CN114014919B - OsNramp5 mutant and screening method and application thereof - Google Patents

Abstract

The invention relates to the field of plant biotechnology breeding, in particular to an OsNramp5 mutant and a screening method and application thereof. The OsNramp5 mutant has the nucleotide sequence comprising SEQ ID NO.1 or SEQ ID NO.2 as the coding sequence of the OsNramp5 mutant. According to the invention, the plant genome variation rate is accelerated by adopting radiation mutagenesis, and the variation of OsNramp5 allele is detected from a radiation mutagenesis population by applying a high-throughput molecular screening technology in the seedling stage of M2 generation of mutagenized plants, so that the screening efficiency and the screening accuracy of OsNramp5 mutants are improved. Compared with wild plants, the OsNramp5 mutant screened by the invention has the advantage that the cadmium content in seeds of the OsNramp5 mutant plants is reduced by over 90%.

Description

OsNramp5 mutant and screening method and application thereof

Technical Field

The invention relates to the field of plant breeding, in particular to an OsNramp5 mutant and a screening method and application thereof.

Background

The radiation mutation breeding of crops plays an important role in ensuring the food safety of the world and increasing the nutrition supply. According to the latest statistics of FAO/IAEA mutant variety databases in the United nations, the total number of plant mutant varieties which are bred on 214 plant varieties and are commercially registered reaches 3299 in more than 60 countries, the number of new varieties which are bred directly or indirectly through a mutation breeding technology in China is 1050, the number of the bred mutant varieties accounts for nearly one third of the number of the bred mutant varieties in the world, and the nuclear radiation mutation breeding technology as a traditional biological breeding technology has great social and economic effects by 2019. Under long-term natural environmental conditions, the organism itself interacts with the external environment, and in order to adapt to the change of the environment, the genetic materials in the organism undergo a certain spontaneous mutation, however, the frequency is very low, about 10^-5～10^-8Next, the process is repeated.

In south and southeast asia, indica rice is the main type of planting production and consumption, and in general, the Cd content in the tender shoots and grains of standard indica rice varieties is higher than that of japonica rice varieties. Therefore, the control of the accumulation of cadmium in the indica rice grains has important significance on the food safety and health of people. The creation of rice germplasm resources with low cadmium accumulation and the breeding of rice varieties with cadmium accumulation are the most fundamental and economic methods for realizing safe production in cadmium-polluted rice fields. The industrialization of gene editing products presents a policy obstacle of "transgenic safety management" and may present a risk of infringing the original intellectual property rights of gene editing techniques from abroad. Therefore, a new rice variety with low cadmium accumulation and complete independent intellectual property rights and without involving a transgenic technology needs to be cultured urgently, and the radiation mutagenesis technology becomes an effective way for creating low cadmium accumulation germplasm resources of rice.

Disclosure of Invention

In view of the defects of the prior art, the invention aims to provide the OsNramp5 mutant, the screening method and the application thereof, realize the precise identification and high-throughput screening of OsNramp5 allelic variation in a mutagenic population, realize the high-throughput screening and identifying method of important target character genetic variation, and break through the bottleneck of industrialization of rice gene editing biological products.

To achieve the above objects and other related objects, the present invention is achieved by the following technical solutions.

One of the purposes of the invention is to provide an OsNramp5 mutant, wherein the nucleotide sequence for coding the OsNramp5 mutant comprises a sequence shown as SEQ ID NO.1 or a sequence shown as SEQ ID NO. 2.

The second object of the present invention is to provide biomaterials related to the OsNramp5 mutant as described, including any one of the following:

A) a recombinant expression vector containing the nucleotide as described;

B) a bioengineering bacterium containing the nucleotide or the recombinant expression vector of A);

C) a transgenic plant cell containing the nucleotide as described, or a transgenic plant containing the recombinant expression vector of A);

D) a protein encoded by a nucleotide as described.

The invention also aims to provide the application of the OsNramp5 mutant or the biological material in molecular assisted breeding of plants.

The fourth purpose of the invention is to provide the application of the OsNramp5 mutant or the biological material in breeding and/or preparing low-cadmium-accumulation plants.

The fifth purpose of the invention is to provide a method for screening OsNramp5 mutant, which comprises the following steps:

1) after radiation mutagenesis is carried out on plants, genome DNA is extracted, and a DNA library is constructed according to the genome DNA;

2) designing a probe according to OsNramp5 gene of wild rice; hybridizing the probe to the DNA library;

3) enriching the hybridized product by using magnetic beads to construct an enriched library;

4) and identifying and analyzing the enrichment library, and screening a target gene with a difference with the sequence of the OsNramp5 gene of the wild rice to obtain the OsNramp5 mutant.

According to the technical scheme of the invention, in the step 1), the radiation mutagenesis comprises gamma ray mutagenesis and heavy ion mutagenesis.

In a preferred embodiment, the mutagenic source of said gamma-ray mutagenesis is selected from the group consisting of⁶⁰Co-gamma rays.

In a more preferred embodiment, the dose of the gamma rays is 200-500 Gy. Preferably, the dose of the gamma ray is 80-150 Gy. In particular, the dose of gamma rays is 300 Gy.

In a more preferred embodiment, the dose rate of the gamma rays is 2-16 Gy/min. Preferably, the dose rate of the gamma rays is 8-12 Gy/min. Specifically, the dose rate of the gamma rays is 8 Gy/min.

In a preferred embodiment, the mutagenesis source for heavy ion mutagenesis is selected from the group consisting of¹²C⁺⁶Heavy ions.

In a more preferred embodiment, the dose of heavy ion mutagenesis is 80-150 Gy. Preferably, the¹²C⁺⁶The dosage of the heavy ions is 100-140 Gy. The described¹²C⁺⁶The dose of heavy ions was 120 Gy.

In a more preferred embodiment, the dosage rate of heavy ion mutagenesis is 0.8-2.2 Gy/min. Preferably, the dosage rate of microgravity mutagenesis is 1.2-1.7 Gy/Min. Specifically, the dosage rate of microgravity mutagenesis is 1.5 Gy/min.

