CN110106198A - Upland cotton transformation event 18C006-10-13 and its specificity identification method - Google Patents
Upland cotton transformation event 18C006-10-13 and its specificity identification method Download PDFInfo
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Abstract
The invention discloses upland cotton transformation event 18C006-10-13 and its specificity identification methods.The present invention will be in Cry51Aa1.C006-1 channel genes upland cotton CCRI24, it is overexpressed gene in upland cotton CCRI24, obtain transgene cotton, the transgene cotton is the 63328845-63329028 interdigit that exogenous dna fragment shown in sequence 1 is inserted into purpose cotton gene group D12 chromosome, the base sequence for replacing the 63328845-63329028 interdigit 182bp of D12 chromosome, obtained cotton.Test proves that not only anti-mirid pentatomid aspect has highly resistance characteristic to the transgene cotton, but also plant height, the first fruit branch length, fruit branch number, knot bell number and ginning outturn are also above upland cotton CCRI24.The present invention is to cultivate that there is the transgene cotton of anti-mirid pentatomid to lay the foundation, and has important application value.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to upland cotton transformation event 18C006-10-13 and its specificity mirror
Determine method.
Background technique
Transformation event is point being made of foreign gene in the upstream and downstream flanking region and foreign gene of genomic insertion site
Minor structure.In general, the available transformant group of gene transformation plant, this transformant group includes a large amount of independent
Event, wherein each event is unique.The dyeing that expression of the foreign gene in plant is inserted by foreign gene
The influence of body position.This may originate from the influence of transcriptional regulatory element near chromatin Structure or integration site.It is mutually homogenic
Expression in different transformation events has very big difference, is also likely to be present difference on the space of expression or time mode
It is different.And the insertion of foreign gene may also will affect the expression of endogenous gene.Therefore, each separate transformation events plant receptor
The influence of object is all different.Energy effective expression foreign gene is obtained, while the plant for not influencing plant economical character itself turns
Change event has important application value in cultivating genetically modified crops new varieties.
China is maximum Cotton Production state and country of consumption in the world, is also the largest textile and clothing producing country and goes out
Mouth state.Cotton total output accounts for 25% of World cotton production or so.The ups and downs of Cotton Production, the development to Chinese national economy,
Or even the raising of living standards of the people all generates certain influence.Cotton be by pest damage crops the most serious it
One, at least 300 kinds of Cotton in China pest.Transgenic cotton against pests (i.e. bollworm resisting) cultivation before, with cotten aphid, bollworm,
It is the most serious that cotton spider mites and four major pest of pink bollworm endanger cotton, every year because caused by insect pest output of cotton loss up to 15%~
20%, the annual pesticide expense per hectare for pest control investment is up to 1200-1800 member.With the extensive plantation of BT Insect Resistant Cotton
With being greatly reduced for field pesticides dosage, the lepidoptera pests such as bollworm, but the ecology in cotton field are fundamentally effectively controlled
A series of variations have occurred in system therewith, while the harm of bollworm has obtained effectively preventing, cotten aphid, mirid pentatomid, cigarette
The non-target insects quantity such as aleyrodid is significantly increased, and is gradually increasing the primary pest for cotton field, especially mirid pentatomid, the life to cotton
Production causes huge loss.
2007, Huang great Fang was by the Cry51Aa1 albumen being separated in Bt bacterial strain F14-1 and encodes the albumen
Cry51Aa1 gene order is committed to NCBI GenBank, and the number of calling the roll of the contestants in athletic events is DQ836184, and Cry51Aa1 albumen is one kind by Su Yun
One of the parasporal crystal that golden bacillus generates, it is with important application prospects in field of biological control.The present inventor
Rite-directed mutagenesis and the research of external virulence test are carried out to Cry51Aa1 gene, screening, which obtains, has relatively strong anti-mirid pentatomid special
The Cry51Aa1.C006-1 gene of property.
Summary of the invention
The object of the present invention is to provide a kind of transgene cotton breeding methods with anti-mirid pentatomid characteristic.
The present invention protects a kind of breeding method of transgene cotton first, for exogenous dna fragment is inserted into purpose cotton base
Because organizing the 63328845-63329028 interdigit of D12 chromosome, the 63328845- of D12 chromosome is replaced
The base sequence of 63329028 interdigit 182bp, obtains transgene cotton；
The mirid pentatomid resistance and/or plant height of the transgene cotton and/or the first fruit branch length and/or fruit branch number and/or
Knot bell number and/or ginning outturn are above the purpose cotton；The Single boll weight of the transgene cotton is lower than the purpose cotton；
The exogenous dna fragment can be the DNA molecular containing Cry51Aa1.C006-1 gene.It is described
The 2614-3534 nucleotide from 5 ' ends of sequence 1 in the nucleotide sequence of Cry51Aa1.C006-1 gene such as sequence table
It is shown.
In above-mentioned breeding method, the exogenous dna fragment can be by described in the importing of the recombinant vector containing exogenous dna fragment
Purpose cotton.The recombinant vector containing exogenous dna fragment can be the EcoRI that expression cassette A is inserted into pCambia2301 carrier
It between HindIII restriction enzyme site, and keeps the other sequences of pCambia2301 carrier constant, obtains recombinant vector；Expression cassette A
Nucleotide sequence as shown in sequence 1 2186-3822 from 5 ' ends in sequence table.It is described containing exogenous dna fragment
The nucleotide sequence of recombinant vector can be as shown in sequence 4 in sequence table.
In above-mentioned breeding method, the nucleotide sequence of the exogenous dna fragment can be as shown in sequence 1 in sequence table.
Exogenous DNA molecule shown in sequence 1 in sequence table is concretely inserted into purpose cotton gene by the breeding method
63328845-63329028 interdigit (5 ' of exogenous DNA molecule shown in sequence 1 in sequence table of group D12 chromosome
End is close to the 63329028th of D12 chromosome, and 3 ' ends of exogenous DNA molecule shown in sequence 1 are tight in sequence table
The 63328845th of neighbour's D12 chromosome), replace the 63328845-63329028 interdigit of D12 chromosome
The base sequence of 182bp, obtains transgene cotton.
The seed of any of the above-described transgene cotton is CCTCC in the deposit number of China typical culture collection center
NO: P201822。
The present invention also protects whether derive from the transgene cotton or the method for its offspring for plant identification sample, wraps
It includes following steps: whether containing DNA fragmentation A in the genomic DNA of the detection plant sample to be measured；The DNA fragmentation A is by upper
Any exogenous dna fragment is stated in the upstream flanking fragment, the exogenous dna fragment and the external source of the transgene cotton
DNA segment is formed in the upstream flanking fragment of the transgene cotton；
If the genomic DNA of the plant sample to be measured contains the DNA fragmentation A, the plant sample to be measured be or
Candidate is the transgene cotton or its offspring；
If the genomic DNA of the plant sample to be measured does not contain the DNA fragmentation A, the plant sample to be measured is not
For or candidate be not the transgene cotton or its offspring.
Any of the above-described exogenous dna fragment can be the purpose cotton in the upstream flanking fragment of the transgene cotton
The length that genome D12 chromosome extends from the 63329028th nucleotide to its 3 ' direction is appointing for 0-5Kb
It anticipates a DNA fragmentation.
Any of the above-described exogenous dna fragment can be the purpose cotton in the downstream flanking fragment of the transgene cotton
The length that genome D12 chromosome extends from the 63328845th nucleotide to its 5 ' direction is appointing for 0-5Kb
It anticipates a DNA fragmentation.
The nucleotide sequence of any of the above-described upstream flanking sequence can be as shown in sequence 2 in sequence table.
The nucleotide sequence of any of the above-described downstream flanking sequence can be as shown in sequence 3 in sequence table.
Above, the transgene cotton is higher than the purpose cotton to the resistance of mirid pentatomid and can embody are as follows: with purpose cotton
Flower is compared, and the plant height of transgene cotton, the first fruit branch length, fruit branch number, knot bell number and ginning outturn are significantly increased.Therefore, turn base
Because Resistance Strain of Cotton mirid pentatomid characteristic dramatically increases, it can be used for cultivating anti-mirid pentatomid cotton in production.
In the above method, described " whether containing DNA fragmentation A in the genomic DNA of the detection plant sample to be measured "
Method can be S1) or S2) or S3):
S1) direct Sequencing；
S2 PCR amplification) is carried out with the genomic DNA that primer pair 1 and/or primer pair 2 treat measuring plants sample, is then carried out
Following judgement: if there is purpose amplified production, the plant sample to be measured is or candidate is the transgene cotton or its offspring；
If not having purpose amplified production, the plant sample to be measured is not or candidate is not the transgene cotton or its offspring；
The primer pair 1 can be held and close to upstream flanking sequence described in its for that can expand by the exogenous dna fragment 5 '
The primer pair of the DNA molecular first of part or all of segment composition；Its corresponding purpose amplified production is the DNA molecular first；
The primer pair 2 can be held and close to flank sequence in downstream described in its for that can expand containing the exogenous dna fragment 3 '
The primer pair of the DNA molecular second of some or all of column composition；Its corresponding purpose amplified production is the DNA molecular second.
S3) probe of the DNA molecular first described in energy specific bond or the DNA molecular second is to the plant sample to be measured
Genomic DNA carries out Southern blot hybridization, then makes the following judgment: if hybridized fragment can be obtained, the plant to be measured
Object sample is or candidate is the transgene cotton or its offspring；If hybridized fragment cannot be obtained, the plant sample to be measured
It is not or candidate is not the transgene cotton or its offspring.
The primer pair 1 specifically can be in the single strand dna as shown in sequence 5 in sequence table and sequence table shown in sequence 7
Single strand dna composition.The primer pair 2 specifically can be in single strand dna and sequence table as shown in sequence 6 in sequence table
The composition of single strand dna shown in sequence 8.
The present invention also protects a kind of for identifying that whether plant sample to be measured derived from that the cultural method obtains turns base
Because of cotton or the kit of its offspring, including any of the above-described primer pair 1 and/or primer pair 2.
A kind of acquisition mirid pentatomid resistance is also protected to improve by the present invention and/or plant height improves and/or the first fruit branch length improves
And/or the method for the cotton that fruit branch number improves and/or knot bell number improves and/or ginning outturn improves and/or Single boll weight reduces, it may include
Following steps:
(1) transgene cotton is obtained according to any of the above-described cultural method；
(2) transgene cotton is selfed or is hybridized, obtain breeding offspring, identified according to any of the above-described method
The breeding offspring, obtains target plant.
In the above method, the seed of the transgene cotton is in the deposit number of China typical culture collection center
CCTCC NO: P201822。
Any one of the present invention also protects D1)-D6).
D1) application of the transgene cotton obtained using any of the above-described breeding method in cotton breeding.
D2) application of any of the above-described exogenous dna fragment in regulation Resistance Strain of Cotton mirid pentatomid.
