CN110106198B - Upland cotton transformation event C006-10-13 and specificity identification method thereof - Google Patents
Upland cotton transformation event C006-10-13 and specificity identification method thereof Download PDFInfo
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- CN110106198B CN110106198B CN201811416535.1A CN201811416535A CN110106198B CN 110106198 B CN110106198 B CN 110106198B CN 201811416535 A CN201811416535 A CN 201811416535A CN 110106198 B CN110106198 B CN 110106198B
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- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
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Abstract
The invention discloses a upland cotton transformation event C006-10-13 and a specificity identification method thereof. The invention leads Cry51Aa1.C006-1 gene into upland cotton CCRI24, leads the gene to be over-expressed in the upland cotton CCRI24 to obtain transgenic cotton, wherein the transgenic cotton is obtained by inserting an exogenous DNA fragment shown in a sequence 1 into the 63328845-63329028 th position of the D12 th chromosome of a target cotton genome and replacing the base sequence of 182bp between the 63328845-63329028 th position of the D12 th chromosome. Experiments prove that the transgenic cotton not only has high resistance in the aspect of lygus lucorum resistance, but also has higher plant height, first fruit branch length, fruit branch number, boll number and clothes score than cotton terrestris CCRI24. The invention lays a foundation for cultivating transgenic cotton with anti-lygus lucorum and has important application value.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a upland cotton transformation event C006-10-13 and a specificity identification method thereof.
Background
The transformation event is a molecular structure consisting of the foreign gene and the foreign gene in the upstream and downstream flanking regions of the genomic insertion site. Generally, transformation of a plant with an exogenous gene results in a population of transformants comprising a plurality of independent events, each of which is unique. Expression of the foreign gene in the plant is affected by the chromosomal location into which the foreign gene is inserted. This may result from the chromatin structure or the influence of transcriptional regulatory elements near the integration site. The expression levels of the same gene in different transformation events vary greatly, and there may also be differences in the spatial or temporal pattern of expression. Moreover, the insertion of foreign genes may also affect the expression of endogenous genes. Thus, the effect of each independent transformation event on the recipient plant is different. The obtained plant transformation event which can effectively express the exogenous gene and does not influence the agronomic characters of the plant has important application value in cultivating new varieties of transgenic crops.
China is the largest cotton producing and consuming country in the world, and is also the largest textile and garment producing and exporting country. The total cotton yield accounts for about 25% of the world cotton yield. The growth and decline of cotton production have certain influence on the development of national economy of China and even the improvement of the living standard of people. Cotton is one of crops with the most serious harm of pests, and at least 300 cotton pests exist in China. Before the transgenic pest-resistant cotton (i.e. the cotton bollworm resistance cotton) is cultivated, the cotton is most seriously damaged by four pests, namely cotton aphid, cotton bollworm, cotton leaf mite and cotton red bollworm, the yield loss of the cotton caused by the pests reaches 15 to 20 percent every year, and the pesticide cost for controlling the pests reaches 1200 to 1800 yuan per hectare every year. Along with the wide planting of the BT insect-resistant cotton and the great reduction of the using amount of field pesticides, lepidoptera pests such as cotton bollworms and the like are fundamentally and effectively controlled, but an ecological system of a cotton field is changed in series, so that the harm of the cotton bollworms is effectively prevented and treated, the number of non-target pests such as cotton aphids, stinkbug weevils and bemisia tabaci is greatly increased and gradually increased to main pests of the cotton field, particularly the stinkbug weevils, and great loss is caused to the production of cotton.
In 2007, cry51Aa1 protein separated from Bt strain F14-1 and Cry51Aa1 gene sequence encoding the protein are submitted to NCBI GenBank with a registration number of DQ836184, and the Cry51Aa1 protein is one of parasporal crystals generated by Bacillus thuringiensis, and has important application prospects in the field of biological control. The inventor of the invention carries out site-directed mutagenesis and in vitro toxicity test research on the Cry51Aa1 gene, and screens and obtains the Cry51Aa1.C006-1 gene with stronger lygus lucorum resistance.
Disclosure of Invention
The invention aims to provide a transgenic cotton breeding method with a lygus lucorum resistance characteristic.
The invention firstly protects a method for cultivating transgenic cotton, which is characterized in that an exogenous DNA fragment is inserted into the No. 63328845-63329028 locus of the No. D12 chromosome of a target cotton genome to replace the base sequence of 182bp between the No. 63328845-63329028 locus of the No. D12 chromosome, so as to obtain the transgenic cotton;
the transgenic cotton has higher lygus resistance and/or plant height and/or first fruit branch length and/or fruit branch number and/or boll number and/or coat score than the target cotton; the single-boll weight of the transgenic cotton is lower than that of the target cotton;
the exogenous DNA fragment can be a DNA molecule containing a Cry51Aa1.C006-1 gene. The nucleotide sequence of the Cry51Aa1.C006-1 gene is shown as 2614-3534 th nucleotides from the 5' end of a sequence 1 in a sequence table.
