CN107177565A - Anti- tomato stem wilt gene mLCB2b and its application - Google Patents
Anti- tomato stem wilt gene mLCB2b and its application Download PDFInfo
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- C12N15/8282—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for fungal resistance
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- C12Y203/00—Acyltransferases (2.3)
- C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
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Abstract
The invention discloses a kind of albumen mLCB2b for improving tomato to stem wilt resistance caused by alternaric bacteria, the present invention further simultaneously discloses the gene mLCB2b for encoding above-mentioned protein；Gene mLCB2b can be used to improve resistance of the tomato to alternaric bacteria；That is, for building transgene tomato, render transgenic tomato is improved to the resistance of alternaric bacteria.
Description
Technical field
The invention belongs to biological technical field.Specifically related to one anti-tomato stem wilt gene mLCB2b and its application.
Background technology
In recent years, with the expansion of tomato planting area, the generation of tomato stem wilt is also increasingly severe, its main harm
The stem and fruit of tomato, have a strong impact on the yield and quality of tomato.Alternaric bacteria (Alternaria alternata
F.sp.Lycopersici, AAL) be tomato stem wilt pathogen, it mainly by produce have pathogenic mycotoxin
AAL-toxin realizes the murder by poisoning to tomato tissue cell.For how to improve resistance of the tomato to alternaric bacteria, traditional agriculture
Chemical regulation method used in production does not obviously meet the production theory of current environmental protection, and by excavating resistant gene
The resistance of tomato itself is improved, will be a kind of permanently effective method.
《Arabidopsis is studied the resistance mechanism of the similar class mycotoxin of sphingolipid with tomato》One text, with map based cloning
Method clones FB1 resistant genes in the anti-FB1 mutant of arabidopsis, and its function is analyzed, and discloses and plants from molecular level
Resistance mechanism of the thing to the FB1 PCD induced；And lose the FB1 resistant genes of arabidopsis in the tomato susceptible to AAL-Toxin
Conversion is passed, change and its mechanism of the analysis transgene tomato to AAL-Toxin sensitiveness probe into FB1 resistant genes in difference
Floristics is the study mechanism of the anti-stem wilt of tomato and makes the susceptible product of tomato by genetic engineering to the universality of SMAT resistances
Plant and confrontation stem wilt resistance offer theoretical foundation and technical support are provided.
The content of the invention
The technical problem to be solved in the present invention is to provide a kind of anti-tomato stem wilt gene mLCB2b and its application.
In order to solve the above-mentioned technical problem, the present invention provides a kind of raising tomato to stem wilt resistance caused by alternaric bacteria
Albumen mLCB2b, the amino acid sequence such as SEQ ID No of the protein：Shown in 3.
The present invention is also simultaneously there is provided the gene mLCB2b, the nucleotide sequence such as SEQ of gai gene for encoding above-mentioned protein
ID No：1、SEQ ID No：Shown in 2.
The present invention is also simultaneously there is provided plant expression vector, Escherichia coli, Agrobacterium and mistake containing above-mentioned encoding gene
Express the transfer-gen plant of the gene.
The primer pair of the present invention also simultaneously there is provided amplification said gene, including the primer pair being made up of F1 and R1, and
The primer pair being made up of F2 and R2；
F1：5’-ATGATTACGATCCCATACCT-3’；
F2：5’-ATCCTGGATACGCTCCGATCTGCT T-3’；
R1：5’-AGGATCTGGAGATTGTTGGTGCGCC-3’；
R2：5’-ACGTCTTGTTGTTATCGTCA-3’.
The purposes of the present invention also simultaneously there is provided said gene mLCB2b：For improving resistance of the tomato to alternaric bacteria.
It is used as the improvement of gene mLCB2b purposes：For building transgene tomato, the transgene tomato is to rod method
The resistance of bacterium is improved.
Tomato is improved to the gene of stem wilt resistance the invention provides one, is named as mLCB2b, is compiled in arabidopsis
The gene LCB2b of code serine palmitoyltransferase (Serine Palmitoyl Transferase, SPT) subunit is by fixed point
Mutation is obtained, the long 285bp of its ORFs, sequence such as SEQ ID NO：2, a small peptide containing 94 amino acid is encoded,
Sequence such as SEQ ID NO：Shown in 3.
