CN102876689B - Tea tree FPS (famesyl diphosphate synthase) gene and application thereof - Google Patents
Tea tree FPS (famesyl diphosphate synthase) gene and application thereof Download PDFInfo
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- CN102876689B CN102876689B CN201210239772.1A CN201210239772A CN102876689B CN 102876689 B CN102876689 B CN 102876689B CN 201210239772 A CN201210239772 A CN 201210239772A CN 102876689 B CN102876689 B CN 102876689B
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Abstract
The invention discloses a tea tree FPS gene which has a nucleotide sequence shown in SEQ ID NO.1. The invention further discloses a protein encoded by the tea tree FPS gene, which has an amino acid sequence shown in SEQ ID NO.2. The invention further discloses an application of the tea tree FPS gene; and the gene expression relates to the tea tree disease-resistant stress, and the disease resistance of a tea tree can be improved through inducing high expression.
Description
Technical field
The invention belongs to tea tree genetically engineered field; Specifically, the present invention relates to point analysis of variance of tea tree farnesyl pyrophosphate synthase (famesyl diphosphate synthase, FPS) gene, also relate to and utilize this genetic expression intensity of adjusting to carry out disease for tea plant prevention.
Background technology
The generation of disease for tea plant and popular be the interactional result of tea tree and germ, often cause tea yield and quality to decline, when serious, also can cause tree vigo(u)r decline.
Invade from germ the process that disease symptom appears in tea tree, generally can be divided into stage of invasion, incubation period and 3 stages of period of disease.Stage of invasion, refers to from germ contact tea tree to the stage of setting up nutrition or parasitism with tea tree; Incubation period refers to from germ and tea tree sets up parasitism to the process that occurs obvious disease symptom; Period of disease refers to that the tea tree that catches an illness reflects the stage that pathological change and germ produce propagulum in formalness.Stage of invasion is the important stage that affects Occurrence and development of disease, this in stage germ sprout at tea tree tissue surface, produce holdfast, and by pore or wound invasion tea tree tissue space, tea tree cell produces a series of stress reaction for germ invasion, suppressing germ such as the synthetic plant protecting chemical of induction adheres to and grows, produce o-quinone material poisoning germ by polyphenoloxidase, form callose and stop that germ deeply, produce the contiguous plant of its sense material notice etc., this in stage pathogenic bacteria and the germ of having made decision mutually of tea tree can infect tea tree, determine in other words tea tree disease resistance power.
Disease for tea plant occurs and the popular impact that is mainly subject to the factors such as germ kind, tea tree breed, upgrowth situation and etap and environmental factor, and wherein germ and tea tree breed are main factor of determinations, and environment mainly plays fall out effect.Disease for tea plant conventionally strong at pathogenecity, tea tree breed resistance is poor, envrionment conditions is when suitable, just can occur with popular.
Disease for tea plant prevention and control mainly contain three kinds of approach: occur by seed selection resistance tea tree breed, agricultural chemicals control germ and spread and build good environment and suppress disease generation.Wherein seed selection resistant variety is optimization approach, but this approach is because mechanism is indefinite, causes difficult; Agricultural chemicals control has less investment, instant effect feature, but easily causes product and environmental pollution and bring out germ resistance; There is eco-friendly advantage by the agronomic measures factor of controling environment, but it is slow to take effect.How, by modern molecular biology means, specify the reaction mechanism of tea tree to disease, propose corresponding disease measure of control and be at present and even one of the important research content in Tea Science field in for some time from now on.
Summary of the invention
The technical problem to be solved in the present invention is to provide the gene order of a kind of tea tree farnesyl pyrophosphate synthase (famesyl diphosphate synthase, FPS) and the protein of coding thereof, by inducing this genetic expression can improve tea tree disease resistance.
In order to solve the problems of the technologies described above, the invention provides a kind of tea tree FPS gene, it has the nucleotide sequence shown in SEQ ID No:1.
The present invention also provides the protein of above-mentioned FPS genes encoding, and it has the aminoacid sequence shown in SEQ ID NO:2.
The present invention also provides above-mentioned FPS gene purposes: this genetic expression with tea tree is disease-resistant stress be relevant, induction high expression level can improve tea tree disease resistance.
The present invention also provides a kind of method that improves tea tree disease resistance simultaneously, comprises with the gene transformation tea tree cell with the nucleotide sequence shown in SEQ ID No:1, then the tea tree cell culture after transforming is become to plant.
The present invention makes the practical problemss such as mechanism is indefinite mutually mainly at present disease generating process tea tree and germ, by disease for tea plant generation blade and healthy leaves differential gene are screened and are cloned, and activity of gene expression and disease resistance correlation research, specify the relation between activity of gene expression rise of the present invention and disease for tea plant generation.For having created condition by regulating this activity of gene expression to carry out disease for tea plant control, simultaneously also for the initiative of carrying out high disease-resistant new tea material by genetic engineering means is laid a good foundation.
The present invention has adopted following technical scheme to realize: adopt Using Suppression Subtractive Hybridization (suppression subtractive hybridization, SSH) and RACE technical point from responding relevant FPS gene to disease for tea plant, it has the nucleotide sequence shown in SEQ ID No:1; A kind of protein sequence of tea tree FPS total length is provided, and it has sequence as shown in SEQ ID No:2.
The present invention also provides tea tree FPS gene expression characteristics, proves that the present invention improves FPS activity of gene expression and can improve tea tree disease resistance.
