by tobacco being transcribed the group sequencing result, utilize the tobacco that obtains 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, its Protein sequence is shown in the SEQ ID No:1 in the 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 to suppress the transgenic cell line that NtJAZ10 expresses in tobacco cell, the transgenic tobacco cells that obtains system has the special phenotype that the nicotine content relative comparison obviously reduces.As seen, utilize transgenic technology, disturb, reticent or knock out the NtJAZ10 gene and can obtain the transgene tobacco that nicotine content reduces, this provides a kind of effective technique means so that low Nicotine content tobacco breeding becomes possibility for reducing the tobacco nicotine content.

In example of the present invention, at first, for making up 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 the RNAi binary expression vector that is configured to NtJAZ10 among the 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, change this plasmid over to Agrobacterium LBA4404, and via Agrobacterium this plasmid is changed in the tobacco BY-2 suspension cell.And then, detect by the efficient of Real-timePCR method to RNAi.Result's demonstration, the expression of NtJAZ10 is by establishment (Fig. 3) in the transgenic cell line that obtains.

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 the 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.

Description of drawings

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 that increases 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 expression under the processing at JA; Take Actin as confidential reference items.

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; Among the figure, RB, LB show the border, the left and right sides 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, utilizing gas chromatography mass spectrometry analytical technique of mass spectrum (GC-MS) to carry out nicotine content detects, nicotine content decrease behind the Antisense Suppression NtJAZ10, WT are the contrast of tobacco wild-type BY-2 cell.

Embodiment

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

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, 4 days suspension cell of growth is reached without 2, in the MS substratum of 4-D, after 24 hours, adding 50 μ M Jasmonates (final concentration) processes, collected sample at 0,0.5,2,6,12,24 hour respectively, in the centrifuge tube of the precooling of packing into.Behind the liquid nitrogen flash freezer, transfer in-80 ℃ of cryogenic refrigerators and preserve, to be used for the Real-time pcr analysis.

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

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

Glasswork used in the RNA related experiment process is before use in 180 ℃ of baking 8h.Plastics comprise various types of rifle heads and centrifuge tube, spend the night with 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 soaking 30min in the dehydrated alcohol, then at 30%H ₂O ₂Middle immersion 30min, the DEPC with sterilization processes water flushing 5 times at last.

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

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

■ RNA quality examination

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

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

Digest residual DNA in the RNA sample by the DNase without RNase, 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 as 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 place on ice rapidly behind 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.Mixing reaction system I and II, 42 ℃ of reverse transcription reaction 60min process 5min for 70 ℃, place 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 uses 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 use pENTR/D-TOPO cloning kit test kit that clone's gene is linked to each other 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 and transform, and 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 overnight incubation to the 1-5mlLB/Kan substratum, next day, centrifugal 13000rpm 1min collected thalline.Add 250 μ L solution I (please check first and whether added RNaseA), behind the vibration mixing, 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 mixing white flocks can occur at this moment.The centrifugal 10min of 12000rpm, carefully supernatant is transferred to (adsorption column adds in the collection tube) in the adsorption column with pipettor, room temperature was placed 2 minutes, the centrifugal 1min of 12000rpm, outwell the waste liquid in the collection tube, adsorption column is relay in the recovery collector, add 500 μ l rinsing liquids (please checking first whether added dehydrated alcohol before using), centrifugal 1 minute of 12000rpm, abandon waste liquid, adsorption column is put into collection tube, repeat once, then the 12000rpm sky is from 2min.Drying at room temperature is 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 obtains plasmid.

The PCR of plasmid pENTY-NtJAZ10 identifies: by PCR the total length of NtJAZ10 is carried out electrophoresis and identify, 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 in the Nicotine route of synthesis induces expression under the processing as positive control at JA, and is 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).

