CN114468037B - Efficacy and application of gibberellin in delaying softening and aging of okra after picking - Google Patents
Efficacy and application of gibberellin in delaying softening and aging of okra after picking Download PDFInfo
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
- A23B7/00—Preservation or chemical ripening of fruit or vegetables
- A23B7/14—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10
- A23B7/153—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10 in the form of liquids or solids
- A23B7/154—Organic compounds; Microorganisms; Enzymes
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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Abstract
The invention provides effects and applications of gibberellin in delaying softening and aging of okra after picking, and provides applications of gibberellin in maintaining hardness and weightlessness of okra, inhibiting degradation of cell walls, maintaining total pectin content, inhibiting generation of soluble pectin, inhibiting expression of okra AePG1, aePG2, aePG3, aePG4, aePG5, aePG6, aePME1, aePME2, aePL1, aePL2, aePL3, aePL4, aePL5, aecx, aeα -Af1, aeα -Af2, aeα -Af3, aeα -Af4, aeα -Af5, aeβ -Gal1, aeβ -Gal2, aeβ -Gal3, aeβ -Gal4, aeβ -Gal5, aeβ -Gal6 genes, and provides important conditions for delaying aging by measuring the hardness of okra, aescular wall expression and the cell wall of okra, and analyzing the time of the cell wall of the okra by measuring the gene expression of the okra, and analyzing the cell wall degradation of the cell wall and the cell loss, and the degradation of the cell loss of the okra.
Description
Technical Field
The invention belongs to the technical field of vegetable preservation, and particularly relates to an effect of gibberellin in delaying softening and aging of okra after picking and application of gibberellin.
Background
Okra is an annual herb, belonging to the family malvaceae and growing widely in tropical and subtropical countries. The fruit has tender meat quality, smooth taste, unique flavor, and has the effects of promoting digestion, reducing blood lipid, protecting liver, delaying aging, protecting vision and the like because the special high vitamin C, dietary fiber, calcium and low saturated fatty acid are considered as ideal health food. At present, along with the continuous improvement of the attention of the society to green and organic vegetables, okra is one of vegetables with nutrition and health care functions, and has received more and more attention. Okra belongs to the genus okra of the family Malvaceae, is a fruit and vegetable type, is praised as a finger of a fairy due to the fine shape and quite similar fingers, and has higher commodity potential value and processing additional value. Okra is a non-respiratory jump type fruit, and has high moisture content, large surface area, easy water loss, aging and softening, and serious influence on commodity value. Therefore, the method for researching the storage and fresh-keeping of okra and delaying softening and aging after picking has a certain practical application value.
Softening of the fruit indicates that the fruit has developed and matured and even has begun to age. Studies have shown that okra has continuously reduced hardness in the storage process, the weight loss rate is continuously increased, and the cell walls are degraded to soften and age fruits. Gibberellin (GA) is also reported to have a positive effect on delaying softening of certain fruits, for example, CN111657344A discloses a method for maintaining hardness and fresh keeping of strawberries by adopting gibberellin treatment, but no report on preventing softening and aging of okra after picking is provided at present, and no report on delaying weight loss, softening and cell wall degradation of okra during storage of gibberellin and on regulating and controlling enzyme genes related to cell wall degradation and fruit softening during storage of okra is provided.
Disclosure of Invention
In order to solve the problems, the invention provides the effects and the application of gibberellin for delaying post-harvest softening and aging of okra, provides the application of gibberellin for maintaining the hardness and the weight loss rate of okra and inhibiting the degradation of okra cell walls, provides the application of gibberellin for maintaining the total pectin content of okra or inhibiting a soluble pectin generation preparation, and provides the application of gibberellin for inhibiting the expression of the genes of the enzyme AePG1, aePG2, aePG3, aePG4, aePG5, aePG6, the gene of the fruit esterase AePG1 or AeME 2 of okra, the gene of the enzyme AePL1, aePL2, aePL3, aePL4, the gene of the enzyme AeCX, the gene of the enzyme Aeα -L-arabinofuranosidase Af1, the gene of the enzyme Aeα -Af2, the enzyme Aeα -Af3, the gene of the enzyme Aeα -Af4, the gene of the enzyme Aeα -Af5, the gene of the enzyme Aeβ -galactosidase Aeβ -1, the gene of Galβ - β -Gal- β -5, and the gene of Galβ - β -Gal-5. According to the invention, the okra is treated by gibberellin, the hardness, the weight loss rate and the content change of cell wall components of the okra in the storage process are measured, the expression condition of cell wall degradation genes of the okra in the storage process is researched by utilizing a qT-PCR technology, important genes involved in the degradation of the cell wall of the okra are screened out, and the expression difference of the genes between a gibberellin treatment group and a control group is analyzed, so that theoretical basis is provided for softening and aging of the okra in the storage process of the okra in gibberellin treatment and prolonging the fresh-keeping time.
In one aspect, the invention provides the use of gibberellin for the preparation of an okra cell wall degradation-inhibiting formulation.
The invention also provides the application of gibberellin in preparation of okra hardness maintenance or weight loss rate maintenance.
Further, the mass percentage of the gibberellin is 0.001% -0.05% (10 mg/L-500 mg/L).
Most fruits exhibit a rapid decrease in hardness upon softening; the fruit loss rate of water loss is increased; the color of the surface of the fruit changes; degradation of protopectin and increase of water-soluble pectin. In recent years, research on softening of picked fruits is mainly focused on the content of cell wall substances and the action of related enzymes, and many researches show that sugar and other substances contained in the cell wall are continuously degraded in the storage process of the fruits, so that the cell wall structure is damaged, and finally, the fruits are softened and rotten. Pectin, cellulose and hemicellulose are main components forming cell walls, and are crosslinked together through glycosidic bonds and hydrogen bonds, so that the cell wall structure can be maintained to be in a relatively stable state, and the substances are hydrolyzed during storage of the fruits, so that the cell wall structure is changed, and softening and aging of the fruits are caused.
Okra contains a large amount of pectic substances in tender okra pods, and the highest variety pectin content can reach 24.05% by measuring the pectin content of okra of different varieties and different tissue parts. The hardness of okra is continuously reduced in the storage process, the weight loss rate is continuously increased, and the cell walls are degraded to soften and age fruits.
It can be appreciated that the gibberellin provided by the present invention has the use of inhibiting okra cell wall degradation, maintaining okra hardness, or maintaining weight loss rate for various okra. In some embodiments, okra is preferably used as okra.
According to the invention, a great deal of researches show that the okra treated by gibberellin can inhibit the loss of water during the storage period, so that okra cells have better water holding capacity, can effectively control the weight loss of fruits, can effectively relieve the reduction of hardness and the aging of the fruits, and can greatly increase the storage and fresh-keeping time of okra; and gibberellin can also delay the degradation of okra cell walls, inhibit the reduction of cellulose and total pectin content in the okra cell walls during storage, inhibit the generation of soluble pectin, and maintain the integrity of the cell walls, thereby delaying the softening and aging of fruits.
In another aspect, the invention provides the use of gibberellin for the preparation of a formulation that maintains okra cellulose content.
Further, the mass percentage of the gibberellin is 0.001% -0.05%.
Research shows that okra treated by gibberellin can effectively inhibit degradation of cell walls, maintain cellulose content in the cell walls and inhibit degradation of cellulose.
In yet another aspect, the present invention provides the use of gibberellin to prepare a formulation that maintains total pectin content of okra, or inhibits soluble pectin formation.
Further, the mass percentage of the gibberellin is 0.001% -0.05%.
The okra has continuously reduced hardness, continuously increased weight loss rate, continuously degraded protopectin, continuously increased soluble pectin, and degraded cell wall, so that the fruit is softened and aged.
According to a great deal of research, the gibberellin treatment can inhibit degradation of protopectin and generation of soluble pectin to a certain extent, and delay degradation of cell walls, so that the integrity of the cell walls is maintained, and softening and aging of fruits in the storage process are delayed.
In yet another aspect, the present invention provides the use of gibberellin for the preparation of a formulation that inhibits any one or more of the okra polygalacturonase AePG1, aePG2, aePG3, aePG4, aePG5, aePG6 genes.
Further, the sequences of the genes of the AePG1, aePG2, aePG3, aePG4, aePG5 and AePG6 are respectively shown as Seq ID No.1, seq ID No.2, seq ID No.3, seq ID No.4, seq ID No.5 and Seq ID No. 6; the mass percentage of the gibberellin is 0.001% -0.05%.
Enzymes involved in cell wall metabolism are up to 30 more, but PG, PME, PL, cx, α -Af, β -Gal, etc. are generally considered to be closely related to degradation of cell walls and softening of fruits. The synergistic effect of PG, PME and PL, collectively referred to as pectase, is widely believed to lead to the breakdown of pectin in post-harvest fruits and vegetables, playing an important role in determining pectin content.
The polygalacturonase PG of okra is a main enzyme related to softening of okra fruits, the change of the enzyme activity is closely related to the change of the cell wall structure of pulp in the storage process of the picked fruits, the hardness change of the fruits is further influenced, the measurement of the enzyme activity has important significance for revealing the postharvest softening mechanism of the fruits, and the change trend of the PG enzyme gene expression is consistent with the change trend of the PG enzyme activity.
The related genes of okra polygalacturonase PG, including AePG1, aePG2, aePG3, aePG4, aePG5 and AePG6, are screened by transcriptome sequencing. The research shows that gibberellin can inhibit the expression of AePG1, aePG2, aePG3, aePG4, aePG5 and AePG6, thereby delaying the occurrence of fruit softening and aging.
In some modes, the okra is soaked in a 0.001% -0.05% gibberellin solution, so that the expression of any one or more genes of the okra AePG1, aePG2, aePG3, aePG4, aePG5 and AePG6 can be inhibited.
In yet another aspect, the invention provides the use of gibberellin for the preparation of an agent that inhibits expression of an okra pectin fruit esterase AePME1 or AePME2 gene.
Further, the sequences of the AePME1 and AePME2 genes are respectively shown as Seq ID No.7 and Seq ID No. 8; the mass percentage of the gibberellin is 0.001% -0.05%.
The pectate esterase PME of okra is a cell wall-associated pectate hydrolase protein that softens, drops and ages okra fruits by de-esterifying pectin to produce water-soluble pectic acids and methyl esters.
The related genes of okra pectin fruit esterase PME, including AePME1 and AePME2, are screened by transcriptome sequencing. The research shows that gibberellin can inhibit the expression of AePME1 and AePME2, thereby delaying the occurrence of fruit softening and aging.
In yet another aspect, the invention provides the use of gibberellin to prepare a formulation that inhibits the expression of any one or more of the okra pectin lyase AePL1, aePL2, aePL3, aePL4, aePL5 genes.
Further, the sequences of the AePL1, aePL2, aePL3, aePL4 and AePL5 genes are respectively shown as Seq ID No.9, seq ID No.10, seq ID No.11, seq ID No.12 and Seq ID No. 13; the mass percentage of the gibberellin is 0.001% -0.05%.
The pectin lyase PL of okra can catalyze the elimination and cleavage of pectin molecular chains, and the pectin polymer is cleaved through trans-elimination, so that okra fruits are softened and aged.
The related genes of okra pectin lyase PL are screened by transcriptome sequencing, wherein the related genes comprise AePL1, aePL2, aePL3, aePL4 and AePL5. The research shows that gibberellin can inhibit the expression of AePL1, aePL2, aePL3, aePL4 and AePL5, thereby delaying the occurrence of fruit softening and aging.
In yet another aspect, the present invention provides the use of gibberellin for the preparation of an agent that inhibits expression of the Aecx gene of okra cellulase.
Further, the sequence of the AeCx gene is shown in Seq ID No.14, respectively; the mass percentage of the gibberellin is 0.001% -0.05%.
The cellulase Cx of okra can degrade carboxymethyl cellulose and plays an important role in fruit ripening and softening, but can soften and age okra fruits after fruit picking.
The related genes of the okra cellulase Cx, including the AeCX, are screened by transcriptome sequencing. The research shows that gibberellin can inhibit the expression of Aecx, thereby delaying the occurrence of fruit softening and aging.
In yet another aspect, the present invention provides the use of gibberellin for the preparation of a formulation for inhibiting the expression of any one or more of the okra α -L-arabinofuranosidase Aeα -Af1, aeα -Af2, aeα -Af3, aeα -Af4, aeα -Af5 genes.
Further, the sequences of the Aeα -Af1, aeα -Af2, aeα -Af3, aeα -Af4 and Aeα -Af5 genes are respectively shown as Seq ID No.15, seq ID No.16, seq ID No.17, seq ID No.18 and Seq ID No. 19; the mass percentage of the gibberellin is 0.001% -0.05%.
The alpha-L-arabinofuranosidase alpha-Af of okra is an important enzyme for degrading cell wall polysaccharide, has a very important role in mature softening of fruits, but can soften and age okra fruits after fruit harvest.
The related genes of the okra alpha-L-arabinofuranosidase alpha-Af are screened by transcriptome sequencing, and comprise the Ae alpha-Af 1, the Ae alpha-Af 2, the Ae alpha-Af 3, the Ae alpha-Af 4 and the Ae alpha-Af 5. The research shows that gibberellin can inhibit the expression of Aeα -Af1, aeα -Af2, aeα -Af3, aeα -Af4 and Aeα -Af5, thereby delaying the occurrence of fruit softening and aging.
In yet another aspect, the present invention provides the use of gibberellin for the preparation of a gene expression formulation that inhibits any one or more of the Abelmoschus esculentus β -Gal1, aeβ -Gal2, aeβ -Gal3, aeβ -Gal4, aeβ -Gal5, aeβ -Gal6 genes.
Further, the sequence of the Aeβ -Gal1, aeβ -Gal2, aeβ -Gal3, aeβ -Gal4, aeβ -Gal5, aeβ -Gal6 genes is shown as Seq ID No.20, seq ID No.21, seq ID No.22, seq ID No.23, seq ID No.24, seq ID No.15, respectively; the mass percentage of the gibberellin is 0.001% -0.05%.
Beta-galactosidase beta-Gal of okra can degrade cell wall components, so that cell wall structures are damaged, and fruits are softened, but the fruits of okra are softened and aged after being picked.
The related genes of the okra beta-galactosidase beta-Gal are screened by transcriptome sequencing, and comprise Ae beta-Gal 1, ae beta-Gal 2, ae beta-Gal 3, ae beta-Gal 4, ae beta-Gal 5 and Ae beta-Gal 6. The research shows that gibberellin can inhibit the expression of Aeβ -Gal1, aeβ -Gal2, aeβ -Gal3, aeβ -Gal4, aeβ -Gal5 and Aeβ -Gal6, thereby delaying the occurrence of fruit softening and aging.
In yet another aspect, the present invention provides a use of a gene for promoting softening senescence of okra, the gene comprising AePG1, aePG2, aePG3, aePG4, aePG5, aePG6, aePME1, aePME2, aePL1, aePL2, aePL3, aePL4, aePL5, aecx, aeα -Af1, aeα -Af2, aeα -Af3, aeα -Af4, aeα -Af5, aeβ -Gal1, aeβ -Gal2, aeβ -Gal3, aeβ -Gal4, aeβ -Gal5, aeβ -Gal6, the sequence of which is shown as Seq ID No.1 to Seq ID No.25, respectively.
The invention firstly screens related genes for inhibiting okra softening and aging from okra genes through a large number of transcription sequencing works, defines specific sequences of the related genes, and surveys the variation trend of the expression condition of each gene in the okra storage period.
The application of gibberellin provided by the invention for delaying softening and aging after harvest of okra has the following beneficial effects:
1. the gibberellin is used for maintaining the hardness of okra or the weight loss rate, and can be used for preparing a preparation for maintaining the hardness of okra or the weight loss rate;
2. the gibberellin is found to be used for inhibiting the degradation of okra cell walls and can be used for preparing a preparation for inhibiting the degradation of okra cell walls;
3. the gibberellin is found to be used for maintaining the content of the okra cellulose, maintaining the total pectin content of the okra or inhibiting the generation of the soluble pectin, and can be used for preparing a preparation for maintaining the content of the okra cellulose, maintaining the total pectin content of the okra or inhibiting the generation of the soluble pectin;
4. the gibberellin is found to be used for inhibiting the expression of the genes of the okra polygalacturonases AePG1, aePG2, aePG3, aePG4, aePG5 and AePG6 and can be used for preparing preparations for inhibiting the expression of the genes of the okra polygalacturonases AePG1, aePG2, aePG3, aePG4, aePG5 and AePG 6;
5. the application of gibberellin in inhibiting the expression of the genes of the Abelmoschus esculentus pectin fruit esterases AePME1 and AePME2 is found, and the gibberellin can be used for preparing preparations for inhibiting the expression of the genes of the Abelmoschus esculentus pectin fruit esterases AePME1 and AePME 2;
6. The application of gibberellin in inhibiting the expression of the genes of the okra pectin lyase AePL1, aePL2, aePL3, aePL4 and AePL5 is found, and the gibberellin can be used for preparing preparations for inhibiting the expression of the genes of the okra pectin lyase AePL1, aePL2, aePL3, aePL4 and AePL 5;
7. the application of gibberellin in inhibiting the expression of the Abelmoschus esculentus cellulase Aecx gene is found, and the gibberellin can be used for preparing a preparation for inhibiting the expression of the Abelmoschus esculentus cellulase Aecx gene;
8. the gibberellin is found to be applied to inhibiting the expression of the okra alpha-L-arabinofuranosidases Aealpha-Af 1, aealpha-Af 2, aealpha-Af 3, aealpha-Af 4 and Aealpha-Af 5, and can be used for preparing preparations for inhibiting the expression of the okra alpha-L-arabinofuranosidases Aealpha-Af 1, aealpha-Af 2, aealpha-Af 3, aealpha-Af 4 and Aealpha-Af 5;
9. the gibberellin is found to be applied to inhibiting the expression of Abelmoschus esculentus beta-Gal 1, abelmoschus esculentus beta-Gal 2, abelmoschus esculentus beta-Gal 3, abelmoschus esculentus beta-Gal 4, abelmoschus esculentus beta-Gal 5 and Abelmoschus esculentus beta-Gal 6, and can be used for preparing preparations for inhibiting the expression of Abelmoschus esculentus beta-Gal 1, abelmoschus esculentus beta-Gal 2, abelmoschus esculentus beta-Gal 3, abelmoschus esculentus beta-Gal 4, abelmoschus escu 5 and Abelmoschus esculentus beta-Gal 6;
10. provides a theoretical basis for gibberellin treatment and softening aging and prolonging fresh-keeping time in the okra storage process.