Realize concrete technological step of the present invention as follows:
One, tea tree FPS gene isolation and analysis
In tea tree breed garden, gather with the infection process blade of kind and the healthy leaves of identical ripening degree, be placed in respectively after the abundant grinding of liquid nitrogen, with TRIZOL(Invitrogen company) extract total RNA, and process with UNIQ-10 pillar mRNA extracting and purifying test kit (Shanghai Sheng Gong biotechnology company limited), obtain respectively the mRNA sample of sick, strong blade.Respectively with disease, 2 μ g mRNA of strong leaf are initiator, adopt PCR-Select cDNA Subtraction Kit(Clontech Laboratories, Inc) carrying out double-stranded cDNA synthesizes, enzyme is cut, joint connects, subtractive hybridization and amplification, concrete operations are with reference to this test kit operation instruction, obtain the differential gene fragment of up-regulated expression in sick leaf, insert the precious biotechnology (Dalian) of TaKaRa pMD 18-T Vector(company limited), and transform the precious biotechnology (Dalian) of JM109(company limited) competence intestinal bacteria, through blue hickie screening, the amplification of picking hickie, carry out bacterium liquid PCR with M13 universal primer (sequence is as shown in SEQ ID No:3 and SEQ ID No:4), the positive colony of Insert Fragment will be had, entrust Shanghai Ying Weijieji company to check order.Through and American National bioinformation center NCBI(http: //www.ncbi.nlm.nih.gov/) the Blastn instrument that provides compares discovery online, wherein has the intermediate sequence height homology of the plant FPS genes such as 1 long 244bp differential fragment and the bark of eucommia (Eucommia ulmoides) and grape (Vitis vinifera).According to the fragment sequence of the 244bp obtaining, design 5 ' end RACE sequence specific primers SEQ ID No:5 and SEQ ID No:6(and estimated the about 530bp of product length), and 3 ' end RACE sequence specific primers SEQ ID No:7 and SEQ ID No:8(estimate that product length is 880bp), cause harm the mRNA of blade as initiator taking above-mentioned germ, adopt TaKaRa 5'-Full RACE Core Set, TaKaRa 3'-Full RACE Core Set and TaKaRa Taq (precious biotechnology (Dalian) company limited) carry out FPS 5 ' end and 3 ' terminal sequence clone (concrete operations reference reagent box operation instruction), to obtaining 5 ' RACE and 3 ' RACE product carries out 1.5% agarose gel electrophoresis, with the precious biotechnology (Dalian) of TaKaRa MiniBEST Agarose Gel DNA Extraction Kit(company limited) to the target stripe purifying of tapping rubber, the target stripe of purifying is inserted to TaKaRa pMD
18-T Vector, and transform JM109 competence intestinal bacteria, through blue hickie screening, the amplification of picking hickie, carry out bacterium liquid PCR with M13 universal primer (SEQ ID No:3 and SEQ ID No:4), the positive colony of Insert Fragment will be had, entrust Shanghai Ying Weijieji company to check order, obtained 5 ' end 524bp(and the intermediate segment overlapping head of district 118) RACE product and 3 ' hold the overlapping head of district 101bp of 870bp(and intermediate segment) RACE product, through comparing with NCBI known array, 5 ' end of the plant FPS genes such as above-mentioned sequence and the bark of eucommia and Radix Glycyrrhizae (Glycyrrhiza uralensis) and 3 ' end height homology.Splice obtaining tea tree FPS gene 5 ' end, intermediate sequence and 3 ' terminal sequence with SeqMan software (DNASTAR, Inc.) in DNAStar bag, obtained tea tree FPS full length gene sequence.This gene length 1419bp, as shown in SEQ ID No:1 (underlined is the sequence of corresponding SEQ ID No:2, and last three bases of underscore are terminator codon); There is 1029bp(84
th-1112
th) open reading frame, coding 342 amino-acid residues protein, as shown in SEQ ID No. 2.Through comparing discovery with ncbi database, tea tree FPS aminoacid sequence and Radix Glycyrrhizae (Glycyrrhiza uralensis), ginseng (Panax ginseng) FPS have 86% consistence and 93% similarity.Prove that the diversity sequence of high expression level is tea tree FPS gene in sick leaf.According to PredictProtein(http: //www.predictprotein.org/) analyze and show, tea tree FPS is positioned in tenuigenin, there is poly isopentene group synthase (polyprenyl synthase) signature sequence [LIVMFY] Gx2[FYL] Q[LIVM] xDD[LIVMFY] x[DNG] (x is arbitrary amino acid residue), belong to farnesyl tetra-sodium (FPP)/geranyl geranyl tetra-sodium (GGPP) synthase family.Studies show that, FPS is an important enzyme in plant cytoplasm isoprene pathways metabolism, main responsible catalysis dimethyl propylene thiazolinyl tetra-sodium (DMAPP) is end to end with isopentenyl pyrophosphate (IPP), generate the geranyl tetra-sodium (GPP) containing 10 carbon, and then again with IPP through end to end, the synthetic farnesene base tetra-sodium (farnesyl pyrophosphate, FPP) containing 15 carbon.FPP is not only many important meta-bolitess as the synthetic precursor of steroidal, dolichol, ubiquinone etc.; Under the effect of sesquiterpene cyclase, generate the sesquiterpene derivative including plant protecting chemical and volatile oil component, and plant protecting chemical and volatile oil component play an important role in plant autoimmunization and sick worm defensive raction simultaneously.In addition, the intermediate GPP in FPS catalytic process etc. can also enter chloroplast(id) (plastid) and participate in the synthetic of monoterpene, diterpene, tetraterpene compound.As can be seen here, in the sick leaf of " Dragon Well tea 43 " tea tree, to raise may be body to one of defensive stress reaction of one that pathogen infection rose in FPS genetic expression, limits pathogen growth and further infect so that body produces more biocidal property material.
Two, FPS genetic expression intensity and disease association analysis
According to the tea tree FPS gene order of above-mentioned acquisition, adopt PrimerSelect design in DNAStar software package (DNAStar Inc.) to express primer, sequence (is estimated product length 406bp) as shown in SEQ ID No:9 and SEQ ID No:10, simultaneously with tea tree Actin muscle (Actin, ACT) gene (
http:// www.ncbi.nlm.nih.gov/ nuccore/FJ355923) be reference gene, be designed for the primer that act gene is expressed, sequence (is estimated product 344bp) as shown in SEQ ID No:11 and SEQ ID No:12.