According to the technical scheme of the invention, the method in the step 1) comprises the following steps:

a) carrying out radiation mutagenesis on seeds of the plants, and planting to obtain M1 generation seeds;

b) planting the M1 generation seeds to obtain M2 generation seeds;

c) planting the M2 generation seeds, taking leaves on each planted individual plant, and mixing the leaves of each individual plant;

d) extracting the genomic DNA from the mixed leaves;

e) and (2) fragmenting the genome DNA, adding A tail to the fragmented DNA fragment, connecting a sequencing adaptor, and amplifying to obtain the DNA library.

In a preferred embodiment, in step b), after the M1 generation seeds are planted in individual plants, each ear seed of each individual plant is harvested according to the ear harvest method to form the M2 generation seeds.

In a preferred embodiment, in step c), the M2 generation seeds are planted in single plants, each 100-1000 plants are used as a sample group, each sample group is numbered, and the leaves of the plants in each sample group are mixed to extract DNA.

In a preferred embodiment, in the step c), when the leaves of each planted individual plant are taken, the leaves of different parts of the same individual plant are selected and mixed in equal amount; equal mixing of different individuals.

According to the technical scheme, in the step 2), the probes comprise a plurality of probes designed according to the trait genes.

According to the technical scheme of the invention, in the step 2), the sequence of the probe comprises a sequence shown as SEQ ID NO. 4-SEQ ID NO. 23.

According to the technical scheme, the probe is a biotin-labeled probe.

According to the technical scheme of the invention, the magnetic beads are streptavidin-labeled magnetic beads.

According to the technical scheme of the invention, the plants comprise angiosperms and gymnosperm.

In a preferred embodiment, the plant includes dicotyledons and monocotyledons.

In a preferred embodiment, the plants include herbaceous plants and woody plants.

In a preferred embodiment, the plant arabidopsis thaliana, tobacco, rice, corn, sorghum, barley, wheat, millet, soybean, tomato, potato, quinoa, lettuce, rape, cabbage, strawberry. More specifically, the plant is rice.

The sixth purpose of the invention is to provide a plant with low cadmium accumulation, which comprises the OsNramp5 mutant or the biological material.

Compared with the prior art, the invention has the following beneficial effects:

according to the invention, the plant genome variation rate is accelerated by adopting radiation mutagenesis, the variation of OsNramp5 allele is detected from a radiation mutagenesis population by applying a high-throughput molecular screening technology in the seedling stage of M2 generations of mutagenized plants, the defects that the agronomic trait phenotype identification is easily influenced by the subjective judgment of breeders, is difficult to identify by naked eyes, is inaccurate in identification, high in cost, long in period and the like are overcome, and the efficiency and the accuracy of screening the OsNramp5 mutant are improved.

Compared with wild plants, the OsNramp5 mutant screened by the invention has the advantage that the cadmium content in seeds of the plants containing the OsNramp5 mutant is reduced by over 90 percent.

Drawings

FIG. 1 is a partial sequence alignment diagram of OsNramp5 gene and OsNramp5 mutant gene in wild-type rice in example 2.

FIG. 2 is a second drawing showing the alignment of partial sequences of OsNramp5 gene and OsNramp5 mutant gene in wild-type rice in example 2 of the present invention.

Detailed Description

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be readily apparent to those skilled in the art from the disclosure of the present invention.

Before the present embodiments are further described, it is to be understood that the scope of the invention is not to be limited to the specific embodiments described below; it is also to be understood that the terminology used in the examples herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention. Test methods in which specific conditions are not noted in the following examples are generally performed under conventional conditions or conditions recommended by each manufacturer.

When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any number between the two endpoints are optional unless otherwise specified in the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.

In the examples described below in this application, Serapure magnetic beads were Invitrogen

M-270Streptavidin beads。

In the following examples of the present application, the Tiangen NG301 kit and NG303 kit were purchased from Tiangen Biochemical technology Ltd.

In the following examples of the present application,

hybridization and Wash Kit, purchased from Integrated DNA Technologies, Inc.

In the examples described below, the KAPA library amplification kit was purchased from Shanghai Jieyi Biotech, Inc.

In the examples described below in this application, KAPA HiFi HotStart ReadyMix PCR Kit, purchased from Roche.

In the examples described below, the MiniSeq sequencing kit was purchased from Illumina.

Example 1

In the embodiment, according to breeding target agronomic traits, a wild rice OsNramp5 Gene is searched in a Gene bank database, and a probe is designed aiming at the Gene by using Oligo6.0 software, wherein the nucleotide sequence of the wild rice OsNramp5 Gene comprises a sequence shown as SEQ ID NO. 3. The probe labeled with biotin was synthesized by Alberson Biotechnology Ltd.

A probe is designed according to the OsNramp5 gene of wild rice, the probe comprises Os07t 0257200-1-Os 07t0257200-20, and the sequence of the probe comprises the sequences shown in SEQ ID NO. 4-SEQ ID NO. 23.

Os07t0257200-1

GTCACTACCACCATTCTCTTCTTCGTCTACTTCCAGCTAGCCTGAGCTCTAGCTTAGCTCTAGCTTAGCCTGAAGAAGCTAAGAGAGGAAGCAACAATGGAGATTGAGAGAGAGAGCAGT(SEQ ID NO.4)

Os07t0257200-2

GAGAGAGGGAGCATCAGCTGGAGAGCTAGTGCGGCACATGATCAAGATGCCAAGAAGCTCGACGCAGATGATCAGCTGCTAATGAAGGTCAGTTGCTACCTTAATTATCACAAAGTTGTC(SEQ ID NO.5)

Os07t0257200-3

GTTAGTTTCTCAGTTTTGGTAAATTGTGTACAGGAGCCTGCATGGAAAAGGTTCCTTGCCCATGTTGGTCCTGGATTCATGGTGTCTTTAGCCTACTTGGATCCTGGCAATTGTGAGAAT(SEQ ID NO.6)

Os07t0257200-4

TGTCACAAAATGAAACAGTGGAAACCGATCTGCAAGCCGGAGCCAACCACAGATATGAGGTACGTACAGTCGACTTTATTTTACTGTTTACGCACTGAATGACTGAAAGCATGAGTAACT(SEQ ID NO.7)