Drawings
FIG. 1 is a graph showing the effect of gibberellin treatment and control on okra weightlessness in example 1, wherein the differences at levels of 0.05, 0.01 and 0.001 are significant;
Fig. 2 is a graph showing the effect of gibberellin treatment and control on okra hardness in example 2, wherein the differences at levels of 0.05, 0.01, and 0.001 are significant, respectively;
FIG. 3 is a graph showing the effect of gibberellin treatment and control on okra cell wall composition levels in example 3, wherein the differences are significant at levels of 0.05, 0.01, and 0.001, respectively;
FIG. 4 shows the alignment of the polygalacturonase PG-related gene sequences of AePG1 and hibiscus in example 4;
FIG. 5 shows the alignment of the polygalacturonase PG-related gene sequences of AePG2 and hibiscus in example 4;
FIG. 6 shows the alignment of AePG3 and polygalacturonase PG related gene sequences of hibiscus in example 4;
FIG. 7 shows the alignment of the polygalacturonase PG-related gene sequences of AePG4 and hibiscus in example 4;
FIG. 8 shows the alignment of AePG5 and polygalacturonase PG related gene sequences of hibiscus in example 4;
FIG. 9 shows the alignment of AePG6 and polygalacturonase PG related gene sequences of hibiscus in example 4;
fig. 10 is a graph showing the effect of gibberellin treatment on gene expression of okra aeli 1, aePG2, aePG3, aePG4, aePG5, and AePG6 in example 4, wherein the differences at 0.05, 0.01, and 0.001 levels are shown to be significant;
FIG. 11 is a graph showing the effect of gibberellin treatment and control on the gene expression of okra AePME1 and AePME2 in example 5, wherein the differences are significant at 0.05, 0.01, and 0.001 levels, respectively;
FIG. 12 shows the result of comparison of the gene sequences related to pectin lyase PL of hibiscus and cotton with AePL2 in example 6;
FIG. 13 shows the result of comparison of the gene sequences related to pectin lyase PL of AePL3 and hibiscus and cotton in example 6;
fig. 14 is a graph showing the effect of gibberellin treatment on gene expression of okra aeli 1, aePL2, aePL3, aePL4, and AePL5 in example 6, wherein the differences at 0.05, 0.01, and 0.001 levels, respectively;
FIG. 15 is a graph showing the effect of gibberellin treatment and control on the expression of okra Aecx gene in example 7, wherein the differences at the levels of 0.05, 0.01 and 0.001 are significant;
FIG. 16 shows the results of an alignment of Aeα -Af1 with the α -L-arabinofuranosidase α -Af-related gene sequences of hibiscus and cotton in example 8;
FIG. 17 shows the result of comparison of the gene sequences related to Aeα -Af2 and α -L-arabinofuranosidase α -Af of hibiscus and cotton in example 8;
FIG. 18 shows the result of comparison of the gene sequences related to Aeα -Af3 and α -L-arabinofuranosidase α -Af of hibiscus and cotton in example 8;
FIG. 19 shows the result of comparison of the gene sequences related to Aeα -Af4 and α -L-arabinofuranosidase α -Af of hibiscus and cotton in example 8;
FIG. 20 shows the result of comparison of the gene sequences related to Aeα -Af5 and α -L-arabinofuranosidase α -Af of hibiscus and cotton in example 8;
FIG. 21 is a graph showing the effect of gibberellin treatment on expression of the genes of Abelmoschus esculentus Aeα -Af1, aeα -Af2, aeα -Af3, aeα -Af4 and Aeα -Af5 in example 8 compared to the control, wherein the differences are significant at the levels of 0.05, 0.01 and 0.001, respectively;
FIG. 22 shows the results of an alignment of Ae beta-Gal 1 with respect to the β -Gal gene of hibiscus, cotton, durian, and cocoa;
FIG. 23 shows the results of an alignment of Ae beta-Gal 2 with respect to the β -Gal gene of hibiscus, cotton, durian, and cocoa;
FIG. 24 shows the results of an alignment of Ae beta-Gal 3 with respect to the β -Gal gene of hibiscus, cotton, durian, and cocoa;
FIG. 25 shows the results of an alignment of Ae beta-Gal 4 with respect to the β -Gal gene of hibiscus, cotton, durian, and cocoa;
FIG. 26 shows the results of an alignment of Ae beta-Gal 5 with respect to the β -Gal gene of hibiscus, cotton, durian, and cocoa;
Fig. 27 is a graph showing the effect of gibberellin treatment on gene expression of okra Ae β -Gal1, ae β -Gal2, ae β -Gal3, ae β -Gal4, ae β -Gal5, and Ae β -Gal6 in example 9 versus control, wherein the differences are significant at the 0.05, 0.01, and 0.001 levels, respectively.
Detailed Description
The following description of the preferred embodiments of the present invention in further detail with reference to the accompanying drawings, it should be noted that the following embodiments are intended to facilitate an understanding of the present invention, and are not intended to limit the invention in any way, and all of the features disclosed in the embodiments of the present invention, or all of the steps in the methods or processes disclosed, can be combined in any way, except mutually exclusive features and/or steps.
Example 1 Effect of gibberellin treatment on okra loss in weight
In the embodiment, newly picked okra (purchased from the state district heart-linked vegetable market of Ningbo city) is adopted, and 200 okra which are relatively uniform in size, full in size and free of black spots and damages are divided into 2 groups (control group and treatment group), and 100 okra are selected from each group. Preparing gibberellin aqueous solution with the mass percentage content of 0.01% (100 mg/L), and soaking okra in the gibberellin aqueous solution for 15min. After the soaking, naturally air-drying 2 groups of okra at normal temperature, and finally storing in a constant temperature and humidity incubator at 25+/-1 ℃ and 80-90% relative humidity for 12 days. Samples were taken before (day 0) and after 3, 6, 9, and 12 days of storage, respectively, and the weight loss change of okra in the control group and the treated group was measured, and the weight loss rate was calculated.
The detection method of the weight loss rate of okra comprises the following steps: a mass loss method is adopted. 10 fruits are selected, taken out at the same time every day, weighed by a balance, and measured repeatedly three times. The weight loss rate, which is the percentage of the mass difference between the sample before and after storage, is calculated as follows:
the change in the weight loss rate of the okra in the two groups is shown in fig. 1, wherein the changes in the weight loss rate of the okra in the two groups are shown as significant at the levels of 0.05, 0.01 and 0.001 respectively.
As can be seen from fig. 1, the loss rates of okra in the treatment group and the control group increased continuously with the increase of the storage time until the end of the storage, the loss rate of okra in the treatment group increased to 2.9% and the loss rate of okra in the control group increased to 3.3%, and the loss rate of okra in the control group was always higher than that in the treatment group during the whole storage period and had significance (P < 0.05) at 9d and 12 d. Therefore, the gibberellin treatment can well inhibit the loss of the okra moisture during the storage period, especially in the later period of the storage, so that the okra cells have good water holding capacity, and the weight loss of fruits is effectively controlled.
In this example, experiments were performed using 0.001%, 0.003%, 0.005%, 0.007%, 0.01%, 0.03%, and 0.05% gibberellin aqueous solutions, respectively, to achieve a certain effect of preventing okra from water loss and delaying the increase of the weight loss rate, and the description is omitted herein because of the limitation of the space.
Example 2 Effect of gibberellin treatment on okra hardness
In the embodiment, newly picked okra (purchased from the state district heart-linked vegetable market of Ningbo city) is adopted, and 200 okra which are relatively uniform in size, full in size and free of black spots and damages are divided into 2 groups (control group and treatment group), and 100 okra are selected from each group. Preparing gibberellin aqueous solution with the mass percentage content of 0.01%, and soaking okra in the gibberellin aqueous solution for 15min. After the soaking, naturally air-drying 2 groups of okra at normal temperature, and finally storing in a constant temperature and humidity incubator at 25+/-1 ℃ and 80-90% relative humidity for 12 days. Samples were taken before (day 0) and after 3, 6, 9, 12 days of storage, respectively, and the hardness change of okra in the control group and the treated group was measured.
The method for measuring the hardness of okra comprises the following steps: the hardness of okra fruit was measured using TMS-Touch physical Analyzer (FTC Co., USA). 8 fruits were selected, and the measurement was performed at the maximum transverse diameter of the waist of the fruit, and the measurement was repeated three times, and the result was represented by N.
The hardness changes of the okra of the two groups are shown in fig. 2, wherein the changes of the okra at the levels of 0.05, 0.01 and 0.001 are respectively obvious.
As can be seen from FIG. 2, the hardness of the control group was continuously lowered during the storage period of 0 to 12d, the hardness at the end of the storage period was 17.2N, and the hardness was lowered by 32.1% as compared with the hardness before the storage. The hardness of the fruits of the treatment group was kept well in the early stage of storage, and the hardness at the end of storage was 20.4N, which was reduced by 19.4% compared with the hardness before storage. The hardness of fruits in the treated group was significantly higher than that in the control group (P < 0.05) throughout the whole storage period. The results show that the gibberellin treatment can effectively delay the reduction of hardness and the aging of fruits during the storage period of okra, and the storage and fresh-keeping time of okra is prolonged to a certain extent.
In this example, experiments were performed using 0.001%, 0.003%, 0.005%, 0.007%, 0.01%, 0.03%, and 0.05% gibberellin aqueous solutions, respectively, to achieve a certain effect of delaying hardness reduction and fruit senescence during okra storage, and therefore, the present invention is not limited by the space.
Example 3 Effect of gibberellin treatment on okra cell wall component content
In the embodiment, newly picked okra (purchased from the state district heart-linked vegetable market of Ningbo city) is adopted, and 200 okra which are relatively uniform in size, full in size and free of black spots and damages are divided into 2 groups (control group and treatment group), and 100 okra are selected from each group. Preparing gibberellin aqueous solution with the mass percentage content of 0.01%, and soaking okra in the gibberellin aqueous solution for 15min. After the soaking, naturally air-drying 2 groups of okra at normal temperature, and finally storing in a constant temperature and humidity incubator at 25+/-1 ℃ and 80-90% relative humidity for 12 days. Samples were taken before (day 0) and after 3, 6, 9, 12 days of storage, respectively, and the changes in the cell wall contents of okra in the control group and the treated group were measured. The change of the cell wall component content mainly comprises the change of protopectin, soluble pectin and cellulose content during the storage period of okra. Protopectin is gradually decomposed during the storage period of okra, soluble pectin is generated, cellulose is also gradually degraded, and fruits are gradually aged and softened.
The detection method of the pectic substances of okra comprises the following steps: the carbazole colorimetric method is adopted for measurement. And drawing and calculating a standard curve of pectin content by taking the mass of the treated galacturonic acid as an abscissa and the absorbance value at the wavelength of 530nm as an ordinate. And respectively adding 1.0mL of extracted protopectin and 1.0mL of soluble pectin into a 25mL graduated test tube, and measuring the content of protopectin and soluble pectin in the sample according to the operation steps of a standard curve, wherein the total content of protopectin and soluble pectin is the total pectin content after repeating the three times of measurement.
The method for measuring the cellulose content of okra comprises the following steps: and (3) measuring by adopting an anthrone colorimetric method. And drawing and calculating a standard curve of the cellulose content by taking the treated cellulose content as an abscissa and the absorbance value at the wavelength of 620nm as an ordinate. The diluted 1.0mL solution is added into a 25mL graduated test tube, the cellulose content in the sample is measured according to the operation steps of a standard curve, and the measurement is repeated three times.
The change in cell wall component content of the okra in the two groups is shown in fig. 3, wherein the changes are shown in the levels of 0.05, 0.01 and 0.001.
As can be seen from FIG. 3, the protopectin content of okra in the pre-storage treatment group and the control group is slowly reduced without significant difference (P > 0.05), but the protopectin content of okra in the post-storage treatment group and the control group is greatly reduced, the reduction rate of the control group is far higher than that of the treatment group, the protopectin content of okra in the treatment group is significantly higher than that of the control group (P < 0.05) at 9d and 12d, and the protopectin content of okra in the treatment group and the control group is respectively reduced by 31.27% and 50.04% at the end of storage, so that the reduction of the protopectin content of okra can be effectively delayed during storage after gibberellin treatment.
The soluble pectin content of okra in the treatment group and the control group gradually increases during the storage period, and the rising rate of the control group is higher than that of the treatment group. The control group had a greater soluble pectin content than the treatment group throughout the storage period, and there was a significant difference at 6d, 9d and 12d (P < 0.05); at the end of storage, the soluble pectin content in the treated group and the control group was 5.24mg/g and 7.08mg/g, respectively; it can be seen that okra can effectively inhibit the formation of soluble pectin during storage after being treated by gibberellin.
The total pectin content of the okra of the two groups generally showed a small increase trend, but the total pectin content of the control group suddenly decreased at 9d, and the total pectin content of the treatment group and the control group did not significantly differ during storage (P > 0.05). The main reason is that the total pectin is the total content of protopectin and soluble pectin, and the protopectin is gradually degraded into the soluble pectin during the storage period of okra, so that the protopectin content is continuously reduced, the soluble pectin content is continuously increased, and the fruits are gradually softened and aged, but the change of the total pectin content is not obvious, so that the total pectin content of two groups of okra has no obvious difference.
The change in cellulose content during storage of okra after exogenous GA treatment is shown in FIG. 3. In addition to the small increase in the treatment group at 3d, the cellulose content of the treatment group and the control group showed different degrees of decrease trend during the whole storage period, the decrease rate of the treatment group was lower than that of the control group, the cellulose content of the treatment group was also higher than that of the control group during the whole storage period, and the cellulose content of the treatment group was significantly higher than that of the control group (P < 0.05) at 9d and 12d, until the cellulose contents of the treatment group and the control group were decreased by 19.5% and 38.5%, respectively, and it was found that okra after gibberellin treatment was effective in inhibiting the degradation of cellulose during the storage period.
The results show that gibberellin treatment can inhibit degradation of protopectin and generation of soluble pectin to a certain extent, inhibit degradation of cellulose, and delay degradation of cell walls, so that the integrity of the cell walls is maintained, and softening and aging of fruits in the storage process are delayed.
In this example, experiments were performed using aqueous solutions of 0.001%, 0.003%, 0.005%, 0.007%, 0.01%, 0.03%, and 0.05% gibberellin, respectively, which have effects of inhibiting degradation of protopectin, formation of soluble pectin, and degradation of cellulose during storage of okra to some extent, and are not illustrated herein.
Example 4 Effect of gibberellin treatment on expression of okra polygalacturonase PG-related Gene
In the embodiment, newly picked okra (purchased from the state district heart-linked vegetable market of Ningbo city) is adopted, and 200 okra which are relatively uniform in size, full in size and free of black spots and damages are divided into 2 groups (control group and treatment group), and 100 okra are selected from each group. Preparing gibberellin aqueous solution with the mass percentage content of 0.01%, and soaking okra in the gibberellin aqueous solution for 15min. After the soaking, naturally air-drying 2 groups of okra at normal temperature, and finally storing in a constant temperature and humidity incubator at 25+/-1 ℃ and 80-90% relative humidity for 12 days. Samples were taken before (day 0) and after 3, 6, 9, and 12 days of storage, respectively, and changes in expression of polygalacturonase PG-related genes AePG1, aePG2, aePG3, aePG4, aePG5, and AePG6 of okra in the control group and the treatment group were measured.
The related genes of polygalacturonase PG of okra (which are subjected to transcriptome sequencing by Kyoto-Dio Biotechnology Co., ltd.) are selected by transcriptome sequencing, and comprise AePG1, aePG2, aePG3, aePG4, aePG5 and AePG6, and the sequences of the genes are respectively shown as Seq ID No. 1-Seq ID No. 6. The sequences of the AePG1, the AePG2, the AePG3, the AePG4, the AePG5 and the AePG6 are respectively compared with the polygalacturonase PG related gene sequences of cotton and hibiscus on NCBI, and the results are respectively shown in figures 4-9, wherein the comparison result of the AePG1 is shown in figure 4, the comparison result of the AePG2 is shown in figure 5, the comparison result of the AePG3 is shown in figure 6, the comparison result of the AePG4 is shown in figure 7, the comparison result of the AePG5 is shown in figure 8, the comparison result of the AePG6 is shown in figure 9, and the results are highly similar, so that the AePG1, the AePG2, the AePG3, the AePG4 and the AePG6 genes are truly the polygalacturonase PG related genes of okra.
The total RNA extraction and cDNA synthesis method comprises the following steps: about 0.2g of jelly sample was weighed, and total RNA of okra was extracted with column type plant RNA extraction kit 2.0 (Enzernine Gene technologies Co., ltd., beijing). The quality of the extracted RNA is detected by using a nucleic acid protein instrument and an agarose electrophoresis instrument. The cDNA was reverse transcribed using a HiFiScript cDNA Synthesis Kit kit (Beijing kang Biometrics Co.).
In the embodiment, the change condition of the expression of the AePG1, the AePG2, the AePG3, the AePG4, the AePG5 and the AePG6 in okra is analyzed through real-time quantitative PCR, specific primers of the AePG1, the AePG2, the AePG3, the AePG4, the AePG5 and the AePG6 are designed by a Beacon Designer 7, and the AeACT is taken as an internal reference gene, wherein the primer sequences are shown in table 1. The three-step amplification was performed using a Real-Time PCR instrument under the samer flag, usa, according to the DyNAmo Flash SYBR Green qPCR kit instructions of samer feier, usa. The following program settings were made: pre-denaturation at 95℃for 7min; this step was performed for 39 cycles of 95℃15S,45℃30s anneal, 75℃15s extension. Each sample was subjected to 4 biological replicates and the relative expression level was 2 -ΔCT The method performs computational analysis.
TABLE 1 fluorescent quantitative PCR
The expression of the genes of AePG1, aePG2, aePG3, aePG4, aePG5 and AePG6 of the two groups of okra is shown in fig. 10, wherein the expression of the genes is shown as significant at the levels of 0.05, 0.01 and 0.001.
As can be seen from fig. 10, the control group showed a tendency of ascending followed by descending of the AePG1, aePG2, aePG3, aePG4, and AePG5, except for the gradual ascending trend of the AePG6 during storage. The expression modes of the AePG s genes in the treatment group are various during the storage period, wherein the AePG1 and the AePG4 are expressed and risen in the early storage period, the expression in the mid storage period is reduced, the AePG2 is in a rising trend after the storage period, and the AePG3, the AePG5 and the AePG6 are in a rising trend before the storage period. Gibberellin treatment promotes the expression of AePG4 and AePG5 at several time points during storage while inhibiting the expression of AePG1, aePG2, aePG3, aePG6 throughout storage, and significantly inhibits the expression (P < 0.05) at both 9d and 12d, and the expression intensity in these four genes is much higher than AePG4 and AePG5, so gibberellin treatment is an inhibitory effect on the AePGs gene of okra as a whole.
In this example, experiments were performed using 0.001%, 0.003%, 0.005%, 0.007%, 0.01%, 0.03%, and 0.05% gibberellin aqueous solutions, respectively, which have a certain effect of inhibiting the expression of okra AePG genes, and therefore, the present invention is not limited by the space.
Example 5 Effect of gibberellin treatment on okra pectin fruit esterase PME-related Gene expression
In the embodiment, newly picked okra (purchased from the state district heart-linked vegetable market of Ningbo city) is adopted, and 200 okra which are relatively uniform in size, full in size and free of black spots and damages are divided into 2 groups (control group and treatment group), and 100 okra are selected from each group. Preparing gibberellin aqueous solution with the mass percentage content of 0.01%, and soaking okra in the gibberellin aqueous solution for 15min. After the soaking, naturally air-drying 2 groups of okra at normal temperature, and finally storing in a constant temperature and humidity incubator at 25+/-1 ℃ and 80-90% relative humidity for 12 days. Samples were taken before (day 0) and after 3, 6, 9, 12 days of storage, respectively, and changes in the expression of the pectate esterase PME-associated genes AePME1 or AePME2 of okra in the control group and the treatment group were determined.
The relevant genes for pectate esterase PME of okra (delegated to Kyoto Dio Biotechnology Co., ltd.) were selected by transcriptome sequencing in this example, including AePME1 and AePME2, whose sequences are shown in Seq ID No.7 and Seq ID No.8, respectively. The sequences of the AePME1 and the AePME2 are compared with the sequences of the pectin fruit esterase PME related genes of cotton, cocoa and hibiscus on NCBI, and the comparison results show that the sequences are highly similar, so that the AePME1 and the AePME2 are proved to be the pectin fruit esterase PME related genes of okra.
The total RNA extraction and cDNA synthesis method comprises the following steps: about 0.2g of jelly sample was weighed, and total RNA of okra was extracted with column type plant RNA extraction kit 2.0 (Enzernine Gene technologies Co., ltd., beijing). The quality of the extracted RNA is detected by using a nucleic acid protein instrument and an agarose electrophoresis instrument. The cDNA was reverse transcribed using a HiFiScript cDNA Synthesis Kit kit (Beijing kang Biometrics Co.).
In the embodiment, the change condition of the expression of the AePME1 and the AePME2 in okra is analyzed by real-time quantitative PCR, specific primers of the AePME1 and the AePME2 are designed by a Beacon Designer 7, and the sequence of the primers is shown in table 2 by taking the AeACT as an internal reference gene. The three-step amplification was performed using a Real-Time PCR instrument under the samer flag, usa, according to the DyNAmo Flash SYBR Green qPCR kit instructions of samer feier, usa. The following program settings were made: pre-denaturation at 95℃for 7min; this step was performed for 39 cycles of 95℃15S,45℃30s anneal, 75℃15s extension. Each sample was subjected to 4 biological replicates and the relative expression level was 2 -ΔCT The method performs computational analysis.