D3) any of the above-described exogenous dna fragment is in regulation cotton plant height and/or the first fruit branch length and/or fruit branch number
And/or the application in knot bell number and/or ginning outturn and/or Single boll weight.
D4) any of the above-described exogenous dna fragment is identifying whether plant sample to be measured derives from any of the above-described training
The transgene cotton of the method for supporting acquisition or the application in its offspring.
D5) any of the above-described primer pair 1 and/or primer pair 2 are identifying whether plant sample to be measured derives from above-mentioned
The transgene cotton of the one breeding method acquisition or the application in its offspring.
D6) any of the above-described upstream flanking sequence and/or downstream flanking sequence are identifying whether plant sample to be measured comes
Application in the transgene cotton or its offspring that any of the above-described breeding method obtains.
Above, the purpose cotton can be upland cotton.The upland cotton concretely upland cotton CCRI24.
The present invention makes Cry51Aa1.C006-1 gene exist in Cry51Aa1.C006-1 channel genes upland cotton CCRI24
It is overexpressed in upland cotton CCRI24, obtains transgene cotton 18C006-10-13, transgene cotton 18C006-10-13 is will be outer
Source DNA segment is inserted into the 63328845-63329028 interdigit of purpose cotton gene group D12 chromosome, replaces the
The base sequence of the 63328845-63329028 interdigit 182bp of D12 chromosome, obtained cotton.Test proves, turns base
There is highly resistance characteristic in terms of because of cotton 18C006-10-13 not only anti-mirid pentatomid, and plant height, the first fruit branch length, fruit branch number,
Bell number and ginning outturn are tied also above upland cotton CCRI24.The present invention is to cultivate that there is the transgene cotton of anti-mirid pentatomid to lay the foundation,
With important application value.
Detailed description of the invention
The sequence that Fig. 1 is 18C006-10-13 is analyzed.
Fig. 2 is the Detached-leaf test of transgene cotton 18C006-10-13 and upland cotton CCRI24.
Fig. 3 is the field for turning Cry51Aa1.C006-1 cotton homozygous lines 18C006-10-13 and upland cotton CCRI24 in T5 generation
Between growing state.
Fig. 4 is the Net evaluation of transgene cotton 18C006-10-13 and upland cotton CCRI24.
Specific embodiment
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.
Experimental method in following embodiments is unless otherwise specified conventional method.
Test material as used in the following examples is unless otherwise specified to buy from routine biochemistry reagent shop
It arrives.
Quantitative test in following embodiment, is respectively provided with three repeated experiments, and results are averaged.
Upland cotton CCRI24 in following embodiments is in document " PAG1, a cotton brassinosteroid
It is disclosed in catabolism gene, modulates fiber elongation ", the public can be from Chinese Academy of Agricultural Sciences cotton
Flower research institute obtains.
Agrobacterium LBA4404 in following embodiments is in document " Constitutive expression of the
virulence genes improves the efficiency of plant transformation by
It is disclosed in Agrobacterium ", the public can obtain from the Chinese Academy of Agriculture Science and Technologys Cotton Research Institute.
The nucleotide sequence of primer involved in following embodiments is shown in Table 1.
Table 1
| Nucleotide sequence (5 ' -3 ') | Position in sequence table | |
| Primer 5 | gagcttggatcagattgtcg | Sequence 5 |
| Primer 6 | actatagggcacgcgtggt | - |
| Primer 7 | gtttcgctcatgtgttgagc | Sequence 6 |
| Primer 8 | gccgtcgttttacaacgtc | - |
| Primer 9 | cgatcgcccttcccaac | - |
| Primer 10 | gaggatctcgtcgtgacac | - |
| Primer 11 | cgacaagctcgagtttctc | - |
| Primer 12 | gtaatacgactcactatagggcacgcgtggtntcgastwtsgwgtt | - |
| Primer 13 | gtaatacgactcactatagggcacgcgtggtngtcgaswganawgaa | - |
| Primer 14 | gtaatacgactcactatagggcacgcgtggtwgtgnagwancanaga | - |
| Primer 15 | gtaatacgactcactatagggcacgcgtggtagwgnagwancawagg | - |
| Primer 16 | gtaatacgactcactatagggcacgcgtggtngtawaasgtntscaa | - |
| Primer 17 | gtaatacgactcactatagggcacgcgtggtngacgaswganawgac | - |
| Primer 18 | gtaatacgactcactatagggcacgcgtggtngacgaswganawgaa | - |
| Primer 19 | gtaatacgactcactatagggcacgcgtggtgtncgaswcanawgtt | - |
| Primer 20 | gtaatacgactcactatagggcacgcgtggtncagctwsctntsctt | - |
| Primer 21 | gtaatacgactcactatagggc | - |
| Primer 22 | cttgtctgatcgatgtgaac | Sequence 7 |
| Primer 23 | cacctcagaagttgaagcctg | Sequence 8 |
Note: "-" expression is not present；N is a or c or g or t, and s is c or g, and w is a or t.
Embodiment 1, the acquisition for turning Cry51Aa1.C006-1 cotton and Analysis of agronomic characters
One, turn the acquisition of Cry51Aa1.C006-1 cotton
1, the building of recombinant vector
By expression cassette A insertion pCambia2301 carrier, (plasmid is purchased from NTCC Type Tissue Collection subordinate's
BioVector plasmid vector bacterium cell gene collection, article No. are as follows: Biovectorpcambia1300) EcoRI and
It between HindIII restriction enzyme site, and keeps the other sequences of pCambia2301 carrier constant, obtains recombinant vector (nucleotide sequence
As shown in sequence 4 in sequence table).The 2186- from 5 ' ends of sequence 1 in the nucleotide sequence of above-mentioned expression cassette A such as sequence table
Shown in 3822.Expression cassette A from upstream successively include the CAMV35S from cauliflower mosaic virus promoter,
Cry51Aa1.C006-1 gene and NOS terminator；Wherein, sequence 1 in the nucleotide sequence of CAMV35S promoter such as sequence table
From 5 ' ends shown in 2192-2537 nucleotide；Sequence in the nucleotide sequence of Cry51Aa1.C006-1 gene such as sequence table
Column 1 are from 5 ' ends shown in 2614-3534 nucleotide；In the nucleotide sequence of NOS terminator such as sequence table sequence 1 from
It rises shown in 3569-3821 nucleotide 5 ' ends.
PCambia2301 carrier itself includes expression cassette B and expression cassette C.Expression cassette B successively includes cauliflower from upstream
Enhancing promoter, KanR antibiotic marker genes and the Ploy A terminator of the CAMV35S of mosaic virus.Expression cassette C is from upstream
Play promoter, GUS marker gene and the NOS terminator of the successively CAMV35S including cauliflower mosaic virus.
2, the acquisition of recombinant bacterium
The recombinant vector that step 1 is obtained imports in Agrobacterium LBA4404, obtains recombinant bacterium.
3, turn the acquisition of Cry51Aa1.C006-1 cotton
Using the explant (1500 for the recombinant bacterium conversion upland cotton CCRI24 that the method for mediated by agriculture bacillus obtains step 2
It is a), induced synthesis callus is cultivated on culture medium containing kanamycin, selects the callus of conversion, callus
Embryo callus is formed on kanamycin screening culture medium, the embryo callus subculture for selecting survival converts to form regeneration cotton,
Finally in 50 T0 generations of acquisition, turn Cry51Aa1.C006-1 cotton altogether, and are used for screening.
4, turn the identification of Cry51Aa1.C006-1 cotton
(1) Cry51Aa1.C006-1 cotton seeds are turned in chamber planting to the T1 of harvest generation, carry out greenhouse efficiency analysis and
Analysis of molecules.T1 is extracted for the DNA for turning Cry51Aa1.C006-1 cotton leaf, using primer: 5 '-
ACCCATCCAAGGTTGATACC-3 ' and 5 '-TTGATAGGTGAAGTCGGAGC-3 ' carries out PCR amplification, and testing goal gene is
No presence, and the segregation ratio of statistical material.According to mendel's law, 12 T1 singly copied generations turn are obtained
Cry51Aa1.C006-1 cotton；
(2) in 12 that above-mentioned steps (1) the obtain T1 singly copied generations, are turned using Taqman PCR and Southern blot
Cry51Aa1.C006-1 cotton is further verified.Specific step is as follows: in the T1 generation that the early flowering season singly copies 12 of acquisition
The Cry51Aa1.C006-1 gene expression amount for turning Cry51Aa1.C006-1 cotton material and upland cotton CCRI24 material is divided
Analysis shows that 12 T1 singly copied generations turn to have 9 T1 generations to turn Cry51Aa1.C006-1 cotton in Cry51Aa1.C006-1 cotton
Expression quantity greatly improved compared with upland cotton CCRI24.
(3) turn the agriculture of Cry51Aa1.C006-1 cotton material in 9 T1 generation that florescence measurement above-mentioned steps (2) obtains
Skill character and mirid pentatomid resistance.Turn the result shows that 9 T1 generations turn 9 T1 generations in Cry51Aa1.C006-1 cotton
The Some Agronomic Traits (such as plant height, the first fruit branch length, fruit branch number, knot bell number) and mirid pentatomid of Cry51Aa1.C006-1 cotton
Resistance is significantly improved compared with upland cotton CCRI24.
(4) 9 T2 generations turn are assessed in the pairs of plot of same position in the field experiment of First Year field
Agronomic and quality traits (plant height, the first fruit branch length, fruit branch number, knot the bell number, Single boll weight, clothing of Cry51Aa1.C006-1 cotton
Point, wherein fiber quality includes length, specific strength, mic value, elongation, uniformity) and mirid pentatomid resistance.The result shows that: 9
In a T2 generation, turns to share 2 T2 in Cry51Aa1.C006-1 cotton for the Some Agronomic Traits for turning Cry51Aa1.C006-1 cotton
(plant height, the first fruit branch length, fruit branch number, knot bell number, ginning outturn) and mirid pentatomid resistance are considerably better than upland cotton CCRI24.
(5) above-mentioned steps (4) obtain 2 are assessed in the pairs of plot of same position in the field experiment of second year field
A T2 generation turn Cry51Aa1.C006-1 cotton agronomic and quality traits (plant height, the first fruit branch length, fruit branch number, knot bell number,
Single boll weight, ginning outturn, wherein fiber quality includes length, specific strength, mic value, elongation, uniformity) and mirid pentatomid resistance.
The result shows that: in 2 T2 generations, turn to share 1 T2 in Cry51Aa1.C006-1 cotton for the part for turning Cry51Aa1.C006-1 cotton
Economical character (plant height, the first fruit branch length, fruit branch number, knot bell number, ginning outturn) and mirid pentatomid resistance are considerably better than upland cotton
The seed that T2 generation turns Cry51Aa1.C006-1 cotton is named as 18C006-10-13, T2 generation is turned by CCRI24
Cry51Aa1.C006-1 cotton 18C006-10-13 is selfed, until obtaining T5 generation turns Cry51Aa1.C006-1 cotton homozygosis
Strain 18C006-10-13.