Os07t0257200-5

AGCTGCTCTGGGTGATTCTGATTGGACTCATCTTCGCACTTATCATACAGTCGCTAGCAGCTAATCTTGGAGTGGTTACAGGTGACTAAATGACACGCAATTAAACTGTAATTACCAACT(SEQ ID NO.8)

Os07t0257200-6

TCCTAGGGAGGCATCTGGCTGAGATCTGCAAGAGTGAGTACCCCAAGTTCGTCAAGATTTTCCTATGGCTGCTGGCAGAGTTGGCCGTCATCGCTGCAGATATCCCAGAAGGTATATACT(SEQ ID NO.9)

Os07t0257200-7

TGCAGTTATAGGGACGGCCTTTGCTTTCAACATATTGTTCCATATTCCGGTGTGGGTCGGCGTCCTCATCACCGGCACCAGCACTCTACTGCTTCTTGGCCTCCAAAAATACGGGGTATA(SEQ ID NO.10)

Os07t0257200-8

TTTCTGATATCGATGCTGGTGTTCGTGATGGCGGCGTGCTTCTTCGGGGAGCTGAGCATCGTGAAGCCtttGGCGAAGGAGGTGATGAAGGGGCTCTTCATCCCCAGGCTCAACGGCGAC(SEQ ID NO.11)

Os07t0257200-9

ACGCAACAATCAATCAATTAATTCATACAACTAATCAGTTAATTAACTACTGCATGCATTTATATATATGCAGCCACAATCTGTTCTTGCATTCTGCCTTGGTGCTATCGAGGAAGACAC(SEQ ID NO.12)

Os07t0257200-10

AGCCACAATCTGTTCTTGCATTCTGCCTTGGTGCTATCGAGGAAGACACCGGCATCAGTCAGAGGAATCAAGGTAGCTATATTCAGCCTGGGCAGATCGAGATAGGTATAGGGTAGCTAG(SEQ ID NO.13)

Os07t0257200-11

ATATATGCAGCCACAATCTGTTCTTGCATTCTGCCTTGGTGCTATCGAGGAAGACACCGGCATCAGTCAGAGGAATCAAGGTAGCTATATTCAGCCTGGGCAGATCGAGATAGGTATAGG(SEQ ID NO.14)

Os07t0257200-12

ATCCAGGCTAATAATTAATATGTACTATATGGTGTGTGAATTGCCACGTAGGACGGGT

GCAGGTTCTTCCTGTACGAGAGCGGGTTCGCGCTGTTCGTGGCGCTGCTGATAAACATCGCC(SEQ ID NO.15)

Os07t0257200-13

AGAGGACGCCGACAAGTGCGCCAACCTCAGCCTCGACACCTCCTCCTTCCTTCTCAAGGTCATTCATTCATTATTACTTCCTACTCCTTAATATTAAAAATTTTTAAATGGATTAGACGT(SEQ ID NO.16)

Os07t0257200-14

TCAGAACGTGCTGGGCAAGTCGAGTGCGATCGTGTACGGCGTGGCACTGTTGGCATCTGGGCAGAGCTCCACTATTACCGGCACATACGCTGGACAGTACATCATGCAGGTAATTAATTA(SEQ ID NO.17)

Os07t0257200-15

GTGCATGCAGATGATACTGTCCTTCGAGCTGCCGTTTGCTCTCATCCCTCTTCTCAAGTTCAGCAGCAGTAAGAGCAAGATGGGGCCCCACAAGAACTCTATCTATGTAAGATCGTACAC(SEQ ID NO.18)

Os07t0257200-16

GATAATAGTGTTCTCGTGGTTCCTGGGTCTGCTCATCATCGGCATCAACATGTACTTCCTGAGCACGAGCTTCGTCGGCTGGCTCATCCACAACGACCTCCCCAAGTACGCCAACGTGCT(SEQ ID NO.19)

Os07t0257200-17

CTCGTCTACATCGTCGCtGTtGTtTACCTCACCATCAGGAAGGACTCCGTCGTCACCTTCGTCGCCGACTtCTCtCTCtGCtGCtGTtGTtGACGCCGAGAAGGCCGACGCCGGCGACCT(SEQ ID NO.20)

Os07t0257200-18

AGCCCTTGCCGTACCGCGACGACCTGGCCGACATCCCGCTCCCAAGGTAGAGAAGAAGAAGATCGACATGCATACGTATGGTATAGCGTATGTATACGTACGTATATACGCGTACATTGT(SEQ ID NO.21)

Os07t0257200-19

TGTATACGTACGTATATACGCGTACATTGTGAATATACGTACTTACGTACGTATATGTGCATCCTCTATGGATGCCACGTGCGTGCGTCGGAGCCGTTCGTTTATATTTGTGCGGTCCAG(SEQ ID NO.22)

Os07t0257200-20

ATCGCTCGCAGATCGATCGATCGACAAGGGTGTGCAATTAATATATAAGCATTTGAGGGGAGGTGGCTCATGTATCCTTGTAATTTATATGTACGATATGATTTATCATAATTCAGGGCA(SEQ ID NO.23)

Example 2

In this example, OsNramp5 mutants were screened, including the following:

1. subjecting plants to radiation mutagenesis treatment comprising:

1) 2000 dry rice seeds of XF1822, using heavy ions¹²C⁺⁶Performing radiation mutagenesis treatment, wherein the dose is 120Gy, the dose rate is 1.5Gy/Min, and planting is performed to obtain M1 generation seeds.

2) And (3) planting the plants raised with the seeds of the M1 generation in separate plants, strictly selfing and fructifying the M1 generation, and harvesting each ear seed of each single plant according to an ear harvest method to obtain seeds of the M2 generation.

3) Planting 14000 plants in each plant division of M2 seed seedlings, wherein each group is 100 plants, and each group is numbered; taking each sample group as a unit, when the seedling grows to have two leaves and one heart, taking an equal amount of leaves of each individual plant by using a puncher with the diameter of 6mm, mixing the leaves of the individual plants in equal amount, and mixing the plants in equal amount to obtain 140 sample groups.