TABLE 2 fluorescent quantitative PCR
The expression of the AePME1 and AePME2 genes of the two okra groups is shown in fig. 11, wherein the expression of the AePME1 and the AePME2 shows that the difference at the level of 0.05, 0.01 and 0.001 is significant.
As can be seen from fig. 11, the expression patterns of the okra in the treatment group and the control group tended to rise and then fall during the storage period, except that the control group began to fall after rising to 9d and the control group began to fall after rising to 6d, and the expression amount of the AePME1 in the control group was significantly higher than that in the treatment group (P < 0.05) during the whole storage period, and it was seen that gibberellin treatment had a significant inhibitory effect on the AePME1 gene expression of okra.
The expression pattern of the AePME2 in the treatment group during storage was slowly increased with the extension of the storage time, and the expression pattern was also slowly increased in the early storage period but was sharply increased when the later storage period was reached in the control group, so that the expression of the AePME2 in the control group was significantly higher than that in the treatment group (P < 0.05). It can be seen that gibberellin treatment has an obvious inhibition effect on AePME2 gene expression of okra.
Thus, gibberellin treatment effectively inhibited the expression of AePME1 and AePME 2.
In this example, experiments were performed using 0.001%, 0.003%, 0.005%, 0.007%, 0.01%, 0.03%, and 0.05% gibberellin aqueous solutions, respectively, which can achieve a certain effect of inhibiting the expression of the okra AePME1 and AePME2 genes, and the description thereof will not be given here.
Example 6 Effect of gibberellin treatment on okra pectin lyase PL-related Gene expression
In the embodiment, newly picked okra (purchased from the state district heart-linked vegetable market of Ningbo city) is adopted, and 200 okra which are relatively uniform in size, full in size and free of black spots and damages are divided into 2 groups (control group and treatment group), and 100 okra are selected from each group. Preparing gibberellin aqueous solution with the mass percentage content of 0.01%, and soaking okra in the gibberellin aqueous solution for 15min. After the soaking, naturally air-drying 2 groups of okra at normal temperature, and finally storing in a constant temperature and humidity incubator at 25+/-1 ℃ and 80-90% relative humidity for 12 days. Samples were taken before (day 0) and after 3, 6, 9, 12 days of storage, respectively, and changes in pectin lyase PL-related gene AePL1, aePL2, aePL3, aePL4, aePL5 expression in control and treatment groups of okra were measured.
The relevant genes of pectin lyase PL of okra (delegated to Kyoto Dio Biotechnology Co., ltd.) were selected by transcriptome sequencing in this example, including AePL1, aePL2, aePL3, aePL4 and AePL5, the sequences of which are shown in Seq ID No.9 to Seq ID No.13, respectively. The sequences of the AePL1, the AePL2, the AePL3, the AePL4 and the AePL5 are aligned with the pectin lyase PL related gene sequences of hibiscus and cotton on NCBI (the partial alignment results are shown in figures 12-13 because of limited space, the alignment results of the AePL2 are shown in figure 12, the alignment results of the AePL3 are shown in figure 13), the results all prove that the sequences are highly similar, and the genes of the AePL1, the AePL2, the AePL3, the AePL4 and the AePL5 are really the pectin lyase PL related genes of okra.
The total RNA extraction and cDNA synthesis method comprises the following steps: about 0.2g of jelly sample was weighed, and total RNA of okra was extracted with column type plant RNA extraction kit 2.0 (Enzernine Gene technologies Co., ltd., beijing). The quality of the extracted RNA is detected by using a nucleic acid protein instrument and an agarose electrophoresis instrument. The cDNA was reverse transcribed using a HiFiScript cDNA Synthesis Kit kit (Beijing kang Biometrics Co.).
In the embodiment, the change condition of the expression of the AePL1, the AePL2, the AePL3, the AePL4 and the AePL5 in the okra is analyzed by real-time quantitative PCR, specific primers of the AePL1, the AePL2, the AePL3, the AePL4 and the AePL5 are designed by Beacon Designer 7, and the AeACT is used as an internal reference gene, wherein the primer sequences are shown in table 3. The three-step amplification was performed using a Real-Time PCR instrument under the samer flag, usa, according to the DyNAmo Flash SYBR Green qPCR kit instructions of samer feier, usa. The following program settings were made: pre-denaturation at 95℃for 7min; this step was performed for 39 cycles of 95℃15S,45℃30s anneal, 75℃15s extension. Each sample was subjected to 4 biological replicates and the relative expression level was 2 -ΔCT The method performs computational analysis.
TABLE 3 fluorescent quantitative PCR
The expression of the AePL1, aePL2, aePL3, aePL4 and AePL5 genes of the two okra groups are shown in fig. 14, wherein the expression of the genes is shown as significant at the 0.05, 0.01 and 0.001 levels, respectively.
As can be seen from fig. 14, the expression levels of AePL3, aePL4 and AePL5 all tended to rise and fall in the control group, but the expression level of AePL3 in the treatment group was almost unchanged at the earlier stage of storage but increased sharply at 12d, and significantly lower than that in the control group (P < 0.05) at 6d, 9d and 12d, the expression level of AePL4 was increased gradually to 9d and then decreased sharply, and significantly lower than that in the control group (P < 0.05) at 3d, 6d and 12d, and the expression pattern of AePL5 in the control group was the same as that in the treatment group and also tended to rise and fall first and significantly lower than that in the control group (P < 0.05) at 6d and 9d, so that gibberellin treatment had a significant inhibitory effect on the expression of AePL3, aePL4 and AePL5 genes of okra.
The AePL1 is in an expression mode of ascending and then descending and then ascending in a control group, and is in a gradually ascending expression mode in the control group, the expression quantity of the AePL1 is higher than that of the control group in the whole storage period than that of the control group in a treatment group, and significance (P is less than 0.05) exists at 9d and 12d, so that the gibberellin treatment has obvious inhibition effect on the AePL1 gene expression of okra.
The expression patterns of the AePL2 in the control group and the treatment group are continuously increased along with the extension of the storage time, the difference is that the control group is rapidly increased from 6d, the treatment group is slowly increased to 9d and then is rapidly increased, the expression quantity of the AePL2 in the whole storage period is higher than that of the control group, and the expression quantity of the AePL2 in the control group is remarkably (P is 0.05) in 6d, 9d and 12d, so that the gibberellin treatment has remarkable inhibition effect on the AePL2 gene expression of okra. .
Thus, gibberellin treatment has a significant inhibitory effect on the AePLs genes during storage, particularly during mid-to late-storage.
In this example, experiments were performed using 0.001%, 0.003%, 0.005%, 0.007%, 0.01%, 0.03%, and 0.05% gibberellin aqueous solutions, respectively, which can achieve a certain effect of inhibiting the expression of the okra AePME1 and AePME2 genes, and the description thereof will not be given here.
Example 7 Effect of gibberellin treatment on okra cellulase Cx-related Gene expression
In the embodiment, newly picked okra (purchased from the state district heart-linked vegetable market of Ningbo city) is adopted, and 200 okra which are relatively uniform in size, full in size and free of black spots and damages are divided into 2 groups (control group and treatment group), and 100 okra are selected from each group. Preparing gibberellin aqueous solution with the mass percentage content of 0.01%, and soaking okra in the gibberellin aqueous solution for 15min. After the soaking, naturally air-drying 2 groups of okra at normal temperature, and finally storing in a constant temperature and humidity incubator at 25+/-1 ℃ and 80-90% relative humidity for 12 days. Samples were taken before treatment (day 0) and after treatment for 3, 6, 9, and 12 days, respectively, and changes in cellulase Cx-related gene AeCX expression of okra in the control group and the treatment group were measured.
The relevant genes of cellulase Cx from okra (designated Guangzhou Diao Biotechnology Co., ltd.) were selected by transcriptome sequencing in this example, including AeCX, the sequences of which are shown in Seq ID No.14, respectively. The sequence of the Aecx is compared with the sequence of the cellulase Cx related gene of hibiscus on NCBI, and the comparison result is highly similar, so that the Aecx gene is proved to be truly the cellulase Cx related gene of okra.
The total RNA extraction and cDNA synthesis method comprises the following steps: about 0.2g of jelly sample was weighed, and total RNA of okra was extracted with column type plant RNA extraction kit 2.0 (Enzernine Gene technologies Co., ltd., beijing). The quality of the extracted RNA is detected by using a nucleic acid protein instrument and an agarose electrophoresis instrument. The cDNA was reverse transcribed using a HiFiScript cDNA Synthesis Kit kit (Beijing kang Biometrics Co.).
In this example, the change of the expression of AeCx in okra was analyzed by real-time quantitative PCR, the specific primer of AeCx was designed by Beacon Designer 7, aeACT was used as the reference gene, and the primer sequence is shown in table 4. The three-step amplification was performed using a Real-Time PCR instrument under the samer flag, usa, according to the DyNAmo Flash SYBR Green qPCR kit instructions of samer feier, usa. The following program settings were made: pre-denaturation at 95℃for 7min; this step was performed for 39 cycles of 95℃15S,45℃30s anneal, 75℃15s extension. Each sample was subjected to 4 biological replicates and the relative expression level was 2 -ΔCT The method performs computational analysis.
TABLE 4 fluorescent quantitative PCR
The AeCx gene expression profiles of the two okra groups are shown in fig. 15, wherein the expression of AeCx gene is shown as significant at the levels of 0.05, 0.01 and 0.001, respectively.
As can be seen from FIG. 15, the expression pattern of the control group Aecx gene was continuously up-regulated with the increase of the storage time after gibberellin treatment, and the expression pattern of the control group Aecx gene was also continuously up-regulated with the increase of the storage time after slightly decreasing the treatment group Aecx gene from 0 to 3 d. The control group AeCx gene expression level was significantly higher than that of the treatment group (P < 0.05) throughout the storage period. Therefore, gibberellin treatment can effectively inhibit the expression of the Abelmoschus esculentus AeCX gene.
In this example, experiments were performed using aqueous solutions of 0.001%, 0.003%, 0.005%, 0.007%, 0.01%, 0.03%, and 0.05% gibberellin, respectively, which have a certain effect of inhibiting the expression of the abelmoschus esculentus aex gene, and the description thereof will not be given here.
Example 8 Effect of gibberellin treatment on expression of okra alpha-L-arabinofuranosidase alpha-Af-related Gene
In the embodiment, newly picked okra (purchased from the state district heart-linked vegetable market of Ningbo city) is adopted, and 200 okra which are relatively uniform in size, full in size and free of black spots and damages are divided into 2 groups (control group and treatment group), and 100 okra are selected from each group. Preparing gibberellin aqueous solution with the mass percentage content of 0.01%, and soaking okra in the gibberellin aqueous solution for 15min. After the soaking, naturally air-drying 2 groups of okra at normal temperature, and finally storing in a constant temperature and humidity incubator at 25+/-1 ℃ and 80-90% relative humidity for 12 days. Samples were taken before (day 0) and after 3, 6, 9, 12 days of storage, respectively, and changes in the expression of the control and treatment groups of the α -L-arabinofuranosidase α -Af-related genes Aeα -Af1, aeα -Af2, aeα -Af3, aeα -Af4, and Aeα -Af5 of okra were measured.
The related genes of the alpha-L-arabinofuranosidase alpha-Af of okra (which are designated as genome sequencing by Kyoto Dio Biotechnology Co., ltd.) are selected by transcriptome sequencing in this example, and comprise Ae alpha-Af 1, ae alpha-Af 2, ae alpha-Af 3, ae alpha-Af 4 and Ae alpha-Af 5, the sequences of which are respectively shown as Seq ID No. 15-Seq ID No. 19. The sequences of the Ae alpha-Af 1, ae alpha-Af 2, ae alpha-Af 3, ae alpha-Af 4 and Ae alpha-Af 5 are aligned with the alpha-L-arabinofuranosidase alpha-Af related gene sequences of hibiscus and cotton on NCBI, the results are shown in figures 16-20, the alignment of Ae alpha-Af 1 is shown in figure 16, the alignment of Ae alpha-Af 2 is shown in figure 17, the alignment of Ae alpha-Af 3 is shown in figure 18, the alignment of Ae alpha-Af 4 is shown in figure 19, the alignment of Ae alpha-Af 5 is shown in figure 20, and the Ae alpha-Af 1, ae alpha-Af 2, ae alpha-Af 3 and Ae alpha-Af 5 genes are proved to be the alpha-L-arabinofuranosidase alpha-Af related genes of okra.
The total RNA extraction and cDNA synthesis method comprises the following steps: about 0.2g of jelly sample was weighed, and total RNA of okra was extracted with column type plant RNA extraction kit 2.0 (Enzernine Gene technologies Co., ltd., beijing). The quality of the extracted RNA is detected by using a nucleic acid protein instrument and an agarose electrophoresis instrument. The cDNA was reverse transcribed using a HiFiScript cDNA Synthesis Kit kit (Beijing kang Biometrics Co.).
In this example, the changes in the expression of Aeα -Af1, aeα -Af2, aeα -Af3, aeα -Af4 and Aeα -Af5 in okra were analyzed by real-time quantitative PCR, and specific primers for Aeα -Af1, aeα -Af2, aeα -Af3, aeα -Af4 and Aeα -Af5 were designed by Beacon Designer 7, and AeACT was used as an internal reference gene, and the primer sequences are shown in Table 5. The three-step amplification was performed using a Real-Time PCR instrument under the samer flag, usa, according to the DyNAmo Flash SYBR Green qPCR kit instructions of samer feier, usa. The following program settings were made: pre-denaturation at 95℃for 7min; this step was performed for 39 cycles of 95℃15S,45℃30s anneal, 75℃15s extension. Each sample was subjected to 4 biological replicates and the relative expression level was 2 -ΔCT The method performs computational analysis.
TABLE 5 fluorescent quantitative PCR
The expression of the Ae a-Af 1, ae a-Af 2, ae a-Af 3, ae a-Af 4, and Ae a-Af 5 genes of the two okra groups are shown in fig. 21, wherein the expression of the Ae a-Af 1, ae a-Af 2, ae a-Af 3, ae a-Af 4, and Ae a-Af 5 are shown as significant at the 0.05, 0.01, and 0.001 levels, respectively.
As can be seen from fig. 21, the control group showed a trend of ascending and then descending except for the trend of change of Ae α -Af2 in the storage period, in which Ae α -Af1, ae α -Af4, ae α -Af5 were all down-regulated after 9d reached the peak of the expression level, and the expression level of Ae α -Af3 was gradually decreased after 6 d. The treatment group showed a trend of ascending and then descending in all the other expression patterns except for the small decrease in Aeα -Af4 at 3d and then gradually ascending. The Aeα -Af2 gene expression level was higher in the treated group than in the control group except for 6d, and the Aeα -Afs gene expression level was higher in the control group than in the treated group throughout the storage time, and the control group was significantly higher in the treated group (P < 0.05) at 9d and 12 d. Because of the lower expression levels of Aeα -Af2 compared to other Aeα -Afs, gibberellin treatment generally has an inhibitory effect on Aeα -Afs.
In this example, experiments were performed using aqueous solutions of 0.001%, 0.003%, 0.005%, 0.007%, 0.01%, 0.03%, and 0.05% gibberellin, respectively, which gave a certain effect of inhibiting the expression of the Abelmoschus esculentus Aeα -Afs gene, and therefore, the description is not given here.
Example 9 Effect of gibberellin treatment on okra beta-galactosidase beta-Gal-related Gene expression
In the embodiment, newly picked okra (purchased from the state district heart-linked vegetable market of Ningbo city) is adopted, and 200 okra which are relatively uniform in size, full in size and free of black spots and damages are divided into 2 groups (control group and treatment group), and 100 okra are selected from each group. Preparing gibberellin aqueous solution with the mass percentage content of 0.01%, and soaking okra in the gibberellin aqueous solution for 15min. After the soaking, naturally air-drying 2 groups of okra at normal temperature, and finally storing in a constant temperature and humidity incubator at 25+/-1 ℃ and 80-90% relative humidity for 12 days. Samples were taken before (day 0) and after 3, 6, 9, 12 days of storage, and changes in the expression of the β -galactosidase β -Gal related genes Ae β -Gal1, ae β -Gal2, ae β -Gal3, ae β -Gal4, ae β -Gal5, and Ae β -Gal6 of okra in the control and treatment groups were determined.
The related genes of beta-galactosidase beta-Gal of okra (which were subjected to transcriptome sequencing by Kyoto Guangzhou Diao Biotechnology Co., ltd.) were selected by transcriptome sequencing in this example, and the sequences of the genes include Aebeta-Gal 1, aebeta-Gal 2, aebeta-Gal 3, aebeta-Gal 4, aebeta-Gal 5 and Aebeta-Gal 6 are shown as Seq ID No.20 to Seq ID No.25, respectively. The sequences of Aeβ -Gal1, aeβ -Gal2, aeβ -Gal3, aeβ -Gal4, aeβ -Gal5 and Aeβ -Gal6 were aligned on NCBI with the related gene sequences of Hibiscus syriacus, cotton, durian, and cocoa β -galactosidase β -Gal (the results of the partial alignment are shown in FIGS. 22-26, where the results of the alignment of Aeβ -Gal1 are shown in FIGS. 22, the results of the alignment of Aeβ -Gal2 are shown in FIG. 23, the results of the alignment of Aeβ -Gal3 are shown in FIG. 24, the results of the alignment of Aeβ -Gal4 are shown in FIG. 25, and the results of the alignment of Aeβ -Gal5 are shown in FIG. 26), and the results of the alignment of Aeβ -Gal1, aeβ -Gal2, aeβ -Gal3, aeβ -Gal4, aeβ -Gal5 and Aeβ -Gal6 are proved to be highly similar.
The total RNA extraction and cDNA synthesis method comprises the following steps: about 0.2g of jelly sample was weighed, and total RNA of okra was extracted with column type plant RNA extraction kit 2.0 (Enzernine Gene technologies Co., ltd., beijing). The quality of the extracted RNA is detected by using a nucleic acid protein instrument and an agarose electrophoresis instrument. The cDNA was reverse transcribed using a HiFiScript cDNA Synthesis Kit kit (Beijing kang Biometrics Co.).
In this example, the changes in the expression of Aeβ -Gal1, aeβ -Gal2, aeβ 0-Gal3, aeβ 1-Gal4, aeβ -Gal5 and Aeβ -Gal6 in okra were analyzed by real-time quantitative PCR, specific primers for Aeβ -Gal1, aeβ -Gal2, aeβ -Gal3, aeβ -Gal4, aeβ -Gal5 and Aeβ -Gal6 were designed by Beacon Designer 7 with AeACT as the internal reference gene, and the primer sequences are shown in Table 6. The three-step amplification was performed using a Real-Time PCR instrument under the samer flag, usa, according to the DyNAmo Flash SYBR Green qPCR kit instructions of samer feier, usa. The following program settings were made: pre-denaturation at 95℃for 7min; this step was performed for 39 cycles of 95℃15S,45℃30s anneal, 75℃15s extension. Each sample was subjected to 4 biological replicates and the relative expression level was 2 -ΔCT The method performs computational analysis.
TABLE 6 fluorescent quantitative PCR
The aeβ -Gal1, aeβ -Gal2, aeβ -Gal3, aeβ -Gal4, aeβ -Gal5 and aeβ -Gal6 gene expression of two groups of okra are shown in fig. 27, wherein the differences are significant at the 0.05, 0.01 and 0.001 levels, respectively.