In T5 generation, is turned Cry51Aa1.C006-1 cotton seeds (Gossypiumhirsutum L.) by October 30th, 2018
18C006-10-13 is preserved in China typical culture collection center, and classification naming is upland cotton seed C006-10-13, preservation
Number is CCTCC NO:P201822.
Two, turn the Analysis of agronomic characters of Cry51Aa1.C006-1 cotton
1, cotton fleahopper is as Resistance Identification method
Resistance Identification method with reference to 2015 publication with implement professional standard " Resistance Strain of Cotton mirid pentatomid identification method " in
Magnificent people's republic's agricultural industry criteria (NY/T 2676-2015) " ".
(1) Detached-leaf test (see Fig. 2, C006-10-13 18C006-10-13, CCRI24 are upland cotton CCRI24)
Cotton branch with small flower bud is inserted into the transparent plastic cup for filling 2% agar, if then by 3 mirid pentatomids
Worm is accessed wherein, and top covers the inverted plastic cup for pricking hole, prevents steam from generating, and sealed rim of a cup with sealed membrane,
Prevent fleahopper from escaping.It is to be tested when carrying out to the 6th day, it carries out borer population living and is developed into the investigation of borer population.
(2) Net evaluation (see Fig. 4, C006-10-13 18C006-10-13, CCRI24 are upland cotton CCRI24)
1) cotton seeds should meet requirement of the GB 4407.1 to seed quality；
2) identification carries out in solarium, and solarium's specification length × width × height is respectively 20m × 3m × 2m, and nylon wire is 80 mesh；
3) identification method:
A. experimental design is with management: random alignment plantation compares cotton material with sense worm for examination cotton material in solarium, often
1 row of material kind, spacing in the rows 0.25m, line-spacing 0.80m.Each solarium is primary repetition, totally 3 repetitions.Cotton is planted in solarium
The same crop field of training management mode, 7d-10d sprays the short chemical pesticide cleaning solarium of residual effect before testing, and does not apply during test any
Chemical pesticide.
B. it sows: pressing local cotton conventional seed planting period for examination cotton material and sense worm control material and application rate carries out
Sowing.
C. test worm is raised: for -5 age of the 1 age mirid pentatomid mixed population of the artificial feedings such as indoor cowpea, tucket.
D. test worm release period and burst size: in solarium cotton growth to -6 leaf phase of 4 leaf when discharge mirid pentatomid, when release, is selected
The strong individual of energy is selected, every plant of cotton discharges 1.
E. result records: after test worm discharges 7d-10d, each 10 plants of material random searching of each solarium records cotton plant top
The victimization state of 5 tender leafs, at the same investigate cotton cotton buds and bolls quantity and total cotton buds and bolls number.Cotton leaf victimization state is divided into 5
Rank, i.e., 0 grade, 1 grade, 2 grades, 3 grades and 4 grades, grade scale is as follows: 0 grade is the non-hazardous shape of blade；1 grade is slightly aggrieved for blade,
0% < injured area≤5%；2 grades aggrieved for medium blade, 5% < injured area≤20%；3 grades are seriously aggrieved for blade,
20% < injured area≤50%；4 grades are endangered for blade with clock synchronization kind, injured area > 50%.
F. identification of indicator: blade hazard index (the hazard level mean value of every plant of 5, cotton top blade), blade harm refer to
Number decline rate (%), square percentage of injury (%) and square are killed decline rate (%).
Resistance Identification the results are shown in Table 2 (CK is upland cotton CCRI24).The result shows that compared with upland cotton CCRI24,
The mirid pentatomid resistance of 18C006-10-13 significantly improves.
The mirid pentatomid resistance of table 2. 18C006-10-13 and upland cotton CCRI24
| Cotton to be measured | Larval mortality (%) | Corrected mortality (%) | It is developed into borer population (head) | It is developed to adult ratio (%) |
| 18C006-10-13 | 61.90±15.31 | 54.28±18.37 | 0.4±0.3 | 14.29±9.91 |
| CK | 16.67±7.45 | - | 0.8±0.4 | 27.78±13.38 |
2, economical character
In T5 generation, is turned into Cry51Aa1.C006-1 cotton homozygous lines 18C006-10-13 and upland cotton CCRI24 carries out other
The investigation and measurement of economical character.Plot experiment is designed in Earthquake of Anyang station in Henan, 3 cells are set, 3 cell settings are identical,
Cell row long 8m, every row plant 30 plants, line space 80cm, and each material plants 3 rows, cell totally 6 row.Using Students '
Test turns the plant height of Cry51Aa1.C006-1 cotton homozygous lines 18C006-10-13 and upland cotton CCRI24, first to T5 generation
Fruit branch length, fruit branch number, knot bell number, ginning outturn are statisticallyd analyze respectively.
Field growing situation is shown in that (C006-10-13 is to turn Cry51Aa1.C006-1 cotton homozygous lines in T5 generation to Fig. 3
18C006-10-13, CCRI24 are upland cotton CCRI24).Statistical result is shown in Table 3.The result shows that compared with upland cotton CCRI24,
In T5 generation, turns the plant height of Cry51Aa1.C006-1 cotton homozygous lines 18C006-10-13, the first fruit branch length, fruit branch number, knot bell
Number, ginning outturn are significantly increased, and Single boll weight significantly reduces.
The economical character of table 3. transgene cotton 18C006-10-13 and upland cotton CCRI24 compares
3, otherwise influence
Since promoter is constitutive promoter, the not only table in chlorenchyma (including stem, leaf, flower bud, growing point)
It reaches, hetero-organization also will receive certain influence.In order to study other than improveing mirid pentatomid resistance, Cry51Aa1.C006-1 gene
Whether its hetero-organization is had an impact, Cry51Aa1.C006-1 cotton homozygous lines 18C006-10-13 and land are turned to T5 generation
Fibre length, specific strength, mic value, uniformity index and the elongation of cotton CCRI24 is detected.
Statistical result is shown in Table 4.The result shows that it is pure that in T5 generation, turns Cry51Aa1.C006-1 cotton compared with upland cotton CCRI24
The mic value and elongation for closing strain 18C006-10-13 increase but without significant differences, and fibre length, fracture are than strong
Degree, uniformity index have reduction but without significant differences.
The fiber quality data of table 4. transgene cotton 18C006-10-13 and upland cotton CCRI24 compares
Three, the phenetic analysis of the DNA sequence dna of 18C006-10-13
Be connected side using the insert of the method analysis 18C006-10-13 genome of molecular biology and with insert
The genome sequence of wing connection.Specific step is as follows:
1, the genome of cotton is extracted.Under greenhouse or field condition, the young tender leaf agreement that contracts a film or TV play to an actor or actress of the cotton of 18C006-10-13 is taken
100mg is placed in the EP pipe of 2.0ml, is managed using liquid nitrogen frozen EP, is then ground using freeze grinding instrument, using Qiagen public affairs
The scheme provided in DNeasy Plant Mini Kit (50) (article No. 69104) is provided and extracts genomic DNA.This method can be through this
Field technical staff improves to extract DNA from any tissue (including but not limited to seed).
2, fusion primer is carried out according to the PCR Master MIX (P213-AA) of Nuo Weizan company and nido combines
FPNI-PCR (Wang et al.2011).According to known transgenic insertions sequence, separately designed at its 5 ' end and 3 ' ends
(it is primer that three nested primers at 5 ' ends are three nested primers that primer 8, primer 9 and primer 5,3 ' are held to three nested primers
10, primer 11 and primer 7).The PCR primer first round designed in cotton gene group is fusion primer, totally 9 (primer 12-
Primer 2 0), this 9 primers carry out first round amplification with primer 8 and primer 10 respectively.The second wheel of design in cotton gene group
PCR amplification primer (primer 2 1), primer 21 carry out the second wheel amplification with primer 9, primer 11 respectively.It is designed in cotton gene group
Third round PCR amplification primer (primer 6), primer 6 carry out third round amplification with primer 5, primer 7 respectively, pass through 3 wheel PCR.Benefit
With agarose gel electrophoresis separation from reaction generate amplicon, then using QIAGEN gel purification kit (Qiagen,
Valencia, CA) purified, according to Dalian treasured biological life Science and Technology Ltd. pMD-19T-Simple (article No.:
D104A) scheme provided in kit enters the amplicons cloned of gel-purified in T cloning vector, and converts bacillus coli DH 5
In α, transformant is screened on the plate of ammonia benzyl resistance, and carry out bacterium colony PCR, positive colony carries out monoclonal sequencing.
Sequencing result shows: transgene cotton 18C006-10-13 is by exogenous DNA molecule shown in sequence 1 in sequence table
It is inserted into 63328845-63329028 interdigit (1 institute of sequence in sequence table of upland cotton CCRI24 genome D12 chromosome
5 ' ends of the exogenous DNA molecule shown are outer shown in sequence 1 in sequence table close to the 63329028th of D12 chromosome
The 63328845th close to D12 chromosome of 3 ' ends of source DNA molecule), replace the of D12 chromosome
The base sequence of 63328845-63329028 interdigit 182bp, obtained transgene cotton, and from the 63329028th upstream and
Nucleotides sequence close to the upstream flanking fragment of the 63329028th nucleotide is classified as sequence 2, from the 63328845th downstream and
Nucleotides sequence close to the downstream flanking fragment of the 63328845th nucleotide is classified as sequence 3 (Fig. 1).
Embodiment 2, the acquisition of 18C006-10-13 breeding characters of progenies and identification and its Analysis of agronomic characters
One, 18C006-10-13 breeds the acquisition of characters of progenies
1, hybridize
1 day afternoon removed the pollen of the first cotton by manpower work or artificial action before flowering, and used ceratuba
Colored column cap is entangled, prevents foreign pollen from contacting with column cap, the next morning when the pollen loose powder of the second cotton, passes through manpower
Work collects the pollen of second of cotton, and the pollen is contacted with the style of the first plant or column cap, i.e. completion hybrid process.
Wherein, when the first cotton is the 18C006-10-13 in embodiment, the second cotton is other cottons or the second cotton is
18C006-10-13, the first cotton are other cottons.
Term of opening bolls harvest hybridization bell, and cotton is dried naturally, it crimps, and suede, i.e. acquisition cenospecies are dragged using the concentrated sulfuric acid
Son.Plantation, obtains hybrid generation, and as 18C006-10-13 breeds offspring.
2, it is selfed
1 day before flowering, the bud of 18C006-10-13 was directly clamped using Grafting clip or bud is bundled using filament,
Foreign pollen contacts with the column cap of 18C006-10-13 when preventing from blooming；Or entangled entire cotton plants by mesh bag, it can
Effectively to prevent from pollinating by insect etc., the self-pollination of cotton can be realized.It can complete to be selfed through the above steps.