2. Extracting the genomic DNA of the sample group obtained in the step 1, fragmenting the extracted genomic DNA, adding A tail to the fragmented DNA fragment, connecting a sequencing adaptor, purifying and amplifying to obtain a DNA library, wherein the DNA library comprises the following steps:

2.1 extraction of DNA from the group of samples by the CTAB method

Numbering the 140 sample groups obtained in the step 1, wherein the numbers are POOL NO.1-POOLNO.140 in sequence; each individual plant in each sample group is also numbered, taking the sample group POOL NO.1 as an example, and the numbers are POOL NO.1-1 to POOL NO.1-100 in sequence.

After DNA is extracted from each sample group by CTAB, the concentration is more than or equal to 20 ng/mu L and the total amount is more than or equal to 1 mu g by the quantification of Qubit fluorescence; the purity of the sample is as follows: OD260/280 is 1.7-2.0, and OD260/230 is more than or equal to 1.8; agarose gel electrophoresis is used for detecting the integrity of genome DNA, and the electrophoresis main band is required to be more than 2000 bp. In this example, the proportion of DNA detected to be less than 2000bp is less than 50%.

2.2 DNA fragmentation/DNA fragment plus A Tail

DNA fragmentation, end repair, DNA fragment plus a tail were performed using the tiangen NG301 kit.

Respectively mixing 140 sample groups obtained in the step 2.1 with 10 XFEA Reaction Buffer and 5 XFEA enzyme in the kit, preparing DNA fragmentation/end repair/DNA fragment adding A tail according to the table 1, putting on ice, melting, reversing, uniformly mixing, centrifuging, and then reacting according to the table 2.

TABLE 1

Components	Per reaction volume (. mu.L)
		Step 2.1 sample group DNA	X
10×FEA Reaction Buffer	5
		5×FEA enzyme Mix	10
ddH2O	35-X

TABLE 2

Temperature of	Time
		Thermal cover 70 deg.C	On
4℃	1min
		32℃	16min
65℃	30min
		4℃	Hold

2.3 Joint connection

Linker ligation was performed using the tengen NG303 kit. Linker-ligated reaction systems were prepared according to Table 3 below, centrifuged, and ligated according to Table 4 to give ligation products.

TABLE 3

TABLE 4

Temperature of	Time
		Hot lid	Off
20℃	15min
		4℃	Hold

2.4 DNA purification

1) Putting 100 mu L of the ligation product obtained in the step 2.3 and 130 mu L of Serapure magnetic beads into a PCR tube, fully and uniformly mixing, and standing for 2min at room temperature;

2) placing the PCR tube on a magnetic frame, carrying out magnetic bead adsorption, standing for 5min, and discarding the supernatant;

3) then 200. mu.L of 75% ethanol is added, and the supernatant is discarded;

4) repeating the step 3) in the step 2.4 once, discarding supernatant as much as possible, and standing at room temperature for 5min until the residual ethanol is completely volatilized;

5) adding 23uL of eluent (10mM Tris-HCl, pH 8.0-pH 8.5) for elution, fully mixing, standing at room temperature for 5min, then placing on a magnetic frame for standing for 2min, taking 23uL of supernatant, and storing in a new PCR tube to obtain a purified DNA library, namely a purified ligation product.

2.5 amplification of DNA libraries

2.5.1 amplification of DNA libraries

And (3) taking the purified DNA library obtained in the step (2.4) as a template, preparing a reaction system according to the table 5, mixing uniformly in a vortex manner, centrifuging, and performing PCR amplification according to the table 6 to obtain a DNA library amplification product. The KAPA library amplification kit is adopted for amplification.

TABLE 5

Components	Reaction volume (μ L)
		KAPA HiFi HotStart ReadyMix(2X)	25
TruSeq Primer 1.0	1.5
		TruSeq Primer 2.0	1.5
Step 2.4 obtaining the purified ligation product	22
		Total amount:	50

TABLE 6

2.5.2 purification after amplification of DNA library

(1) Mixing 50 μ L of the DNA library amplification product obtained in step 2.5.1 and 55 μ L of Serapure magnetic beads in a PCR tube, and standing at room temperature for 2 min;

(2) placing the PCR tube on a magnetic frame, standing for 5min, and removing the supernatant;

(3) then adding 200 mu L of 75% ethanol, standing for half a minute, and removing the supernatant;

(4) repeating the step (3) in the step 2.5.2 once, discarding the supernatant as much as possible, and standing at room temperature for 5min until the residual ethanol is completely volatilized;

(5) and adding 27 mu L of ultrapure water for elution, fully and uniformly mixing, standing at room temperature for 5min, then placing on a magnetic rack for standing for 2min, and storing the supernatant meeting the standard requirements of the library into a new PCR tube for probe capture.

After purification, quantification was performed using the Qubit HS. The DNA library met the criteria of: the bands were concentrated at 200-400bp, at a concentration greater than 5 ng/. mu.L. And (4) the qualified DNA library enters a hybridization capture link.

3. Hybridization of probes and libraries

Use of

Hybridization and Wash Kit Hybridization of probes and libraries.

Concentrating, drying and centrifuging the qualified DNA library purified in the step 2.5.2 by using a vacuum concentrator, wherein the vacuum concentrator is set to be 65 ℃; the reaction system was then prepared as in Table 7 and placed in a PCR tube.

TABLE 7

Adding the components in the following table 8 into the reaction system in the table 7, blowing and beating the components up and down by using a gun head, uniformly mixing the components, flushing the tube wall, and incubating for 5-10min at room temperature; centrifuging, and incubating at 95 deg.C for 10 min; then adding the probe constructed in the embodiment 1 and mixing uniformly; then incubated overnight at 65 deg.C (instrument lid temperature set at 75 deg.C) for no more than 15 h.

TABLE 8

4. Enriching hybridized products by adopting magnetic beads to construct an enriched library

In enrichment, 1 × Bead Wash Buffer was prepared according to table 9, and 10 × Wash Buffer I and Stringent Wash Buffer were prepared according to table 10.