As can be seen from fig. 27, the control groups all tended to rise and then fall during storage, down-regulating after 9d reached the highest peak of expression level. The Aeβ -Gal1 gene and the Aeβ -Gal2 gene were hardly expressed in the control group during the whole storage period compared with the control group, so that the Aeβ -Gal1 and Aeβ -Gal2 expression amounts in the control group were significantly higher than those in the treatment group (P < 0.05) at 6d, 9d and 12 d. Aeβ -Gal3, aeβ -Gal4 and Aeβ -Gal6 were on an ascending trend in the treated group, and the expression levels were significantly lower at 9d and 12d than in the control group (P < 0.05). The Aeβ -Gal5 gene had a tendency to rise sharply and then fall slowly in the treated group, and was significantly lower in expression (P < 0.05) at 9d than in the control group. Overall, aeβ -Gals were expressed higher in the control group than in the treatment group throughout the storage period of okra, especially in the late storage period. Thus, gibberellin treatment has an inhibitory effect on the Aeβ -Gals gene during storage, particularly late in storage.
In this example, experiments were performed using aqueous solutions of 0.001%, 0.003%, 0.005%, 0.007%, 0.01%, 0.03%, and 0.05% gibberellin, respectively, which gave a certain effect of inhibiting the expression of the Abelmoschus esculentus Aeα -Afs gene, and therefore, the description is not given here.
The application of the present invention is not limited thereto. Such as by its range of application in environmental protection. Various changes and modifications may be made by one skilled in the art without departing from the spirit and scope of the invention, and the scope of the invention should be assessed accordingly to that of the appended claims.
Sequence listing
<110> Zhejiang Wanli college
<120> efficacy of gibberellin in delaying softening aging after harvest of okra and application thereof
<160> 77
<170> SIPOSequenceListing 1.0
<210> 1
<211> 4512
<212> DNA
<213> Artificial sequence (Artificial Sequence)
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caacagcagc agaagcacgg ctctgttgat atcacaacct ccccatattg gcatattccc 120
tgtacaccca cactctctag agtgagacca atacctatgt attttagaat ggaactggct 180
ctggtttgtg ccaacattgt ctaattcatg cccacaaatc aaaatggggg tgcttacact 240
gacaaatcca cttcttttag cttccagaaa cgtgggacag ggacgaacac ataattttat 300
gtcaagggtg tgaattatta aagagttata ttaaaaattt tggggtttga aattttcgga 360
cctcttggtc catccttgat ggagccttgt tgattcaact tccatgagat ctttaggtga 420
agaataacaa gtgacattcc ctaaatgtca aaaactgact tgattaatgg ctatagtacc 480
taatacatcg gccaaaccta ggatagtggg accggttttg ctctataagt agagggtacc 540
ctttcattga atcccttaag ttctcattga agttaaaagt ttaggctcaa tatgcccttc 600
catcaagttc ttctcttgtt tttcttcatc ttttcttgtc ttctctttgt tatcattcaa 660
accagacatc atcgtagcca cttggaacac gaggaattcc gcattgtatc tcacgtttct 720
gtccctcctt cacctgcacc agaaccagct gttgctggtc caaggtacag tgtcttcaac 780
gtgcttgatt tcggtgctat tggggacggt gtcaccgatg atactcaagc atttaagctg 840
gcttgggaca cagcttgcca aatcgaatcg gcgattcttc tggttccaga tggttattcc 900
ttcatgttac aatctacagt cttagcaggt ccttgtagga ctgttggcct tgtgtttcag 960
gtgacaaaac attttgccat ttcagttttg cattgagctt attatgttat agtcaaccat 1020
tgatttgttt cgacttcgag tgggaagatc gacggaacca taatgccacc tgatggacct 1080
gattcttggc caagaaatat cagcaaaagg caatggctgg tcttctacag aatcaatggg 1140
atgttaatgc aaggtggtgg ccttgtagat ggaaaaggag aaaaatggtg gaatcttccc 1200
tgcaaacctc acaaagtaat cgaatttagt cacgtttaac atgccagtta cttactccac 1260
aaccaaagtc cataactgac ataatatatc tgctaagcag ggaatcaatg gaacaacaat 1320
gtctggtccc tgtgacagcc ctgtggtaag tacaactgag ctagaactta aaatctttct 1380
ttgattttct gaagatttat cactgtggtt ttttgatgca ggctataagg tttttcatga 1440
gctcgaactt gactgttaaa ggactcataa tcaagaacag cccgcaggtc catttcaaat 1500
tcgatgcctg ccacggcgtt cgtgtagaat cactgactat caaagcaccg gccaaaagtc 1560
ccaacaccga tggaattcac atcgagaaca caaataacgt gcagatatac aattccttcg 1620
tatccaacgg tgaattttct tcatcatttc agtacatctt gagcatatat gagatcaaaa 1680
attcgaaata gtgaacttac atgccttcca tgtatgtact gttaggtgat gattgtgtat 1740
caatcggagc cggatgctat gatgttgata taaagaacat aacttgtggt ccaagtcatg 1800
gaataaggtc tgcaaaacgg ttacaagcat aggctgcaga gtttccgttt gagttcttaa 1860
catgtcaaac atcttaggcc ggctgaagga ggctacttat ggctctttta gttcacatca 1920
tcatctagtt tcagttctat aaatgtgtaa catcttttta tttcttattg cagcattggt 1980
agtctcgggg ttaatcattc acgagcctgt gtttcgaaca tcactgtttc cgattcggta 2040
gtacaacatt ctgaaaacgg catccgcatt aaaacctggc aaggcggctc gggctgtgtg 2100
tcgaaagtcc aataccgaaa catcagcgtg gaagctgttc gtaacccgat tatcatagac 2160
caatactatt gtctcaccaa aaactgcagt aaccaaacaa gtgcagttgt gattaatgac 2220
atctcctacg ttaatattaa gggctcatat gatgttagaa gtccaccgat gagttttgct 2280
tgtagtgact caactccatg cataaacctt acactggcac aggtagagct gcagcccgcc 2340
accccgggac gcatcgtggc aaatccctat tgctggaatg ctcatggaac tgtcctcaca 2400
caaactattc caccagttta ttgcttgatg gaggaaacta atgtcaccat tgatgacaca 2460
aacatatgca gataggtgtt gaatcagatt gttatgtaaa atatacaaga tcaatcaatt 2520
gtatcaagtt gtatcaagta tccaaggact acatcaaaaa aaatgatgga ttcttaagtt 2580
gtatgaaacc caatccagtc ctagacaaaa tgtaaccttt ttatgggatc tcaaaactat 2640
gattcatgca caatggttga ctcgacccat ccatccctta attaggcttt ctaaaagagg 2700
tgtaatagac ctactaacat agcatataca agataatata agccctccat ccaacacaac 2760
atgaaaagac attaaactaa gctgacatac cacagaaatc gtctagtacg aaactcgctt 2820
aagcatcacg ggtgaaggtt gaaatgggtt agtgaaagtt aaccatgcaa aacctcccca 2880
gaatgttata gtccaatcct aattcatttt agtttcttct ctacatcaac agatggttca 2940
accaaacttt aatcattaac ataccaaaca ttaatctaaa tacttaaaag tgagtagaat 3000
aggcaaatgt ttctggtaat agaagcaaac aggaatttat tgacatatta acataaggat 3060
tcagagaaat ttccaagcac cagaggaagt atttagagaa acaaaagaaa tacatatgat 3120
gtggcgtatg tgctgaaata cattttttat tcatatcctt tttattaaag cctttcaata 3180
aacatgcatc ctcccatcca ccctcacata caaccttaca actgtaccaa tttctggaca 3240
taaagacaag tggatgcagg atgttacgga catagtgaag caagtatctc caacaaggaa 3300
gtggcgaact tcatttgggt ctcctcgtct atggtcaaga agagagggac atggacaaag 3360
agagatttga ccccactctg ctcagcaaat cgaagggagt gatagtaaac ataattgcac 3420
acaaatcggc ctgcatcatc agaggtcatc acttcaaatc ccttcttttt taaggccgtt 3480
gtaatctcct caacaggaag agaagtctgc acataggtag cgaaggaaaa tatttaagga 3540
tggaggtaag agatgaaagg tatcaggttc aagtgtaaat tttaaccatg ttaagcaaca 3600
aaagatactt taacaaaaag gatactacct ctcgtacacg tgaaattcca ccatctgcag 3660
gaactatggg cactttctgc aatcaaaaca tggcgtaagg aaaaggaaga aagtttgaga 3720
agaggcagac ataccaaact gcacatgcac cataaaagtg gaaaagggat atatttaacc 3780
tgaggtttcc atcccatttc atcagggcaa cggaaagtag cttcatttgc agcttgacgc 3840
tcaatagcaa atctacttgc accactattg actccaaaat gtagctgaag aagattgtaa 3900
cagtaactaa gatatgtgaa gcttgaattt gtcatgcaaa gagtttgatt gcaccaatga 3960
caagtaaata agataacatc tgttgaaatc cccaataaac aatccataaa atggacctta 4020
ttgcaagtgt gctttgttca tgtactgaga tagcaaagaa agcacaaaga atttaccggg 4080
aaagaaacgg acaaaccttt gttttagctg attggctaat catagattcc aatcccatag 4140
gccataacgt gcatttttac tatgaattta actggtacat tatccacatc acattacaga 4200
tattttttac ccttttcaca tttggcactt tttgagaata catgaatgga aggttattac 4260
gcaagagtta taataaaaaa aaataagacc ctttttatgc attaagcagg taccggaaaa 4320
ggactcagcg gttcagccta ctttacacaa agctaaaacg aatcatttca tgttgaagat 4380
tcattgctgc aatcaccatt gacatgaaag taagcaatta ctagacaact gaaagtgatt 4440
aactaaccca aataattctc ccaaaagaag attcagaatc cttcccagtt aaggcagatc 4500
gcaatgtctg gt 4512
<210> 2
<211> 1756
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
aagaagtttt acatggatga tttatagaat acatccaaac acggcattgc atttacttta 60
tatataatat agactaactg gtcataagat aattaagaag gaaacagaaa catttagggc 120
tgactcaaaa gttcccaact atgagaactc ttgctacaat acaacagcct actcaggttt 180
tgctttaatg gattcattca tatatgcttg accctatagg agcacctttt gagcaccact 240
ttatcaatgg acaaaggctc agccggaaag tcgcaggctg tgattttctc cgagccttga 300
tcaggcaata ggtcacatgg gcgagggctc accccacttg ttatgccctc aacatcagtg 360
caagtccatg gcactttctt tgctttggct gccattccaa ttgtgacatt cgatatgcag 420
atttgagtaa acggatcacc ttcgatgcct tccaatctgg ctgccatcga cacattatct 480
gccctgatgt ctctgtagtt gatcccttga accacaggca atgcattggg atcgtagtga 540
ttatcggggt gttgtttata gttccctgtc atccaaaaca cccatttcat ggtgtgcaga 600
gacattcttt tgacgtatat gtctttcacg aaccctcctc tcccccttgc cgtcttgatc 660
ctgacccctg attccgtctt aattgcaatg atgtcttctg ctctcacatc ctggattccg 720
cccgacatct cactccccaa cgcaatggct gcactgtaag gagaaatgca tgtcatcctc 780
ctgataacta agtgtttggt aggcatccca aatgaaatcc catactcatc ccaaccactc 840
ttcactgcta tacagtcatc tcccgagact atgtaagtgt cctcaattct aacattggtg 900
caagaatctg accataaaaa aacatgttaa aaaccaagca gaacatttga cacgttaata 960
accaagattc tcttgtgtgt tcatgcctgg attgatgcca tcggtgttcg gagatttgac 1020
aggagcgagg attgtaatgc cttgtatgag aatgttgctg ctataaaccg gatgaacgtt 1080
ccaagatgga gagttgacaa gagtaacatt ggagatttga atattgtccg agtacatgaa 1140
ttcaattagg taaggtctgg tatatttcaa cttcccattg tggaatttct gccaccagaa 1200
tgagccttga ccgtcgatag taccattgga ccctgtaacg atgacatcag tgagattagt 1260
tccaaatatg aggctggtaa acctaccccc agctgcatcc cttcctcgac cataggatgg 1320
caaaggcttc aaaacaggcc attcgttcat atcctgagaa gcaagaagaa cagcatcttt 1380
gtgaagaaag agggtgaagt ggctggtgag gctaaagctc ccggtcagcc attttccagc 1440
aggaacataa agctgagctc cgcccttgga tgcaaacttg ctcaaattct caacagcttc 1500
cttaaatgct ttagtattcg atgttttgcc atcaccaact cctccaaaat ctgtgattga 1560
tgcactgtgt cctctgcaac ttatagcagt gtactcaaag gacttcacca ccttcccttt 1620
tttcctggcc tctgctcctc ttggacccaa tgcaaatgcc accaaaacaa gggctatggc 1680
tatgctgaca actttgatcc tcattggatt cttccctaac cacaccattg ttggaaaatg 1740
atgaaagact aaaact 1756
<210> 3
<211> 1898
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
cagggaagtc ctgttcttgc ggtggttttt ctctcctcct cttcttcctt tttttctcct 60
cccccttccc tgtttttaat tcatttaggg tttggtatat cgttttttct ttttgcaatt 120
cctcactttc cttgtctggt catctcttgc cgaattcttt gacagatgaa gatgccagta 180
gccttgctgg tgctgctggc gttgtgcaat gcgatctatg tcaatggagg agggagtaat 240
cttcaatgtg atgataagct gacattgcaa ccgagacccc atagtgtatc catcttggaa 300
ttcggtgccg tcggagatgg caagactttg aataccattg cctttcagaa tgctattttc 360
tatctcaagt cttttgcgga taagggtggc gcccagcttt acgtacctcc tggacaatgg 420
cttactggaa gtttcaatct cactagccac ctcactctct tcttggagaa gggtgctgtc 480
attcttggat ctcaggatcc atctcattgg gatattgttg aacccttacc ttcatatggt 540
cgaggaattg aacttccagg aggaagatat cgaagtttgg tgaatgggta tatgttgcac 600
gatgtagtaa taaccggtga taatggaacc atagatgggc agggctcggt ttggtgggaa 660
tggttcacat ctcattcttt aaattatact cgccctcacc ttgtggaatt tgtttcatct 720
gaagatgtac atgtttcaaa cattactttc ctgaatgccc ctgcgtataa cattcatcca 780
gtctactgca gcaatgtacg catacacaac ataacagcct atgcaccacc aagatcacca 840
tatactgttg gcatagtccc agattcctct gataatgttt gcatcgaaga ctgcgacatt 900
agtatgggcc atgatgctat tgcactcaag agtggctggg atgaatacgg cattgcctat 960
ggaagaccta ccacaaatgt ccacatccga tcagttcagc ttcagtcatc tgctggtgcg 1020
tcccttgcct ttggtagtga aatgtctggt ggcattgtcg atgtccaagt agaacatgtc 1080
cgtctctacg actcgttaac tggtattgag tttagaacaa ctaggggtag gggtggttac 1140
atcaaagaga tcatgatatc agaagctgac atattgaacg ttagcacggc attcgttgcc 1200
aatggtaatt acgggaccca tccggacgaa aaatttgatc tcgacgcact cccgtcagtt 1260
aagaaaatta tgttgcagaa cattattggc cgaaaaatca cattggccgg aaacttcact 1320
gggatcaaac aatccccatt tacttctata tgtatatcaa atgtaactct atcaatcagt 1380
cccactagtt ccatatcttg gacatgttca aacgtttctg gctattcaga gtctgtcttt 1440
ccagaaccat gcgaggagct taagaacgcg aatacatctt cctcatgttt ctccattctg 1500
atgaacagtg gtagtaatgc tccagcaaca gcgattttat gataagtatt tcttccaatt 1560
agaagtacaa caacacacga ataaatcgga aatctccgtc tgtctatttc ttttcaataa 1620
tccaacatgt ctagaatagc ttcgtctctt ttgagtcggt attttagcat gtcctgcaga 1680
ttgtttgtat agcttgattt ctttattctt gtgtaaggaa aatttttgtc atccaaagaa 1740
ggggagttgg gggttatgtt tagtgtttga tgtctcttga attctttggc ttttgttttt 1800
tccttttaat atttatagtg ttgatgcaga cacaggtaca atcactctgt ataatcatca 1860
tttttcactt gctgaatcaa aacaccaggt tcaaagga 1898
<210> 4
<211> 1728
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
ctcttctcat tttaattatg gtgtattata ttcgagaatt aaacttcgca agtattgcgt 60
tgacatagta acccacatat aaggggtcga gtaaagtaaa agatgattag aagttgccgc 120
tgatagaaca tgtctttaac tcgacatctt cgatgggcag cctatcctca gggaaaggac 180
actcgatttc ctttcgaggc aacaaaccac atggttcggg gctcacacgg ttggtaactc 240
cctgaacctc ggtgcagttc cattgcactt ctttcggctt tgcggtcagc ttgatgtcga 300
cgtttgaaat gcaaatgtcg gtgaaaggat gcttcccgat tccatcgagt tgtgccgaga 360
gtgtcacatt atctgccacg atgtccctgt aattgattcc tttgattacc gggaacgcgt 420
tcgggtcgaa cttgtcatgc ggatgcgagc cataatcgcc tgtcatccag aacacgtact 480
tcattgtatt caatgtcatg tttctaacgt aaatgttttt aacataagct cctcttccga 540
ctccggtttt gatcctcacg ccggattctg tattaatcgc tgtaatctct tccgctcgta 600
cgtcttcgat cccaccggac atctcgctcc caagagcaat ggtggcactg ctaggagaaa 660
tgcaggtgaa ccttctgatc actagttgct tggttggcat gttgaatttg ataccgtatt 720
cgtcccaacc acttttaacg gcgatgcagt catccccgga gacaacaaaa ctgtcttcga 780
ttcgggtatt tgtacatgaa tctgggtcta ttccatctgt attaggggaa tcaactggtg 840
caagaacagt caaatgttgg attattatat cactgctgta tacgggatga acattccatg 900
atggtgagtt aaccaacgtt agattcgata tctggacttg tttcgaatac atgatctcaa 960
tcatgtaggg tctggttaca gtcattttgt tctccttgaa ctgcttccac cagtatgcgc 1020
cttggccgtc gatggtgcca ttattacctt gcgttacaag acatgtaatt tccccgcatt 1080
tagttaaatc gaatcgattg aacctgtatc gataattaat ttgagatggt gttttggtta 1140
cctgtaatga caacatctgt gaggttggtg ccgaaaataa ggctgctgag ccttccacca 1200
ggagcatctc ttccctgccc gtatgatggc agaagtgcaa caggaggcca ctctgattca 1260
tcctgagatc caagaatgac ggcatcttta tggacaaaaa gcgtgaaacg gctggtgaga 1320
ttgaagctcc cagttaacca cttccctggc ggcacaataa gctgtgcgcc accgtccgcc 1380
gcaacatggc tgagatttgt aatcgcagct ttgaacgcct tcgtgtttga tgtttttcca 1440
tcgccgatcc cgccaaaatc cgtcaaaacc gcactgtgtt tgcgacaatt tagagccgaa 1500
aacgtgatgc tattcgtcgc aaccccagca gctccacttt ccactacatt tgatctcagt 1560
aattccaata aaaacacggc tgaaattaat ttctccaaca ccttggattt tcttgaaaac 1620
tccattgctg agatgcgagg tactataaac ttgacaaatg tgaaactatt ctgagaacaa 1680
aaggattcac gagaaaatca ttgtttgttt gtgagaactg attttgta 1728
<210> 5
<211> 2158
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 5
ggacaccatt cacttttgct tccttggtgg tcaaaccgaa actaatatac atgctgaagc 60
ttacctgatt tgtgatattt aggactcgat tgtggttgac attaccccaa tcggatgtta 120
tttaggctct tatatgacga gaaacagaca gaacttcctt cttgatacta tataatggaa 180
gttcaagacc tcagtgcaaa gattgtggat caatgaccct cagtgcaaaa cctgggaaaa 240