In kind garden, gather the blade that different varieties disease occurs, the healthy leaves that simultaneously gathers the equal ripening degree of same breed is contrast, extract respectively the disease of these kinds, the strong total RNA of Ye, synthetic cDNA the first chain, adopt the primer pair disease of design, strong sample carries out RT-PCR, through agarose gel electrophoresis and object band photodensitometry, and with the relative expression's intensity that is compared to FPS gene of FPS gene object band optical density(OD) and reference act gene object band optical density(OD), result shows, no matter what different varieties was suffered from is identical disease or different diseases, in the sick leaf of these kinds, FPS gene relative expression intensity is all significantly higher than the healthy leaves of same kind.Illustrate that it is tea tree for one of general response of one of germ invasion that FPS genetic expression is raised, and exists close causalnexus relation between the two.
Further, process tea leaf with methyl jasmonate (this compound is known, important plant hormone or the signaling molecule relevant to damage stress reaction), simulation infection process damage signal, and adopt the impact of above-mentioned RT-PCR technical Analysis methyl jasmonate on FPS genetic expression, result shows, after methyl jasmonate treatment, tea leaf FPS gene relative expression intensity significantly raises, later stage disease survey shows simultaneously, significantly declines through the disease for tea plant occurrence degree of methyl jasmonate treatment.Illustrate that FPS genetic expression can be subject to germ invasion equivalent damage signal induction, by external source signal induction FPS gene high expression, can improve tea tree disease resistance.
On this basis, design for cloning the whole open reading frame of tea tree FPS gene, and embedding and have the primer pair of NdeI and SacI restriction enzyme site, its sequence is as shown in SEQ ID No:13 and SEQ ID No:14, extract tea leaf mRNA, and obtain tea tree FPS gene open reading frame sequence with RT-PCR method, be inserted into TaKaRa pMD
18-T Vector, and transform the amplification of JM109 competence intestinal bacteria, after sequence verification, extract the restructuring pMD containing FPS gene open reading frame sequence with UNIQ-10 pillar plasmid a small amount of extraction agent box (the Shanghai biological company limited of raw work)
18-T Vector, and by this plasmid called after pMD
18-T-FPS.To combine enzyme NdeI and SacI (precious biotechnology (Dalian) company limited) to pMD
18-T-FPS plasmid carries out double digestion and obtains FPS gene open reading frame, and insert the precious biotechnology (Dalian) of the eucaryon binary expression vector TaKaRa pRI 201-AN DNA(company limited that same enzyme cuts), transform after the amplification of TG1 competence intestinal bacteria, extracting restructuring with UNIQ-10 pillar plasmid a small amount of extraction agent box has the binary expression vector of FPS gene open reading frame sequence, and by its called after pRI-201-AN-DNA-FPS.Adopt alternate freezing and thawing method that pRI-201-AN-DNA-FPS is led to the wild Agrobacterium rhizogenes 15834 of people.Adopt Agrobacterium infestation method by external source FPS gene " the Zhejiang agriculture 25 " aseptic seedling stem sections under the driving of CaMV35S strong promoter.Turn FPS gene induction of hairy roots callus from what produce, by this callus called after FPS transgenosis callus.With similar method, the root of hair and the callus that produce to infect the induction of " Zhejiang agriculture 25 " aseptic seedling stem sections without the wild Agrobacterium rhizogenes 15834 of external source FPS gene, by this callus called after contrast callus.Adopt needle-punching method that tea tree anthrax bacteria (Gloeosporium theae-sinesis Miyake) is inoculated on two kinds of callus pieces, under 12h illumination/12h dark, 28 DEG C of conditions, cultivate 3-5d, observe Lesion size.Adopt primer SEQ ID No:9 and SEQ ID No:10(FPS gene simultaneously) and SEQ ID No:11 and the SEQ ID No:12 FPS gene relative expression intensity after to two kinds of callus inoculation germs analyze.Result shows, turns FPS gene callus piece scab and is significantly less than the scab on contrast callus piece, and turn FPS genetic expression intensity in FPS gene callus piece and be also significantly higher than contrast callus.Obviously, turn in the callus of FPS gene, under the driving of CaMV35S strong promoter, the FPS gene that mass expressing external proceeds to, and the anti-anthrax ability of callus piece is significantly improved.
As can be seen here, it is one of tea tree a kind of defensive reaction to germ invasion that FPS gene raises, and improves FPS gene expression dose and can improve the defensive ability/resistance ability of tea tree to germ.
Brief description of the drawings
Below in conjunction with accompanying drawing, the specific embodiment of the present invention is described in further detail.
Fig. 1 is " Dragon Well tea 43 " tea tree health (left side) and the blade comparison diagram of suffering from anthrax (right side);
Fig. 2 is " Zhejiang agriculture 25 " tea tree health (left side) and the blade comparison diagram of suffering from anthrax (right side);
Fig. 3 is " turtledove hole " tea tree health (left side) and the blade comparison diagram of suffering from moire blight (right side);
Fig. 4 is the sick strong blade FPS expression conditions comparison diagram of tea tree;
In Fig. 4: ACT is tea tree Actin muscle reference gene, FPS is tea tree farnesene pyrophosphate synthase gene; 1 is that " Dragon Well tea 43 " germ infringement leaf, 2 is " Dragon Well tea 43 " healthy leaf, and 3 is that " Zhejiang agriculture 25 " germ infringement leaf, 4 is " Zhejiang agriculture 25 " healthy leaf, and 5 is that " turtledove hole " germ infringement leaf, 6 is " turtledove hole " healthy leaf;
Fig. 5 is that " Dragon Well tea 43 " tea tree sprays FPS expression conditions comparison diagram after methyl jasmonate;
In Fig. 5: ACT is tea tree Actin muscle reference gene, FPS is tea tree farnesene pyrophosphate synthase gene; 1 for spray clear water 6h rear blade, 2 is spray methyl jasmonate 6h rear blade, and 3 is that spray clear water 24h rear blade, 4 is spray methyl jasmonate 24h rear blade.
Fig. 6 turns FPS gene tea tree callus and the non-FPS of turning gene tea tree callus anthrax scab comparison diagram;
In Fig. 6: the left side is for turning FPS gene tea tree callus, and the right is the non-FPS gene tea tree callus that turns, and arrow indication is anthrax scab.