TABLE 9

	Concentrated buffer(μL)	Nuclease-Free Water(μL)
			2×Bead Wash Buffer	250	250
10×Wash Buffer I*	30	270
			10×Wash BufferⅡ	20	180
10×Wash BufferⅢ	20	180
			10×Stringent Wash Buffer	40	360

TABLE 10

4.1 enrichment of hybridized product from step 3 with magnetic beads

1) And cleaning the streptavidin marked magnetic beads by adopting 1 multiplied Bead Wash Buffer to obtain pure magnetic beads.

2) And (3) transferring the hybridization product obtained in the step (3) to a PCR tube containing magnetic beads, uniformly mixing, placing in a shaking instrument, and incubating for 45min at 65 ℃ to enable the DNA to be combined on the magnetic beads, thereby obtaining the magnetic beads enriched with the DNA. In the incubation process, shaking for 3s after 5min to mix evenly; and then vortex and shake for 3s every 8min to ensure that the magnetic beads are still in a suspended state, and the rotation speed of the shaking instrument is adjusted to 1500.

4.2 washing the beads to remove unhybridized product

And (4) transferring the magnetic beads enriched with the DNA obtained in the step (4.1) into a centrifugal tube, placing the centrifugal tube on a magnetic frame, separating the magnetic beads from the supernatant, and removing the supernatant.

Then 200. mu.L of preheated 1 XStingent Wash Buffer was added, blown up and down 10 times to mix well, incubated in a water bath at 65 ℃ for 3min, placed on a magnetic rack to separate the beads from the supernatant and the supernatant was removed.

Then adding 200 mu L of room temperature 1 XWash Buffer I, blowing and beating for 10 times, mixing uniformly, and then vortex and shaking for 1 min; placing on a magnetic frame to completely separate the magnetic beads from the supernatant, and removing the supernatant; adding 200 μ L of room temperature 1 × Wash Buffer II (1 × Wash Buffer II is obtained by diluting 10 times of 10 × Wash Buffer II in Table 9), and vortex shaking for 1 min; placing the centrifugal tube on a magnetic frame to completely separate the magnetic beads from the supernatant and removing the supernatant; 200. mu.L of room temperature 1 XWash Buffer III (1 XWash Buffer III is obtained by diluting 10 times of 10 XWash Buffer III in Table 9 with water), pipetting and mixing or vortexing for 30 seconds, placing the centrifuge tube on a magnetic frame to completely separate the magnetic beads from the supernatant, and removing the supernatant.

Taking the centrifugal tube from the magnetic frame, adding 22 mu L of nucleic-Free Water into the centrifugal tube, blowing up and down for 10 times, and re-suspending the magnetic beads to obtain the magnetic beads enriched with the hybridization products.

4.3 obtaining the target gene and carrying out PCR amplification on the target gene

4.3.1 prepare the PCR mixture as in Table 11 below, shake briefly to mix well to ensure that the beads are suspended in the solution. The reaction mixture was then placed in a PCR apparatus (BioRad T100) and amplification was carried out according to the reaction procedure in Table 12 while maintaining the temperature of the lid of the apparatus at 105 ℃ to obtain PCR reaction products. The Kit used for amplification was KAPA HiFi HotStart ReadyMix PCR Kit, purchased from Roche.

TABLE 11

Components	Reaction volume (. mu.L)
		KAPA HiFi HotStart ReadyMix(2X)	25
TruSeq Primer 1.0	1.5
		TruSeq Primer 2.0	1.5
Step 4.2 magnetic beads enriched with hybridization products	22
		Total amount:	50

TABLE 12

Experimental stoppable point: the PCR reaction solution can be stored at 4 ℃ overnight.

After the reaction, the amount of the Qubit HS was determined, and the band of the captured DNA was confirmed by electrophoresis. The total amount is more than 125 ng/. mu.L.

4.3.2 post-amplification purification

1) Adding 65 mu L of Serapure magnetic beads into 50 mu L of PCR reaction product obtained in the step 4.3.1, fully and uniformly mixing, and standing for 2min at room temperature;

2) placing the PCR tube on a magnetic frame, standing for 5min, and removing the supernatant;

3) adding 200 mu L of 75% ethanol into each tube on a magnetic frame, standing for half a minute, and removing the supernatant;

4) repeating the step 3) in the step 4.3.2 once, discarding supernatant as much as possible, and standing at room temperature for 5min until the residual ethanol is completely volatilized;

5) adding 25 μ L EB for elution, mixing, standing at room temperature for 5min, standing in magnetic frame for 2min, and collecting supernatant to obtain enriched library.

After purification, quantification was performed using Qubit HS. The concentration is required to be more than 5 ng/mu L, and the band is concentrated at 200-400bp for subsequent sequencing.

5. Sequencing the enriched library obtained in the step 43.2, performing bioinformatics analysis on the sequencing result, and screening out target genes with variation with the OsNramp5 gene sequence of the wild rice, wherein the variation conditions comprise point mutation, gene fragment insertion and gene fragment deletion.

Sequencing is completed on a MiniSeq 500 sequencer platform by adopting a MiniSeq sequencing kit, and the specific flow refers to the standard flow of the specification.

Table 13 shows the detected gene mutation. It was found that the sample group genes of POOL No.8 and POOL No.3 had a difference in gene sequence from wild-type OsNramp5 gene, that 8874008 and 8874013 of the sample group gene of POOL No.8 had a deletion of CCGATG, and that 8908787 and 890879 of the sample group gene of POOL No.3 had a deletion of ACCAAAATTAACAT, respectively.

TABLE 13 genetic variation

6. According to the sequencing data result of the step 5, the sequence of the gene with the differential site utilizes Primer Premier 5.0 software to design molecular primers, the genomic DNA of 100M 2 generation single plant samples in the sample groups of POOL NO.8 and POOL NO.3 is respectively subjected to PCR amplification, and the PCR amplification products are directly sequenced.