Term of opening bolls harvest hybridization bell, and cotton is dried naturally, it crimps, and suede, i.e. acquisition selfed seed are dragged using the concentrated sulfuric acid
Son, plantation obtain self progeny, and as 18C006-10-13 breeds offspring.
Two, 18C006-10-13 breeds identification and its Analysis of agronomic characters of characters of progenies
(1) 18C006-10-13 breeds the identification method of characters of progenies
Using the genomic DNA of 18C006-10-13 breeding offspring as template, PCR amplification is carried out using specific primer, if
(amplicon refers to that one or one section is closed using PCR amplification to the amplicon that PCR amplified production contains 18C006-10-13
At DNA molecular), illustrate 18C006-10-13 breeding offspring have character identical with 18C006-10-13, if PCR amplification
Product is free of the amplicon of 18C006-10-13, and it is identical with 18C006-10-13 to illustrate that 18C006-10-13 breeding offspring does not have
Character.Specific identification method is as follows:
1, identification method 1
(1) using the genomic DNA of 18C006-10-13 breeding offspring as template, drawn using what primer 5 and primer 22 formed
Object obtains pcr amplification product to PCR amplification is carried out.
PCR amplification system is as follows: 2 × MASTRE PCR MIX, 10 μ l, primer 5 (10 μM) 0.5 μ l, primer 22 (10 μM)
0.5 μ l, template DNA (50ng/ μ l) 1 μ l, 8 μ l of ultrapure water, 20 μ l of total volume.
PCR reaction condition is as follows: 94 DEG C of 5min；94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 45s, 32 circulations；72℃5min.
(2) pcr amplification product for obtaining step (1) carries out agarose gel electrophoresis, then makes the following judgment: if
PCR amplified production contains the DNA fragmentation of about 500bp, then illustrates that 18C006-10-13 breeding offspring contains 18C006-10-13's
Amplicon, 18C006-10-13, which breeds offspring, has 18C006-10-13 character；Otherwise do not have 18C006-10-13 character.
2, identification method 2
(1) using the genomic DNA of 18C006-10-13 breeding offspring as template, drawn using what primer 7 and primer 23 formed
Object obtains pcr amplification product to PCR amplification is carried out.
PCR amplification system is as follows: 2 × MASTRE PCR MIX, 10 μ l, primer 7 (10 μM) 0.5 μ l, primer 23 (10 μM)
0.5 μ l, template DNA (50ng/ μ l) 1 μ l, 8 μ l of ultrapure water, 20 μ l of total volume.
PCR reaction condition is as follows: 94 DEG C of 5min；94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 1min, 32 circulations；72℃5min.
(2) pcr amplification product for obtaining step (1) carries out agarose gel electrophoresis, then makes the following judgment: if
PCR amplified production contains the DNA fragmentation of about 900bp, then illustrates that 18C006-10-13 breeding offspring contains 18C006-10-13's
Amplicon, 18C006-10-13, which breeds offspring, has 18C006-10-13 character；Otherwise do not have 18C006-10-13 character.
(2) 18C006-10-13 breeds the anti-mirid pentatomid and Analysis of agronomic characters of characters of progenies
There is 18C006-10- according to what is obtained in the step two in embodiment 11 method detection above-mentioned steps (one)
The anti-mirid pentatomid and Analysis of agronomic characters of 18C006-10-13 the breeding offspring and upland cotton CCRI24 of 13 amplicons.
Analysis of agronomic characters the results are shown in Table 5.The result shows that compared with upland cotton CCRI24, after 18C006-10-13 breeding
The plant height in generation, the first fruit branch length, fruit branch number, knot bell number and ginning outturn are significantly increased.
The economical character of table 5. transgene cotton 18C006-10-13 and upland cotton CCRI24 compares
Mirid pentatomid Resistance Identification the results are shown in Table 6.The result shows that compared with upland cotton CCRI24,18C006-10-13 breeding
The anti-mirid pentatomid of offspring significantly improves, and blade extent of injury is significantly reduced.
The mirid pentatomid resistance of table 6. transgene cotton 18C006-10-13 and acceptor material CCR124
<110>the Chinese Academy of Agriculture Science and Technologys Cotton Research Institute
<120>upland cotton transformation event 18C006-10-13 and its specificity identification method
<160> 8
<170> PatentIn version 3.5
<210> 1
<211> 5965
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 1
tggcaggata tattgtggtg taaacaaatt gacgcttaga caacttaata acacattgcg 60
gacgttttta atgtactgaa ttaacgccga attaattcgg gggatctgga ttttagtact 120
ggattttggt tttaggaatt agaaatttta ttgatagaag tattttacaa atacaaatac 180
atactaaggg tttcttatat gctcaacaca tgagcgaaac cctataggaa ccctaattcc 240
cttatctggg aactactcac acattattat ggagaaactc gagcttgtcg atcgactcta 300
gctagaggat cgatccgaac cccagagtcc cgctcagaag aactcgtcaa gaaggcgata 360
gaaggcgatg cgctgcgaat cgggagcggc gataccgtaa agcacgagga agcggtcagc 420
ccattcgccg ccaagctctt cagcaatatc acgggtagcc aacgctatgt cctgatagcg 480
gtccgccaca cccagccggc cacagtcgat gaatccagaa aagcggccat tttccaccat 540
gatattcggc aagcaggcat cgccatgtgt cacgacgaga tcctcgccgt cgggcatgcg 600
cgccttgagc ctggcgaaca gttcggctgg cgcgagcccc tgatgctctt cgtccagatc 660
atcctgatcg acaagaccgg cttccatccg agtacgtgct cgctcgatgc gatgtttcgc 720
ttggtggtcg aatgggcagg tagccggatc aagcgtatgc agccgccgca ttgcatcagc 780
catgatggat actttctcgg caggagcaag gtgagatgac aggagatcct gccccggcac 840
ttcgcccaat agcagccagt cccttcccgc ttcagtgaca acgtcgagca cagctgcgca 900
aggaacgccc gtcgtggcca gccacgatag ccgcgctgcc tcgtcctgga gttcattcag 960
ggcaccggac aggtcggtct tgacaaaaag aaccgggcgc ccctgcgctg acagccggaa 1020
cacggcggca tcagagcagc cgattgtctg ttgtgcccag tcatagccga atagcctctc 1080
cacccaagcg gccggagaac ctgcgtgcaa tccatcttgt tcaatcccca tggtcgatcg 1140
acagatctgc gaaagctcga gagagataga tttgtagaga gagactggtg atttcagcgt 1200
gtcctctcca aatgaaatga acttccttat atagaggaag gtcttgcgaa ggatagtggg 1260
attgtgcgtc atcccttacg tcagtggaga tatcacatca atccacttgc tttgaagacg 1320
tggttggaac gtcttctttt tccacgatgc tcctcgtggg tgggggtcca tctttgggac 1380
cactgtcggc agaggcatct tgaacgatag cctttccttt atcgcaatga tggcatttgt 1440
aggtgccacc ttccttttct actgtccttt tgatgaagtg acagatagct gggcaatgga 1500
atccgaggag gtttcccgat attacccttt gttgaaaagt ctcaatagcc ctttggtctt 1560
ctgagactgt atctttgata ttcttggagt agacgagagt gtcgtgctcc accatgttat 1620
cacatcaatc cacttgcttt gaagacgtgg ttggaacgtc ttctttttcc acgatgctcc 1680
tcgtgggtgg gggtccatct ttgggaccac tgtcggcaga ggcatcttga acgatagcct 1740
ttcctttatc gcaatgatgg catttgtagg tgccaccttc cttttctact gtccttttga 1800
tgaagtgaca gatagctggg caatggaatc cgaggaggtt tcccgatatt accctttgtt 1860
gaaaagtctc aatagccctt tggtcttctg agactgtatc tttgatattc ttggagtaga 1920
cgagagtgtc gtgctccacc atgttggcaa gctgctctag ccaatacgca aaccgcctct 1980
ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc 2040
gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt 2100
acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac 2160
aggaaacagc tatgaccatg attacgaatt ctgagacttt tcaacaaagg gtaatatccg 2220
gaaacctcct cggattccat tgcccagcta tctgtcactt tattgtgaag atagtggaaa 2280
aggaaggtgg ctcctacaaa tgccatcatt gcgataaagg aaaggccatc gttgaagatg 2340
cctctgccga cagtggtccc aaagatggac ccccacccac gaggagcatc gtggaaaaag 2400
aagacgttcc aaccacgtct tcaaagcaag tggattgatg tgatatctcc actgacgtaa 2460
gggatgacgc acaatcccac tatccttcgc aagacccttc ctctatataa ggaagttcat 2520
ttcatttgga gagaacagcc ccctgagaac tggtaccatc tagacgaaaa acaaccaagg 2580
aatttgttcg ttcggtgact tttgctccaa ctcatggcca ttcttgattt aaaatctctt 2640
gtgctcaatg caattaatta ctggggtcca aagaacaata atggtatcca gggtggtgat 2700
tttggttatc ctattagcga aaaacaaatt gatacaagta ttattacgtc aacccatcca 2760
aggttgatac cccatgatct gaccattcca caaaacttgg agacaatttt cacaaccact 2820
caggtattaa ctaataacac tgatttgcag caatcccaaa cagtgtcctt cgctaagaag 2880
actactacca ccactgctac ctccacaacg aacggctgga cagaaggcgg aaaaatttca 2940
gatacgttgg aggaaaaggt ttcagtttca attcccttta tcggggaagg tggagggaag 3000
aatagcacca ccattgaggc taacttcgct cataatagtt ccaccaccac agcccaacag 3060
gccagcactg acatcgaatg gaacattagc caaccagtgc tcgtgcctcc cagaaagcag 3120
gtagttgcaa cactcgttat aatgggcggt aatttcacta tccctatgga cctgatgaca 3180
actatagatt caacagagca ctattctggc tatcctatac ttacttggat aagtagccca 3240
gataactctt ataacgggcc gtttatgtcc tggtacttcg ctaattggcc taacttacca 3300
agtggatttg ggcctttgaa ctccgacaat acggttactt atactggatc tgtggtttct 3360
caagtctctg ctggagttta cgcaaccgtc agatttgacc aatacgatat ccacaatctt 3420