gaagaacatc cttccaatga tcattgtttc ttacataaaa caaattttat aacagcaaat 300
tcatgttaca taaacgaaat tgatagcata actttatgtt ctgtcacaat tttctattta 360
aactgtttgg cacttgggaa gctgtcctct ggaaaattgc aggcattgtc tggcccttga 420
tctgttagca gattgcaggg ctgaggggta acattacttg ataccccagc aatctcagtg 480
caattccatt gtatcttctt tggcttttgt gttaatccaa tggtagcatt tgatatgcaa 540
attccagaaa atggatgacc tggaattccc tccaatctag ctgccattgt cacattctct 600
gcaacgatat ctcggaaatt gatattctgg ataacaggga ttgcattagg atcataatca 660
ttgtccggat gagaaccata gttccctgcc atccagaaaa ccattttcat agttttcatg 720
gtcattcctc tcacatatat gtctttgaca tagtttcctc gtcccacagc ggttttgatc 780
ctaacagccg actcggaatt aatgcatgtg atgtcttcag cctttacgtc ctcaattcca 840
ccagacatct cactccctag tgcaatcact gcactgaatg gtgaaataca tgtgagcctt 900
ctgatcacca gttgcttggt tggcatacca aacttgatac catactcatc ccaaccactc 960
ttaacagcta cacaatcatc tccagagaca atgtagcagt cctctatccg ggtatttgtg 1020
caagaatctg ggttgatccc atcagtgtta ggagaagtta ctggtgcaag gatagttaat 1080
ccctgaacaa cgacattgct gctataaaca ggatgaacat tccaggaagg agagttcatc 1140
aatgtgagat tagagatttg aacttcatct gaatacatga tttcaataag gtaaggcctg 1200
gtgtatttca gctctccctt gtgaaatttg ttccaccagg taacaccctg accatcgata 1260
gtgccatttt cacctgttat gacaacgtca gtgaggttag ttccgaaaag aagactgata 1320
tatcttcctc cgtcggcatc ccttcctcga ccataggacg ggagagggtc aatgactgcc 1380
cattcactct catcctggaa caagtttcag acacaagaaa cagtaaacat aaaccacgtc 1440
ttaaattttg ttagtcggca ttaaaaacgc attgtgatga aggtatttag ttcccagttc 1500
ttcgtggctt tatctgaaag gtccatatca ggggaccatg atttattcaa gctacgtttg 1560
aaatgaggtt cttcaaggga ttcccaaaat tagcataggc ctgacaatga gcagttgaga 1620
accacagaat tctagaaaaa gaactcatga agtgggtcct ttgccttgtg gtctcccttg 1680
tttctgtaag gtctagcctt aggataaaaa atggccattt tgaaacagtc atctggccct 1740
aatatccttc attttctcat aaaatcagtg ttaaatcttg ttgtttctat acatgttgcc 1800
attaattggc attgcgagct tctaagttct aacctgtgag gcaagaagag tagcatcttt 1860
gtggatatag agagtgaaat ggctggtaag attgaaacta ccagtcagcc acattcctgg 1920
aggaacaagc agtagcgatc cgccatctga tgcatactgg ctgaggttga aaatggcagc 1980
ctgaaaagcc tttgtatttg acgtcactcc atcaccaacg cctccaaagt ctgtcaatga 2040
agcactgtac ttcctgcttt caacaccctt tatgtcgatc aaaaccagaa ctgttccaag 2100
tagcactatt gtaccaagaa cctggtttct tcttggggtc gtccacattt ccattaac 2158
<210> 6
<211> 2171
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 6
cggaaaacct cacttcactt ctataaattc agttgtcgga acgcacacaa acaagcacga 60
taaatgtgtt ctgtttagct tagctcgacc tggaaatgat ttccctatgg agtttgctct 120
tactttcatt tctacaagtc tcgtctttcg atgtaactaa tttcctttta atttactgaa 180
aaattagtat tttttcagtg ataaacaaac atttgatcgt gtcttgttgt caaataatat 240
tttgcaggtg acttacggtg gtgccggtga ttcatcggtg tcggaaaacc catttacacc 300
aaaagcttcg ttcattcgtt attggaacaa acagttgcac agccaagtac ccagcttttt 360
actctccaaa gtgtccccat taaccgccgt cgactccgcc agtttcgcca aactcgccac 420
ccaaaacaac ctcgattcta gtctctcttc cttttgctcc tccgctaatc tcttttgctt 480
ccctgattta tcgttgggtc ctgagaaaga ctggggagac gttaacttcg cccgctacgg 540
caacaaaagc ttcgtcaatt atggatccaa tagtccttgg ggactcgaca ccttcaagaa 600
gtactcagaa aacggtaacg ttgtcgctaa ttcgttccgc agatacagcc ggaactccga 660
ggatcacaag gatcgattct ccacttacgg cggtgggtca aatgtcgtcg accaaagctt 720
caacaactac ggttcctggt ctaaaggcgg tttcggtacc ttcaacagct acaacgacga 780
agtcaacgtc cctaacctcc gattcaattc gtactccgat cattccaagt ggcatgggca 840
cgaattcgcg tcctacacaa aggacgccaa ctccgggagg caacagttta ttaaatacgg 900
caaagatggc gactgggttc gtaatgactt cagtagttac gcaaacggat ccaatgcagt 960
gaactcgggt ttccatacct acgggaagaa agcgagcgga gctgatgata ctttcacttc 1020
gtacgctttg gattccaatg atccggtgat aggtttcaag agttacggac gcagcggtag 1080
tttctccgtc gacagtttct caaattacca gggccaaggc gaatgggata agagcccttt 1140
caaatcttac gataaaaatt ccaagtggtc ggaagctgat ttcagcaact accggaaaac 1200
ttggaaccaa ggagccgaag atttcaccga atacggactc ggagctgagg atccgtccat 1260
aagatttaag tcctacatag agaacaccac tttcaaagat tatgcaaaaa atggtataac 1320
gttcagtgaa tacaaccaga ctatagattc aaacggaacc gatcaagtta acgccggccg 1380
ccgcggtgta aacaagtggg tggagcccgg aaaattcttc cgagaaaata tgttaaagca 1440
aggaacggtt atgccgatgc cggacattag agacaagtta ccggcgaggt cgtttttgcc 1500
acggattatc ctttacaaat taccgttttc cacatcaaag atcagtgaac tgaaacggat 1560
attccacgcc gggaataact cgtcgctcga gtccataatg cttgacgctt taggcgactg 1620
cgaacgagcc ccgagtctcg gcgagaccaa gcggtgtgtg ggctcggccg aggacatgct 1680
cgacttcgcc tcctctgttt tggggcggaa cgtggttgtt cggacgacgg aaaacgtgaa 1740
cggatcaaat acgaatataa cgatcggagc tatacgtaaa atcaacggcg ggaatgtgat 1800
gaaatcagtt tcatgtcacc agagtttgta cccgtattta ctttactact gccattcggt 1860
tcccaaggtt cgggtctacg aagctgatat cctggatccg aattcaatgg agaagattaa 1920
caatggtgtc gccatctgcc atttggatac atccgattgg agtccgggtc atggagcgtt 1980
tctagctttg ggttcgggtc cgggtcaaat cgaagtttgc cactggatat ttgagaatga 2040
tatgacttgg acagtagccg atacctttta aggagtatta ttaattaaat ataaaattat 2100
taaaaaggag tattaattaa gtataatatt attttatata aacgataata ttgaaaataa 2160
agatgtctta t 2171
<210> 7
<211> 949
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 7
gcttaaactc agatatattt tcaagttcag acacttgaac tttatattcc cttgatacaa 60
tatagtacac aaatcaagaa ataaagtaat acatcacaat ctgcaatctc ctgctatctt 120
aaacttcagt tggggacaga gttttcccat tgccctgttt tgatgataat cttacataca 180
aatcccaaag caacataaac ataaagaaac ctaggagaaa cctgtgtcct tcataccaga 240
aatccacatc tgtttctgtc tgaaaattta ggcaaattag tatccttgag atgaggcata 300
gttggcgatg agagccagag cattgcttgt caaatgtgca attttaacaa tacgtttcct 360
caccacagtc tttgcatatc cattcatgtt gttccctgca aatccatcca tgcaagtatc 420
ctcatctgtc agtgccgcgc taacccaggt ttggacatcg ctcatcgtag gcagaaagtt 480
ggagctactt atgtgaccga gttcccctat agacttttgc agctgatcga cggaatcgcc 540
catcacctcg atgcaatcag ccatggcggc agctactctc ggttccaagc catgggttct 600
tgacaccttt atcaacaacc ttgaagttga tttgcttgct ctgagtgtta cattgagggc 660
ggtatgcact aacagtctag ggctggtgtt gattttggtt gcgtagatgg agagtgaccg 720
gtaacataac ctgggataag tggtggagtt gcatgaagat ttgacgtact ccatgtttgt 780
ttggctgtga aatggtggag ttgctaaggt taaagctatg tatgaagcga attgaagggt 840
aatgaagagg atggccaggc catggagaga agatgaacct gtcatgttta actcgggatt 900
cagtaggatt agtggaaatt tggtggtttc agagaggtga agccatggt 949
<210> 8
<211> 1022
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 8
ctccgtgaga gccgcaacca ccagtttttt tttttctata aattaagatc cctttctttc 60
catatatggt cgtcaaccct ctcgaagcat ttctgctgaa atcaaggaga aagagaagga 120
aatggcaaaa attagttcat ggttgcaagt ctcagccatc tttattttcg ccctctttat 180
atctctcccg tgctccgccg caagaaaact ccacacctct gaaacggaca cagacttcat 240
caaaacatgg tgtgcttcaa caagctacac agacttatgt ttgtccacct tttcaagcta 300
cgcatctgaa atccatggca atccccaaat gttagccacc aagtccctct tcgtcacgct 360
taacaccaca cgttccgctt cgaaaacgct taacgagctc ctcaaaaaac atggattaaa 420
gcctagagaa gttgcggctg tccaggactg tgtggaggaa gtaagtgatt ccatcgacga 480
agtcaaaagc tgtattggtg aaatggatga gagtacaggc aaaagctttg ctttccgaat 540
gaatgatatc gaaacatggg tgagtgcggc tttgaccgac gacgatacct gcatggatgg 600
cttctccgag actgccatgg atggagatgt caaagataag gtgagggctg tgatcgagaa 660
agtcgcacgc ttgaccagca tttctttggc ttttgtcaat cgctatgctg gtgccaaaaa 720
ataacccata gtttcatacc acttgaaaac gtattgatca gatttgtatt ttttatttct 780
tatgaattat tttggtcttg caatatatat ggaaagactg tcaaccataa ccttgaatca 840
gtgttgttgt tctatgtctg tggacaaaga cagctgtgat agcttaactg gttttgttat 900
tgaccactgg ctggcgactg gcgtgctagt ttctctcacg aaagcacaat gctatgctgg 960
tagcctggta gctatatatc aatcctacag gcaatgcttc cctttcatgt tccaccaaaa 1020
ct 1022
<210> 9
<211> 3855
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 9
cagtctcaac aatttgcctt atctgatcta ctttggtcta taaaaacaaa gaaaaaatct 60
tcttagcttc tacatcagct ttcaatggct tccaccatta cacgaattct cttcttcttc 120
ttcttcaccg tcttctactt ctcttcaact tccgctgatg tttcttcaac aaattctctg 180
aacccaaatc tcacttccat acgaagtttc tgcaaatcaa ctccataccc agattcttgt 240
ttcgattcat tgaagctttc catttcagta aacatcagtc ccgacatact tttctacttc 300
cttcaaaccc tcaacactgc attatccgaa gctgcaaagc taacgaacct attctccaac 360
ggcggacatg gaaacattat tgtcgagaaa caaagaggaa cactgcaaga ttgcaaggaa 420
ctccatgaac tgacactgtc tttgttgaaa aaaacagtgt tgagaatcaa agatgcagat 480
tcgaggaaga tagctgatac caaagtttat ctcagtgcag ctcttacgaa caaggataca 540
tgtttagaag ggttggattc cgcatttggt ccattgaaat ctgttattgt aaaatctatg 600
attagtactt acaagcatgt aagtaactct ctttcaatgc tgcctaagtc aggaaaacag 660
catccaaacc ggaggctctc tcgtcaggtt ttgcggagga tcgatgacga atatgatcca 720
agcgaagtgc tgattgttgc ggcagatggg acggggaatt tcagtaccat tactgaggct 780
ataaactttg ctcctaataa gagcaaagag agggtgatga tctatgttag ggaaggggtt 840
tacgaagaaa atgtggagat cccaagccgt aaaaccaaca ttgttttgct cggagatggc 900
agtgatgtca ctttcattac tgggaaacga agtgtgggtg atggctggac cacattccgg 960
tctgcaactg ttggtaatct ttgaaacttg gatttgaatt ttgattttga tcaaattcta 1020
aatgcctgat gcgtaagaca acaatgaaga accatgtcgg aatccgatga ttacagataa 1080
attcatcgat tttaactggt tgcacttgta attctatgtc gcagctgtat ctggggaagg 1140
ctttttggct cgggatataa ccattgagaa cagggcaggg ccagagaagc atcaagcagt 1200
agcattgaga gtgaatgcag atttcgctgc attctacaga tgcagcatca atggttacca 1260
ggacacatta tatgtacatt cttttcgaca attttaccgt gaatgcaata tctctgggac 1320
cgtagattat atattcggga acgctgccgc ggtgttccag gcatgtaaca tagtctccag 1380
gatgccgatg ccaggtcagt tcactgtcat cacagctcaa tctcgtgatg ccctggatga 1440
gtacaccggg atttcaatac agaactgctc gattctcgcc actgatgaac tgtacgggaa 1500
ttcaagcagt gtcaaaagct atctagggag accatggagg gcgtattcca cggcagtttt 1560
tcttgaaacc tacatcgatg atttcatcga tcctaatgga tggaggaagt ggtctagtga 1620
tgaagacttg gaaacccttt attacagaga gtacaataat tacggaccag gttccgccac 1680
agataaccga gtcgcctggc ctggatatca tgtaatggat tatgagagtg cctataattt 1740
cacagtctcg gagttcatca cgggtgaagc ttggctggat tctacttcgt ttccatatga 1800
ttatgggatt tgaattccac cggcttctat tattgagggc tgagggagat tttcactttt 1860
acaggtcaga ttcacgtgga tgtggagtct ttttcttgtg gcgggttcca gttttcaaag 1920
cttctgtttg tactttagaa ctgtaaaaat gaaaaattcc aagcaggaat tgtccatggc 1980
gagcttgatt gaactgtaaa ctctgtacat tgctttggct ttagctttgc atgtacaaca 2040
tggtaccccg tgaagatgta tgataggtaa tcacgtgagt ctttggcctt gaaatcaggg 2100
tcagaagact tcggcctgtt tacggtccct agtaggtcag cagtggtgag aagaatcgga 2160
gtggaactgt aggttggtat ccatccttgt cgcaacgatg ttcccttcta aagacaggct 2220
gacacatgcc cctgcaaaat ttgctacaaa atagatagtt aaatgcacaa actggtcaaa 2280
agaagagtac aaattgcggg atggaatacc tttgctgcaa cagtttaact ctagtttttg 2340
cacaaatagt ttatctcgat tctttacagt gaaacactgg gtaaagaaac agctcgtgtt 2400
ctggttgagc aagaccatta aaatgaaagc ttaatctagc tgggaagtat gatctagaag 2460
aacaaatgaa gttattgtct tgtatgaatg atgaaaaata gatcaaacct ggcaatagct 2520
tctcaaggta tttgatgctt tgtagatctc atgttccatg gacaaaccaa tcggcaagtt 2580
caaccaacct agagtacacg tccaatccat gaggtcaagt tttgctactt gcgcagcatc 2640
gccactggat tttataagaa cacattgtaa cggatcaagc atgtcataga aagaatcaca 2700
cagcctcggg gaatttctat ccctgaattt aacaggtagc aagcgccttc ccttagaaca 2760
cgatctacag aaaatccctc cacaaaacct gcactgcact gaagcaaact gatgtagaac 2820
tatccggaaa ccactccgga ggttcagctg ccaaaacctc cttataggca ttataattta 2880
tcccacttga ctccaaaagt ggaatggcac tcggcatata aacagaggac tgcaaacaag 2940
tatcccccat tgtttccaga ccccagaaat gatcgacact gctgatcggc agaaacattc 3000
aaggcgcctt tattactccc atttccgacc gaaattaacc cactcagcac attcttcaag 3060
ttcacctctg gttccaagcc atttgacatt gacatagata ctcatcactc aaatcatatc 3120
ctccatcgat gaactcatca gaatcagcag gaaactgata aagatcattt tttttatcga 3180
cttcgaccct agtgaggtta catttttatc ttctggcaca gtccaaactg gatcaatata 3240
atcatcatca acaaattgat acgcattatt aatattattc tccttttcta cctctgaaag 3300
cgacggatag gaaaccctac ccccccaaag cttgccatta aaccacacca ccaagaatct 3360
agttcttaaa tccaacaagt agtttcacag aaattcaaaa atcaaaaccc gtcttaatcg 3420
cctattaaaa accctaaaca acaaataact gcaatttcca gcaaattaaa tcacgaaaca 3480
acagtacgtt taatagaaga cccaacaaaa ttcttcgttt cctattcata cttccaacgg 3540
aattttatga caaatcaaaa tacaataact tcgtttagaa tttcacgaat cacgatcttg 3600
atttttcatc ctaactaatt gtttgaggga tgaaaatgaa aatagaaatt gagacaaaaa 3660
ccgtaccttc ttcgcacgca tatatgatta ccagatttga cgccttaatt tcacgaggaa 3720
tatgaaatgc aatcaaatgt aaagaagtaa aacaataata aagagggaaa tataacaaaa 3780
tcaaaacttt aggagctggc aatggcacta accgatgggt gatctttcga tcttaatttc 3840
catggcgcgg aaaat 3855
<210> 10
<211> 525
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 10
acaaggccgg accgacccga accagaacac tggtattata atccacaact ctagagttac 60
ggcagcttcc gatttaaggc cagtgcaagg ctcggtcaaa acgtacctag gtaggccgtg 120
gaaagaatat tcgaggacgg ttttcatgaa aacatttctc gatagcctaa ttaaccctgc 180
cggttggatg gaatgggacg gtaatttcgc tctgaacacg ttgtattatg ccgagtatat 240
gaacaccggt cccggttcgt ccacgtctaa tagagtcaaa tggggaggtt accgtgttct 300
taagagtgca agcgaagcgt caaggtttac tgtcggtaat tttctcgccg gaaattcttg 360
gttgccggac actggtgtgc ctttcacttc gggtctttaa ttaatttgtt gcaattgtat 420
atgtatgatt atgaataaat ttaattgcta tacttaatta ggatattatg tcaactcttc 480
tgatgtatcc agcaacatat ttaacaatat atatataaag ttgtc 525
<210> 11
<211> 1383
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 11
tcttccaatt gtattataaa acttcttttt cacttttcag acagccatca aacagttttg 60
tactgattac agtttgagaa attgaaaagt tttaaacctt tgagagtacc ccagacatct 120
atataaagag acccagagat gcagcactga tatcccatat aaaccctacc atttccaaag 180
aatcaaaccc tatttatggc attagcacaa cctcaaatct tcataacaat tctcttgatt 240
ccctgttttg tttttcctgt cgtgttaggc tacaggacca acgatgtgag gtcatggtgc 300
agtaagacac caaacccggt gccttgtgag tattttttag ccaatgatca aaagaatacg 360
ccaataaaag acgaccctga tttcctcaaa atctccatgc acctagcctt agagcgtgcc 420
actcgtgccg aaagcaacac gaatttgtta ggaccgaagt gccgaaacag gcgcgaaaag 480
gctgcatggt ctgattgttt aaagctctat gagctaaccg tcattagtct caacaaaact 540
gttgattcca cccccaattt cgacaaagac gacgctcaaa catggctcag cacggcttta 600
acgaacctcg aaacatgccg gaacgggttt atcgaactcg gtgtcccgga ttatcttctt 660
cccatgatgt caaataatgt gtccaagcta attagtaaca ctctggcact caacaaggca 720
actttcaagg aaccgacgta caaagaaggg ttcccgacgt gggtgaaacc cggtgacagg 780
aagctattgc agtcgtcgtc gccggcttct caggcaaata tagtcgtagc gaaggatggt 840
tcgggggatt ataggacgat aaaagacgcg atatctgccg cttcaagtag gtccgggagt 900
ggaaggttcg tgatatacgt gaaggctggt acgtacaagg aaaacgttga gatcggaacc 960
aagcaaaaaa atattatgat ggtgggcgat ggcataggga aaactattat cacagggagc 1020
aaaagcgttg gaggaggaac cactacgttc gattcagcca ctgttgctgt tgtcggagat 1080
gggttcattg ctcgtgacat tacgttcagg aacaccgccg gtccccaaaa ccaccaggca 1140
gtggctctcc gttcaggctc cgatctctcc gtattctacc gatgcagctt tgaagggtac 1200
caagacactc tttacgtcca ttccgataga caattttatc gagaatgcga catctacgga 1260
actgtcgatt tcatatttgg caacgcagct gttgtgttac aaaattgcaa catatatgca 1320
cgcaatcccc cgaataaaac caacactgtg accgcacaag gccggaccga cccgaaccag 1380