In the above breeding method, the foreign DNA fragment may be introduced into the cotton of interest by a recombinant vector containing the foreign DNA fragment. The recombinant vector containing the exogenous DNA fragment can be obtained by inserting the expression cassette A between EcoRI and HindIII enzyme cutting sites of the pCambia2301 vector and keeping other sequences of the pCambia2301 vector unchanged; the nucleotide sequence of the expression cassette A is shown as 2186-3822 th from the 5' end of the sequence 1 in the sequence table. The nucleotide sequence of the recombinant vector containing the exogenous DNA fragment can be shown as a sequence 4 in a sequence table.
In the above breeding method, the nucleotide sequence of the exogenous DNA fragment can be shown as sequence 1 in the sequence table.
The cultivation method specifically comprises the steps of inserting the exogenous DNA molecule shown in the sequence 1 in the sequence table into the position 63328845-63329028 of the chromosome D12 of the target cotton genome (the 5 'end of the exogenous DNA molecule shown in the sequence 1 in the sequence table is adjacent to the position 63329028 of the chromosome D12, and the 3' end of the exogenous DNA molecule shown in the sequence 1 in the sequence table is adjacent to the position 63328845 of the chromosome D12), replacing a base sequence of 182bp between the position 63328845-63329028 of the chromosome D12, and obtaining the transgenic cotton.
The preservation number of the seeds of any one of the transgenic cotton in the China center for type culture Collection is CCTCC NO: P201822.
The invention also provides a method for identifying whether a plant sample is derived from the transgenic cotton or the progeny thereof, which comprises the following steps: detecting whether the genome DNA of the plant sample to be detected contains a DNA fragment A; the DNA fragment A consists of any one of the exogenous DNA fragments on the upstream flank of the transgenic cotton, the exogenous DNA fragment and the exogenous DNA fragment on the upstream flank of the transgenic cotton;
if the genomic DNA of the plant sample to be detected contains the DNA fragment A, the plant sample to be detected is or is selected as the transgenic cotton or the descendant thereof;
and if the genomic DNA of the plant sample to be tested does not contain the DNA fragment A, the plant sample to be tested is not or is not candidate to be the transgenic cotton or the descendant thereof.
The upstream flanking segment of any one of the exogenous DNA segments in the transgenic cotton can be any DNA segment which is obtained by extending the chromosome D12 of the target cotton genome from nucleotide 63329028 to the 3' direction of the chromosome and has the length of 0-5 Kb.
The downstream flanking segment of any one of the exogenous DNA segments in the transgenic cotton can be any one DNA segment with the length of 0-5Kb obtained by extending the chromosome D12 of the target cotton genome from nucleotide 63328845 to the 5' direction.
The nucleotide sequence of any upstream flanking sequence can be shown as a sequence 2 in a sequence table.
The nucleotide sequence of any one of the downstream flanking sequences can be shown as a sequence 3 in a sequence table.
Hereinbefore, the transgenic cotton may exhibit a higher resistance to plant bugs than the cotton of interest: compared with the target cotton, the plant height, the first fruit branch length, the fruit branch number, the boll number and the coat length of the transgenic cotton are all obviously improved. Therefore, the transgenic cotton has obviously improved lygus resistant property, and can be used for cultivating lygus resistant cotton in production.
In the above method, the method for detecting whether the genomic DNA of the plant sample to be tested contains the DNA fragment a may be S1) or S2) or S3):
s1) directly sequencing;
s2) carrying out PCR amplification on the genome DNA of the plant sample to be detected by using the primer pair 1 and/or the primer pair 2, and then carrying out judgment as follows: if the target amplification product is obtained, the plant sample to be detected is or is selected as the transgenic cotton or the descendant of the transgenic cotton; if the target amplification product does not exist, the plant sample to be tested is not or is not a candidate to be the transgenic cotton or the descendant thereof;
the primer pair 1 can be a primer pair capable of amplifying a DNA molecule A consisting of the 5' end of the exogenous DNA fragment and a part or all of the fragment of the upstream flanking sequence of the exogenous DNA fragment; the corresponding target amplification product is the DNA molecule A;
the primer pair 2 can be a primer pair which can amplify a DNA molecule B comprising the 3' end of the exogenous DNA fragment and part or all of the downstream flanking sequence of the exogenous DNA fragment; the corresponding target amplification product is the DNA molecule B.
S3) carrying out Southern blot hybridization on the genomic DNA of the plant sample to be detected by using a probe capable of specifically binding the DNA molecule A or the DNA molecule B, and then carrying out the following judgment: if the hybrid fragment can be obtained, the plant sample to be detected is or is selected as the transgenic cotton or the descendant thereof; and if the hybrid fragment cannot be obtained, the plant sample to be tested is not or is not a candidate to be the transgenic cotton or the descendant thereof.