cgaactatcg agaaaacatg gtacgcacgt cacgccacac tccataacgg aaagaaaatc 3480
tccattaaca atgtcactga aatggctccg acttcaccta tcaaaaccaa ttaagtgtga 3540
attacaggtg accagctcga atttccccga tcgttcaaac atttggcaat aaagtttctt 3600
aagattgaat cctgttgccg gtcttgcgat gattatcata taatttctgt tgaattacgt 3660
taagcatgta ataattaaca tgtaatgcat gacgttattt atgagatggg tttttatgat 3720
tagagtcccg caattataca tttaatacgc gatagaaaac aaaatatagc gcgcaaacta 3780
ggataaatta tcgcgcgcgg tgtcatctat gttactagat caagcttggc actggccgtc 3840
gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca 3900
catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa 3960
cagttgcgca gcctgaatgg cgaatgctag agcagcttga gcttggatca gattgtcgtt 4020
tcccgccttc agtttagctt catggagtca aagattcaaa tagaggacct aacagaactc 4080
gccgtaaaga ctggcgaaca gttcatacag agtctcttac gactcaatga caagaagaaa 4140
atcttcgtca acatggtgga gcacgacaca cttgtctact ccaaaaatat caaagataca 4200
gtctcagaag accaaagggc aattgagact tttcaacaaa gggtaatatc cggaaacctc 4260
ctcggattcc attgcccagc tatctgtcac tttattgtga agatagtgga aaaggaaggt 4320
ggctcctaca aatgccatca ttgcgataaa ggaaaggcca tcgttgaaga tgcctctgcc 4380
gacagtggtc ccaaagatgg acccccaccc acgaggagca tcgtggaaaa agaagacgtt 4440
ccaaccacgt cttcaaagca agtggattga tgtgatatct ccactgacgt aagggatgac 4500
gcacaatccc actatccttc gcaagaccct tcctctatat aaggaagttc atttcatttg 4560
gagagaacac gggggactct tgaccatggt agatctgagg gtaaatttct agtttttctc 4620
cttcattttc ttggttagga cccttttctc tttttatttt tttgagcttt gatctttctt 4680
taaactgatc tattttttaa ttgattggtt atggtgtaaa tattacatag ctttaactga 4740
taatctgatt actttatttc gtgtgtctat gatgatgatg atagttacag aaccgacgac 4800
tcgtccgtcc tgtagaaacc ccaacccgtg aaatcaaaaa actcgacggc ctgtgggcat 4860
tcagtctgga tcgcgaaaac tgtggaattg atcagcgttg gtgggaaagc gcgttacaag 4920
aaagccgggc aattgctgtg ccaggcagtt ttaacgatca gttcgccgat gcagatattc 4980
gtaattatgc gggcaacgtc tggtatcagc gcgaagtctt tataccgaaa ggttgggcag 5040
gccagcgtat cgtgctgcgt ttcgatgcgg tcactcatta cggcaaagtg tgggtcaata 5100
atcaggaagt gatggagcat cagggcggct atacgccatt tgaagccgat gtcacgccgt 5160
atgttattgc cgggaaaagt gtacgtatca ccgtttgtgt gaacaacgaa ctgaactggc 5220
agactatccc gccgggaatg gtgattaccg acgaaaacgg caagaaaaag cagtcttact 5280
tccatgattt ctttaactat gccggaatcc atcgcagcgt aatgctctac accacgccga 5340
acacctgggt ggacgatatc accgtggtga cgcatgtcgc gcaagactgt aaccacgcgt 5400
ctgttgactg gcaggtggtg gccaatggtg atgtcagcgt tgaactgcgt gatgcggatc 5460
aacaggtggt tgcaactgga caaggcacta gcgggacttt gcaagtggtg aatccgcacc 5520
tctggcaacc gggtgaaggt tatctctatg aactcgaagt cacagccaaa agccagacag 5580
agtctgatat ctacccgctt cgcgtcggca tccggtcagt ggcagtgaag ggccaacagt 5640
tcctgattaa ccacaaaccg ttctacttta ctggctttgg tcgtcatgaa gatgcggact 5700
tacgtggcaa aggattcgat aacgtgctga tggtgcacga ccacgcatta atggactgga 5760
ttggggccaa ctcctaccgt acctcgcatt acccttacgc tgaagagatg ctcgactggg 5820
cagatgaaca tggcatcgtg gtgattgatg aaactgctgc tgtcggcttt cagctgtctt 5880
taggcattgg tttcgaagcg ggcaacaagc cgaaagaact gtacagcgaa gaggcagtca 5940
acggggaaac tcagcaagcg cacttacagg cgattaaaga gctgatagcg cgtgacaaaa 6000
accacccaag cgtggtgatg tggagtattg ccaacgaacc ggatacccgt ccgcaaggtg 6060
cacgggaata tttcgcgcca ctggcggaag caacgcgtaa actcgacccg acgcgtccga 6120
tcacctgcgt caatgtaatg ttctgcgacg ctcacaccga taccatcagc gatctctttg 6180
atgtgctgtg cctgaaccgt tattacggat ggtatgtcca aagcggcgat ttggaaacgg 6240
cagagaaggt actggaaaaa gaacttctgg cctggcagga gaaactgcat cagccgatta 6300
tcatcaccga atacggcgtg gatacgttag ccgggctgca ctcaatgtac accgacatgt 6360
ggagtgaaga gtatcagtgt gcatggctgg atatgtatca ccgcgtcttt gatcgcgtca 6420
gcgccgtcgt cggtgaacag gtatggaatt tcgccgattt tgcgacctcg caaggcatat 6480
tgcgcgttgg cggtaacaag aaagggatct tcactcgcga ccgcaaaccg aagtcggcgg 6540
cttttctgct gcaaaaacgc tggactggca tgaacttcgg tgaaaaaccg cagcagggag 6600
gcaaacaagc tagccaccac caccaccacc acgtgtgaat tacaggtgac cagctcgaat 6660
ttccccgatc gttcaaacat ttggcaataa agtttcttaa gattgaatcc tgttgccggt 6720
cttgcgatga ttatcatata atttctgttg aattacgtta agcatgtaat aattaacatg 6780
taatgcatga cgttatttat gagatgggtt tttatgatta gagtcccgca attatacatt 6840
taatacgcga tagaaaacaa aatatagcgc gcaaactagg ataaattatc gcgcgcggtg 6900
tcatctatgt tactagatcg ggaattaaac tatcagtgtt tgacaggata tattggcggg 6960
taaac 6965
<210> 2
<211> 2000
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 2
tcaaaattaa ctatagtgcc gttacctccg tcagttcaaa agaattttag tccaaccaga 60
agctgtcatg tcaccagcca cctctatctt ttagaaaatt acataaaatt ttaaaaaaac 120
tgggaaaaaa gaaaaaaata tatttttaat taaatttatt atagtaatta aatttccaat 180
ctgtttccat ctactcaatt ggggtttaat tgttttcaaa atggcatcat attcctttcc 240
ccttctaaac cctctaaatt attagttttt taaatttatt aaatttaatt attttttaaa 300
agaatttcat gtaaattttt taaaatgtgg atagtgacat ggcagcttct ggatggaatt 360
tttttttttg gactaacgat gttaaggttt gaaaatagtt gaggttaaat gaatagacaa 420
aaaagagttg cacatttaag aaaataggta taattaagaa gacaattttt taattaagtc 480
taaaatatat cttgtctgat cgatgtgaac aatccgattc gataataaaa aaaaatcaaa 540
gatttaaact cactttacta aattctaaat ttatgtcgac ttaaaattaa ctaaatctaa 600
aacatttaaa ctaaacctca taattctcca accgaattca aaaaatgatt gtataaaatc 660
gttaaacgat gataagatat gtgatttcca ttctcacata attgcattta tataaaaatt 720
cgatcatcct tcattgatga attgctttta tatttttata tatataattt aattattaca 780
tgtaatgcat aatcgacagt tgacttattt tataaatgtg gtacgtggcg ttaattgaat 840
ggacgtagat gtactataat tatttcctct ctctcaactc aaaaaaatag acaaattaat 900
cctcataagt tatatcaatg agaaatctga tttttttact gaaaatttca ttaaattact 960
attaaaaagt ggttcatgca tgtcagcacg aggtacatat ggcatgtcat gtgtaactgt 1020
ttgtttattc catcaactac atcagttttt aatagtaaaa atgaattaat tttttaacaa 1080
aatggataag tttgcttttt aatctaacat acaaagacta atttgtcatt ttcttgtgaa 1140
taaaatgaaa aaatacaatc taattcttaa tataaggact tctataatac atttacctaa 1200
aaagtgttca taaattgaac catcattact caattctaaa atatttttca agttggagtc 1260
aacttaattc actaaaccga tacatcttaa taaaataaat aaaatattaa aaaatataat 1320
tttatactaa tatttatata atttaaaatc tatttaattt aaactaatat tgaatttcaa 1380
ttaattcaaa attattaaag tccacccaaa accaactcaa caaccgtgga ccggtatagt 1440
cagaacccat tcgagctcaa aaaacccggc agctactata caaaacaatc ttttacgtta 1500
tccgtaacaa atttatcctt ttattgaaaa ttatatcggt attagtaccg ccgtagatgg 1560
aggccgccgc cgccgccacc gccactttgg ttggatcttc tttacattta catccaccac 1620
cttcaagctc cgttaccaga tcgactgttt acgctgccag acctcgcctc tctttcccga 1680
ttaaggtttc aatttgctta ctgttctttt catttatacc agttttgggt tttatccttt 1740
ttttttctcg ttttttaaac ttatattccg agtttagatc cttaatttca tataattatt 1800
tgaaagggaa gattttgatg catgtatttg taaacacacc aaccgaagaa cacatttctc 1860
tgctgattat gatttctttt ttttaataaa tgtagggttt atgttaatga tttatcatgt 1920
ttattgaatg gaaacttcat ttccagtttg tgtctatgga gtctattaaa cagatttttt 1980
ttttacatta aattaccaca 2000
<210> 3
<211> 2001
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 3
tacaagagaa ccttttccca tatcatgccc ttgatgttat tttgaccttc ttcctcataa 60
gatccatctt cgtataccca aaaatgagta tccctggggc attgtacctt tgccagctga 120
aataaaatta aagtacaaat cgaatcaaac cattatacaa agtagtgcgc atgctgaaaa 180
aaaagataaa aaactgttcc aaccatcaga attaggaatc atgctttaga gagtgaaaaa 240
atattaccag gatgacctta aattttggat gaaaagtatc aactatattc taatttctaa 300
gcatgactcc ataaatgaat tcttttatct tctcatacac caaaccatga caaactatca 360
ccagaagtat aatttatgta tatgtgtagc atttcaattg ttaactccaa taacttgagt 420
ttatgcattt tccaataaac atcatcaaaa gtaaaatgaa ggttttgctt aataaaaggc 480
aagcacacaa aaaaattagt ctttgatcta agaaattctt tactctcatg ttgtttagag 540
tgcagcaatg aagaaatggc atgcaataag aagaaaagaa gtaatttaag taccttcaac 600
actctaagct cagtctttgt gatttcgcgg ccattgatga aaacccttgt attcccattg 660
caagcatcct ttttcagctt accaccaata ttcaacttgg aactaattat actgtcaggc 720
ttttcccctt cctaaaattc caattcaaag gtcaaacaca tgaaactgat aataataata 780
ataataataa caagaacaat ctattttgaa tacagaaatt tacctttccc caaaatccag 840
aatccttatc ataccaatac ctgccaggct tcaacttctg aggtggtact ggacacccaa 900
aaagctcagc caattcttca tgattcaatt gtctcccatt cacaaccaac tgctcaggcc 960
tcaattgatt ggccgcgcat tccttctcag ccttcattat ttgctgaacc tctaagggac 1020
tacaaacttt cctcaatagc ctggaactct tcccaagtct agacctctta gcttcatcaa 1080
taggcttttt aatgcaactc acacacttcc ttccttcagg cattgacccc atagccttaa 1140
gcaaacagtt gttacaatac ctcgcatcac aaaccaaaca cgcctctctt tccttcaatc 1200
tactccgttt gccacacctg ctacaaatcc ctctcttctt ctgcttcact atccccctcg 1260
tttccacatt ttccaataaa acaccacgac ccgccacgct ggattgcgaa gaagtgtacc 1320
catcatcatc atcatcttct tcttcttctt cttcatcatc atcattttcc gaatctttcg 1380
gcgtattaaa cgtcacaaca acattcttct tatcagggtg ctgctgctga gactgctccg 1440