The present invention relates to the method that rite-directed mutagenesis (base insertion) is carried out to the gene LCB2b in arabidopsis.
Answering for anti-stem wilt material is being obtained by formulating mLCB2b genetically modified plants the invention provides the gene
With.Its key step is as follows：
(1) structure of mLCB2b expression vectors：
MLCB2b ORFs is connected into plant expression vector, it is driven by strong promoter.
(2) acquisition of the Agrobacterium of conversion mLCB2b genes：
MLCB2b expression vectors are imported in Agrobacterium by heat shock method.
(3) mLCB2b is overexpressed the acquisition of transfer-gen plant：
By agriculture bacillus mediated transgenic method, mLCB2b transfer-gen plant is obtained.
(4) identification of transfer-gen plant：
Identified by DNA, gene expression amount is determined, screening mLCB2b is overexpressed transfer-gen plant.
(5) acquisition of transfer-gen plant homozygous line：
Using the expression quantity of antibiotic resistance and gene as index, the separation situation of each transgenic line offspring is analyzed, is chosen
Strain containing single copy insertion.Reserved seed for planting afterwards by individual plant, obtain the homozygous line that characters of progenies is not separated.
(6) Resistance Identification of transfer-gen plant：
Using the transgenic line of homozygosis as material, alternaric bacteria is inoculated with by live body, incidence is analyzed, transgenosis is found
The disease symptom of plant substantially relatively compares light；So that it is determined that mLCB2b improve tomato to the function in terms of alternaric bacteria resistance.
The overexpression of gene of the present invention significantly improves tomato to stem wilt pathogenic bacteria alternaric bacteria (Alternaria
Alternata f.sp.lycopersici) resistance, the wilting lesser extent of Transgenic Tomato Plants, the Relative electro-conductivity of blade
Rate and bacteria biomass are also significantly smaller.
Brief description of the drawings
The embodiment to the present invention is described in further detail below in conjunction with the accompanying drawings.
Fig. 1 is plant expression vector pGWB17-35S-mLCB2b Vector map；
Fig. 2 is that mLCB2b is overexpressed transfer-gen plant and to impinging upon the overall incidence after inoculation alternaric bacteria；
Fig. 3 is that mLCB2b is overexpressed transfer-gen plant and to impinging upon the local blade figure after inoculation alternaric bacteria；
Fig. 4 is that mLCB2b is overexpressed transfer-gen plant and compares the relative conductivity and bacterium relative biomass of incidence of leaf；
In Fig. 4, left figure is is overexpressed transfer-gen plant and compares the relative conductivity of incidence of leaf, and right figure turns to be overexpressed
Gene plant and the bacterium relative biomass for compareing incidence of leaf；
Fig. 5 is the local blade figure and blade that FBR41-D is overexpressed after transfer-gen plant and its control inoculation alternaric bacteria
Relative conductivity；
In Fig. 5, left figure is inoculated with the local blade figure after alternaric bacteria to be overexpressed transfer-gen plant and control, and right figure was
Express transgenic plant and the relative conductivity for compareing incidence of leaf.