The primer pair 1 can be composed of a single-stranded DNA molecule shown in a sequence 5 in a sequence table and a single-stranded DNA molecule shown in a sequence 7 in the sequence table. The primer pair 2 can be composed of a single-stranded DNA molecule shown in a sequence 6 in a sequence table and a single-stranded DNA molecule shown in a sequence 8 in the sequence table.
The invention also provides a kit for identifying whether a plant sample to be detected is derived from the transgenic cotton or the descendant thereof obtained by the culture method, which comprises any one of the primer pair 1 and/or the primer pair 2.
The invention also protects a method for obtaining cotton with increased plant stinkbug resistance and/or increased plant height and/or increased first fruit branch length and/or increased fruit branch number and/or increased boll number and/or increased coat and/or reduced single boll weight, which may comprise the steps of:
(1) Obtaining transgenic cotton according to any of the above described culturing methods;
(2) Selfing or hybridizing the transgenic cotton to obtain breeding progeny, and identifying the breeding progeny according to any one of the methods to obtain a target plant.
In the method, the preservation number of the seeds of the transgenic cotton in China center for type culture Collection is CCTCC NO: P201822.
The present invention also protects any one of D1) to D6).
D1 Application of the transgenic cotton obtained by adopting any one of the breeding methods in cotton breeding.
D2 Application of any one of the exogenous DNA fragments in regulation and control of cotton plant bug resistance.
D3 Use of any one of the above exogenous DNA fragments in regulating cotton plant height and/or first fruit branch length and/or fruit branch number and/or boll number and/or clothes fraction and/or single boll weight.
D4 Use of any one of the above exogenous DNA fragments in identifying whether a plant sample to be tested is derived from transgenic cotton or progeny thereof obtained by any one of the above culture methods.
D5 Application of any one of the primer pair 1 and/or the primer pair 2 in identifying whether a plant sample to be detected is derived from the transgenic cotton obtained by any one of the breeding methods or progeny thereof.
D6 Use of any of the upstream flanking sequences and/or the downstream flanking sequences described above in the identification of whether a plant sample to be tested is derived from transgenic cotton or progeny thereof obtained by any of the breeding methods described above.
Above, the target cotton may be upland cotton. The upland cotton can be particularly the upland cotton CCRI24.
The invention leads Cry51Aa1.C006-1 gene into upland cotton CCRI24, so that the Cry51Aa1.C006-1 gene is over-expressed in upland cotton CCRI24 to obtain transgenic cotton C006-10-13, wherein the transgenic cotton C006-10-13 is cotton obtained by inserting exogenous DNA fragment into the position 63328845-63329028 of the D12 chromosome of a target cotton genome and replacing 182bp base sequence between 63328845-63329028 of the D12 chromosome. The test proves that the transgenic cotton C006-10-13 not only has high resistance in the aspect of resisting plant bugs, but also has higher plant height, first fruit branch length, fruit branch number, boll number and clothes content than the cotton CCRI24. The invention lays a foundation for cultivating transgenic cotton with anti-lygus lucorum and has important application value.
Drawings
FIG. 1 shows the sequence analysis of C006-10-13.
FIG. 2 is the ex vivo identification of transgenic cotton C006-10-13 and upland cotton CCRI24.
FIG. 3 shows the field growth of the T5 generation-transformed Cry51Aa1.C006-1 cotton homozygous line C006-10-13 and upland cotton CCRI24.
FIG. 4 shows the net-room identification of transgenic cotton C006-10-13 and cotton CCRI24.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention.
The experimental procedures in the following examples are conventional unless otherwise specified.
The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.
The quantitative tests in the following examples, all set up three replicates and the results averaged.
The cotton CCRI24 of the following examples is disclosed in "PAG1, a cotton branched carbohydrate metabolism gene, models fiber electrophoresis", publicly available from the Cotton research institute of the Chinese academy of agricultural sciences ".
Agrobacterium LBA4404 in the following examples is disclosed in the literature "structural expression of the viral genes improvements of plant transformation by Agrobacterium", publicly available from the Cotton research institute of the national academy of agricultural sciences.
The nucleotide sequences of the primers referred to in the examples below are shown in Table 1.
TABLE 1
Note: "-" indicates not present; n is a or c or g or t, s is c or g, and w is a or t.