Molecular primers were designed from the gene of mutant No. 92 in sample group No. POOL No. 8:

a forward primer: 5'-ATGATCCATGTATGTGCAG-3' (SEQ ID NO.24)

Reverse primer: 5'-ACTACTACCTAGCTAGCTAC-3' (SEQ ID NO.25)

Molecular primers were designed based on the gene of the mutant numbered 97 in the sample group numbered POOL NO. 3:

a forward primer: 5'-ATGCTGACCGAAGCGATGATG-3' (SEQ ID NO.26)

Reverse primer: 5'-ATGACAAGAACCATCGCCATC-3' (SEQ ID NO.27)

PCR amplification was performed as per Table 14.

TABLE 14PCR amplification System

The nucleotide sequence of the OsNramp5 mutant obtained from the POOL NO.3 sample group is shown as SEQ ID NO.1 by sequencing, and the sequence pair of the mutant and the wild type gene part is shown as a figure 1; the nucleotide sequence of the OsNramp5 mutant obtained from POOL NO.8 sample group comprises the sequence shown in SEQ ID NO.2, and the sequence pair of the mutant and the wild-type gene part is shown in FIG. 2.

7. And (4) planting the single plants with the genome sequence difference in the step (6) to a field, strictly selfing to propagate offspring, and taking each tillered leaf blade in the tillering stage for mutation verification again. Finally, the mutant which has stable heredity and excellent comprehensive character and accords with the breeding target character is selected.

The content of effective cadmium in the field is 0.98mg/Kg, and the cadmium content is measured by collecting seeds after the rice is ripe. The content of cadmium is measured according to GB 5009.15-2014. The cadmium content in the seeds of the mutant and wild type rice is shown in table 1.

FIG. 1 is a second schematic diagram showing the alignment of the partial sequences of the wild-type OsNramp5 gene and the OsNramp5 mutant gene in example 2.

As seen from FIG. 1, 13 bases ACCAAAATTAACA were deleted in OsNramp5 gene of No. 97 plant in POOL NO.3 sample group, compared with wild-type OsNramp 5.

FIG. 2 is a diagram showing the alignment of partial sequences of the wild-type OsNramp5 gene and OsNramp5 mutant gene in example 2.

As is clear from FIG. 2, in the sample group numbered POOL No.8, the OsNramp5 gene of the No. 92 plant had a deletion of 6 bases GCCGAT as compared with wild-type OsNramp 5.

TABLE 1

Name(s)	Cadmium content of polished rice
		XF1822	0.632mg.Cd.Kg-1
NO.8-92	0.038mg.Cd.Kg-1
		NO.3-97	0.026mg.Cd.Kg-1

As can be seen from Table 1, the cadmium content in the seeds of OsNramp5 mutant rice is significantly reduced by more than 90% compared with that of wild rice.

The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Those skilled in the art can modify or change the above-described embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.

Sequence listing

<110> research institute for nuclear agriculture and space flight breeding in Hunan province