gaggcggcgg tggcggcgac ttatcaacct cgatctcaga atcataatcc ccgtttcgag 1500
aatcaatctg agatcttgaa gctgaagagg gtctctgact ttctaaagga cttccatttc 1560
taacagcata ccgattcttt ttaaaactag agaatttagg tttactaggg attgaagcag 1620
cgacaggaat agaagatata tcagaagcag aaagggaatc gagatctagt ggatcaacac 1680
gaggaacatc ataaggaata ggaggacctt catattcaat agcgatcgag taatcgagat 1740
gatcctcgtc cggtaacgga gctccgaccg gtaacattct tctaattaca tcttcccatg 1800
ctctgttctc ttcttcagct tctttagctg ccatggtgag tcagattccg agtttctgaa 1860
tctctgagtt cgagtccgag cttccacctc aggtggcagc tacggcagcc gttagggagt 1920
tggcagaaag taaacggagg aaaaattaac gctgaagaat cagtccgtta tatttgacgc 1980
tttcagagta tatgactcag g 2001
<210> 4
<211> 13218
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 4
tggcaggata tattgtggtg taaacaaatt gacgcttaga caacttaata acacattgcg 60
gacgttttta atgtactgaa ttaacgccga attaattcgg gggatctgga ttttagtact 120
ggattttggt tttaggaatt agaaatttta ttgatagaag tattttacaa atacaaatac 180
atactaaggg tttcttatat gctcaacaca tgagcgaaac cctataggaa ccctaattcc 240
cttatctggg aactactcac acattattat ggagaaactc gagcttgtcg atcgactcta 300
gctagaggat cgatccgaac cccagagtcc cgctcagaag aactcgtcaa gaaggcgata 360
gaaggcgatg cgctgcgaat cgggagcggc gataccgtaa agcacgagga agcggtcagc 420
ccattcgccg ccaagctctt cagcaatatc acgggtagcc aacgctatgt cctgatagcg 480
gtccgccaca cccagccggc cacagtcgat gaatccagaa aagcggccat tttccaccat 540
gatattcggc aagcaggcat cgccatgtgt cacgacgaga tcctcgccgt cgggcatgcg 600
cgccttgagc ctggcgaaca gttcggctgg cgcgagcccc tgatgctctt cgtccagatc 660
atcctgatcg acaagaccgg cttccatccg agtacgtgct cgctcgatgc gatgtttcgc 720
ttggtggtcg aatgggcagg tagccggatc aagcgtatgc agccgccgca ttgcatcagc 780
catgatggat actttctcgg caggagcaag gtgagatgac aggagatcct gccccggcac 840
ttcgcccaat agcagccagt cccttcccgc ttcagtgaca acgtcgagca cagctgcgca 900
aggaacgccc gtcgtggcca gccacgatag ccgcgctgcc tcgtcctgga gttcattcag 960
ggcaccggac aggtcggtct tgacaaaaag aaccgggcgc ccctgcgctg acagccggaa 1020
cacggcggca tcagagcagc cgattgtctg ttgtgcccag tcatagccga atagcctctc 1080
cacccaagcg gccggagaac ctgcgtgcaa tccatcttgt tcaatcccca tggtcgatcg 1140
acagatctgc gaaagctcga gagagataga tttgtagaga gagactggtg atttcagcgt 1200
gtcctctcca aatgaaatga acttccttat atagaggaag gtcttgcgaa ggatagtggg 1260
attgtgcgtc atcccttacg tcagtggaga tatcacatca atccacttgc tttgaagacg 1320
tggttggaac gtcttctttt tccacgatgc tcctcgtggg tgggggtcca tctttgggac 1380
cactgtcggc agaggcatct tgaacgatag cctttccttt atcgcaatga tggcatttgt 1440
aggtgccacc ttccttttct actgtccttt tgatgaagtg acagatagct gggcaatgga 1500
atccgaggag gtttcccgat attacccttt gttgaaaagt ctcaatagcc ctttggtctt 1560
ctgagactgt atctttgata ttcttggagt agacgagagt gtcgtgctcc accatgttat 1620
cacatcaatc cacttgcttt gaagacgtgg ttggaacgtc ttctttttcc acgatgctcc 1680
tcgtgggtgg gggtccatct ttgggaccac tgtcggcaga ggcatcttga acgatagcct 1740
ttcctttatc gcaatgatgg catttgtagg tgccaccttc cttttctact gtccttttga 1800
tgaagtgaca gatagctggg caatggaatc cgaggaggtt tcccgatatt accctttgtt 1860
gaaaagtctc aatagccctt tggtcttctg agactgtatc tttgatattc ttggagtaga 1920
cgagagtgtc gtgctccacc atgttggcaa gctgctctag ccaatacgca aaccgcctct 1980
ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc 2040
gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt 2100
acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac 2160
aggaaacagc tatgaccatg attacgaatt ctgagacttt tcaacaaagg gtaatatccg 2220
gaaacctcct cggattccat tgcccagcta tctgtcactt tattgtgaag atagtggaaa 2280
aggaaggtgg ctcctacaaa tgccatcatt gcgataaagg aaaggccatc gttgaagatg 2340
cctctgccga cagtggtccc aaagatggac ccccacccac gaggagcatc gtggaaaaag 2400
aagacgttcc aaccacgtct tcaaagcaag tggattgatg tgatatctcc actgacgtaa 2460
gggatgacgc acaatcccac tatccttcgc aagacccttc ctctatataa ggaagttcat 2520
ttcatttgga gagaacagcc ccctgagaac tggtaccatc tagacgaaaa acaaccaagg 2580
aatttgttcg ttcggtgact tttgctccaa ctcatggcca ttcttgattt aaaatctctt 2640
gtgctcaatg caattaatta ctggggtcca aagaacaata atggtatcca gggtggtgat 2700
tttggttatc ctattagcga aaaacaaatt gatacaagta ttattacgtc aacccatcca 2760
aggttgatac cccatgatct gaccattcca caaaacttgg agacaatttt cacaaccact 2820
caggtattaa ctaataacac tgatttgcag caatcccaaa cagtgtcctt cgctaagaag 2880
actactacca ccactgctac ctccacaacg aacggctgga cagaaggcgg aaaaatttca 2940
gatacgttgg aggaaaaggt ttcagtttca attcccttta tcggggaagg tggagggaag 3000
aatagcacca ccattgaggc taacttcgct cataatagtt ccaccaccac agcccaacag 3060
gccagcactg acatcgaatg gaacattagc caaccagtgc tcgtgcctcc cagaaagcag 3120
gtagttgcaa cactcgttat aatgggcggt aatttcacta tccctatgga cctgatgaca 3180
actatagatt caacagagca ctattctggc tatcctatac ttacttggat aagtagccca 3240
gataactctt ataacgggcc gtttatgtcc tggtacttcg ctaattggcc taacttacca 3300
agtggatttg ggcctttgaa ctccgacaat acggttactt atactggatc tgtggtttct 3360
caagtctctg ctggagttta cgcaaccgtc agatttgacc aatacgatat ccacaatctt 3420
cgaactatcg agaaaacatg gtacgcacgt cacgccacac tccataacgg aaagaaaatc 3480
tccattaaca atgtcactga aatggctccg acttcaccta tcaaaaccaa ttaagtgtga 3540
attacaggtg accagctcga atttccccga tcgttcaaac atttggcaat aaagtttctt 3600
aagattgaat cctgttgccg gtcttgcgat gattatcata taatttctgt tgaattacgt 3660
taagcatgta ataattaaca tgtaatgcat gacgttattt atgagatggg tttttatgat 3720
tagagtcccg caattataca tttaatacgc gatagaaaac aaaatatagc gcgcaaacta 3780
ggataaatta tcgcgcgcgg tgtcatctat gttactagat caagcttggc actggccgtc 3840
gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca 3900
catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa 3960
cagttgcgca gcctgaatgg cgaatgctag agcagcttga gcttggatca gattgtcgtt 4020
tcccgccttc agtttagctt catggagtca aagattcaaa tagaggacct aacagaactc 4080
gccgtaaaga ctggcgaaca gttcatacag agtctcttac gactcaatga caagaagaaa 4140
atcttcgtca acatggtgga gcacgacaca cttgtctact ccaaaaatat caaagataca 4200
gtctcagaag accaaagggc aattgagact tttcaacaaa gggtaatatc cggaaacctc 4260
ctcggattcc attgcccagc tatctgtcac tttattgtga agatagtgga aaaggaaggt 4320
ggctcctaca aatgccatca ttgcgataaa ggaaaggcca tcgttgaaga tgcctctgcc 4380
gacagtggtc ccaaagatgg acccccaccc acgaggagca tcgtggaaaa agaagacgtt 4440
ccaaccacgt cttcaaagca agtggattga tgtgatatct ccactgacgt aagggatgac 4500
gcacaatccc actatccttc gcaagaccct tcctctatat aaggaagttc atttcatttg 4560
gagagaacac gggggactct tgaccatggt agatctgagg gtaaatttct agtttttctc 4620
cttcattttc ttggttagga cccttttctc tttttatttt tttgagcttt gatctttctt 4680
taaactgatc tattttttaa ttgattggtt atggtgtaaa tattacatag ctttaactga 4740
taatctgatt actttatttc gtgtgtctat gatgatgatg atagttacag aaccgacgac 4800
tcgtccgtcc tgtagaaacc ccaacccgtg aaatcaaaaa actcgacggc ctgtgggcat 4860
tcagtctgga tcgcgaaaac tgtggaattg atcagcgttg gtgggaaagc gcgttacaag 4920
aaagccgggc aattgctgtg ccaggcagtt ttaacgatca gttcgccgat gcagatattc 4980
gtaattatgc gggcaacgtc tggtatcagc gcgaagtctt tataccgaaa ggttgggcag 5040
gccagcgtat cgtgctgcgt ttcgatgcgg tcactcatta cggcaaagtg tgggtcaata 5100
atcaggaagt gatggagcat cagggcggct atacgccatt tgaagccgat gtcacgccgt 5160
atgttattgc cgggaaaagt gtacgtatca ccgtttgtgt gaacaacgaa ctgaactggc 5220
agactatccc gccgggaatg gtgattaccg acgaaaacgg caagaaaaag cagtcttact 5280
tccatgattt ctttaactat gccggaatcc atcgcagcgt aatgctctac accacgccga 5340
acacctgggt ggacgatatc accgtggtga cgcatgtcgc gcaagactgt aaccacgcgt 5400
ctgttgactg gcaggtggtg gccaatggtg atgtcagcgt tgaactgcgt gatgcggatc 5460
aacaggtggt tgcaactgga caaggcacta gcgggacttt gcaagtggtg aatccgcacc 5520
tctggcaacc gggtgaaggt tatctctatg aactcgaagt cacagccaaa agccagacag 5580
agtctgatat ctacccgctt cgcgtcggca tccggtcagt ggcagtgaag ggccaacagt 5640
tcctgattaa ccacaaaccg ttctacttta ctggctttgg tcgtcatgaa gatgcggact 5700
tacgtggcaa aggattcgat aacgtgctga tggtgcacga ccacgcatta atggactgga 5760
ttggggccaa ctcctaccgt acctcgcatt acccttacgc tgaagagatg ctcgactggg 5820
cagatgaaca tggcatcgtg gtgattgatg aaactgctgc tgtcggcttt cagctgtctt 5880
taggcattgg tttcgaagcg ggcaacaagc cgaaagaact gtacagcgaa gaggcagtca 5940
acggggaaac tcagcaagcg cacttacagg cgattaaaga gctgatagcg cgtgacaaaa 6000
accacccaag cgtggtgatg tggagtattg ccaacgaacc ggatacccgt ccgcaaggtg 6060
cacgggaata tttcgcgcca ctggcggaag caacgcgtaa actcgacccg acgcgtccga 6120
tcacctgcgt caatgtaatg ttctgcgacg ctcacaccga taccatcagc gatctctttg 6180
atgtgctgtg cctgaaccgt tattacggat ggtatgtcca aagcggcgat ttggaaacgg 6240
cagagaaggt actggaaaaa gaacttctgg cctggcagga gaaactgcat cagccgatta 6300
tcatcaccga atacggcgtg gatacgttag ccgggctgca ctcaatgtac accgacatgt 6360
ggagtgaaga gtatcagtgt gcatggctgg atatgtatca ccgcgtcttt gatcgcgtca 6420
gcgccgtcgt cggtgaacag gtatggaatt tcgccgattt tgcgacctcg caaggcatat 6480
tgcgcgttgg cggtaacaag aaagggatct tcactcgcga ccgcaaaccg aagtcggcgg 6540
cttttctgct gcaaaaacgc tggactggca tgaacttcgg tgaaaaaccg cagcagggag 6600