Take Actin as confidential reference items, 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 the ■ 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, go that reverse transcription is cDNA behind the DNA, with 5 times of cDNA dilutions and be stored in-20 ℃ stand-by.Add 10 μ L2 * ABI powerSYBR greenPCR master mix in 8 quantitative PCR pipes, each 1 μ L of upstream and downstream primer (10 μ M) and cDNA adds dd H ₂O supplies reaction system to 20 μ L, and mixing is also of short duration centrifugal.Above-mentioned reaction system is placed ABI7500 real-time fluorescence quantitative PCR instrument, carry out the PCR reaction by normal process.Cycling condition is: 50 ℃ of 2min, 94 ℃ of denaturation 10min; 95 ℃ of sex change 15sec anneal and extension 1min 40 circulations for 60 ℃; Add at last a solubility curve and measure circulation.Application software ABI7500Software v2.0 analyzed experimental result after reaction finished.Key enzyme PMT in the Nicotine route of synthesis is as positive control, with ACTIN(GenBank:ACH69153.1) be expressed as confidential reference items.The 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 NtJAZ10 gene is induced by Jasmonate and expressed, and along with the JA prolongation of action time, the expression amount of gene increases gradually, reach maximum value at 2h, 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 higher fragment of specificity (design of primers is as follows), take 2 hours specimen material of Jasmonate processing as template, carry out the PCR clone, obtain the dna fragmentation as RNAi, connect the ENTY carrier and also check order.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 among the 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).

Using the Agrobacterium infestation method changes the NtJAZ10 gene silencing transgene carrier (NtJAZ10-RNAi) that builds in the tobacco BY-2 suspension cell over to.

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

The Molecular Identification of ■ Transgenic Tobacco clone

BY-2 suspension cell screening and culturing on the hygromycin resistance substratum after Agrobacterium infected, 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 Screening and Identification, the positive callus of acquisition.Concrete operations are carried out suspension cell for the positive callus after will screening, and carry out Jasmonate and process, collecting 0hour and 12hour sample extraction RNA and reverse transcription is cDNA, detects the expression (Fig. 3) of NtJAZ10 by Real-time PCR, and screening obtains transgenic positive clone.

■ Transgenic Tobacco clone nicotine content detects

Positive callus after the screening is carried out suspension cell, and carry out Jasmonate and process, 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 for subsequent use, the Liquid nitrogen precooler drill bit grinds 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 the methylene dichloride of adding 5ml and the mixed solvent (volume ratio 3:1) of methyl alcohol, jolt 5min.Leave standstill, treat layering.Take off layer organic phase 2.5ml in new centrifuge tube, add glacial acetic acid 20 μ l, mixing.With nitrogen organic phase is dried up, add 0.2ml methylene dichloride dissolving determinand, the whirlpool mixing, 2000rpm, 5min is centrifugal, and sucking-off solution is put into chromatotube and is treated machine testing.

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

Claims (10)

1. the JAZ albumen that grows tobacco is following proteins (i) or (ii):

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

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

2. the JAZ protein gene that grows tobacco, called after NtJAZ10, coding following proteins (i) or (ii):

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

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

3. tobacco JAZ protein gene as claimed in claim 2 is characterized in that, described tobacco JAZ protein gene is cDNA sequence or the genomic dna sequence of described protein gene.

4. tobacco JAZ protein gene as claimed in claim 3 is characterized in that, the sequence of described tobacco JAZ protein gene is shown in SEQ ID No:2 in the sequence table.

5. the application of tobacco JAZ protein gene claimed in claim 2 in reducing the tobacco nicotine content.

6. application as claimed in claim 5 is characterized in that, by transgenic technology disturb, reticent or knock out described tobacco JAZ protein gene, obtain the Transformation of tobacco plant that nicotine content reduces.

7. application as claimed in claim 6 is characterized in that, makes up RNAi carrier and the transformation of tobacco of described tobacco JAZ protein gene, obtains the transgene tobacco that nicotine content reduces.

8. application as claimed in claim 7, it is characterized in that, the RNAi carrier of described tobacco JAZ protein gene makes up by following method:, as homing sequence this specific nucleic acid fragment is inserted in the plant expression vector with positive and negative both direction with the specific nucleic acid fragment of described tobacco JAZ protein gene.

9. application as claimed in claim 8 is characterized in that, the specific nucleic acid fragment of described tobacco JAZ protein gene is the 1-513 position nucleotide sequence of SEQ IDNo:2 in the sequence table.

10. application as claimed in claim 8, 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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