aac 1383
<210> 12
<211> 2312
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 12
cctctatacc aaatgattta tacaaatact aaagatttaa tttatacaat gacctattat 60
tatacaagtt acaactcgtg taataattaa ttattatttt cttatgctaa tacttaaata 120
ccaattctaa gctacaaaag caaatcataa acattatatc aataacataa tcacttcata 180
aaacggaagc acatcacata atcatatcaa gtttttaaaa cttaaaacgc aaagcatatc 240
atgacataac acatcagatt gataagtcac caagcattct ggcagtaacc agaatgtttg 300
gcattttatt gctcgacaat ccaacggaca cagacacaac tagacaccat taaggaggca 360
tgcttgatct tcttttattt aatagctacg atggcatgaa ctccgtttat agagggaacc 420
acctgcacgg ttactcatca tagtcatcat ggcgcttatg gtggtggtgc ttatagcggt 480
gatgacgctt ctcttcatca tcctcatcac catcaccatc actgttgtcg ccaccctaca 540
acattagata ggttcaatgg aatgagtttt gagagggagg atgataatca aagcataaac 600
aagtatggat ataacatcat agttcaccac aagattccaa tttttccaag atatgatgat 660
ataaaccttt gagaacttaa attacctccc aaatcgacat gatgaagatg ctaaaaaaga 720
atcaaaacta tcaacacatc aaaggaataa aaataagtag ttgaaattgg agaaagctca 780
agcaagagat gatggattct taaatctgat ccagagaata tacaattcaa ccaacaccaa 840
gctaccagaa tggatgagct acattgcatc acagataact tgagactgag actagcagga 900
atgattaatg tataatcata ttctgttaat actagttcat ataatgcatg aggtctaaat 960
ggttctatat cttggattag tctgtcctaa cacagtttca actctaaggc aatggtttaa 1020
ttactaacta tgttattcgg attggggttc gggtgtcgga tacaaatata tgtccccaaa 1080
tccatgtatt ggggcattca agaaatataa ggagtaacat agattcataa gcaaatcaca 1140
catcttcaac catatcctac acaattggca gaagtcatct acgaaacaat cagtcaaaag 1200
accaaacgga acttacatca tacacaagta agactacaaa tcatattaaa gctatcaact 1260
ttagttattt ttaccgtaaa aattaaatca acttaaagat actcacatat ttcttgaggc 1320
catagccttc ttcatcgccg tagccatggc tcttcctctt ttcatcgtct gaatcatcat 1380
tgtggttcct gccataactc ggcttcctat atccttcttc ctcttcctct gatctcccat 1440
aactcgattt cctctcatac cctaacccat attcgggttc tgtcctacct ccatagcccg 1500
atccatactc gccctcagcc tcagtcctcc gtccatatcc agatccatat tcagatccac 1560
caccttcata ctctgccctt cgatcatacc cagatccata ttctccatgc tcagctttcc 1620
tcccaaatcc agatccatag tcggatcctt gctccccata ctcaggtctt ccaccatacc 1680
cagatccaaa ttctggtggt ggaggctgct catattcggg tccccgtcca tacccggatc 1740
cgaaatcctg atgatgtgaa tgctcatatg ctggcttccg gccatactca tatccatact 1800
ccgaaggagg ttgctcgaat tcaggtcttc gtccataacc agactcatat tcggatccgg 1860
gttgttcgta ctccggtttt cgtccataac cagattcaaa ttgcccgtat tcaggtttcc 1920
ttccataacc agactcatgc tgagccccgt actgatcaga tccaacacca caaacgggtg 1980
caggttcgaa acgaccaggt cggggcttgg gtctgacata gctactgtat tcagtttgga 2040
gagcttcgtc ggcataagca aggtgttcag caccggatga gaaatcagga cggcagtaat 2100
caaagtcacc atcagacggt gtggctgaag ggtagcaagt ctcttcgcag ggttccaatg 2160
gacgaccata ggtcaattcg agactgtatc caccaccgta aggcgtcgga tcgaactcgt 2220
cgaattcgtc gacgtcatca tcgccggccc tcgaatagta cggcatcttt tgacttacct 2280
actgaaatga aataagattg tgctgaaaat gg 2312
<210> 13
<211> 2966
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 13
attttctctg caactctttc acctaccaaa atagcttcga attttcttcg tattctctga 60
aggaaaaatt ccgggaaaat gaacccgacc cgaaagaaca gttttagctt gattcggatt 120
ctctgtaaaa accgacccat cacccgctcc tctacctccg agtcgtcttc ttgtttgttc 180
ctacctgaaa agatcgtttc taaagataaa aattaagcct tttttttgtt tttgttttgt 240
ttatttgcta aaacccaaaa tgtctccgtt tcaatcgacc caattgtttc agtcgaacaa 300
gaacatcctt agtttaacag aagatatact cgttcggatc tacgagattc ttgtctctga 360
gtcggatcgg aaatcgtttc ggctcgtatg cagagagttc catcgaatcg actcgctaac 420
tcggaagcat ctccgagtac tccggatcga gttcttgccg attcttctcg ataagtaccc 480
ccgactgcaa tctctcgatc tctccgtttg tccccgcatt gaagatgggg tggttccctt 540
gctcttatct cgggttggcc cgggctcgaa ttcgctgagt ttgggtaatt ggactcgggg 600
gctgaagagt ctagtgctga gtcgagctac tgggttgaga ttctcgggtt tggagatgct 660
ggtaagtgca tgtccgtgtt tggagagcgt ggacgtgtcg tattgttgtg ggtttggaga 720
cagggaagct gctgctctgt cgtccgccgt tggattgagg gagttgaaca tggataagtg 780
cttgcgactg accgatgtcg gattggcgaa gatcgctgtc gggtgttcga agttagaaaa 840
attaagcttg aagtggtgta tggagataac tgatcttggt gtcgatcttt tgtgtaagaa 900
atgcgtagat ttgaagtatc ttgacgtctc ttatctcaag gtgactaatg aatcacttca 960
ttctattgct tctctgctga aactggaggc tttgggtttg atggcgtgtc ctttgatcga 1020
tgatgtcgga ttacagttta ttgagcacgg atgcccttta ctaaaggtca ttgatgtatc 1080
caggtgcgaa ggtctgagtt cgtctggttt aatctttgtt agaggtcatg gtaatcttct 1140
ggagctaaat gcaggttact gtctttcagg gctttcgacc gcccttctgc atcagataaa 1200
gaacttaaag tgccttgaag tgattagaat tgatggggct agaatttccg agtctagctt 1260
ccaagtaatt agcaccaact gcaagtcatt ggtagaggtc gggctcagca aatgtgtggg 1320
ggtgaccaac atgggtgtaa tgagacttgt atctggctgc attaatttaa gagtcttaaa 1380
cctaacctgt tgccgttcca ttactgatac tgcagtttct gctatagctg tctcatgcag 1440
aaaccttgca tgcctcaagc tagagtcttg tagtatgatt accgagaagg gtctttgtca 1500
gctcgggtcc ttttgcttgc tactggagga aattgatcta acggattgtt gtggtgtaga 1560
tgacaaaggg ttggaatact tatctaaatg ttctgagcta ttatgcttga aactggggct 1620
ttgcacaaac atatcggata aaggactctc ctatatcggt tctaactgca aaaagattca 1680
tgaactagat ctataccggt gtaccggcat tggtgatgat ggcttagacg ctttatccag 1740
tggttgcaag aagttgagga aactcaacgt gtcatactgc gatgaagttt ccgacagagg 1800
gctaggttat attggtcgtc tagaggaact ttctgatctg gaaatgcggg cacttcataa 1860
aattactggt gtgggtttgg aagcagttgt agctagatgt aagagacttg cagatttgga 1920
tatgaaacat tgtgagaaag ttgatgattc gggctttttg gcacttgccc actactcaag 1980
gaaccttcga cagataaatt tgagctactg tggaatttct gatacggcat tgtgcatggt 2040
gatgtggaac ttgacccggc tgcaggaggc aaagcttgtg cacctcggca atgtcacagt 2100
tgaagggttc gaacttgcac ttggagcctg ctgtgaacgg gtaaaaaagg tcaagctgtt 2160
ggccactcta agattcatgc tttcatcgga gatcctcgaa accctgcatg ctagaggttg 2220
catcatgaga tgggattaga tgaatacatc ttgatgttca tcaggaatat aataaataaa 2280
ctcctcatgc cttgtccatt ttaaagttgt ggctaatata gcctcttctg catgttttct 2340
tcgatagtta aacatgaatt tcgaagttca tagtctgctc tggatagcag gtttaagttc 2400
aactacagct tggacttgga ctgaccacga aatatttatt attctatcat tttaagtatg 2460
ttatttttca tctctgaatg cttcaactgg tgaaaattac agatatgctt attaaaggtc 2520
attttacaga ggcaattggc tacttggttc ccttggcaat aaacatgtct gactcgaacc 2580
tgccaatctc ggcacaacat tctgattact gctcgtccaa ctgttgtaat gacaatcatc 2640
gtactttttg gtgcacggct gcacggccgt cttgtctgat actagctctg caaatatctt 2700
caccattgga aggatcattg atggccggtt atggttacct gcaaccggcg tcaaggtcac 2760
ctgtgggatt tgagcaatca tgttgaaaaa cctttagttc cctttgcatg tatgaatcct 2820
gaccaagtta ccaccttaat aggcaactag tttctaacca tatttttttt ccttttgtca 2880
aaaggaaagt tggtagagga tgtgggatcc gattatttga tatcagttct tttgttactc 2940
cactagtaat tcaatcaata attttt 2966
<210> 14
<211> 2265
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 14
cccaatttac cattaaaaaa cacagtgtaa ataattgcct ttattggtca aatcaactag 60
atttcgtatg accaccacca taatttcttt tcaatttcta tacatttgga tataaataaa 120
tctaaaacca aatgcttcat atcaaacaat tccttcccat tgatcaacag gcaaaatata 180
acaattcact gaaaatgaat acttttttat atggagtcga tcaaaaggtt cgggattaac 240
ccctgtccag gcaagtactg caacaacgac cgttcctcac tcgtgctgct ccgggtgctg 300
tccacaagct tgaaccactg gctcgaaggg tcgcaactgt tgtctttact taggcatttg 360
cagctgtttg tgatgatggt gttgtcggat gctatatcga ggcagatgga cttgccatct 420
ccaaacttag ccgagagatg catcttggaa tcggatatca tgtcccattt cgaattcgag 480
tcagtacaga agataccaag tttcgttaat gtcccggatc cgtcggcttg taagcagaag 540
tatgttcctt ttactactaa tgtcttctgg ggtgtgtaac tccaagcctc ggagtcggta 600
cagggaccta accgtaaagg gtcgaagaat gattttcttg tgatacaaag ccctgttaga 660
ggatggaaga tcactttgtg tagtttggat tctgatagac ctggacctgt atatgtcacc 720
gtggaataac agaagatgtt catcaagtgt tcgatcgaca tcgattttcg atgaaaagag 780
gttaatacat gttaacttac ctcgaaaagg aggctgcaga gctgaaattc tttgtaggaa 840
actcgaattt ctgatttcgc accagttata attcaagatt ccgtagtatt cgtttaaccc 900
gatgacacct tctctaagat aatagcttcc aaccagtgtc cacaatgccc aatccagatc 960
aagctcagct gccacactca agaaacagtt caaataccta ttgtcattca cattggttcc 1020
tcgttcgtcg attccgaact cgctaatgaa caacgggtat ccttgatcaa ccaagtatcc 1080
tgatgtcctt accatgtctc ttgccactct gccacaaact tggttcgggt tgccggtcac 1140
ccatgcctgt ccatccgaga atccatacca atgcgcctcg aacactagtt tcccactaaa 1200
tgtaagattt accggtcggt ttcggatgaa cgacaagtcc ctgtcgtaac tcaatccaga 1260
aagtataact aaaacatctg ggtttgccga gtgcactgcc tcagctcctt tctgcatgta 1320
cttgtaccaa tcgtttacgt tttgtttcgg ccctctgagt tcgttcctca agctcatacc 1380
gaccacattg gtgacaccat tgaataaggt ggccatccga gtcagtccgg tgatccatag 1440
atccgggttg aagtacttgt cgccgaagaa accattgcca tcgaaattac tacagcacca 1500
accgggatta cttatgtgat tgtcaagtat gaccatcaca ttgttttccc ctaagcttga 1560
caccactgtc tgaaatataa ataccaacaa tttcatttta ttattattat ttcatttcaa 1620
gaatttccaa aataatcatt tagcataaaa taatgtgagg ataaaggcta actggctacc 1680
taccacgcat tacgaaacac gtattggata aaccttaatc aaccaatgcc gtaaagtttg 1740
agacttgttt attcaatgtt tagttgagtg tggggtctca aaagggttca cattttcctc 1800
tttcctaggc aacccattcg gagaaaacaa ggattcggaa aatttgtcat acctggtaag 1860
ctttgatgaa ggatacatcg atgatcgaag ggttgtttgc ttggattccg acgatcgatt 1920
caagtagtcc gagttgttga aacgattgac gaacggtgag agatcccaac gaatcattag 1980
tgatcagaaa cagtggccaa gtgaacctaa cacagttgaa ccccatggac acaatccgtt 2040
taccaatcgt ccccatggac tgcttgctga gcccttcggc aagcaccggt tccaagtgcg 2100
acacccaatt cacacacgcc agcttcacac gtcgtcctcc ctcatcgacg atccaacgtg 2160
agttcgtcga caaaggaagc gacgtgacaa taggaaagag tagtaagatg atgagaatgg 2220
aggaggggat gaagcttccc atggattttt tcttttactt ttacc 2265
<210> 15
<211> 3348
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 15
gattagagag attgtatatc aaatattttt ccatgttcta aacatattga tacttcttgg 60
caaggaaatg gaaatggaaa tggaaatgga taggcttgag cagagctagg gaccacaaga 120
aatggttaac gtaatccaag gggggataga gatatggatt aaggagaaaa gttcaaaact 180
tgacttgttt gatgttttgg agttgcttct aatccatctt ctaccatcag acttactatt 240
ctaggacttc ggcagaaggc ggacatgaca aggtttggaa gttacaaagt ataaagtaca 300
tccttttgct gacgtgaaag acaggcaacc caatcccttg aagctaagct tccaaacttt 360
catcgacaca gcatacgcac acagctccgt ctcttcctct cccaggcgat gggttattgc 420
aagcttcctt gcagtgtgct ttttctatgc ctcttcatca gttttctcct tccctatcaa 480
tctttggctg ataaagaaaa cctgtcagct aagctgttta tagatgcttc cgaacggtca 540
ggccatccaa tacctcaaac actcttcgga gtattctttg aggagatcaa ccatgctggt 600
gctggtggtc tgtgggcaga gcttgtaagc aacagaggtt ttgaagccgg gggctctaac 660
actccttcaa acattgaccc atggtccata attggggatg aatcatctat aattgtgtca 720
acggacctgt catcatgttt tgaacgtaac aaggtggctc tcagaatgga ggtgctctgc 780
aatagtgagg gcacttatac ctgtccatct gggggagttg gtatatataa cccagggttt 840
tggggcatga atattgagca agggaagagc tacaaggtcg tattttatgt ccgttcaaca 900
gaagcaattg acatttctgt atcttttacg agctcaaatg ggttgcagac actggcttcc 960
accaaaataa tagcttctgc ttctgatgtt tcaaactgga aaaagatgga ggttctgtta 1020
gaagcaaaag aaacaaacca caattcaagg ctgcaactaa caacaacaaa gaagggtgtg 1080
atatggtttg atcaagtgtc agctatgcct ttggacactt acaaggtttt gtgtttcttt 1140
cttaagtgtg acatattgaa aagtgacaat tttatgtttt tagttcaact gccaagtgcc 1200
aatatttctt gattgcatag aaactgataa ttaagataat tttaggatga ggattgcttg 1260
gagtgcttaa attggttgtc ctggcaggaa tatgtttata tatgtcatct aaaacgatga 1320
tgaggatttc taaactaaaa atatgtactt gatggtttgg aaccctgtgg atatatcttg 1380
aacaagcatg taagtgtaca gaaaactcat gcctaaagtg atttctatga gcgggcaacc 1440
tgtataaacc tatcaaatgg taacacgatc aaaatagcta ttcaaagttt tagatctcat 1500
tttgcaactt gtcatacatt tgagatcaat aagtagcact tctcttgcgt aggctctgtt 1560
catctgcatg ttaacaatgc caaaaatacc ttttccaatt tcgggtttgg tgttttttat 1620
agattatata tactataaag ttgttaccaa tcttttagtt tcaaaaataa aaacagaaat 1680
gggaaacaat gctaaccgga tggaatacta atttcaaggt ttcttttgga tagagtgagt 1740
tatttgcaaa tgtttattag tttatcttaa ggagattgca attttctgga tttgctattg 1800
catgatcttg ttcacaattt catgcagtca tatgtatctt atatagtcta atcttttatc 1860
cggtaatggt tttggaacag gggcatggtt ttcgaaatga gctctttcaa atgcttgcag 1920
atataaaacc tcaatttgtt agatttccag gtggctgttt tgtggaaggt gaatggttaa 1980
gaaatgcttt tcgctggaaa gcaagtgttg gaccttggga agaaagacct gggcattttg 2040
gtgacgtttg gaagtactgg actgatgatg gacttggtta ctttgaattc cttcaattag 2100
cagaggacct tggtgcatcg ccaatatggg tgttcaacaa tggaattagt cacaatgatc 2160
aagttgatac atccagtgtt ttcccttttg tgcaagaagc ccttgacggc cttgagtttg 2220
ctagaggtga ttctagtact aggtggggtg ctgctcgagc tgcaatggga caccctgaac 2280
ctttcgattt gagatatgtt gctattggga atgaggattg ctggaagaag aattatcgag 2340
gaaattacct caagttctat gatgctataa agcgggccta cccagatatc aaaataattt 2400
caaactgtga tggatcgtct cgcccactcg accaccctgc tgatctttat gattttcatg 2460
tttatacatc tgccaacaac ttgttttcca tgtccaatca ttttgatcgt acatcaagac 2520
agggtccaaa ggcctttgtt agtgaatatg ctgtgaccgg gaaagatgct ggcactggaa 2580
gtcttttagc agcacttgct gaggctggat tccttattgg tctggaaaag aacagcgata 2640
ttgtgcagat ggctagctat gcaccacttt ttgtaaattc caatgacagg cgatggaacc 2700
cagatgcaat tgtcttcaac tcctttcagg cctatgggac tcctagctac tgggtgcaac 2760
gttttttcac agagtcaagt ggagcaacct ttcttaatgc aacactgcaa aaagattcct 2820
ctacttccct tgtagcatct gcaattactt ggaaaacttc agatggtcaa acctacataa 2880
ggataaaggc tgtaaacttt gggagcaatt cagtgaacct ccaaatttct gttggtggat 2940
tggatccaaa ctcagtaaag ctgtctggat caacaaagac cattctcaca tctgctaatc 3000
tgatggatga gaattccttc aaggagccaa agaagatcgc ccccattcaa accctgctgg 3060
aacaagcaga ccaggagatg agtgtcctac ttttacccca ttcattcact tcatttgatt 3120
tgttaaagga gtctgtgagt cttagaatta cagaagatga ttggtccctg aaatcttcca 3180
tctgaaataa atgatatggc tgtgtcatgt gtgtattaga ataatgatag tttgccaata 3240
gtctctggtt gatatggagt ctttgacttc aatctatgct atgtatctgg tttagtgaaa 3300
ctataactat aactataact atatatatat atatatatat atatattg 3348
<210> 16
<211> 1150
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 16
gctttactga gtttggatcc aatccaccaa cagaaatttg gaggttcact gaattgctcc 60
caaagtttac agcctggatc cagttgatat cttttcaatt agttagacaa tggtagaagt 120
aagcaagtaa ccaaaaaaat tgatttgtcg cggaagggac cggatgaaat catcttcaga 180
attcataatt agtttactct tctgaccaac aaacctacaa cccctagaaa aaaaaatacc 240
caaaatggca aaggatttta atttatgtta ggtttcatcc aaggatgtca tataggttgg 300
gatagatata atcatgccta acttgcaatc ttttctaact cgagaaggct ttcgatgtat 360
cagtcttttt ataacctgaa atcatgtctg ctaaaccatt atcctattcc tagattgcat 420
tcaagagcca aatacccatg agaaacataa ggggaatata tgtacttgca aacacatgca 480
aacagtgcag ccatatatca agaggtcata tatatgagtt agtacatgca aacagtgcag 540
ccaaaaactg ccagtgaaaa gaaaaaagtc cacaagagat aatggcatgt gttccttaga 600
aatttaaata ataaataaac tggacgcatg ctatcatccc tacccagcat gtggaaagtt 660
tttcgatcca ctggcagtgt acccgacaat aattctatat atataaccag aaaatgttct 720
acgtcctaca cattcaggga aatcaagcaa ttatcctttt tcttaatgtg taaatgcatc 780
tatactgaaa cctattttgt gtgttatttg ttaagcatca cacgccctgg gtgacaaatg 840
aggttttgga ggacatggag ttcttgttag gcagaaggca aaatgaatca aatgtagcta 900