Embodiment
With reference to specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in
This：
First, the clone of mLCB2b genes and preservation
MLCB2b genes are encoding serine palmitoyl transferase (the Serine Palmitoyl in arabidopsis
Transferase, SPT) subunit gene LCB2b rite-directed mutagenesises.Key step is as follows：Arabidopsis is extracted with Trizol methods
Wild type Col-0 total serum IgE, LCB2b cDNA is obtained using RT-PCR method.To make LCB2b genes in initiation codon
Start to insert ATCCT after 126 bit bases, using the method for segmentation amplification during amplification gene：First, according to LCB2b open readings
Before frame (ORF) before 126bp sequences Design primer F1 (5 '-ATGATTACGATCCCATACCT-3 ') (sequence enters SEQ ID NO：
4) with rear primer R1 (5 '-AGGATCTGGAGATTGTTGGTGCGCC-3 ', italic insertion base) (sequence such as SEQ ID NO：
5), amplification obtains 131bp DNA fragmentation；Then, according to primer before LCB2b gene ORF 127bp to 280bp sequences Design
F2 (5 '-ATCCTGGATACGCTCCGATCTGCT T-3 ', italic is insertion base) (sequence such as SEQ ID NO：6), and after draw
Thing R2 (5 '-ACGTCTTGTTGTTATCGTCA-3 ') (sequence such as SEQ ID NO：7), amplification obtains 156bp DNA fragmentation；Most
Afterwards, the DNA fragmentation of purifying is blended together as template, with F1 and R2 as front and rear primer, enters performing PCR amplification, obtain
282bp DNA fragmentation (remarks：Due to the insertion of 5 bases frameshift mutation occurs for LCB2b genes, causes translation to terminate in advance,
Therefore we have cloned the sequence before first terminator TAA according to LCB2b sequences).The fragment is connected to entry vector
On pQBV3, heat-shock transformed bacillus coli DH 5 alpha is coated on the LB plate overnights containing 25 μ g/ml chloramphenicol, afterwards picking list
Bacterium colony, bacterium colony PCR is carried out with the primer M13F on carrier and gene rear primer R2, is chosen positive colony and is shaken bacterium and extract matter
Grain, send company to be sequenced, final successful clone obtains mLCB2b genes, is named as pQBV3-mLCB2b.
The nucleotide sequence of mLCB2b genes such as SEQ ID No：1、SEQ ID No：Shown in 2；The albumen of the gene code
MLCB2b amino acid sequence such as SEQ ID No：Shown in 3.
2nd, the structure of plant expression vector
PQBV3-mLCB2b will be connected with and plant expression vector pGWB17 is blended in same system, system composition is as follows：
The system is reacted into 2h under the conditions of 25 DEG C, 0.5ul Preoteinase K solution are added, at 37 DEG C
10min is reacted, so as to terminate LR reactions.Afterwards, it is the system is heat-shock transformed to 100ul bacillus coli DH 5 alpha competent cells
In, it is coated on the LB culture mediums containing 50mg/L kanamycins overnight, afterwards picking single bacterium colony, with the leading of mLCB2b genes
Thing F1 (SEQ ID NO：4) the primer myc-R and on carrier carries out bacterium colony PCR detections, chooses positive colony and shakes bacterium and extract matter
Grain, send company to be sequenced, finally gives pGWB17-35S-mLCB2b.The carrier contains strong promoter-tobacco mosaic virus (TMV)
(CaMV) 35S promoter, can start expression of the mLCB2b in plant with composing type, and the mLCB2b albumen given expression to carries myc
Label.
3rd, the acquisition of the Agrobacterium of conversion mLCB2b genes
(1) prepared by Agrobacterium LBA4404 competence
Agrobacterium strains LBA4404 is coated on containing 500ug/ml streptomycin sulphates (SM) and 50ug/ml rifampins
(Rif) on YEB solid mediums, 28 DEG C of dark conditions are activated 2 to 3 days.Single bacterium after picking activation falls within 5ml and contained
Bacterium 16h is shaken in 500ug/ml SM and 50ug/ml Rif YEB fluid nutrient mediums.According to 1:100 ratio is by incubated overnight
Bacterium is added in the YEB fluid nutrient mediums that 100ml contains 500ug/ml SM and 50ug/ml Rif and shaken greatly, until OD600To 0.5-
1.0.Bacterium solution is dispensed, 15min on ice is placed in, afterwards in 4000rpm, 10min is centrifuged at 4 DEG C, precipitation is stayed, with 1ml precoolings
20mM CaCl2It is resuspended, packing is quick-frozen, that is, obtains Agrobacterium competence.
(2) conversion of expression vector
1ug pGWB17-35S-mLCB2b vector plasmids are drawn in 100ul agrobacterium strains LBA4404 competent cell
In, gently mix, more than 30min is being placed on ice, 3min in liquid nitrogen, 37 DEG C of water-bath 5min are placed on afterwards, 1ml YEB are being added molten
Liquid, is placed under 28 DEG C, 200rpm and dark condition and activates 2-4h.The bacterium solution of activation is centrifuged, is resuspended with 100ul YEB, uniformly
It is coated on the YEB culture mediums containing 500ug/ml SM, 50ug/ml Rif and 50ug/ml Kan, 2- is cultivated under the same terms
3d.Finally, picking single bacterium colony, carries out bacterium colony PCR identifications, and picking positive single bacterium colony shakes bacterium, and -80 DEG C save backup.