Example 1 acquisition of transgenic Cry51Aa1.C006-1 Cotton and agronomic traits analysis
1. Obtaining of Cry51Aa1.C006-1 transgenic Cotton
1. Construction of recombinant vectors
The expression cassette A was inserted into the EcoRI and HindIII cleavage sites of the pCambia2301 vector (this plasmid was purchased from Biovector plasmid vector strain cell Gene Collection, having the accession number: biovector plasmid 1300, which is a member of the NTCC type culture Collection), while keeping the other sequences of the pCambia2301 vector unchanged, to obtain a recombinant vector (the nucleotide sequence is shown as sequence 4 in the sequence listing). The nucleotide sequence of the expression cassette A is shown as 2186-3822 th from 5' tail end of the sequence 1 in the sequence table. The expression cassette A sequentially comprises a promoter of CAMV35S from the cauliflower mosaic virus, a Cry51Aa1.C006-1 gene and an NOS terminator from the upstream; wherein the nucleotide sequence of the CAMV35S promoter is represented by nucleotides 2192 to 2537 from the 5' end of a sequence 1 in a sequence table; the nucleotide sequence of the Cry51Aa1.C006-1 gene is shown as 2614-3534 th nucleotides from the 5' end of a sequence 1 in a sequence table; the nucleotide sequence of the NOS terminator is shown as 3569-3821 th nucleotides from the 5' tail end of a sequence 1 in a sequence table.
The pCambia2301 vector itself includes expression cassette B and expression cassette C. The expression cassette B comprises an enhanced promoter of CAMV35S of cauliflower mosaic virus, a KanR antibiotic marker gene and a Ploy A terminator in sequence from the upstream. The expression cassette C comprises, in order from the upstream, the cauliflower mosaic virus CAMV35S promoter, GUS marker gene and NOS terminator.
2. Obtaining of recombinant bacteria
And (3) introducing the recombinant vector obtained in the step (1) into agrobacterium LBA4404 to obtain a recombinant bacterium.
3. Obtaining of transgenic Cry51Aa1.C006-1 Cotton
Transforming the recombinant bacteria obtained in the step 2 into explants (1500) of upland cotton CCRI24 by adopting an agrobacterium-mediated method, culturing and inducing the explants on a culture medium containing kanamycin to form callus, selecting the transformed callus, forming embryogenic callus on a kanamycin screening culture medium by the callus, selecting the surviving embryogenic callus, transforming and forming into cotton, and finally obtaining 50T 0 generation transformed Cry51Aa1.C006-1 cotton in total and using the cotton for screening.
4. Identification of Cry51Aa1.C006-1 transgenic cotton
(1) Harvested cotton seeds transformed from the T1 generation to Cry51Aa1.C006-1 were grown in the greenhouse and subjected to greenhouse efficacy and molecular analysis. Extracting DNA of a T1 generation transformed Cry51Aa1.C006-1 cotton leaf, adopting a primer: 5 'ACCCCATCCAAGGGTTGATACC-3' and 5 'TTGATAGGTGAAGGTCGGAGC-3' are subjected to PCR amplification, whether the target gene exists or not is detected, and the separation ratio of the materials is counted. Obtaining 12 single copies of the cotton transformed from the T1 generation to Cry51Aa1.C006-1 according to Mendelian's law of heredity;
(2) Further validation of 12 single copies of the cotton of the T1 generation Cry51Aa1.C006-1 obtained in step (1) above by Taqman PCR and Southern blot. The method comprises the following specific steps: the expression level of the Cry51Aa1.C006-1 gene of 12 single-copy T1 generation-to-Cry51Aa 1.C006-1 cotton materials and upland cotton CCRI24 materials obtained in the initial flowering stage is analyzed, and the expression level of 9T 1 generation-to-Cry51Aa 1.C006-1 cotton in the 12 single-copy T1 generation-to-Cry51Aa 1.C006-1 cotton is greatly improved compared with the upland cotton CCRI24.
(3) And (3) determining the agronomic characters and the lygus resistance of the 9T 1 generation transgenic Cry51Aa1.C006-1 cotton materials obtained in the step (2) at the flowering stage. The result shows that the partial agronomic characters (such as plant height, first fruit branch length, fruit branch number and boll formation number) and the lygus lucorum resistance of 9T 1 generation to Cry51Aa1.C006-1 cotton in 9T 1 generation to Cry51Aa1.C006-1 cotton are obviously improved compared with cotton CCRI24.
(4) Agronomic and quality traits (plant height, first fruit branch length, fruit branch number, boll number, single boll weight, coat, wherein fiber quality includes length, specific strength, micronaire value, elongation, regularity) and lygus resistance were evaluated for 9T 2 transgenic cry51aa1.C006-1 cottons in field trials in the first year in paired plots at the same location. The results show that: the partial agronomic characters (plant height, first fruit branch length, fruit branch number, boll number and clothes) and the lygus lucorum resistance of 2T 2 generation-to-Cry51Aa 1.C006-1 cottons in the 9T 2 generation-to-Cry51Aa 1.C006-1 cottons are obviously better than that of cotton CCRI24 in upland field.