tggtaaaata ttttgagaaa aatgtcatga tcaacacgtt ccagaccttt atccttatgt 960
aggtttgacc atctgaattt ttccaagtaa ttgcagatgc tacaagggaa gtagaggaat 1020
ccttttgcag tgttgagtta agaaaagttg ctccactcga ctctgcgaaa aaacgttgca 1080
cccagtagct aggagtccca tacgcctgaa aggagttgaa gacaattgca tctgggttcc 1140
atcgcctgtc 1150
<210> 17
<211> 1304
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 17
cttttggttt gggttacata tagagtttca gtaaatcaga gacgactaca aagacttgat 60
atcaaccaga gagcatagac tgatgatggt ggactatcat cagtatcata catatttatt 120
tacagacata tatcatgtta tgtttgcata ttactacttc cagtaattca tttatttcag 180
atagcagatt tgagggatga atcatcttct gcaattctaa gactcccaga ctccttcaac 240
aaatcaaatg cagtgaaaga atggggtaaa agtagtacac tcatctcctt gtctgcttgt 300
tccagcagtg tttgaatagg taccaccttc tttggctcct tgaaggaatt ctcatccatc 360
agattagcag atgtgagaac agtctttgtt gatccagaca gctttacgga gtttggatcc 420
aatccaccga tagaaatttg gaggttcact gaactgctcc caaagttcac agcctgtatt 480
tttaaaacag aaaaaaaaag gaccattttg aatgcaagtg gaatgtaagc caataaaatg 540
aaagataatc aacatcttct cgattagtag gacagcggta gaagcgagca tgtaaaggat 600
ttggatactt tgattgacgg gttttaagga acgtgtaaat tctaaaatat gaacattaga 660
gagaagtaaa tttcaaaatg caaagaaatg gaaacagtgc agtcatttaa tgcatgccta 720
tatatcaaga ggtcttatat atatatattt aatatttggt acattgctgg cagaaagtgt 780
ccccttaaaa tatgaaaata aataaatgag atccgtcatc atctaaaccc gactagtgaa 840
acgttttcat tctattggca gtgtatctga tcataatcct atttatataa tgaaaaaggt 900
tcttctaggt actacacatt caaggaaatc aagatatgat cctgttctaa atatgtgtaa 960
atgtatctat agtgaaacct agccatggac ttctagctag ggagaaggca aaatgaatca 1020
aatgaagcta tggggaaaca atttgagaaa aatatcatga tctccatgtt acagaccttt 1080
atccttatgt aggttttacc atcctctgag tttttccaag taattgcaga tgctacaatg 1140
gaagtagagg aatctttttg cagtgttgca ttaagagaag ttgctccact cgactctctg 1200
aaaaaatgtt gcacccaata gctaggagtt ccatatgcct gaaaggagtt gaacacgatt 1260
gcatctgggt tccacctcct gtcattggaa tttacaaaga gtgg 1304
<210> 18
<211> 683
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 18
tttgtggaag gtgaatggtt aagaaatgct tttcgctgga aagcaagtgt tggaccttgg 60
gaagaaagac ctgggcactt tggtgacgtg tggatgtact ggactgatga tggaatgggt 120
tactttgagt ttcttcaatt agcagaggac cttggtgcgt tgccaatatg ggtgttcaac 180
aacggtactt tgcatgtgca tctttctact cattatgtat tttcttttct tttttaaaag 240
ttataactag tttactaatg ccttcggtag ctttaccttt ttccttgggc tttatcaaaa 300
tttacaggaa ttagtcacaa tgatcaagtt gatacatcca gtgttttgcc ttttgtgcaa 360
gaagcccttg atggcattga gtttgctagg ggtgattcga attctacatg gggggctctt 420
cgagctgcaa tgggacaccc tgaacctttt gatttgaaat atgttgctat agggaatgag 480
gattgtggga agaagaatta tcgaggaaat tacctcaagt tctatgatgc tataaaacag 540
gcctacccag atatcaaaac aatttcaaac tgtgatggat cgtctcgccc gctcgaccac 600
ccaactgatc tttatgattt tcatgtttat acatctgcca acaacttgtt ttccatgtcc 660
aatcagtttg atcatacatc acg 683
<210> 19
<211> 930
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 19
tgggacaccc tgaacctttt gatttgagat atgttgctat tgggaatgag gattgctgga 60
agaagaatta tcgaggtctg caaacttacc atacttgcac atgcatgtgt atgtggattt 120
gttagagaga tagtagttat aaatgagaaa cttttatagt tttttgcact tcattaataa 180
cttttttttg tcaaaaaaga ttagttattt agttattgaa ggtaaacttt gcgtggtcca 240
tatgagggtg gatgttgagt ctagcctgtg agtatttggt gttgcgaaat tttgaaaaac 300
agagctatct tagtagacat gaaaagcttt aatccattgc acaaggatta ctggtatgtg 360
tttactactt aggtgggaca gcttgttttc aaaagtgatt ggatccacaa ttgctggttt 420
cttaatttga tgtgtgttca tgtgtgtgac tcagaagcat gaggtttcat caaataagta 480
gttgtcatgg ttttgtaaac tatattattc tcgtggctta gtttcaggaa tatcaatgtt 540
cagttccaca taacctctga ttttgaaaag cgtgcttgaa aatttgtggt tggcaagcaa 600
tgatgaaata caattgtcct gaatcctgat tgtcattttt gaatatacat ttatcactga 660
tgttgctgat cgtatcactt aacaactttt ttgagccaga catgatgaca aattcgattg 720
agcagtaaat tacctcaagt tcaatttaat ttagaaatat ccgtggagat tcattgcccc 780
ctcatttttt tgaactgagc aggaaattac ctcaagttct acaatgctat aaagcgggcc 840
tacccagata tcaaaataat ttcaaactgt gatggatcgt ctcgtccact cgaccaccct 900
gctgatcttt atgattttca tgtttataca 930
<210> 20
<211> 2731
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 20
tttatcatga aaaaaagcta ttgaaatgct tggatttatt aattttacat ttattttcac 60
aaagataagc aataaaattg cttgcctctg gagattcatg cataacttga ttccagcaaa 120
agctagagtg atcctagtaa ttttaagttt gctttataaa gctgtatcag acaaatgata 180
gcttctctat gtacaattaa gcattctttt tggaccattt cacgatcaat gtttactgtt 240
tgccgaacag actgtgttct gtttcactct gttcatactc tattgctttt tatcttcttt 300
gcagtaaggg agtgtgactg taagtatata tgtactgaat atgaactcgc ttgaacttta 360
ttaccaccaa tctatgtctt tagttttcaa ctgcaggcag cttcaactga gagcttcttc 420
acgctcctgc agttgttctt ttaaccaaag caattctaga tggatcacca ccccattctt 480
caaacacaac caaaaggttt ccacttggct tcacccacga acgaggaaca tggtaccatc 540
tttgagaagg ctgtccacag ccagttctac attttttctc agtgtaagtt ccggcataat 600
cacaagcacc acaactgcca ctagctgtat atccaggcca gtggcgtcca atgctctggc 660
cgtttatcca tacttcccct ttccccatgc tactcatatc taaagctaat ggttcattgc 720
ctcccggtgc attgaaagtt gtcttgtacc aagtcatagg ttgtttcttc accaattgtt 780
ctgcttgaac ccattcaaca aaaaagcttc cagaaacagt gtgaaagttt aagccttccc 840
cttttagacc aatcttgtaa gaccatttct gtttagatat gtctcttgtc ccctcattga 900
gacccttcaa tgtaaccggg cctagaacac cagcattcca tgtttcgaaa tgagtaccga 960
cattcgggag tccgactgtg acacttaata aagaaatctt attgatcccg gctcttagct 1020
tgacattctt gctgaatgat agttttggat tgtctaatcc accgtatgcg gtgcctgata 1080
gttgaccgtt gatgaaaaca tgcaaagcat ggcctgctga ccaaatggtg agaagaggat 1140
cttgtccatt cctcaagtcg ttagcgtcga tttttacatc tgtcatgtac cacaaataat 1200
ctgatgcatc tccggtgaga tatacttgtt cccaaagccc gtcatgtaca aatgtatctc 1260
gatcatcagc agtggggctt tcttcattgt atgattgcca tgaaaatgca ctgcttactg 1320
gcaccattgc cggttgagat cttggggatc caaccctggc agtgttgaaa acagcagttt 1380
tgcagtcagg aaggatgctg atggaccacc gcggtaagtc gtattgaacc tttccgaacg 1440
ttacttttac ggagtatttt gtgtcgtagt ttgcaaggaa tgcagcacat gctgacctcg 1500
accagaatac atgagcctcc tggttacgtc cgagagatgt cactgtagga tttgcagaaa 1560
ctaaggctgg ttcacttgat ttgattgctt tatgcagatc tcttagatgt ccccattttg 1620
gttccctttt aagcccgtat tcatcaagag gagcatcgta atcatagcta gtagcaatga 1680
agggaccacc ggcagtccgg ccgaagttgg ttcctccatg gtacatgtaa taattaacaa 1740
atgaaccacc attctgtatg aatcttgcaa ccgaaaatgc tatgtcttct acaggtcttg 1800
tcggaactgc aacaccgaaa cccgtaaacc aaccagtcca gttttcagtc cacatctttg 1860
gcttatactt tgcattggga gtaaagtttt cacagtaaaa tccattgcaa gtgtttatca 1920
cagggtcagg agcatcatct tgcttgcaca taatccacgg gactccggtg ttgagtccaa 1980
ccgccatttg tgcagcccat ttcgtgtaag ctttaccggg agcgccgatt tcccattcga 2040
ccggaccgaa ttcattctca atctgagcca taataattgg tcctccttga gtctgaaaca 2100
gcctctctgc tttcatcatg ttgactatct tagttgtgaa tgtttgcatt gcagccttga 2160
aaggtccatt gtctgtccta aaagcaatgc caggaacata ttttaaccag acaggaaacc 2220
ccccataatt ccattcagca caaacatatg gaccaatccg gagattaaca taaagtccag 2280
cttgttgaac cagtttaaca aatctaacca gatcatatct atcctgaaag taatactgca 2340
aaacaaaaag aaacagaatc tccaaagaaa atcatacaga aaatgaaaga aacatatgaa 2400
ttatgtgcta caaagtgtat tactttgcca cgagaaggct catgtccatt ccagaaaaca 2460
taagtttgta taacatccaa gccaccttct ttagcctttg ctataagatc aggccacatc 2520
tctggtgtac ttctgggata atgaatagaa ccagaaagaa gaattcttct tctgccatta 2580
atggtgatag ctttgctatc ataagaaaca gtagctgaaa ccgagaaaac ccaggaagaa 2640
aacagcatcc ataacatgaa catgtttttt ctgtacctca ttattaacaa gacttgaaga 2700
aatcaatgga gggagtcgtt aaggagtttt g 2731
<210> 21
<211> 3418
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 21
atgaagcttc ccgcgtctga gtttacagaa aaaagtgaag ccattgagag gcgccaaaag 60
ggtccacaca tttcactttc aatcctcagt tcttcactgt ttaaatgcag ctgcaaacca 120
ccaccaaaaa attcaaactt tttttcattt ttcatctccc aaagttgaaa aaaggttaat 180
aggaacacat ttttttttct attatttaag gtttgaaagg aatgcagaat cccaatgtca 240
caaacaagca cataagaaac agctgattgt ttggttttac ttgtttccct ctcccttttc 300
cttttttctt tttttttttc tcgagaataa agtgtacaag ataaagaatc aggaaaaaag 360
ggcattgctt ttgtgtgcaa acaagagtgt ctcatttgtt cattttgtta aagttctttt 420
cttttctgga catggctatg gattccaact ccaagcttcc tataatgctg tatgcttggc 480
tggttgttct gttagcttac tgggtatgtt cagtttctgc tactgtttcc tatgatcata 540
aagctatcac cattaatgga caaagaagga tcctcatttc tggatccatt cactacccaa 600
gaagctctcc tgagatgtgg ccagatctta tacagaaggc taaagaagga ggactagatg 660
tgattcagac atatgttttt tggaatggcc atgaacctgc acctggaaaa tattactttg 720
gaggcaacta tgatttggtc aaatttatta agctagtgaa gcaagcaggc ctctatgttc 780
atctgaggat tggtccttat gtctgtgctg agtggaactt tgggggtttt cctgtttggc 840
tgaagtatgt tcctggcatc agtttcagaa cgaacaatgg acctttcaag gctcaaatgc 900
aaagatttac aaaaaagatt gtggatatga tgaaagctga aaggttgttt gagtctcaag 960
ggggtcctat tattctatct cagattgaga atgaatacgg acccatggaa tacgaactcg 1020
gtgcacctgg taaagcttat agttattggg cagctaaaat ggctttggga cttgggaccg 1080
gtgtcccctg ggtcatgtgc aagcaagatg atgcaccaga ccccattata aacacctgca 1140
atggtttcta ctgtgattac ttttctccta acaaggccta caaaccaaag atgtggactg 1200
aagcctggac aggctggtat acagagtttg gaggggcagt tccttacaga cctgcagaag 1260
acttggcatt ttcagttgca aggtttatac aaaaaggagg atcgttcatt aattattata 1320
tgtatcatgg aggaacaaat tttggccgaa ctgctggtgg tcctttcatt gctactagct 1380
atgattatga tgctcctctt gatgaatacg gactaaagag gcaacccaag tggggccatt 1440
tgaaagattt gcatagagct ataaagcttt gtgaaccagc tttagtaaat ggagatccga 1500
ccgtcatgcg acttggaaac tttcaagagg ctcatgtatt caaatataag gccggaggtt 1560
gcgctgcctt ccttgcaaat tacaacccga gagcttatgc tacagttgcc tttcagaaca 1620
agcactacaa cctgcctcct tggtctatca gcattcttcc tgactgcagg aacactgttt 1680
ataacactgc aagggttgga gcccaaattg cacggaagaa aatggttctc gttcccatgc 1740
acgggggatt ctcttggcag gcatacactg aagggaccac atctgatact gacagttcat 1800
tcacgatggt cgggttgttg gaacagataa atgcaactag agatgcaact gactatttgt 1860
ggtacacaac agatgttaag attggctcga atgaaggatt cttgaggaat ggaaaatctc 1920
ctgttcttac cgtcttatca gctggccatg cattgcacgt ctttgtcaat ggtcaactat 1980
caggaagttc ctatggaagt ttagaatccc ccaaactaac attcagacaa ggcgttaatt 2040
tgagagctgg tatcaataaa atttcgcttc taagcattgc tgttggtctc ccgaatgttg 2100
gtccacactt tgagacatgg aatgctggtg ttcttggccc ggttacattg aatggtctca 2160
acgagggaag gagagatctc tcttggcaga agtggtctta caagattggc cttaagggag 2220
aggccttgaa tcttcattca gtaagtggta gttcatcagt ggagtgggca cagaggtcat 2280
ttgtcacccg aaggcagcca cttacgtggt acaaaacaac tttcaatgct ccagctggaa 2340
cttctccctt ggctttagat atgagtagta tgggcaaagg ccagatttgg ataaatggaa 2400
agagcatcgg acgccactgg cctgcatata aagcttccgg aaattgtggt gtctgtagtt 2460
atgccggaac atttaatgag aagaaatgcg gaactaattg tggagaggcc tctcaaagat 2520
ggtaccatgt tcctcgttca tggctcaacc caacaaggaa tttgttggtc gtgtttgagg 2580
aatggggtgg agacccgaat ggaatttctt tggtccgccg agagactgac agtgtatgtg 2640
ctgatatcta tgaatggcaa ccaaacttaa tgaattatct gatgcaagcc tctggtaaag 2700
tcaacaaacc cctaaggcca aaagtccatt tagagtgtga tgctggtcag aaaatctctg 2760
cagtgaagtt tgccagcttc ggcacaccgg aaggggtctg tggaagctac cgcgaaggaa 2820
gctgtcatgc acaccactct tatgatgcat tcaataggct atgtgtcggg cagaacttct 2880
gtgcagtaac tgtagcacca gagatgttcg gaggagaccc gtgtcctaga gtcatgaaga 2940
aactctctgt ggaggtcgtt tgcagctgac tgaacacatc cgaaataccg aaataaacac 3000
tccaagatct caaccatttt gttttcagct tttttccagg gtcaagttgt acaaatccac 3060
aacacacacc cttggcaaaa agctcattag attgtgaaat gattgcagaa taagctctat 3120
gtatatatac acatatgttt gatcctgcat aacataccaa agaagcgatg gaagtaccat 3180
agaagttgtg catcgagacg atactatacc gagtccatgt gcaatgtgtt ccatcattta 3240
ggataacaag aaaacctctg gtttctactg gtaaaagtct attgttgtaa tctaaaaaac 3300
aatcttttca ttgctctgtt cttgtaagaa cagcttgaga agaaaataaa atcatgccct 3360
ttcatgatct ctgatgtaat tccttttaac tattattaaa actgtgattg acctttca 3418
<210> 22
<211> 3604
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 22
cagataatat caaaatgcag tagctaaaca acaagaagaa gaggaagaag atgatgaaat 60
cagcggcggc gtgcagtgct gttttctggt aacttacagt gttcattttg ttatacaaag 120
cggccaagca acaaacatta tctgagcaaa ggagggaaat aaagtagaaa aaggagacca 180
acctaaagac aaaagcaaaa gaattgttta caatcaagaa gctaagtttt cttcgttttt 240
atatataaaa aagaagggta ctttacgtgt gtgattgtgt tcctgtaaat tttcacaatg 300
aggggaagaa cagagattct ggcgttgttt ttttgtttgg ttactgcaac gaagatgttt 360
gcatccaccg tcacgtacga tcaccgagcg gttgtcatcg acgggaaacg ccgcgttttg 420
gtatccggct ccattcacta ccctcgcagc acccctgata tgtggccaga ccttatacaa 480
aaatccaaag atggaggatt agatgtgatc gaaacttacg ttttctggaa tttacatgaa 540
ccagttagaa accagtataa tttcgaagga agaaacgatt tggttaaatt tgtaaagtta 600
gttgcagcag ctggtcttta tgttcatctg cgcatcgggc catatgtttg tgctgaatgg 660
aactatggtg gatttcctct ttggttgcat tttatacccg gaatcaagtt tcgaactgat 720
aatgaaccat ttaaggctga gatgcagagg ttcacggcta agattgtgga catgatgaag 780
caagagaaac tgtatgcatc acaaggagga cccattattt tgtcacagat tgaaaatgag 840
tatggaaata ttgattcagc ttatggggca gctgcaaaac catatatcaa atgggcagct 900
gatatgtctg tttcattgga taccggagtt ccctgggtca tgtgccagca atctgatgcc 960
cctgatccca ttattaacac ctgcaatgga ttctactgtg accaattcac cccaaattct 1020
aataagaaac caaagatgtg gactgagaat tggaccggat ggtttctttc tttcggtggt 1080
gctgttcctt acagacctgt agaagacatc gcatttgctg ttgcgcggtt ttaccaaaga 1140
ggtggaactt tccaaaacta ttatatgtac catggtggaa caaactttgg caggactaca 1200
ggtggaccct tcattgctac tagttatgat tatgatgctc caattgatga atacggactt 1260
gttagacaac ctaaatgggg tcatctaaga gatgttcata aggctataaa gctttgcgaa 1320
gaagcattga tagccactga tcctactatt tcctccttgg gtccaaactt ggaggctgct 1380
gtatataaaa caggatcagc atgttctgct tttctagcca atgtggacgg taaatctgat 1440
gcaactgttc atttcaatgg gaattcatac gttttacctg catggtctgt cggcatctta 1500
ccagactgca agaatgtagt tctgaatact gccaagatta actctcagac tgtgattcca 1560
agcttcatgc acgaatcttt gaataccaat gctgactcaa tcgggtcagg ctggagttgg 1620
ataaacgaac ctgtgggtat ctcaaaggac agtgcattta gtaaacttgg attgttagag 1680
caaatcaaca ctactgctga taaaagtgac tacttatggt attcgttaag catggatatc 1740
aagggagatg agcctttcct ccaagatgga tctcaaaccg ttctccatat tgaatcactt 1800
gggcattctc ttcatgcttt tataaatggg aaacttgcag ggagcggaac tggcaatagc 1860
ggaaatgtta aggttaaagt ggatatcccc atcacggttg tacctgggaa gaacacaatc 1920
gatcttctga gtttgactgt aggactgcag aactatgggg ccttttttga cctatcaggg 1980
gcaggggtca gcggtccggt gaaattaaat ggtttaaaca atggtagcag catagatctc 2040
tcctcacagc agtggagata tcaggtcagt atctcttttt ttcactatga tgttaatttt 2100
gatcattctc agataaacac tgcatttttt tctttcttga tgttgagact taatattaca 2160
tactttatct cgcggaagat gaaccaaaac cgtaaacgaa gttaactcct aaaagaaaat 2220
ttttgcaaca gtactataat tttccttgtt tctgtaatca tgatatatag attggactta 2280
aaggagaaga ttcaggtcta ccaagtggaa gttcttccca atggatctca caatccggct 2340
tgcctaagaa tcaaccctta atatggtaca aggtaacttt aaattagtcg tgaattattt 2400
gataaggttt ttagcctatc ctttttgcat tggttacttt tatatacttt atttttcttt 2460