<120> OsNramp5 mutant and screening method and application thereof

<160> 27

<170> SIPOSequenceListing 1.0

<210> 1

<211> 7457

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

atccaatcca ccaacttgca ctaattatta attattgaca gagatcaaac tactgctccg 60

aagtctatac agtacactac cgtacgtgtc tctagtttct ggaaaaaaag gtacagctat 120

agtactactc ttccaatact aatatattaa tctaattgct taattaatta attaattaat 180

agtagttgat tcatcacaga aaattgaagg acgttggctc tgccctgaat tatgataaat 240

catatcgtac atataaatta caaggataca tgagccacct cccctcaaat gcttatatat 300

taattgcaca cccttgtcga tcgatcgatc tgcgagcgat ctggaccgca caaatataaa 360

cgaacggctc cgacgcacgc acgtggcatc catagaggat gcacatatac gtacgtaagt 420

acgtatattc acaatgtacg cgtatatacg tacgtataca tacgctatac catacgtatg 480

catgtcgatc ttcttcttct ctaccttggg agcgggatgt cggccaggtc gtcgcggtac 540

ggcaagggct cgtcgtcgtc gacggcgagg tcgccggcgt cggccttctc ggcgtcgacg 600

acggcggcga gggaggagtc ggcgacgaag gtgacgacgg agtccttcct gatggtgagg 660

tagacgacgg cgacgatgta gacgagcatg aacgggaaga cggcggcgcc gacgagcacg 720

ttggcgtact tggggaggtc gttgtggatg agccagccga cgaagctcgt gctcaggaag 780

tacatgttga tgccgatgat gagcagaccc aggaaccacg agaacactat tatctgcatt 840

caattcaatt caatttattt tttgacatac atgacgctaa ttaattcatt gatagcgtca 900

ctaaataacc agtcatcttt gacgggtggt aacttatata tctttttaac gggtatcgac 960

tgatgagcat gcgcaatttt tataggtagt atatatatat atatatacat atatatatat 1020

atatatatat acacatatat atatatacac atatatatat atatacacac acatatatat 1080

atatatatat agccatgtgt gtaataataa gaaataaatt tacatttgtt caattttgtt 1140

gtagatcagt ttatttatgg tccttaaaaa agaattacat tttctaatgt tgacttgtat 1200

gtgtctagaa atgtttaagt agagatacaa cgacatacgt aatgatctat gttaaattat 1260

atttacattt tacttccatg caagaaaaaa tatatatagt gtactgaaga acttggctct 1320

tcgatgtctg taaagatcaa gcaagtacgt tactgcatga ttttgacaca tcatgccgac 1380

gacgcaatac atgattaatc tctggtacta ctgcaagaca gaaaaatgat gcaggacgta 1440

catcttggtc gtcgttttca tatgtacgac gatcctctat atgcatggct ggaccatata 1500

tatacctgtc ctgcattggc ccctgctgtg ctataatact attgcatgag ctaggtagct 1560

aatgattcaa ttcaattcac tgcacatctc gatctatatt atgcacacac tgacctatag 1620

aacatggtgt cctgtagggg ttgcatgcat gcagggtgaa ggaccagcta gctagccaac 1680

tatcatcatt aatttatatt ctttaccaac aattattgaa actctaattt catgcatgga 1740

cagatagatg catatattta tgttacttgt acatttgctg ccaattgaga ttaagaaaca 1800

aactataatt aagcaatgca tatatatagt gtacgatctt acatagatag agttcttgtg 1860

gggccccatc ttgctcttac tgctgctgaa cttgagaaga gggatgagag caaacggcag 1920

ctcgaaggac agtatcatct gcatgcacac aacacgtacg gattaggaac aattaatcat 1980

atatatgtaa ttaagcaaac aagaactagc aatgatcaga tcactacttt gatttgattt 2040

aattatatat aattagtcct tacttattga gttggaagct agtctagcta gatggtacac 2100

tgtattatat atatgcatgg tgctatttta gatcagattg caaaaaaaga aaagatacct 2160

agctagtcag tgacattttg ctaatcagat cactactttg atttggcaat aatctaaaaa 2220

aagaagaaaa agtgcttttg ctctaatgac atatatatat gctcttagta ctatgtcact 2280

aggtacctag tggatacccc actactacct agctagctac ataaatccgc acgatcgact 2340

gcacacacgt gactgtttgg gcatgtgcaa aattaaaaat ttaaacgcat taaattttaa 2400

tcgatcggcg acacgacggc cggccggttg ctcgccggcg ggcgccgccg tatggcgcac 2460

gcagtttgct atagtaaaat agtaataata gtaaagattt gctatagtaa atagtaataa 2520

taaaaatgct gaccgaagcg atgatgatga ggcggccggc gcccctggag ccgccgatga 2580

tggagacgat gaggctcggc gcgatggcga tggttcttgt catcaggttc cgaagccact 2640

tcctcatcct gatgtccaag aaaccctgca catacatgga tcatatcata tatatcaacg 2700

cgacatgatg atgatttatc atatactcct tccgtcatta aatatttgac gccgttgatt 2760

tttctaaaca tgtttaacca ttcatcttat tcaataaatt taagtagtta ttaattcttt 2820

tcttatcatt tgattcattg ttgtatatac tactactact ctagtttttt aaataatatt 2880

cataaaagtt tttgcataag acgaacggtc aaacatattt ataaaaagtc aacgacgtca 2940

aatatttaga gacggagtga gtatatcctg aagatcagtc ctttggagtg aaaaaataaa 3000

tatatatttt cgggtccgat ttatcccgta actgaaatac gcttcatatc ataaatagat 3060

tattaagagg aatgagactc gaactagatc ggttagcccg ccaggtagat taggaaaact 3120

agttgttctc tttctcaacg ttcacttcag aaagaaaatt ttacggtgcg atgggatatc 3180

ctattttttt taatataatt tatagagttt cgttagcata tttttattaa tcaattggtt 3240

ttctaagtat atgtaaacag taaaatcgct ggttattgat gagactgcac cgtgaaatgg 3300

actagaccaa aagagcggag aaatatggac gaaagtggta agtagtggca gcaaggtagc 3360

gtcttaattc gatctgactt taacaaaaac atatggtgaa atccgacgag gatcgccaga 3420

ttacctaatt aataaaggaa tattatatca ctaaatattc gtgatgtaaa caggattagg 3480

tgtgcacccc tacaattcgt cagttaacct ccataattga acggattaac aaattaatta 3540

tgtggcagct agccgtttaa ttaattatag ttcaatcatt taattacgcg ctaagctagc 3600

tggtgatgta caggtacagg tacagttaag ttaattaatt taattaatta cctgcatgat 3660

gtactgtcca gcgtatgtgc cggtaatagt ggagctctgc ccagatgcca acagtgccac 3720

gccgtacacg atcgcactcg acttgcccag cacgttctga accacataaa aaaattgacg 3780

tcacaatttg accataaatt agttactcct cctattcaaa aatataaaca tatctaataa 3840

agttattata ttttaggacg gagaaatcgt gtagactaat tcggtattat tgtggttaat 3900

taaatattga ttaaattgaa cacatacaag ctagtactac gaatctgaat agatttctat 3960

ctgtagtact aggatacgtc taatccattt aaaaattttt aatattaagg agtaggaagt 4020

aataatgaat gaatgacctt gagaaggaag gaggaggtgt cgaggctgag gttggcgcac 4080

ttgtcggcgt cctcttggga gaggttggcg gaggagcagg cggtgccgga gacggagacg 4140

acggcgatgt ttatcagcag cgccacgaac agcgcgaacc cgctctcgta caggaagaac 4200