Embodiment
In tea tree breed garden, pluck " Dragon Well tea 43 " by the cause harm healthy leaves (Fig. 1) of blade and identical ripening degree of anthrax bacteria, be placed in respectively after the abundant grinding of liquid nitrogen, with TRIZOL(Invitrogen company) extract total RNA, and process with UNIQ-10 pillar mRNA extracting and purifying test kit (Shanghai Sheng Gong biotechnology company limited), obtain respectively the mRNA sample of sick, strong blade.Respectively with disease, 2 μ g mRNA of strong leaf are initiator, adopt PCR-Select cDNA Subtraction Kit(Clontech Laboratories, Inc) carrying out double-stranded cDNA synthesizes, enzyme is cut, joint connects, subtractive hybridization and amplification, concrete operations are with reference to this test kit operation instruction, obtain the differential gene fragment of up-regulated expression in " Dragon Well tea 43 " sick leaf, insert the precious biotechnology (Dalian) of TaKaRa pMD 18-T Vector(company limited), and transform the precious biotechnology (Dalian) of JM109(company limited) competence intestinal bacteria, specific experiment operates with reference to carrier and engineering bacteria operation instruction.Through blue hickie screening, picking hickie, transfers in 1ml liquid LB substratum, 37 DEG C of shaking culture 12h; Get bacterium liquid 100 μ l in new centrifuge tube, the centrifugal 2min of 6000rpm, abandons after supernatant, adds 20 μ l and heavily boils off ionized water (ddH
20) dilution, is used as bacterium liquid pcr template.Carry out bacterium liquid PCR with M13 universal primer (sequence is as shown in SEQ ID No:3 and SEQ ID No:4), PCR solution preparation and programming are as shown in table 1.
Table 1
SEQ ID No:3 (M13 upstream primer) 5 '-cgccagggttttcccagtcacgac
SEQ ID No:4 (M13 downstream primer) 5 '-agcggataacaatttcacacagga
PCR product is carried out to 1.5% agarose gel electrophoresis, will have the positive colony of Insert Fragment, entrust Shanghai Ying Weijieji company to check order.Through and American National bioinformation center NCBI(http: //www.ncbi.nlm.nih.gov/) the Blastn instrument that provides compares discovery online, wherein has the intermediate sequence height homology of the plant FPS gene such as differential fragment and the bark of eucommia (Eucommia ulmoides) and grape (Vitis vinifera) of 1 long 244bp.According to the fragment sequence of 244bp obtaining, designed 5 ' end RACE sequence specific primers SEQ ID No:5 and SEQ ID No:6(and estimated the about 530bp of product length) and 3 ' end RACE sequence specific primers SEQ ID No:7 and SEQ ID No:8(estimate that product length is 880bp).
SEQ ID No: 5 (FPS 5’RACE out) 5’-tcatctgtccacaagttgtctg
SEQ ID No: 6 (FPS 5’RACE in) 5’-cagcagatccacataataagg
SEQ ID No:7 (FPS 3’RACE out) 5’-tgccaatgatggcgtaattctc
SEQ ID No:8 (FPS 3’RACE in) 5’-agacaacttgtggacagatgatag 。
Cause harm the mRNA of blade as initiator taking above-mentioned germ, adopt TaKaRa 5'-Full RACE Core Set, TaKaRa 3'-Full RACE Core Set and TaKaRa Taq (precious biotechnology (Dalian) company limited) carry out tea tree FPS gene 5 ' end and 3 ' terminal sequence clone (concrete operations reference reagent box operation instruction), 5 ' the RACE and 3 ' the RACE product that obtain are carried out to 1.5% agarose gel electrophoresis, with the precious biotechnology (Dalian) of TaKaRa MiniBEST Agarose Gel DNA Extraction Kit(company limited) to the target stripe purifying of tapping rubber, the target stripe of purifying is inserted to TaKaRa pMD
18-T Vector, and transform JM109 competence intestinal bacteria, concrete operations are with reference to purification kit, carrier and engineering bacteria operation instruction.Through blue hickie screening, picking hickie, transfers and increases in LB bacteria culture medium, adopts above-mentioned identical method to carry out bacterium liquid PCR with M13 universal primer (SEQ ID No:3 and SEQ ID No:4), and the configuration of PCR reaction soln and programming are as shown in table 1.The positive colony of Insert Fragment will be had, entrust Shanghai Ying Weijieji company to check order, obtained 5 ' end 524bp(and intermediate segment overlapping head of district 118bp) RACE product and 3 ' hold the overlapping head of district 101bp of 870bp(and intermediate segment) RACE product, through comparing with NCBI known array, 5 ' end of the plant FPS genes such as above-mentioned sequence and the bark of eucommia and Radix Glycyrrhizae (Glycyrrhiza uralensis) and 3 ' end height homology.Splice obtaining tea tree FPS gene 5 ' end, intermediate sequence and 3 ' terminal sequence with SeqMan software (DNASTAR, Inc.) in DNAStar bag, obtained tea tree FPS full length gene sequence.This gene length 1419bp, as shown in SEQ ID No:1, has 1029bp(84
th-1112
th) open reading frame, coding 342 amino-acid residues protein, as shown in SEQ ID No. 2.Through comparing discovery with ncbi database, tea tree FPS aminoacid sequence and Radix Glycyrrhizae (Glycyrrhiza uralensis), ginseng (Panax ginseng) FPS have 86% consistence and 93% similarity.Prove that the diversity sequence of high expression level is tea tree FPS gene in sick leaf.According to PredictProtein(http: //www.predictprotein.org/) analyze and show, tea tree FPS is positioned in tenuigenin, has poly isopentene group synthase (polyprenyl synthase) signature sequence [LIVMFY] Gx
2[FYL] Q[LIVM] xDD[LIVMFY] x[DNG] (x is arbitrary amino acid residue), belong to farnesyl tetra-sodium (FPP)/geranyl geranyl tetra-sodium (GGPP) synthase family.Studies show that, FPS is a key enzyme in plant cytoplasm isoprene pathways metabolism, main responsible catalysis dimethyl propylene thiazolinyl tetra-sodium (DMAPP) is end to end with isopentenyl pyrophosphate (IPP), generate the geranyl tetra-sodium (GPP) containing 10 carbon, and then again with IPP through end to end, the synthetic farnesene base tetra-sodium (farnesyl pyrophosphate, FPP) containing 15 carbon.FPP is not only many important meta-bolitess as the synthetic precursor of steroidal, dolichol, ubiquinone etc., simultaneously under the effect of sesquiterpene cyclase, the sesquiterpene derivative of generation including plant protecting chemical and volatile oil component, and plant protecting chemical and volatile oil component play an important role in plant autoimmunization and sick worm defensive raction.In addition the intermediate GPP in FPS catalytic process etc. can also enter chloroplast(id) (plastid) and participate in the synthetic of monoterpene, diterpene, tetraterpene compound.As can be seen here, in the sick leaf of " Dragon Well tea 43 " tea tree, to raise may be body to one of defensive stress reaction of one that pathogen infection rose in FPS genetic expression, limits pathogen growth and further infect so that body produces more biocidal property material.