gcaaacaagc tagccaccac caccaccacc acgtgtgaat tacaggtgac cagctcgaat 6660
ttccccgatc gttcaaacat ttggcaataa agtttcttaa gattgaatcc tgttgccggt 6720
cttgcgatga ttatcatata atttctgttg aattacgtta agcatgtaat aattaacatg 6780
taatgcatga cgttatttat gagatgggtt tttatgatta gagtcccgca attatacatt 6840
taatacgcga tagaaaacaa aatatagcgc gcaaactagg ataaattatc gcgcgcggtg 6900
tcatctatgt tactagatcg ggaattaaac tatcagtgtt tgacaggata tattggcggg 6960
taaacctaag agaaaagagc gtttattaga ataacggata tttaaaaggg cgtgaaaagg 7020
tttatccgtt cgtccatttg tatgtgcatg ccaaccacag ggttcccctc gggatcaaag 7080
tactttgatc caacccctcc gctgctatag tgcagtcggc ttctgacgtt cagtgcagcc 7140
gtcttctgaa aacgacatgt cgcacaagtc ctaagttacg cgacaggctg ccgccctgcc 7200
cttttcctgg cgttttcttg tcgcgtgttt tagtcgcata aagtagaata cttgcgacta 7260
gaaccggaga cattacgcca tgaacaagag cgccgccgct ggcctgctgg gctatgcccg 7320
cgtcagcacc gacgaccagg acttgaccaa ccaacgggcc gaactgcacg cggccggctg 7380
caccaagctg ttttccgaga agatcaccgg caccaggcgc gaccgcccgg agctggccag 7440
gatgcttgac cacctacgcc ctggcgacgt tgtgacagtg accaggctag accgcctggc 7500
ccgcagcacc cgcgacctac tggacattgc cgagcgcatc caggaggccg gcgcgggcct 7560
gcgtagcctg gcagagccgt gggccgacac caccacgccg gccggccgca tggtgttgac 7620
cgtgttcgcc ggcattgccg agttcgagcg ttccctaatc atcgaccgca cccggagcgg 7680
gcgcgaggcc gccaaggccc gaggcgtgaa gtttggcccc cgccctaccc tcaccccggc 7740
acagatcgcg cacgcccgcg agctgatcga ccaggaaggc cgcaccgtga aagaggcggc 7800
tgcactgctt ggcgtgcatc gctcgaccct gtaccgcgca cttgagcgca gcgaggaagt 7860
gacgcccacc gaggccaggc ggcgcggtgc cttccgtgag gacgcattga ccgaggccga 7920
cgccctggcg gccgccgaga atgaacgcca agaggaacaa gcatgaaacc gcaccaggac 7980
ggccaggacg aaccgttttt cattaccgaa gagatcgagg cggagatgat cgcggccggg 8040
tacgtgttcg agccgcccgc gcacgtctca accgtgcggc tgcatgaaat cctggccggt 8100
ttgtctgatg ccaagctggc ggcctggccg gccagcttgg ccgctgaaga aaccgagcgc 8160
cgccgtctaa aaaggtgatg tgtatttgag taaaacagct tgcgtcatgc ggtcgctgcg 8220
tatatgatgc gatgagtaaa taaacaaata cgcaagggga acgcatgaag gttatcgctg 8280
tacttaacca gaaaggcggg tcaggcaaga cgaccatcgc aacccatcta gcccgcgccc 8340
tgcaactcgc cggggccgat gttctgttag tcgattccga tccccagggc agtgcccgcg 8400
attgggcggc cgtgcgggaa gatcaaccgc taaccgttgt cggcatcgac cgcccgacga 8460
ttgaccgcga cgtgaaggcc atcggccggc gcgacttcgt agtgatcgac ggagcgcccc 8520
aggcggcgga cttggctgtg tccgcgatca aggcagccga cttcgtgctg attccggtgc 8580
agccaagccc ttacgacata tgggccaccg ccgacctggt ggagctggtt aagcagcgca 8640
ttgaggtcac ggatggaagg ctacaagcgg cctttgtcgt gtcgcgggcg atcaaaggca 8700
cgcgcatcgg cggtgaggtt gccgaggcgc tggccgggta cgagctgccc attcttgagt 8760
cccgtatcac gcagcgcgtg agctacccag gcactgccgc cgccggcaca accgttcttg 8820
aatcagaacc cgagggcgac gctgcccgcg aggtccaggc gctggccgct gaaattaaat 8880
caaaactcat ttgagttaat gaggtaaaga gaaaatgagc aaaagcacaa acacgctaag 8940
tgccggccgt ccgagcgcac gcagcagcaa ggctgcaacg ttggccagcc tggcagacac 9000
gccagccatg aagcgggtca actttcagtt gccggcggag gatcacacca agctgaagat 9060
gtacgcggta cgccaaggca agaccattac cgagctgcta tctgaataca tcgcgcagct 9120
accagagtaa atgagcaaat gaataaatga gtagatgaat tttagcggct aaaggaggcg 9180
gcatggaaaa tcaagaacaa ccaggcaccg acgccgtgga atgccccatg tgtggaggaa 9240
cgggcggttg gccaggcgta agcggctggg ttgtctgccg gccctgcaat ggcactggaa 9300
cccccaagcc cgaggaatcg gcgtgacggt cgcaaaccat ccggcccggt acaaatcggc 9360
gcggcgctgg gtgatgacct ggtggagaag ttgaaggccg cgcaggccgc ccagcggcaa 9420
cgcatcgagg cagaagcacg ccccggtgaa tcgtggcaag cggccgctga tcgaatccgc 9480
aaagaatccc ggcaaccgcc ggcagccggt gcgccgtcga ttaggaagcc gcccaagggc 9540
gacgagcaac cagatttttt cgttccgatg ctctatgacg tgggcacccg cgatagtcgc 9600
agcatcatgg acgtggccgt tttccgtctg tcgaagcgtg accgacgagc tggcgaggtg 9660
atccgctacg agcttccaga cgggcacgta gaggtttccg cagggccggc cggcatggcc 9720
agtgtgtggg attacgacct ggtactgatg gcggtttccc atctaaccga atccatgaac 9780
cgataccggg aagggaaggg agacaagccc ggccgcgtgt tccgtccaca cgttgcggac 9840
gtactcaagt tctgccggcg agccgatggc ggaaagcaga aagacgacct ggtagaaacc 9900
tgcattcggt taaacaccac gcacgttgcc atgcagcgta cgaagaaggc caagaacggc 9960
cgcctggtga cggtatccga gggtgaagcc ttgattagcc gctacaagat cgtaaagagc 10020
gaaaccgggc ggccggagta catcgagatc gagctagctg attggatgta ccgcgagatc 10080
acagaaggca agaacccgga cgtgctgacg gttcaccccg attacttttt gatcgatccc 10140
ggcatcggcc gttttctcta ccgcctggca cgccgcgccg caggcaaggc agaagccaga 10200
tggttgttca agacgatcta cgaacgcagt ggcagcgccg gagagttcaa gaagttctgt 10260
ttcaccgtgc gcaagctgat cgggtcaaat gacctgccgg agtacgattt gaaggaggag 10320
gcggggcagg ctggcccgat cctagtcatg cgctaccgca acctgatcga gggcgaagca 10380
tccgccggtt cctaatgtac ggagcagatg ctagggcaaa ttgccctagc aggggaaaaa 10440
ggtcgaaaag gtctctttcc tgtggatagc acgtacattg ggaacccaaa gccgtacatt 10500
gggaaccgga acccgtacat tgggaaccca aagccgtaca ttgggaaccg gtcacacatg 10560
taagtgactg atataaaaga gaaaaaaggc gatttttccg cctaaaactc tttaaaactt 10620
attaaaactc ttaaaacccg cctggcctgt gcataactgt ctggccagcg cacagccgaa 10680
gagctgcaaa aagcgcctac ccttcggtcg ctgcgctccc tacgccccgc cgcttcgcgt 10740
cggcctatcg cggccgctgg ccgctcaaaa atggctggcc tacggccagg caatctacca 10800
gggcgcggac aagccgcgcc gtcgccactc gaccgccggc gcccacatca aggcaccctg 10860
cctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt 10920
cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg 10980
tgttggcggg tgtcggggcg cagccatgac ccagtcacgt agcgatagcg gagtgtatac 11040
tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatat gcggtgtgaa 11100
ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc 11160
actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg 11220
gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc 11280
cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc 11340
ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga 11400
ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc 11460
ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat 11520
agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg 11580
cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc 11640
aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga 11700
gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact 11760
agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt 11820
ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag 11880
cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg 11940
tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgca ttctaggtac 12000
taaaacaatt catccagtaa aatataatat tttattttct cccaatcagg cttgatcccc 12060
agtaagtcaa aaaatagctc gacatactgt tcttccccga tatcctccct gatcgaccgg 12120
acgcagaagg caatgtcata ccacttgtcc gccctgccgc ttctcccaag atcaataaag 12180
ccacttactt tgccatcttt cacaaagatg ttgctgtctc ccaggtcgcc gtgggaaaag 12240
acaagttcct cttcgggctt ttccgtcttt aaaaaatcat acagctcgcg cggatcttta 12300
aatggagtgt cttcttccca gttttcgcaa tccacatcgg ccagatcgtt attcagtaag 12360
taatccaatt cggctaagcg gctgtctaag ctattcgtat agggacaatc cgatatgtcg 12420
atggagtgaa agagcctgat gcactccgca tacagctcga taatcttttc agggctttgt 12480
tcatcttcat actcttccga gcaaaggacg ccatcggcct cactcatgag cagattgctc 12540
cagccatcat gccgttcaaa gtgcaggacc tttggaacag gcagctttcc ttccagccat 12600
agcatcatgt ccttttcccg ttccacatca taggtggtcc ctttataccg gctgtccgtc 12660
atttttaaat ataggttttc attttctccc accagcttat ataccttagc aggagacatt 12720
ccttccgtat cttttacgca gcggtatttt tcgatcagtt ttttcaattc cggtgatatt 12780
ctcattttag ccatttatta tttccttcct cttttctaca gtatttaaag ataccccaag 12840
aagctaatta taacaagacg aactccaatt cactgttcct tgcattctaa aaccttaaat 12900
accagaaaac agctttttca aagttgtttt caaagttggc gtataacata gtatcgacgg 12960
agccgatttt gaaaccgcgg tgatcacagg cagcaacgct ctgtcatcgt tacaatcaac 13020
atgctaccct ccgcgagatc atccgtgttt caaacccggc agcttagttg ccgttcttcc 13080
gaatagcatc ggtaacatga gcaaagtctg ccgccttaca acggctctcc cgctgacgcc 13140
gtcccggact gatgggctgc ctgtatcgag tggtgatttt gtgccgagct gccggtcggg 13200
gagctgttgg ctggctgg 13218
<210> 5
<211> 20
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 5
gagcttggat cagattgtcg 20
<210> 6
<211> 20
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 6
gtttcgctca tgtgttgagc 20
<210> 7
<211> 20
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 7
cttgtctgat cgatgtgaac 20
<210> 8
<211> 21
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 8
cacctcagaa gttgaagcct g 21
Claims (10)
1. a kind of breeding method of transgene cotton, for exogenous dna fragment is inserted into purpose cotton gene group D12 chromosome
63328845-63329028 interdigit, replace the 63328845-63329028 interdigit 182bp's of D12 chromosome