(5) The agronomic and quality traits (plant height, first fruit branch length, fruit branch number, boll number, single boll weight, clothes branch, wherein the fiber quality comprises length, specific strength, micronaire value, elongation, uniformity) and lygus lucorum resistance of the 2T 2 generation transgenic cry51aa1.C006-1 cotton obtained in step (4) above were evaluated in paired plots at the same location in a field trial of the second year. The results show that: partial agronomic characters (plant height, first fruit branch length, fruit branch number, boll number and clothes) and stinkbug resistance of 1T 2 generation transformed Cry51Aa1.C006-1 cotton in 2T 2 generation transformed Cry51Aa1.C006-1 cotton are obviously better than that of cotton CCRI24 in upland field, seeds of the T2 generation transformed Cry51Aa1.C006-1 cotton are named as C006-10-13, and the T2 generation transformed CryAa 1.C006-1 cotton C006-10-13 is selfed until a T5 generation transformed Cry511. C006-1 cotton homozygous line C006-10-13 is obtained.
C006-10-13 of a cotton seed (Gossypumiumhirsutum L.) with the generation T5 being changed into Cry51Aa1.C006-1 is preserved in a China center for type culture collection in 30.10.2018, and the variety is named as Gossypium hirsutum C006-10-13, and the preservation number is CCTCC NO: P201822.
2. Agronomic character analysis of Cry51Aa1.C006-1 cotton
1. Cotton plant bug resistance identification method
The resistance identification method refers to the industry standard of 'Cotton anti-lygus trait identification method' agricultural industry Standard of people's republic of China (NY/T2676-2015)', published and implemented in 2015.
(1) In vitro identification (see FIG. 2, CCRI24 is upland cotton CCRI 24)
Inserting the cotton branch with small buds into a transparent plastic cup containing 2% agar, then inoculating 3-head lygus nymphs into the plastic cup, covering the upside of the plastic cup with the holes to prevent water vapor, and sealing the cup opening by using a sealing film to prevent the lygus from escaping. When the test is carried out to the 6 th day, the number of live insects and the number of adult insects are investigated.
(2) Net room authentication (see FIG. 4, CCRI24 is land cotton CCRI 24)
1) The cotton seeds should meet the requirements of GB 4407.1 on seed quality;
2) The identification is carried out in a net room, the length, the width and the height of the specification of the net room are respectively 20m multiplied by 3m multiplied by 2m, and the nylon net is 80 meshes;
3) The identification method comprises the following steps:
a. experiment design and management: the cotton materials to be tested and the insect-susceptible control cotton materials are randomly arranged and planted in the net room, each material is planted with 1 row, the plant spacing is 0.25m, and the row spacing is 0.80m. Each cell was replicated once for a total of 3 replicates. The cotton cultivation management mode in the net room is the same as that of the field, the net room is cleaned by spraying chemical pesticide with short residual effect 7d-10d before the test, and no chemical pesticide is applied during the test period.
b. Sowing: the test cotton material and the insect-sensitive control material are both sown according to the conventional sowing time and sowing quantity of local cotton.
c. Test insect feeding: is a mixed population of 1-5-year-old lygus bugs artificially bred with cowpeas, tender corncobs and the like indoors.
d. Test insect release period and release amount: when the cotton peanuts in the net chamber grow to the 4-6 leaf stage, the lygus bugs are released, and when the cotton peanuts are released, individuals with strong activity are selected, and 1 head of each cotton is released.
e. And (4) recording the result: after the test insects are released for 7-10 days, randomly investigating 10 plants for each material in each net room, recording the damage condition of 5 tender leaves on the upper part of the cotton plant, and simultaneously investigating the number of the cotton buds and the total number of the cotton buds. The cotton leaf damage condition is divided into 5 grades, namely 0 grade, 1 grade, 2 grade, 3 grade and 4 grade, and the grading standard is as follows: the 0 level is the harmless state of the blade; the 1 stage is the slight damage of the blade, and the damaged area is less than or equal to 5 percent when the ratio is more than 0 percent; 2, the damage area is equal to or less than 20 percent after 5 percent; the 3-stage is serious damage of the blade, and the damaged area is less than or equal to 50 percent after 20 percent; the 4-stage is the variety with the damage of the leaves to the same time, and the damage area is more than 50 percent.
f. Identification indexes are as follows: the leaf damage index (mean damage level of 5 leaves on the upper part of each cotton plant), the decline rate (%) of the leaf damage index, the damage rate (%) of the cotton buds and the damage decline rate (%) of the cotton buds.
The results of resistance identification are shown in Table 2 (CK is cotton CCRI 24). The results show that the lygus resistance of C006-10-13 is significantly improved compared with the cotton CCRI24.
C006-10-13 and Gossypium hirsutum CCRI24 for corilagin resistance
| Cotton to be tested | Larval mortality (%) | Corrected mortality (%) | Number of adult (head) developing | Proportion of developed adult (%) |
| C006-10-13 | 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. Agronomic traits
The T5 generation-transformed Cry51Aa1.C006-1 cotton homozygous line C006-10-13 and upland cotton CCRI24 are subjected to other agronomic trait investigation and determination. A plot experiment is designed in Anyang in Henan, 3 plots are set, the 3 plots are completely the same, the plot line length is 8m, 30 plants are planted in each line, the line spacing is 80cm, 3 lines are planted in each material, and the total number of plots is 6. Students' test is adopted to carry out statistical analysis on the plant height, the first fruit branch length, the fruit branch number, the boll number and the clothes score of the T5 generation-transformed Cry51Aa1.C006-1 cotton homozygous line C006-10-13 and the cotton CCRI24 on upland field respectively.