According to the tea tree FPS gene order of above-mentioned acquisition, adopt PrimerSelect design in DNAStar software package (DNAStar Inc.) to express primer, sequence (is estimated product length 406bp) as shown in SEQ ID No:9 and SEQ ID No:10, simultaneously with tea tree Actin muscle (Actin, ACT) gene (
http:// www.ncbi.nlm.nih.gov/ nuccore/FJ355923) be reference gene, be designed for the primer that act gene is expressed, sequence (is estimated product 344bp) as shown in SEQ ID No. 11 and SEQ ID No. 12.
In kind garden, gather suffer from anthrax " Dragon Well tea 43 " (Fig. 1) and " Zhejiang agriculture 25 " (Fig. 2), and " the turtledove hole " of suffering from moire blight disease leaf of kind such as (Fig. 3), the healthy leaves that simultaneously gathers the equal ripening degree of same breed is contrast, respectively get about 100mg, extract the total RNA of blade with TRIZOL reagent (Invitrogen company), detect RNA quality with electrophoretic method, and by each sample rna concentration adjustment to 500 μ g/ml, with synthetic cDNA the first chain of MMLV the first chain cDNA synthetic agent box (Shanghai Sheng Gong biotechnology company limited), concrete operations reference reagent box operation instruction.Taking above-mentioned cDNA the first chain as template, respectively taking SEQ ID No:9 and SEQ ID No:10(FPS genetic expression) and SEQ ID No:11 and SEQ ID No:12(ACT genetic expression) as primer pair, carry out PCR, the preparation of PCR reaction solution and time variable control are in table 2.
Table 2
SEQ ID No:9 (FPS genetic expression upstream primer) 5 '-aaaggaattaaccgatgatgaaa
SEQ ID No:10 (FPS genetic expression downstream primer) 5-tggcaggtaaaatgagtagtaagc
SEQ ID No:11 (reference act gene expression upstream primer) 5 '-tccgttgccctgaagtcct
SEQ ID No:12 (reference act gene expression downstream primer) 5 '-tgaagcgaataagcaatgaaaaat
By PCR product at 1.5%(w/v) carry out electrophoresis on sepharose, with Jetta JD801 gel electrophoresis images analytical system (Jiangsu Jetta development in science and technology company limited), electrophoresis result is made a video recording, as Fig. 4, and read object fragment absorbancy, be compared to this sample FPS gene relative expression intensity with the absorbancy of FPS gene object fragment of same sample and the absorbancy of reference act gene object fragment.
Result shows, in " Dragon Well tea 43 " sick, strong leaf, FPS gene relative expression intensity is respectively 0.35 ± 0.05 and 0.17 ± 0.03, in " Zhejiang agriculture 25 " sick, strong leaf, FPS gene relative expression intensity is respectively 0.45 ± 0.03 and 0.21 ± 0.02, and in " turtledove hole " sick, strong leaf, FPS gene relative expression intensity is 0.56 ± 0.04 and 0.29 ± 0.04.Visible, what suffer from regardless of different varieties is identical disease or different diseases, and in the sick leaves of these kinds, FPS gene relative expression intensity is all compared with the high 1 times of left and right of healthy leaves.Illustrate that it is tea tree for one of general response of one of germ invasion that FPS genetic expression is raised, and exists close causalnexus relation between the two.
In order further to verify the relation between FPS genetic expression and disease, employing can be induced the plant signal molecule methyl jasmonate treatment tea leaf of damage stress reaction, the damage of simulation infection process.Specific practice is, before disease occurs, and taking concentration as 0.2%(w/v, i.e. 2g/1L) methyl jasmonate medicine liquid spray mode processing " Dragon Well tea 43 " tea tree, liquid usage quantity is 70ml/m
2replace 0.2% methyl jasmonate medicine liquid spray as contrast taking clear water, 6h and 26h after spray, gather respectively processing and the contrast blade of identical ripening degree, and adopt method identical in embodiment 2 to extract total RNA, synthetic cDNA the first chain, RT-PCR, electrophoresis and band photodensitometry.Result as shown in Figure 5, after methyl jasmonate treatment 6h and 24, spray clear water contrast and improve approximately 1.2 times by tea leaf FPS gene relative expression intensity.Meanwhile, after 1 month, there is investigation and show in the disease of " Dragon Well tea 43 " tea tree to spray methyl jasmonate and clear water, and the sickness rate that sprays the tea tree anthrax of methyl jasmonate liquid sprays low 75% left and right of contrast of clear water.Illustrate that FPS genetic expression can be subject to germ invasion equivalent damage signal induction, by external source signal induction FPS gene high expression, can improve tea tree disease resistance.
As can be seen here, it is one of tea tree a kind of defensive reaction to germ invasion that FPS gene raises, and improves FPS gene expression dose and can improve the defensive ability/resistance ability of tea tree to germ.