ttagtcctga atgtatacac acacaggaag aagcccattc tctctgtgtg tatatatata 2520
tctctttaaa ccttcaattt ctgatgagtt ttttgaagaa aaacttttgt atgtattgct 2580
acttatttat tttgatccat cgtgaacttc catgatccga ttttgtctat ggtcttttat 2640
tgttcataga agtattgtgg tggatcgaat ctaagctatt aaagctcaaa cttctgtctg 2700
cagacaaatt ttgatgcccc aactgggaat ggtccaattg ctttagactt cactgggatg 2760
gggaagggtg aagcatgggt gaacggtcag agcattggaa gatattggtc gtcctatatc 2820
gctccaaatg gtggctgtac tgacacttgt aattatagag gagcttacag ttcaaacaaa 2880
tgcctcaaga attgtgggaa gccatctcag caattgtacc atgtaccccg ttcatggtta 2940
caaccgagtg gtaacattct cgtattgttt gaggaaattg gcggggatcc gacacagctt 3000
gcttttgcaa cgaaacagat gggaagtttg tgctcacatg tatctgaaac gcacccttta 3060
cctgtagata tgtggacttt ggattcaaaa acaagaagag cttcaaactc tactctatcc 3120
ctcagttgcc caccgaatca ggttatttct tcaatcaaat ttgcaagcta tggaactcct 3180
ctcggcactt gtggcagttt tagccatggc aagtgtagca gcgctaaagc acgctccatc 3240
atacacaagg cttgtgttgg atcgacaagt tgcagtatca atgtctctac tagcacattc 3300
ggtgaccctt gcaagggagt aaagaagagc ttggcagtag aagtttcttg tgcataaaga 3360
aatgtcaata agcatgcgtt actgggtgcg agcaaaggca ttgtccccca gtaagtgttc 3420
aaatcaaata agcttttcag caaagaattc ttctctaggg gggggggggg gttctcatac 3480
tggaaatatg ttattggcag cactggaaaa attaagatta atgtagtatt aatggcattt 3540
ctgcatagta tcttttagca gagcacattc ttataattcg attggacttg aaaaataatt 3600
gaac 3604
<210> 23
<211> 533
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 23
gccacgagaa ggctcatgtc cattccagaa aacataagtt tgtataacat ccaagccccc 60
ttctttagcc tttgctataa gatcaggcca catctctggg gtgcttcttg gataatgaat 120
agacccagaa agaagaattc ttcttcttcc attaatggtg atagctttgc tatcataaga 180
aacagtggct gaaactgaga aaacccagga agaaaacaat agccatagca tgaacatgaa 240
catgtttttt ctgtacatca tattaacaag aatgaagaaa atcaatggag gaagaattgg 300
aagaatattt aaagagtttt gggttttcgt gaagctctct tcttcttctt cttcttcttc 360
ttcttctccc ccctgcaact ggtttttata ggtaattttt taacaaattc tgcttctgtg 420
gtggactcct ctctgacatg aaacttgaac atccattttt aaatgatcaa tcttcgtttt 480
tacttcactt atactattct tttctctcga attcggatat ttttgagttt tgt 533
<210> 24
<211> 716
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 24
tcagtcggtt ttctgatgtt agcttcgttc tttctctact tctgatgaca tattgtccaa 60
ttttcataca tttcagaatg tcggcactca ttttgaaaca tggaacgctg gtgttctagg 120
cccggttaca ttgaagggtc tcaatgaggg gacaagagac atatctaagc agaaatggtc 180
ttacaaggtt tgctttaaga atttcgagta tttgcactta caggcatatt gatgtactaa 240
taggcaatgt tggcatgatg ttttcagatt ggtctgaaag gggaaggctt aaaccttcac 300
actgtttctg gaagctcttc tgttgaatgg gctcaaggag cacaattggt gaagaaacaa 360
cctatgactt ggtacaaggt tagggagttc ccatgtttga ttttaacaga tgccatgtct 420
gaaaaagatc gaatcagata tcaatcttta acatctgttg ttttgcagac aactttcaat 480
gcaccgggag gcaatgaacc attagcttta gatatgagta gcatggggaa aggagaagta 540
tggataaacg gacagagcat cggacgccac tggcctggat atatagcaac tggtggctgt 600
ggtgcttgtg attatgccgg aacttatact gagaaaaaat gtagaactgg ctgtggacag 660
ccttctcaaa gatggtatca tgttccacgt tcgtgggtga agccaagtgg aaacct 716
<210> 25
<211> 3312
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 25
cccaactcca cttctacaat cacatttttg cctatttact attttcttat caatcaaact 60
actacatagg catagccaga gaaaaaagat gaaggcggtg gtgataacaa cagcaggagg 120
tccagatgtg ttgcaagtac aacaagttga tgacccagaa atcagggatg atgaagtcct 180
catcaaggta gaagccaccg ctttgaaccg ggctgatact ctccagagga aaggttctta 240
ccctcctcca aagggtgcta gcccttaccc tggacttgaa tgttcaggca ccgtcctagc 300
tgttggcaac agtgtttctc gctggaaagt tggtgatcag gtaaaattga aaaaataaaa 360
cttctcgtaa aagatgtgat ttttatataa agggtccagt gtttttttca ttggatgggg 420
cttaaagatt ggatttttgt gtttttagaa ttttaggaaa ttgggtttgc gtgatattta 480
tatgcaggtg tgtgctcttc ttaatggagg tggatatgct gagaaagtgg cggttccagc 540
tggacatgtt cttcccatcc cacctggggt ttcactcaaa gatgctgctg gtttgcctga 600
agtggcttgt actgtttggt ccactgtttt tatgatgagt cgcttatcgg ccggggagac 660
tttcctggtc catggtggtt caagtggaat tggaactttt gcaattcaga tagccaagga 720
aaaaggagca acagtattgg ttacagcagg tctatcttca atagtgattg aatcggaaac 780
tttgtgtttt aatttggtcc tatatttaga tgtggtagtg ttgatcttca gggagtgaag 840
aaaaattagc tttctgcaag aatcttggag ccgatgtgtg catcaattac aagacagagg 900
actttgttgc acgtgttaag gaagaaactg gagggaaggg tgttgatgtt attcttgatt 960
gtattggagc atcctacctt cagcgaaacc tggacagctt aaacttcgat ggaaggcttt 1020
gtattattgg cttccagggt ggagcagtca ctggaattaa actcaatact ttacttccaa 1080
agcgactcac cgtgcaaggg gctgcattgc gaccaagaag tgcagaaaat aaagcaacgg 1140
ttgtcagtga agtggagaac aatgtttggc ctgcagttgc agcaggcaag gtgaagccta 1200
tcatctacca atcttttcct ttatccaagg ccggtgagtc tcatcggctc atggaaagta 1260
gtgaacatat tgggaagata cttcttgttc cgtgatgccg gtaaggacct gtcttgtagt 1320
gagaaaatgg ggtgtcattt aacgataaaa tgagtcgctt gtggaacttg tatgtgtcat 1380
gtccttgtgg gctaagttta atctttttac caaaatgata atcatgcaaa accgatttgt 1440
tgtgaaagat ttttagtaca ttccagtgtc atgtatttga gaaagatata atcagcaagc 1500
ttctgtgtta ttaattgcgt gtcaaaataa aaagtattgt gatcaagtaa aaaagcaatg 1560
agattttatt cgctacatat agcttcaatg gcaagggtct tcagcacatc ggggcaaggg 1620
tcaccaaggt tagcagcgga aacaggtatt gagcactgct cctgaccaat gcaagcctgc 1680
aaattcaacc gaggatgccg aagaaatttt gctcaactgt gaaatctgca atctaaaagg 1740
taccatatat gaacaaagaa aggttctctc ttaccttttt aacctttgat agtatgttag 1800
catgacaagc ccctgctttg aatgaaccac aatttccttg tggagatcca aaactggcaa 1860
agttaatcga agtaatgcgc cagccttgtt cacaggtgag ttgagctttg ggactttgag 1920
acctgaagcc tttgtttcgt atccaagaat cagccggccg aggatcatcc tcggatacaa 1980
ttgaacaaat ctcttggccg gttcgtgtca gcactgaaat ctgtgtaggg tcaccaccga 2040
gctcttcatg gagtactaat atgttctcac tgggatgtac ccaagagcgt gggacatgat 2100
atctggacaa aacgaataga gacaattcga gaatacatca aacacagaag tttacatata 2160
ggatcaggat caaatgtttg gtaaaactta caatgtctga gcaggttggc cacaactctg 2220
ttgacatttt gtcgaatcat attcacctct ataatcgcaa ttctcggtac aaccattggg 2280
tggtgagaga taggccggcc agtatcgtcc tatactctgt ccattaaccc aagcttgacc 2340
cttccccatg ctagtgagat ttaaggctaa gggacccttg ccctgtggag cagagaaagg 2400
aattttgtac cagatcaatg aattgttgat aggtggaata ctcccctttg tccacagtga 2460
actatttgct tggattactt catctagtcc aaggtattct ccctcaagcc ccacctaatc 2520
atacaaaaac ttccatggat gatatatgta actcgaaaag tcgataactt gataatatat 2580
gctaggctaa tgttgttgat ctgcagattt taacctgata tgtccattct tcagaagaat 2640
catcatactt gttataccat agatcaaaaa gaaccactga aaaaactccg gctccttgaa 2700
catcgaacca cggcccgaag ttctgaaaca tataaaaaat gcaagtctat caaagtaacc 2760
cttcaggcat gaaggttttg gaataagcta agaaattttg attacctgta gaccaaccat 2820
catgctcaat atatccaatg tattgttccc ctcaacaaga ctaattttct caaagattga 2880
aaagctagca tcatcatgat taccatatcc aaaagctgta aaaaagagag tagaaaacag 2940
aaggaaaaca tcacatagaa gatctgtgac ggtttcgaat tcagcttcaa agttggcgtc 3000
gaagagtgcg gtggagagat atcgagattg gacaggagga taccaaacga acatcattca 3060
catgatgaat aaaagaagaa gaagaaagta cattcaacag caatcaaaac agcaaattac 3120
caacaagctt cttgttaaca aatacaagtg ctgcatgtcc caagctttcg ataagcagga 3180
aggcctcttt aggttggccc ggcgtgatat ttatactgtc aattcaatca agattcgtta 3240
atcaatgaat ataatatgtc atagttgatt tgtgctagat ttcaaacact tcatttactt 3300
ggcagaaagg tt 3312
<210> 26
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 26
cacatacaac cttacaactg 20
<210> 27
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 27
tggagatact tgcttcac 18
<210> 28
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 28
aatgccttgt atgagaat 18
<210> 29
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 29
taccagacct tacctaat 18
<210> 30
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 30
ctcctcctct tcttcctt 18
<210> 31
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 31
ggcatcttca tctgtcaa 18
<210> 32
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 32
gccattatta ccttgcgtta 20
<210> 33
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 33
caccaacctc acagatgt 18
<210> 34
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 34
ctgtgaggca agaagagt 18
<210> 35
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 35
aatgtggctg actggtag 18
<210> 36
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 36
ggttcgtaat gacttcag 18
<210> 37
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 37
ctttcttccc gtaggtat 18
<210> 38
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 38
tcggtgagaa gcacagggtg 20
<210> 39
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 39
ggttgggatg ggtcagaagg 20
<210> 40
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 40
aacctgtgtc cttcatacc 19
<210> 41
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 41
gctctcatcg ccaactat 18
<210> 42
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 42
ggacacagac ttcatcaa 18
<210> 43
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 43
gacttggtgg ctaacatt 18
<210> 44
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 44
ctcttacgaa caaggataca t 21
<210> 45
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 45
gacttaggca gcattgaa 18
<210> 46
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 46
gaagcgtcaa ggtttact 18
<210> 47
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 47
tggatacatc agaagagttg 20
<210> 48
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 48
gcacaacctc aaatcttca 19
<210> 49
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 49
ttggtcctgt agcctaac 18
<210> 50
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 50
ttatggtggt ggtgcttata g 21
<210> 51
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 51
cgacaacagt gatggtgat 19
<210> 52
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 52
tgaagtggtg tatggagat 19
<210> 53
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 53
agaagcaata gaatgaagtg at 22
<210> 54
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 54
cgatgacacc ttctctaa 18
<210> 55
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 55
ggaaccaatg tgaatgac 18
<210> 56
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 56
ggagtctgtg agtcttag 18
<210> 57
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 57
atacatagca tagattgaag tc 22
<210> 58
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 58
tggtagaagt aagcaagtaa 20
<210> 59
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 59
ttgttggtca gaagagtaa 19
<210> 60
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 60
ctccttgtct gcttgttc 18
<210> 61
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 61
ttctcacatc tgctaatctg 20
<210> 62
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 62
aattacctca agttctatga tg 22
<210> 63
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 63
aagttgttgg cagatgta 18
<210> 64
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 64
tattctcgtg gcttagtt 18
<210> 65
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 65
atcaggattc aggacaat 18
<210> 66
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 66
gagcatcgta atcatagc 18
<210> 67
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 67
atacagaatg gtggttca 18
<210> 68
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 68
tgttcttacc gtcttatc 18
<210> 69
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 69
ccttgtctga atgttagt 18
<210> 70
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 70
tgaataccaa tgctgact 18
<210> 71
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 71
tgctctaaca atccaagt 18
<210> 72
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 72
atggaggaag aattggaaga 20
<210> 73
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 73
ggagaagaag aagaagaaga ag 22
<210> 74
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 74
tctctacttc tgatgaca 18
<210> 75
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 75
tcaatatgcc tgtaagtg 18
<210> 76
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 76
caactccact tctacaatca cat 23
<210> 77
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 77
ttatcaccac cgccttca 18
Claims (3)
1. The application of gibberellin in preparing preparations for inhibiting the expression of Abelmoschus esculentus AePME1 or AePME2 genes is characterized in that the sequences of the AePME1 and AePME2 genes are respectively shown as Seq ID No. 7 and Seq ID No. 8.
2. The application of gibberellin in preparing a preparation for inhibiting the expression of any one or more genes of Abelmoschus esculentus AePL1, aePL2, aePL3, aePL4 and AePL5 genes is characterized in that the sequences of the AePL1, aePL2, aePL3, aePL4 and AePL5 genes are respectively shown as Seq ID NO 9, seq ID NO 10, seq ID NO 11, seq ID NO 12 and Seq ID NO 13.
3. Use of gibberellin for the preparation of a preparation for inhibiting the expression of any one or more genes selected from the group consisting of Abelmoschus esculentus Aeβ -Gal1, aeβ -Gal2, aeβ -Gal3, aeβ -Gal4, aeβ -Gal5, aeβ -Gal6, wherein the sequence of the Aeβ -Gal1, aeβ -Gal2, aeβ -Gal3, aeβ -Gal4, aeβ -Gal5, aeβ -Gal6 gene is represented by Seq ID NO:20, seq ID NO:21, seq ID NO:22, seq ID NO:23, seq ID NO:24, and Seq ID NO:25, respectively.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111552597.7A CN114468037B (en) | 2021-12-17 | 2021-12-17 | Efficacy and application of gibberellin in delaying softening and aging of okra after picking |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
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| CN101692835A (en) * | 2009-09-27 | 2010-04-14 | 中国热带农业科学院南亚热带作物研究所 | Fruit retention agent as well as preparation method and use method of Feizixiao litchis |
| CN110564738A (en) * | 2019-09-23 | 2019-12-13 | 中国农业科学院郑州果树研究所 | application of sweet cherry PaPME2 gene in regulating and controlling ripening or softening of sweet cherry fruits |
| CN112040772A (en) * | 2018-04-30 | 2020-12-04 | 斯托尔勒企业公司 | Synergistic formulation containing at least one gibberellin compound and salicylic acid |
| CN114468038A (en) * | 2021-12-17 | 2022-05-13 | 浙江万里学院 | Efficacy and application of gibberellin in inhibiting browning of picked okra |
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| CN101692835A (en) * | 2009-09-27 | 2010-04-14 | 中国热带农业科学院南亚热带作物研究所 | Fruit retention agent as well as preparation method and use method of Feizixiao litchis |
| CN112040772A (en) * | 2018-04-30 | 2020-12-04 | 斯托尔勒企业公司 | Synergistic formulation containing at least one gibberellin compound and salicylic acid |
| CN110564738A (en) * | 2019-09-23 | 2019-12-13 | 中国农业科学院郑州果树研究所 | application of sweet cherry PaPME2 gene in regulating and controlling ripening or softening of sweet cherry fruits |
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