The field growth conditions are shown in figure 3 (C006-10-13 is T5 generation transformed Cry51Aa1.C006-1 cotton homozygous line C006-10-13, CCRI24 is upland cotton CCRI 24). The statistical results are shown in Table 3. The result shows that compared with the cotton CCRI24, the T5 generation-to-Cry51Aa 1.C006-1 cotton homozygous strain C006-10-13 has obviously improved plant height, first fruit branch length, fruit branch number, boll number and clothes rate and obviously reduced single boll weight.
TABLE 3 comparison of agronomic traits in transgenic Cotton C006-10-13 and upland Cotton CCRI24
3. Effects of other aspects
Since the promoter is a constitutive promoter, the promoter is not only expressed in green tissues (including stems, leaves, buds and growing points), but also other tissues are influenced to a certain extent. In order to investigate whether the cry51Aa1.C006-1 gene has an effect on other tissues besides improving the resistance of plant bugs, the fiber length, specific strength, micronaire value, uniformity index and elongation of the T5 generation transgenic cry51Aa1.C006-1 cotton homozygous line C006-10-13 and upland cotton CCRI24 were examined.
The statistical results are shown in Table 4. The result shows that compared with the land cotton CCRI24, the T5 generation-to-Cry51Aa 1.C006-1 cotton homozygous strain C006-10-13 has improved micronaire value and elongation rate but has no significant difference, and the fiber length, the breaking ratio strength and the uniformity index are all reduced but have no significant difference.
TABLE 4 comparison of fiber quality data for transgenic cotton C006-10-13 and upland cotton CCRI24
3. Characterization analysis of C006-10-13 DNA sequence
The insert of the C006-10-13 genome and the genomic sequences flanking the insert were analyzed using molecular biology methods. The method comprises the following specific steps:
1. extracting the genome of the cotton. Under greenhouse or field conditions, about 100mg of young leaves of C006-10-13 cotton were placed in a 2.0ml EP tube, the EP tube was frozen with liquid nitrogen, and then ground with a cryo-grinder, and genomic DNA was extracted using the protocol provided in the Qiagen DNeasy Plant Mini Kit (50) (Cat. 69104). The method may be modified by those skilled in the art to extract DNA from any tissue, including but not limited to seeds.
2. The fusion primer and nested FPNI-PCR (Wang et al.2011) was performed according to PCR Master MIX (P213-AA) from Novowed. According to the known sequence of the transgene insert, three nested primers are respectively designed at the 5 'end and the 3' end (the three nested primers at the 5 'end are primer 8, primer 9 and the three nested primers at the 5,3' end are primer 10, primer 11 and primer 7). The first round of PCR primers designed on the cotton genome are fusion primers, and 9 primers (primer 12-primer 20) are used, and the 9 primers are subjected to first round amplification respectively with primer 8 and primer 10. A second round of PCR amplification primer (primer 21) is designed on the cotton genome, and the primer 21 and the primers 9 and 11 are respectively used for second round amplification. A third round of PCR amplification primer (primer 6) is designed on the cotton genome, and the primer 6 is subjected to third round of amplification respectively with the primer 5 and the primer 7, and then 3 rounds of PCR are carried out. The amplicons generated from the reactions were separated by agarose gel electrophoresis, subsequently purified using a QIAGEN gel purification kit (Qiagen, valencia, CA), cloned into a T-cloning vector according to the protocol provided in the pMD-19T-Simple (cat # D104A) kit of Dalibao BioLife technologies, inc., and transformed into E.coli DH 5. Alpha. Transformants were selected on ampicillin resistant plates and colony PCR was performed, and the positive clones were subjected to monoclonal sequencing.
The sequencing result shows that: the transgenic cotton C006-10-13 is obtained by inserting the exogenous DNA molecule shown in the sequence 1 in the sequence table into the position 63328845-63329028 of the chromosome D12 of the CCRI24 genome of Gossypium hirsutum (the 5 'end of the exogenous DNA molecule shown in the sequence 1 in the sequence table is adjacent to the position 63329028 of the chromosome D12, and the 3' end of the exogenous DNA molecule shown in the sequence 1 in the sequence table is adjacent to the position 63328845 of the chromosome D12), replacing a base sequence of 182bp between the positions 63328845-63329028 of the chromosome D12, wherein the nucleotide sequence of an upstream flanking fragment which is upstream from the position 63329028 and is adjacent to the nucleotide 63329028 is sequence 2, and the nucleotide sequence of a downstream flanking fragment which is downstream from the position 63328845 and is adjacent to nucleotide 63328845 is sequence 3 (FIG. 1).