Adopt MapDraw(DNAStar Inc. in DNAStar software package) open reading frame of tea tree FPS gene to clone, be the Nucleotide between the 84th to 1112 in SEQ ID No:1, carry out the analysis of restriction endonuclease point of contact, find in this open reading frame without NdeI and SacI restriction enzyme site, design to embed and had NdeI and SacI restriction enzyme site, the primer pair of the whole open reading frame of tea tree FPS gene that can increase, its sequence is as shown in SEQ ID No:13 and SEQ ID No:14, 5 ' the end at SEQ ID No:13 has comprised NdeI restriction enzyme site sequence (CATATG) and 2 protection bases (AA), 5 ' the end at SEQ ID No:14 has comprised SacI restriction enzyme site sequence (GAGCTC) and 2 protection bases (AA).Taking the tea leaf mRNA of the purifying in above-described embodiment 1 as initiator, with synthetic cDNA the first chain of MMLV the first chain cDNA synthetic agent box (Shanghai Sheng Gong biotechnology company limited), concrete operations reference reagent box operation instruction.Preparation cumulative volume is 25 μ l PCR reaction solutions, comprising 10 × PCR damping fluid, 2.5 μ l, 25mmol/L MgCl
22 μ l, 10mmol/L dNTP 0.5 μ l, cDNA the first chain 2 μ l, the each 1 μ l of 20 μ mol/L primers (SEQ ID No:13 and SEQ ID No:14), 5U/ μ l Taq archaeal dna polymerase 0.2 μ l, ddH
2o 15.8 μ l; Carry out PCR reaction, its program is: 95 DEG C of denaturation 4min, 1 circulation, 94 sex change 1min, 55 DEG C of annealing 1min, 72 DEG C extend 1.5min, 40 circulations, extend 10min after 72 DEG C.PCR product is carried out to 1.5% agarose gel electrophoresis, under ultraviolet lamp, extract target stripe, with the precious biotechnology (Dalian) of TaKaRa MiniBEST Agarose Gel DNA Extraction Kit(company limited) target stripe is carried out to purifying, the target stripe of purifying is inserted to TaKaRa pMD
18-T Vector, and transform JM109 competence intestinal bacteria, concrete operations are with reference to purification kit, carrier and engineering bacteria operation instruction.Through blue hickie screening, picking hickie, transfers and increases in LB bacteria culture medium, adopts above-mentioned identical method to carry out bacterium liquid PCR with M13 universal primer (SEQ ID No:3 and SEQ ID No:4), and the configuration of PCR reaction soln and programming are as shown in table 1.To there is the positive colony of Insert Fragment, entrust Shanghai Ying Weijieji company to check order, result demonstration, the sequence of acquisition is consistent with the open reading frame sequence of tea tree FPS in embodiment 1.Positive colony after sequence verification is inoculated in LB liquid nutrient medium, 12h is cultivated in 37 DEG C of concussions, the centrifugal 5min of 6000rpm, collects bacterial precipitation, extracts the restructuring pMD containing FPS gene open reading frame sequence with UNIQ-10 pillar plasmid a small amount of extraction agent box (the Shanghai biological company limited of raw work)
18-T Vector, and by this plasmid called after pMD
18-T-FPS.Respectively to combine enzyme NdeI and SacI (precious biotechnology (Dalian) company limited) to pMD
18-T-FPS plasmid carries out double digestion, through electrophoresis, reclaims respectively containing FPS gene object fragment with glue purification test kit, and concrete operations are with reference to restriction endonuclease and test kit related description.Adopt same procedure to the precious biotechnology (Dalian) of eucaryon binary expression vector TaKaRa pRI 201-AN DNA(company limited, the open reading frame of foreign gene is inserted to this expression vector, can realize foreign gene and under the driving of CaMV35S promotor, carry out strong expression) carry out double digestion (NdeI and SacI), electrophoresis and rubber tapping purifying linearized vector.With T4DNA ligase enzyme (precious biotechnology (Dalian) company limited), the FPS gene open reading frame sequence of recovery is connected with linearized vector, ligation system is 25 μ l, comprising 10 × T4 DNA ligase damping fluid, 2.5 μ l, FPS open reading frame sequence 5 μ l(0.3 pmol), linearizing pRI 201-AN DNA vector 2 μ l(0.03 pmol), 2000U T4DNA ligase enzyme 1 μ l and ddH2O 14.5 μ l, under 16 DEG C of conditions, connect and spend the night.In linked system, add the cold dehydrated alcohol of 3mol/L sodium-acetate (pH5.2) 2.5 μ l and 62.5 μ l, place 60min in-20 DEG C, in 12000rpm, 4 DEG C of centrifugal collecting precipitations, and be dissolved in 25 μ l TE damping fluids, and transform TG1 competence intestinal bacteria, and coat containing on 50 μ g/ml kantlex LB solid mediums and carry out to the greatest extent resistance screening, positive colony is transferred in LB liquid nutrient medium, 37 DEG C of amplifications.Carry out bacterium liquid PCR checking with above-mentioned SEQ ID No:13 and SEQ ID No:14 primer, the sequence positive colony that has FPS gene open reading frame to insert is increased, and recombinate and have the binary expression vector of FPS gene open reading frame sequence with the extraction of UNIQ-10 pillar plasmid a small amount of extraction agent box, and by its called after pRI-201-AN-DNA-FPS.Adopt alternate freezing and thawing method [can reference: Hofgen R, Willmitzer L.Nucleic acid Research, 1988,16 (20): 9877.], pRI-201-AN-DNA-FPS is led to the wild Agrobacterium rhizogenes 15834 of people.Infect by expanding numerous restructuring Agrobacterium bacterium liquid that is 0.5 ~ 0.8 to OD600 " Zhejiang agriculture 25 " aseptic seedling stem sections 15 min that cultivate 60 ~ 70 d seedling ages, on the YMB solid medium that adds 100 mmol/L Syringylethanones, cultivate altogether 2d, Induction Transformation on the MS substratum containing 500 mg/L cefotaxime sodiums (Shanghai raw work biological company limited) [can reference: Zhang Guanghui, Liang Yuerong, Lu Jianliang. tea science, 2006, 26 (1): 1-10], by the root of hair that has external source FPS gene obtaining, transfer in containing 2mg/L 2-4-D, 1.5mg/L KT(6-chaff aminopurine), 30g/L sucrose, on the MS substratum of 7.5g/L agar, 12h illumination/12h dark, under 25 DEG C of conditions, cultivate, evoked callus, by this callus called after FPS transgenosis callus.With similar method, infect " Zhejiang agriculture 25 " aseptic seedling stem sections with wild Agrobacterium rhizogenes 15834, and induce root of hair and callus, by this callus called after contrast callus.Above-mentioned two kinds of callus are cut into the thick bulk for 8mm × 4mm × 2mm of length and width, in above-mentioned calli induction media, cultivate 3d(condition the same) with healing wound, afterwards callus piece is taken out and is placed in the culture dish that is lined with moistening filter paper, adopt the method for acupuncture that concentration is about to 10
4the tea tree anthrax bacteria (Gloeosporium theae-sinesis Miyake) of individual/ml is inoculated on callus piece, under 12h illumination/12h dark, 28 DEG C of conditions, cultivates 3-5d.Observe and show, turn FPS gene callus piece scab very little, only for contrasting 1/4 left and right of callus piece, as shown in Figure 6.Callus piece is sampled, and adopt the method in embodiment 2 to carry out FPS gene relative expression strength analysis, result shows, turns in FPS gene callus piece, and FPS genetic expression intensity is contrast the more than 3 times of callus.As can be seen here, turn in the callus of FPS gene, under the driving of CaMV35S strong promoter, the FPS gene that mass expressing external proceeds to, and the anti-anthrax ability of callus piece is significantly improved.