4th, the genetic transformation of tomato
(given from the tomato variety LA12 sensitive to alternaric bacteria by California, USA university Tomato Germplasms center
Send) as genetic transformation material, it is chosen in 1/2MS culture mediums and grows to cotyledon and just flatten the aseptic seedling in period as explant
Body, using Agrobacterium-medialed transformation method, specific method is as follows：
Aseptically, cotyledon is cut, cotyledon is infected using the Agrobacterium containing target gene, is placed on afterwards
The formation of evoked callus on inducing culture containing 1.0mg/L IAA, 1.75mg/L ZT and 75mg/L Kan.By
After the induction of 2-3 weeks, callus is transferred to the training of sprouting containing 1.0mg/L IAA, 1.75mg/L ZT and 50mg/L Kan
Support the generation of induced bud on base.After callus differentiates growing point, the training of taking root containing 50mg/L Kan is transferred into
Support the formation that root is induced on base.About 2-3 time-of-weeks, the resistance seedling for successfully differentiating root is transferred in matrix and cultivates hardening, it
Gene expression amount analysis is carried out to transgenic seedling afterwards, the transfer-gen plant that mLCB2b is overexpressed is primarily determined that.
5th, mLCB2b is overexpressed the acquisition of transfer-gen plant pure lines
The transfer-gen plant progress individual plant of acquisition is reserved seed for planting, 1/ containing 50mg/LKan is multicast to after Seed sterilization is sterilized
On 2MS culture mediums, resistance is selected and non-resistance close to 3:1, i.e., the strain of single copy insertion.The positive seedling of each strain is chosen,
Continue individual plant to reserve seed for planting, select the seed for the individual plant that offspring no longer separates, the experiment material verified as follow-up function is final to obtain
The transgenic line mLCB2b-OX-3-2 and mLCB2b-OX-11-1 of homozygosis.
6th, mLCB2b transfer-gen plants Disease Resistance Identification
(1) culture of alternaric bacteria and the preparation of spore suspension
The alternaric bacteria isolated and purified is placed in PDA culture medium, 25 DEG C are cultivated about 14d, black until being covered with culture medium
The fungal spore of color.A small amount of distilled water is added in the culture dish of culture fungi, 10min is soaked, is then scraped with the blade of sterilizing
Lower fungal spore and mycelia.Mycelia is filtered to remove with double gauze, the spore suspension of black is collected.Suspension is transferred to 50ml
In centrifuge tube, 4000rpm centrifuges 10min under normal temperature, abandons supernatant to remove toxin therein, be resuspended with distilled water, this operation weight
It is multiple 2 times, finally obtain the uniform spore suspension for being free of toxin.The spore concentration of suspension is calculated with blood counting chamber, with steaming
Distilled water is by concentration dilution to 106Individual/ml, the Tween-20 of addition 0.1%, obtain suitable concentration infects liquid.
(2) alternaric bacteria is inoculated with
The spore suspension prepared is squirted into the transgene tomato with 6 true leaves (about 1 month) with sprayer to plant
On the blade of strain and its control, 2d is cultivated under 25 DEG C, super-humid conditions, recovers normal cultivation condition afterwards, is compareed with 0.1%
Tween processing.The observation of disease symptom is carried out, and the relative conductivity and bacteria biomass of blade are surveyed within the 3rd day after inoculation
It is fixed.Compared with control, the wilting degree of the overall plant of transgenic line mLCB2b-OX-3-2 and mLCB2b-OX-11-1 of homozygosis
Relatively light (see Fig. 2), the comparison diagram of blade is as shown in Figure 3；The relative conductivity and bacteria biomass of blade all substantially relatively compare it is small (see
Fig. 4), illustrate to be overexpressed mLCB2b genes, plant pair alternaric bacteria shows resistance.Thus mLCB2b draws in anti-alternaric bacteria
Positive regulating and controlling effect is played in terms of the stem wilt risen.