Example 2, obtaining and identifying of the traits of the offspring bred at C006-10-13 and agronomic trait analysis thereof
1. Obtaining of C006-10-13 breeding offspring character
1. Hybridization of
Removing pollen of the first cotton by manual or artificial action 1 afternoon before flowering, covering the stigma of the flower with a wax tube to prevent the contact of foreign pollen with the stigma, and collecting pollen of the second cotton by manual operation when pollen of the second cotton is scattered in the morning next day, and contacting the pollen with the stigma or stigma of the first plant to complete the hybridization process. Wherein, when the first cotton is C006-10-13 in the embodiment, the second cotton is other cotton, or the second cotton is C006-10-13, and the first cotton is other cotton.
And harvesting the hybrid bolls in the boll opening period, naturally drying the cotton, ginning, and using concentrated sulfuric acid to drag down to obtain the hybrid seeds. Planting to obtain hybrid progeny, namely the C006-10-13 breeding progeny.
2. Selfing
Directly clamping the C006-10-13 flower buds by using a grafting clip or binding the flower buds by using a thin line 1 day before blooming to prevent external pollen from contacting with the C006-10-13 stigma during blooming; or the whole cotton plant is sleeved by the mesh bag, so that pollination by insects and the like can be effectively prevented, and self-pollination of cotton can be realized. The selfing can be completed through the steps.
Harvesting hybrid bolls in a boll opening period, naturally drying cotton in the sun, ginning, and using concentrated sulfuric acid to drag down cotton to obtain selfing seeds, and planting to obtain selfing progeny, namely C006-10-13 breeding progeny.
2. Identification of C006-10-13 breeding progeny character and agronomic character analysis thereof
Method for identifying traits of C006-10-13 breeding offspring
The genome DNA of the C006-10-13 breeding offspring is taken as a template, PCR amplification is carried out by using a specific primer, if the PCR amplification product contains an amplicon of C006-10-13 (the amplicon refers to one or one section of DNA molecule synthesized by adopting the PCR amplification technology), the C006-10-13 breeding offspring has the same character as C006-10-13, and if the PCR amplification product does not contain the amplicon of C006-10-13, the C006-10-13 breeding offspring does not have the same character as C006-10-13. The specific identification method is as follows:
1. identification method 1
(1) And (3) performing PCR amplification by using the genome DNA of the C006-10-13 breeding progeny as a template and adopting a primer pair consisting of a primer 5 and a primer 22 to obtain a PCR amplification product.
The PCR amplification system is as follows: 2 XPASTRE PCR MIX 10. Mu.l, primer 5 (10. Mu.M) 0.5. Mu.l, primer 22 (10. Mu.M) 0.5. Mu.l, template DNA (50 ng/. Mu.l) 1. Mu.l, ultrapure water 8. Mu.l, total volume 20. Mu.l.
The PCR reaction conditions were as follows: 5min at 94 ℃; 30s at 94 ℃, 30s at 55 ℃, 45s at 72 ℃ and 32 cycles; 5min at 72 ℃.
(2) Carrying out agarose gel electrophoresis on the PCR amplification product obtained in the step (1), and then judging as follows: if the PCR amplification product contains a DNA fragment of about 500bp, the C006-10-13 breeding progeny contains the C006-10-13 amplicon, and the C006-10-13 breeding progeny has the C006-10-13 character; otherwise, the C006-10-13 trait is not present.
2. Identification method 2
(1) And (3) performing PCR amplification by using the genome DNA of the C006-10-13 breeding progeny as a template and adopting a primer pair consisting of a primer 7 and a primer 23 to obtain a PCR amplification product.
The PCR amplification system is as follows: 2 XPASTRE PCR MIX 10. Mu.l, primer 7 (10. Mu.M) 0.5. Mu.l, primer 23 (10. Mu.M) 0.5. Mu.l, template DNA (50 ng/. Mu.l) 1. Mu.l, ultrapure water 8. Mu.l, total volume 20. Mu.l.
The PCR reaction conditions were as follows: 5min at 94 ℃; 30s at 94 ℃, 30s at 55 ℃, 1min at 72 ℃ and 32 cycles; 5min at 72 ℃.
(2) Carrying out agarose gel electrophoresis on the PCR amplification product obtained in the step (1), and then judging as follows: if the PCR amplification product contains DNA fragments of about 900bp, the result shows that the C006-10-13 breeding offspring contains the C006-10-13 amplicon, and the C006-10-13 breeding offspring has the C006-10-13 character; otherwise, the C006-10-13 trait is not present.
(II) C006-10-13 breeding progeny trait anti-lygus and agronomic trait analysis
The C006-10-13 breeding progeny with the C006-10-13 amplicon obtained in the above step (one) and the anti-plant bug and agronomic trait analysis of cotton upland CCRI24 were examined according to the method of step 1 in step two of example 1.