SEQ ID No: 13 5’ - aacatatgatgagcgatctaaagtcgaaattc
SEQ ID No: 14 5’ - aagagctcctacttctgcctcttatatatctt
Finally, it is also to be noted that, what more than enumerate is only several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, can also have many distortion.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention, all should think protection scope of the present invention.
<110> Zhejiang University
<120> tea tree FPS gene and application thereof
<160> 14
<210> 1
<211> 1419
<212> DNA
<213> tea tree (
camellia sinensis)
<400> 1
atcacacact ctctctcttc ccatcaaact caaacggacg accaggattc gccatccaat 60
ctctctgtct ctctgtgaaa aca
atgagcg atctaaagtc gaaattcctg gaagtgtaca 120
cttccctgaa atcggagctt ctcaatgacc ctgctttcga gttcaccgat gattctcgtc 180
aatgggtcga ccgaatgcta gactacaatg tccctggagg aaagctgaac cgaggcctct 240
ctgttattga cagctataag ttgctgaaag aagtaaagga attaaccgat gatgaaattt 300
ttcttgcatg tgcccttggg tggtgcattg aatggctcca agcatatttt cttgttcttg 360
atgatattat ggataactct cagacacgcc gaggtcaacc atgttggtac aaactgccaa 420
agattggttt gattgctgcc aatgatggcg taattctccg caaccatatc cctagaattc 480
tcaaaaagca tttcaaagga aagccttatt atgtggatct gctggattta ttcaatgagg 540
tagaatttca gacaacttgt ggacagatga tagatttgat caccacacta gaaggagaga 600
aagatttatc gaagtattca atttctctac accatcggat tgttcagtac aaaaccgctt 660
actactcatt ttacctgcca gtggcatgtg cactgcttat gtcaggcgag gatctggaca 720
atcatactga tgtgaagaac atactcattg aaatgggaac ctattttcaa gtgcaggatg 780
attacctaga ttgttttggt catcctgatg tgattggcaa gattggaaca gatattgaag 840
attttaagtg ttcctggttg gttgtaaaag cattggaact ttgtaacgag gaacaaaaaa 900
agttgttata tgagaactat ggaaaagacg atccaacatg tgtggcaaaa gtgaaggaac 960
tttatgaagc tctcaatctt cagggtgtat ttgcagagta tgagagcaag agttacgaga 1020
agctagtcaa gtccattgaa gctcatgcta gtaaagcagt gcaagcagtg ctgaaatcgt 1080
tcttggcgaa gatatataag aggcagaagt aggaacttca ccaaaaacag gtggttttga 1140
tgggattcct agctgatttg tgtctgtatt ctcctaatgg agggaaatat tttgtttgta 1200
ttttatgtaa ttggctttca tgctttggct tccaaaatct ctctttttct ctctctctct 1260
ctctctccct ctctctctgt tagcagaaga aggtggtacg ttgaatcttt tttgttaatg 1320
atgtctaagc caactgtttc caggataagt tgtttcaatg gtactttaag ctttgtttga 1380
cattgattac ggtaattaac tttttagaaa aaaaaaaaa 1419
<210> 2
<211> 342
<212> PRT
<213> tea tree (
camellia sinensis)
<400> 2
Met Ser Asp Leu Lys Ser Lys Phe Leu Glu Val Tyr Thr Ser Leu
1 5 10 15
Lys Ser Glu Leu Leu Asn Asp Pro Ala Phe Glu Phe Thr Asp Asp
20 25 30
Ser Arg Gln Trp Val Asp Arg Met Leu Asp Tyr Asn Val Pro Gly
35 40 45
Gly Lys Leu Asn Arg Gly Leu Ser Val Ile Asp Ser Tyr Lys Leu
50 55 60
Leu Lys Glu Val Lys Glu Leu Thr Asp Asp Glu Ile Phe Leu Ala
65 70 75
Cys Ala Leu Gly Trp Cys Ile Glu Trp Leu Gln Ala Tyr Phe Leu
80 85 90
Val Leu Asp Asp Ile Met Asp Asn Ser Gln Thr Arg Arg Gly Gln
95 100 105
Pro Cys Trp Tyr Lys Leu Pro Lys Ile Gly Leu Ile Ala Ala Asn
110 115 120
Asp Gly Val Ile Leu Arg Asn His Ile Pro Arg Ile Leu Lys Lys
125 130 135
His Phe Lys Gly Lys Pro Tyr Tyr Val Asp Leu Leu Asp Leu Phe
140 145 150
Asn Glu Val Glu Phe Gln Thr Thr Cys Gly Gln Met Ile Asp Leu
155 160 165
Ile Thr Thr Leu Glu Gly Glu Lys Asp Leu Ser Lys Tyr Ser Ile
170 175 180
Ser Leu His His Arg Ile Val Gln Tyr Lys Thr Ala Tyr Tyr Ser
185 190 195
Phe Tyr Leu Pro Val Ala Cys Ala Leu Leu Met Ser Gly Glu Asp
200 205 210
Leu Asp Asn His Thr Asp Val Lys Asn Ile Leu Ile Glu Met Gly
215 220 225
Thr Tyr Phe Gln Val Gln Asp Asp Tyr Leu Asp Cys Phe Gly His
230 235 240
Pro Asp Val Ile Gly Lys Ile Gly Thr Asp Ile Glu Asp Phe Lys
245 250 255
Cys Ser Trp Leu Val Val Lys Ala Leu Glu Leu Cys Asn Glu Glu
260 265 270
Gln Lys Lys Leu Leu Tyr Glu Asn Tyr Gly Lys Asp Asp Pro Thr
275 280 285
Cys Val Ala Lys Val Lys Glu Leu Tyr Glu Ala Leu Asn Leu Gln
290 295 300
Gly Val Phe Ala Glu Tyr Glu Ser Lys Ser Tyr Glu Lys Leu Val
305 310 315
Lys Ser Ile Glu Ala His Ala Ser Lys Ala Val Gln Ala Val Leu
320 325 330
Lys Ser Phe Leu Ala Lys Ile Tyr Lys Arg Gln Lys
335 340