(3) contrast experiment：
Will《Arabidopsis is studied the resistance mechanism of the similar class mycotoxin of sphingolipid with tomato》In FBR41-D genes
MLCB2b genes are substituted, that is, build pGWB17-35S-FBR41-D over-express vectors, above-mentioned steps three are carried out to step 6.It was found that
Transfer-gen plant is after inoculation alternaric bacteria, and occurring degree is only than compareing lower, comparison diagram such as Fig. 5 left figures institute of blade
Show, the relative conductivity of blade is as shown in Fig. 5 right figures.Comparatively speaking, the transgenic line occurring degree that the present invention is overexpressed
Substantially reduction.Therefore, the resistance for the transgenic line that the present invention is overexpressed is higher.
Finally, in addition it is also necessary to it is noted that listed above is only several specific embodiments of the invention.Obviously, this hair
It is bright to be not limited to above example, there can also be many deformations.One of ordinary skill in the art can be from present disclosure
All deformations for directly exporting or associating, are considered as protection scope of the present invention.
<110>Zhejiang University
<120>Anti- tomato stem wilt gene mLCB2b and its application
<160> 7
<210> 1
<211> 1475
<212> DNA
<213>Arabidopsis（Arabidopsis thaliana）
<400> 1
atgattacga tcccatacct taccgctgta tcaacgtatt tcagctacgg cttgcttttc 60
gcgtttggtc agcttcgcga ttactctcgc ctaatcttcg attggtggcg caccaacaat 120
ctccagatcc tggatacgct ccgatctgct tggcgcatga ggatttctac atccgacgat 180
tgtatcatcg gattcaggac tgttttggac gtcccatttc aagtgcacct gatgcttgga 240
ttgatgtggt tgagagagtc tctgacgata acaacaagac gttaaagcga actacaaaga 300
ctagcaggtg ccttaatttg ggttcctaca attatcttgg atttggttct ttcgatgaat 360
attgcacgcc tcgtgtcatt gagtctctga agaaattttc agcaagtaca tgtagctctc 420
gcgttgatgc aggaactaca tctgtgcatg cagaacttga ggattgtgtt gctaaatatg 480
tcggacaacc tgctgctgtt atttttggca tgggttatgc aacaaactcg gctatcattc 540
ccgtcttgat tggaaaggga gggttgataa taagcgattc tttgaaccat acttcgattg 600
tcaacggcgc tcgaggttca ggagctacta tccgtgtttt ccaacacaac acacctggtc 660
atctggaaaa agtattgaaa gaacaaatcg ccgagggaca acccaggacg catagaccgt 720
ggaagaaaat tattgttgtt gttgagggta tttacagcat ggaaggggaa atttgtcatc 780
tgcccgagat tgtttcaata tgcaagaagt ataaggcata tgtttacttg gacgaagctc 840
acagcattgg agcaattggc aagacaggaa gaggtgtttg tgaactcctt ggagttgaca 900
cttctgatgt ggacataatg atgggaactt tcacgaaatc ttttgggtct tgtggtggct 960
atattgcggg atcaaaggac ctcatccaat atttgaagca ccaatgcccg gctcaccttt 1020
acgccacatc aatttcaact ccttccgcga cacaaatcat atcggcgata aaggttatcc 1080
ttggagagga tggttcaaac agaggggcac aaaagttagc aagaataagg gagaacagca 1140
actttttcag ggctgagttg cagaagatgg ggtttgaagt tcttggagac aatgattcac 1200
cagtcatgcc aataatgctt tacaatccag caaaaattcc agcattctca agggaatgtt 1260
tgcgagaaaa cttggcggtg gtggtcgtcg gtttcccagc tacaccccta ttgctagcca 1320
gggctcgtat ttgcatatcc gcatctcact caagagaaga tcttataaaa gccctacagg 1380
ttataagcaa agcaggtgac cttaccggga tcaaatattt tccggcagct ccaaaaaagc 1440
aagaagtaga gaaaaatggc atcaaattgg attaa 1475
<210> 2
<211> 285
<212> DNA
<213>Arabidopsis（Arabidopsis thaliana）
<400> 2
atgattacga tcccatacct taccgctgta tcaacgtatt tcagctacgg cttgcttttc 60
gcgtttggtc agcttcgcga ttactctcgc ctaatcttcg attggtggcg caccaacaat 120