Base sequence obtains transgene cotton；
The mirid pentatomid resistance and/or plant height of the transgene cotton and/or the first fruit branch length and/or fruit branch number and/or knot bell
Several and/or ginning outturn is above the purpose cotton；The Single boll weight of the transgene cotton is lower than the purpose cotton；
The exogenous dna fragment is the DNA molecular containing Cry51Aa1.C006-1 gene.
2. breeding method as described in claim 1, it is characterised in that: the nucleotide sequence of the exogenous dna fragment such as sequence
In table shown in sequence 1.
3. whether deriving from the transgene cotton or its that breeding method as claimed in claim 1 or 2 obtains for plant identification sample
The method of offspring, includes the following steps:
It detects in the genomic DNA of the plant sample to be measured and whether contains DNA fragmentation A；The DNA fragmentation A is by claim 1
Or exogenous dna fragment described in 2 is in the upstream flanking fragment, the exogenous dna fragment and the external source of the transgene cotton
DNA fragmentation is formed in the upstream flanking fragment of the transgene cotton；
If the genomic DNA of the plant sample to be measured contains the DNA fragmentation A, the plant sample to be measured is or candidate
For the transgene cotton or its offspring；
If the genomic DNA of the plant sample to be measured do not contain the DNA fragmentation A, the plant sample to be measured be not or
Candidate is not the transgene cotton or its offspring.
4. method as claimed in claim 4, it is characterised in that: in the nucleotide sequence of the upstream flanking sequence such as sequence table
Shown in sequence 2；The nucleotide sequence of the downstream flanking sequence is as shown in sequence 3 in sequence table.
5. the method as claimed in claim 3 or 4, it is characterised in that: " the genome of the detection plant sample to be measured
In DNA whether containing DNA fragmentation A " method be S1) S2) or S3):
S1) direct Sequencing；
S2 PCR amplification) is carried out with the genomic DNA that primer pair 1 and/or primer pair 2 treat measuring plants sample, is then carried out as follows
Judgement: if there is purpose amplified production, the plant sample to be measured is or candidate is the transgene cotton or its offspring；If not yet
There is purpose amplified production, then the plant sample to be measured is not or candidate is not the transgene cotton or its offspring；
The primer pair 1 be can expand held by the exogenous dna fragment 5 ' and close to upstream flanking sequence part described in its or
The primer pair of the DNA molecular first of whole segment compositions；Its corresponding purpose amplified production is the DNA molecular first；
The primer pair 2 is that can expand to hold containing the exogenous dna fragment 3 ' and close to the portion of downstream flanking sequence described in its
Point or the primer pair of DNA molecular second that all forms；Its corresponding purpose amplified production is the DNA molecular second；
S3) gene of the probe of the DNA molecular first described in energy specific bond or the DNA molecular second to the plant sample to be measured
Group DNA carries out Southern hybridization, and then make the following judgment: if hybridized fragment can be obtained, the plant sample to be measured is
Or candidate is the transgene cotton or its offspring；If hybridized fragment cannot be obtained, the plant sample to be measured is not or waits
Choosing is not the transgene cotton or its offspring.
6. method as claimed in claim 5, it is characterised in that:
Single stranded DNA shown in sequence 7 point in the single strand dna as shown in sequence 5 in sequence table of primer pair 1 and sequence table
Son composition；
Single stranded DNA shown in sequence 8 point in the single strand dna as shown in sequence 6 in sequence table of primer pair 2 and sequence table
Son composition.
7. a kind of for identifying whether plant sample to be measured derives from the transgenosis that breeding method as claimed in claim 1 or 2 obtains
The kit of cotton or its offspring, including primer pair 1 described in claim 5 or 6 and/or primer pair 2.
8. a kind of acquisition mirid pentatomid resistance improves and/or plant height improves and/or the first fruit branch length improves and/or fruit branch number improves
And/or the method for the cotton that knot bell number improves and/or ginning outturn improves and/or Single boll weight reduces, include the following steps:
(1) transgene cotton is obtained according to method as claimed in claim 1 or 2；
(2) transgene cotton is selfed or is hybridized, obtain breeding offspring, according to any method of claim 3 to 6
It identifies the breeding offspring, obtains target plant.
9. method according to claim 8, it is characterised in that: the deposit number of the seed of the transgene cotton is CCTCC
NO:P201822。
Any one of 10.D1)-D6):
D1) application of the transgene cotton obtained using breeding method as claimed in claim 1 or 2 in cotton breeding；
D2) application of the exogenous dna fragment described in claims 1 or 2 in regulation Resistance Strain of Cotton mirid pentatomid；
D3) exogenous dna fragment described in claims 1 or 2 is in regulation cotton plant height and/or the first fruit branch length and/or fruit branch
Application in number and/or knot bell number and/or ginning outturn and/or Single boll weight；
D4) exogenous dna fragment described in claims 1 or 2 is identifying whether plant sample to be measured derives from claims 1 or 2
The transgene cotton of the breeding method acquisition or the application in its offspring；
D5) primer pair 1 described in claim 5 or 6 and/or primer pair 2 are identifying whether plant sample to be measured derives from right
It is required that the transgene cotton of the 1 or 2 breeding methods acquisitions or the application in its offspring；
D6) whether upstream flanking sequence described in claim 3 or 4 and/or downstream flanking sequence are identifying plant sample to be measured
Application in the transgene cotton or its offspring that breeding method as claimed in claim 1 or 2 obtains.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811416535.1A CN110106198B (en) | 2018-11-26 | 2018-11-26 | Upland cotton transformation event C006-10-13 and specificity identification method thereof |
| PCT/CN2019/086597 WO2020107816A1 (en) | 2018-11-26 | 2019-05-13 | Upland cotton transformation event 18c006-10-13 and specific identification method therefor |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811416535.1A CN110106198B (en) | 2018-11-26 | 2018-11-26 | Upland cotton transformation event C006-10-13 and specificity identification method thereof |
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| CN110106198B CN110106198B (en) | 2023-03-31 |
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| WO (1) | WO2020107816A1 (en) |
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| CN114561401B (en) * | 2022-03-09 | 2023-08-04 | 中国农业科学院棉花研究所 | Upland cotton GhEXO2 and application thereof in aspect of regulating and controlling plant types |
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| US20050216969A1 (en) * | 2004-03-26 | 2005-09-29 | Dow Agrosciences Llc | Cry1F and Cry1AC transgenic cotton lines and event-specific identification thereof |
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| US20180216131A1 (en) * | 2015-03-27 | 2018-08-02 | Institute Of Cotton Research, Chinese Academy Of Agricultural Sciences | Artificially synthesized insect-resistant protein, biological materials associated therewith, and use thereof |
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2018
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| US20050216969A1 (en) * | 2004-03-26 | 2005-09-29 | Dow Agrosciences Llc | Cry1F and Cry1AC transgenic cotton lines and event-specific identification thereof |
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| US20180216131A1 (en) * | 2015-03-27 | 2018-08-02 | Institute Of Cotton Research, Chinese Academy Of Agricultural Sciences | Artificially synthesized insect-resistant protein, biological materials associated therewith, and use thereof |
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| WO2020107816A1 (en) | 2020-06-04 |
| CN110106198B (en) | 2023-03-31 |
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