The results of the agronomic trait analysis are shown in table 5. The result shows that compared with the cotton CCRI24, the plant height, the first fruit branch length, the fruit branch number, the boll number and the clothes score of the C006-10-13 breeding offspring are all obviously improved.
TABLE 5 comparison of agronomic traits in transgenic Cotton C006-10-13 and upland Cotton CCRI24
The results of the lygus resistance identification are shown in table 6. The results show that compared with cotton CCRI24, the plant bug resistance of the offspring bred at C006-10-13 is obviously improved, and the leaf damage degree is also obviously reduced.
TABLE 6 Apolygus resistance of transgenic cotton C006-10-13 and the receptor material CCR124
| Line number | Mortality (%) | Blade damage rating |
| C006-10-13 | 100.00 | 0.00 |
| CK | 37.50 | 0.50 |
<110> Cotton research institute of Chinese academy of agricultural sciences
<120> upland cotton transformation event 18C006-10-13 and method for identifying specificity thereof
<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 (5)
1. A method for identifying whether a plant sample is derived from transgenic cotton or progeny thereof comprising the steps of:
detecting whether the genome DNA of a plant sample to be detected contains a DNA fragment A; the DNA fragment A consists of an exogenous DNA fragment on the upstream flank of the transgenic cotton, the exogenous DNA fragment and an exogenous DNA fragment on the downstream flank of the transgenic cotton;
if the genomic DNA of the plant sample to be detected contains the DNA fragment A, the plant sample to be detected is or is selected as the transgenic cotton or the descendant thereof;
if the genomic DNA of the plant sample to be tested does not contain the DNA fragment A, the plant sample to be tested is not or is not candidate to be the transgenic cotton or the descendant thereof;
the nucleotide sequence of the upstream flanking sequence is shown as a sequence 2 in a sequence table;
the nucleotide sequence of the downstream flanking sequence is shown as a sequence 3 in a sequence table;
the nucleotide sequence of the exogenous DNA fragment is shown as a sequence 1 in a sequence table;
the transgenic cotton is obtained by inserting an exogenous DNA fragment into the chromosome 63328845-63329028 of the D12 th chromosome of a target cotton genome and replacing a base sequence of 182bp between the chromosome 63328845-63329028 of the D12 th chromosome;
the transgenic cotton has higher stinkbug resistance, plant height, first fruit branch length, fruit branch number, boll number and clothes score than the target cotton; the transgenic cotton has a lower single-boll weight than the cotton of interest.
2. The method of claim 1, wherein: the method for detecting whether the genome DNA of the plant sample to be detected contains the DNA fragment A is direct sequencing.
3. A method for obtaining cotton with increased lygus plant resistance, increased plant height, increased first fruit branch length, increased fruit branch number, increased boll bearing number, increased coat-split and reduced single boll weight comprises the following steps:
(1) Obtaining transgenic cotton as claimed in claim 1;
(2) Selfing or crossing the transgenic cotton to obtain breeding progeny, and identifying the breeding progeny according to the method of claim 1 or 2 to obtain a target plant.
4. The method of claim 3, wherein: the preservation number of the transgenic cotton is CCTCC NO: P201822.
5. Use of the method of claim 1 in cotton breeding.
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| 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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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104427862A (en) * | 2012-01-23 | 2015-03-18 | 陶氏益农公司 | Herbicide tolerant cotton event pDAB4468.18.07.1 |
| CN106146628A (en) * | 2015-03-27 | 2016-11-23 | 中国农业科学院棉花研究所 | The insect resistance protein of a kind of synthetic and relevant biological material thereof and application |
| CN106191104A (en) * | 2016-07-18 | 2016-12-07 | 中国农业科学院棉花研究所 | Gossypium hirsutum L. transformation event ICR24001 and specificity identification method thereof |
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| CN101027396B (en) * | 2004-03-26 | 2011-08-03 | 美国陶氏益农公司 | Cry1F and Cry1Ac transgenic cotton lines and event-specific identification thereof |
| AR097292A1 (en) * | 2013-08-09 | 2016-03-02 | Monsanto Technology Llc | ACTIVE PESTICIDE TOXIN AGAINST COLEOPTER AND / OR HEMIPTER INSECTS |
| 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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| CN104427862A (en) * | 2012-01-23 | 2015-03-18 | 陶氏益农公司 | Herbicide tolerant cotton event pDAB4468.18.07.1 |
| CN106146628A (en) * | 2015-03-27 | 2016-11-23 | 中国农业科学院棉花研究所 | The insect resistance protein of a kind of synthetic and relevant biological material thereof and application |
| CN106191104A (en) * | 2016-07-18 | 2016-12-07 | 中国农业科学院棉花研究所 | Gossypium hirsutum L. transformation event ICR24001 and specificity identification method thereof |
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| CN110106198A (en) | 2019-08-09 |
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