<210> 3
<211> 24
<212> DNA
<213> artificial sequence
<220>
<223> M13 upstream primer
<400> 3
cgccagggtt ttcccagtca cgac 24
<210> 4
<211> 24
<212> DNA
<213> artificial sequence
<220>
<223> M13 downstream primer
<400> 4
agcggataac aatttcacac agga 24
<210> 5
<211> 22
<212> DNA
<213> artificial sequence
<220>
<223> FPS 5 ' RACE out primer
<400> 5
tcatctgtcc acaagttgtc tg 22
<210> 6
<211> 21
<212> DNA
<213> artificial sequence
<220>
<223> FPS 5 ' RACE in primer
<400> 6
cagcagatcc acataataag g 21
<210> 7
<211> 22
<212> DNA
<213> artificial sequence
<220>
<223> FPS 3 ' RACE out primer
<400> 7
tgccaatgat ggcgtaattc tc 22
<210> 8
<211> 24
<212> DNA
<213> artificial sequence
<220>
<223> FPS 3 ' RACE in primer
<400> 8
agacaacttg tggacagatg atag 24
<210> 9
<211> 23
<212> DNA
<213> artificial sequence
<220>
<223> FPS genetic expression upstream primer
<400> 9
aaaggaatta accgatgatg aaa 23
<210> 10
<211> 24
<212> DNA
<213> artificial sequence
<220>
<223> FPS genetic expression downstream primer
<400> 10
tggcaggtaa aatgagtagt aagc 24
<210> 11
<211> 19
<212> DNA
<213> artificial sequence
<220>
<223> reference act gene expression upstream primer
<400> 11
tccgttgccc tgaagtcct 19
<210> 12
<211> 24
<212> DNA
<213> artificial sequence
<220>
<223> reference act gene expression downstream primer
<400> 12
tgaagcgaat aagcaatgaa aaat 24
<210> 13
<211> 32
<212> DNA
<213> artificial sequence
<220>
Can the increase upstream primer of the whole open reading frame of tea tree FPS gene of <223>
<400> 13
aacatatgat gagcgatcta aagtcgaaat tc 32
<210> 14
<211> 32
<212> DNA
<213> artificial sequence
<220>
Can the increase downstream primer of the whole open reading frame of tea tree FPS gene of <223>
<400> 14
aagagctcct acttctgcct cttatatatc tt 32
Claims (4)
1. tea tree FPS gene, is characterized in that: be the nucleotide sequence shown in SEQ ID No:1.
2. the protein of tea tree FPS genes encoding as claimed in claim 1, is characterized in that: be the aminoacid sequence shown in SEQ ID NO:2.
3. the purposes of tea tree FPS gene as claimed in claim 1, is characterized in that: this gene and tea tree anthrax and moire blight is disease-resistant stress be relevant, and induce its high expression level can improve the disease resistance of tea tree to tea tree anthrax.
4. improve the method for tea tree to tea tree anthrax disease resistance, it is characterized in that: comprise that using is the gene transformation tea tree cell of the nucleotide sequence shown in SEQ ID No:1, then the tea tree cell culture after transforming is become to plant.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999031254A1 (en) * | 1997-12-16 | 1999-06-24 | Toyota Jidosha Kabushiki Kaisha | Geranyl diphosphate synthase genes |
| WO2002031164A1 (en) * | 2000-10-06 | 2002-04-18 | Sapporo Breweries Limited | Farnesyl pyrophosphate synthase protein, nucleic acid and promoter region thereof |
| CN101798579A (en) * | 2009-07-15 | 2010-08-11 | 复旦大学 | Jatropha curcas farnesyl pyrophosphate synthase protein encoding sequence and application thereof in plants |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999031254A1 (en) * | 1997-12-16 | 1999-06-24 | Toyota Jidosha Kabushiki Kaisha | Geranyl diphosphate synthase genes |
| WO2002031164A1 (en) * | 2000-10-06 | 2002-04-18 | Sapporo Breweries Limited | Farnesyl pyrophosphate synthase protein, nucleic acid and promoter region thereof |
| CN101798579A (en) * | 2009-07-15 | 2010-08-11 | 复旦大学 | Jatropha curcas farnesyl pyrophosphate synthase protein encoding sequence and application thereof in plants |
Non-Patent Citations (2)
| Title |
|---|
| DQ087959 Panax ginseng farnesyl diphosphate synthase (FPS) mRNA, complete cds;Kim,O.T. et al.;《GenBank》;20100804;全文，尤其是核苷酸序列和氨基酸序列 * |
| Kim,O.T. et al..DQ087959 Panax ginseng farnesyl diphosphate synthase (FPS) mRNA, complete cds.《GenBank》.2010, |
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