ctccagatcc tggatacgct ccgatctgct tggcgcatga ggatttctac atccgacgat 180
tgtatcatcg gattcaggac tgttttggac gtcccatttc aagtgcacct gatgcttgga 240
ttgatgtggt tgagagagtc tctgacgata acaacaagac gttaa 285
<210> 3
<211> 94
<212> PRT
<213>Arabidopsis（Arabidopsis thaliana）
<400> 3
Met Ile Thr Ile Pro Tyr Leu Thr Ala Val Ser Thr Tyr Phe Ser Tyr Gly Leu Leu Phe
1 5 10 15 20
Ala Phe Gly Gln Leu Arg Asp Tyr Ser Arg Leu Ile Phe Asp Trp Trp Arg Thr Asn Asn
21 25 30 35 40
Leu Gln Ile Leu Asp Thr Leu Arg Ser Ala Trp Arg MET Arg Ile Ser Thr Ser Asp Asp
41 45 50 55 60
Cys Ile Ile Gly Phe Arg Thr Val Leu Asp Val Pro Phe Gln Val His Leu MET Leu Gly
61 65 70 75 80
Leu MET Trp Leu Arg Glu Ser Leu Thr Ile Thr Thr Arg Arg ***
81 85 90 94
<210> 4
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>Primers F 1
<400> 4
atgattacga tcccatacct 20
<210> 5
<211> 25
<212> DNA
<213>Artificial sequence
<220>
<223>Primer R1
<400> 5
aggatctgga gattgttggt gcgcc 25
<210> 6
<211> 25
<212> DNA
<213>Artificial sequence
<220>
<223>Primers F 2
<400> 6
atcctggata cgctccgatc tgctt 25
<210> 7
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>Primer R2
<400> 7
acgtcttgtt gttatcgtca 20
Claims (6)
1. improve albumen mLCB2b of the tomato to stem wilt resistance caused by alternaric bacteria, it is characterised in that：The ammonia of the protein
Base acid sequence such as SEQ ID No：Shown in 3.
2. encode the gene mLCB2b of protein described in claim 1, it is characterised in that：The nucleotide sequence of the gene is such as
SEQ ID No：1、SEQ ID No：Shown in 2.
3. plant expression vector, Escherichia coli, Agrobacterium containing encoding gene described in claim 2 and it is overexpressed the gene
Transfer-gen plant.
4. the primer pair of amplification gene as claimed in claim 2, it is characterised in that：Including the primer pair being made up of F1 and R1, with
And the primer pair being made up of F2 and R2；
F1：5’-ATGATTACGATCCCATACCT-3’；
F2：5’-ATCCTGGATACGCTCCGATCTGCT T-3’；
R1：5’-AGGATCTGGAGATTGTTGGTGCGCC-3’；
R2：5’-ACGTCTTGTTGTTATCGTCA-3’.
5. gene mLCB2b purposes, it is characterised in that：For improving resistance of the tomato to alternaric bacteria.
6. gene mLCB2b according to claim 5 purposes, it is characterised in that：It is described for building transgene tomato
Transgene tomato is improved to the resistance of alternaric bacteria.
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| CN108034673A (en) * | 2018-02-05 | 2018-05-15 | 范瑶飞 | A kind of method for obtaining anti-wilting laurustinus |
| CN111808873A (en) * | 2020-07-28 | 2020-10-23 | 福建农林大学 | Method for producing long-fruit tomato fruits |
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| CN102653765A (en) * | 2011-03-01 | 2012-09-05 | 中国农业科学院作物科学研究所 | Plant disease-resistant gene and method for improving plant disease resistance |
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| CN102653765A (en) * | 2011-03-01 | 2012-09-05 | 中国农业科学院作物科学研究所 | Plant disease-resistant gene and method for improving plant disease resistance |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108034673A (en) * | 2018-02-05 | 2018-05-15 | 范瑶飞 | A kind of method for obtaining anti-wilting laurustinus |
| CN111808873A (en) * | 2020-07-28 | 2020-10-23 | 福建农林大学 | Method for producing long-fruit tomato fruits |
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