ctgcacccgt cctacgtggc aattcacaca ccatatagta catattaatt attagcctgg 4260

atctttgttc ggtttctttc catgcatgaa aaaattctaa gctgaatcca gtggtactgg 4320

agcccgaaat tatagaagat tagtgctaaa ataaaactta gctaataaag tattggtttt 4380

ctatgaaatt tacatgttaa tttaataaaa tttaaacaag atttaagaga caggactaga 4440

tcgagttcat gtcctagcta ccctatacct atctcgatct gcccaggctg aatatagcta 4500

ccttgattcc tctgactgat gccggtgtct tcctcgatag caccaaggca gaatgcaaga 4560

acagattgtg gctgcatata tataaatgca tgcagtagtt aattaactga ttagttgtat 4620

gaattaattg attgattgtt gcgtatgtat gtatgtatgt aaaagcgtac ggcatgacaa 4680

gagctccgag gagggcaatg gcgtcggcgg tggcgccgtc gccgttgagc ctggggatga 4740

agagcccctt catcacctcc ttcgccggcg gcttcacgat gctcagctcc ccgaagaagc 4800

acgccgccat cacgaacacc agcatcgata tcagaaactc cagcttcctc acctgttttt 4860

tttcaaaaaa aattcatcaa tcaattttca tgcagttcca cagtcagaga aaaaaaactg 4920

aataatgtac atataatatg ccatttgagt acttgacaat cgatccaact agctaatttc 4980

atatactatt ccatttttat aatcgtttct ttgagtgcat cctacgctaa tgcttgtgat 5040

actaaacaaa aacaacttac aaaaaataat ttataccccg tatttttgga ggccaagaag 5100

cagtagagtg ctggtgccgg tgatgaggac gccgacccac accggaatat ggaacaatat 5160

gttgaaagca aaggccgtcc ctataactgc atccacatca aacgacatgt aattaattaa 5220

ttaattataa tcacatcgat ctgtaccttt ctatctgttg atcgatctat ctatctatct 5280

atatatcacc tttgctatca tcatgtgagg tatcttccat aactagttta atttttttaa 5340

aaaagaactt gcatgcatga tcaattaaaa aggcaattta caacggttga ggttaactaa 5400

tcccgatcag caaagcttcg cgcagctccg ttttgcaact gctacaccac tgatctctct 5460

tccatgttgc tagatagata gatatttaga tattgttgtt attgtttaaa cacacatgga 5520

tgtatacagg acaggacaaa tgtgaaatga caagagggtg acaaaatagt tgtttactaa 5580

tctgcgtaag atgatgaacg tactcaccca gtgtttaaaa tctaatcttg atgtgcatca 5640

ttgacacttt tatgggaagc tactgtggac gtggtcactc ctctaaacag ctaaaacgcg 5700

catttgctta tgtttgaaac agattattca acagatatat tattggtaca tattatctac 5760

cttattatat ggatgaatta aatatttttt taaaaaatgg cttagaactt gtattctatg 5820

caatatcgca gaatatattt tttttagaaa aacatattta ctagctagag acatgaaaga 5880

aataatatgt gagaataatc tggtgagtag ctgagaagaa cgtggcttga tggtgattgg 5940

tgagatatta atttgcctat gtttgtggat taggaattcc agtatgtagg cccgtacggt 6000

tggagctaca tatgctagcc agatcaactt gttttcttct actgtcagag agcttaaact 6060

gaattaatta attagctcaa agtcaaagat ggtgacactg ttaaacacaa gtagtttaat 6120

aaattaatta ttcaggctgg accgtgtcaa gttgttacta ataagtatat accttctggg 6180

atatctgcag cgatgacggc caactctgcc agcagccata ggaaaatctt gacgaacttg 6240

gggtactcac tcttgcagat ctcagccaga tgcctcccta ggatgtaaat taatgccggc 6300

catatattag cactttatca agttggtaat tacagtttaa ttgcgtgtca tttagtcacc 6360

tgtaaccact ccaagattag ctgctagcga ctgtatgata agtgcgaaga tgagtccaat 6420

cagaatcacc cagagcagct gcaaattgaa acaacacaat ggacagaaaa gcacatagaa 6480

atcaattaat ggtgcagatt attcactttt ctcttttctc tagatgatgt gagagtgaga 6540

tacatatgca ctggggatta aaagtggttc tgaaaagttt gtcaatttgg agaatttgat 6600

ggagctagca gatgaattaa tggagtgtga gtctgcgagg gacaaggaaa gggacgaaca 6660

cctagaaagc atgaaagtta ctcatgcttt cagtcattca gtgcgtaaac agtaaaataa 6720

agtcgactgt acgtacctca tatctgtggt tggctccggc ttgcagatcg gtttccactg 6780

tttcattttg tgacaaaatt aacagttaca atccagagtt tgtcaaaata aaagtttcag 6840

ttgagagaga gagagagaga gagagagaga gagagagaga gagagagaga gagagagatt 6900

ctcacaattg ccaggatcca agtaggctaa agacaccatg aatccaggac caacatgggc 6960

aaggaacctt ttccatgcag gctcctgtac acaatttacc aaaactgaga aactaacaaa 7020

aagaaacaca gacataattg aggatccaat gttccatcag tccttaatta ccacatacac 7080

acacacacat gaacattcat gagcaatgaa tgccatgcca ggaagtactc tcaagatcta 7140

tctacattaa gctctgctct tgcttctgca gttacagtga acaactgtcc atggtgacag 7200

tggagtgctt acaagaagac aactttgtga taattaaggt agcaactgac cttcattagc 7260

agctgatcat ctgcgtcgag cttcttggca tcttgatcat gtgccgcact agctctccag 7320

ctgatgctcc ctctctcact gctctctctc tcaatctcca ttgttgcttc ctctcttagc 7380

ttcttcaggc taagctagag ctaagctaga gctcaggcta gctggaagta gacgaagaag 7440

agaatggtgg tagtgac 7457

<210> 2

<211> 7464

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atccaatcca ccaacttgca ctaattatta attattgaca gagatcaaac tactgctccg 60

aagtctatac agtacactac cgtacgtgtc tctagtttct ggaaaaaaag gtacagctat 120

agtactactc ttccaatact aatatattaa tctaattgct taattaatta attaattaat 180

agtagttgat tcatcacaga aaattgaagg acgttggctc tgccctgaat tatgataaat 240

catatcgtac atataaatta caaggataca tgagccacct cccctcaaat gcttatatat 300

taattgcaca cccttgtcga tcgatcgatc tgcgagcgat ctggaccgca caaatataaa 360

cgaacggctc cgacgcacgc acgtggcatc catagaggat gcacatatac gtacgtaagt 420

acgtatattc acaatgtacg cgtatatacg tacgtataca tacgctatac catacgtatg 480

catgtcgatc ttcttcttct ctaccttggg agcgggatgt cggccaggtc gtcgcggtac 540

ggcaagggct cgtcgtcgtc gacggcgagg tcgccggcgt cggccttctc ggcgtcgacg 600

acggcggcga gggaggagtc ggcgacgaag gtgacgacgg agtccttcct gatggtgagg 660

tagacgacgg cgacgatgta gacgagcatg aacgggaaga cggcggcgcc gacgagcacg 720

ttggcgtact tggggaggtc gttgtggatg agccagccga cgaagctcgt gctcaggaag 780

tacatgttga tgccgatgat gagcagaccc aggaaccacg agaacactat tatctgcatt 840

caattcaatt caatttattt tttgacatac atgacgctaa ttaattcatt gatagcgtca 900

ctaaataacc agtcatcttt gacgggtggt aacttatata tctttttaac gggtatcgac 960

tgatgagcat gcgcaatttt tataggtagt atatatatat atatatacat atatatatat 1020

atatatatat acacatatat atatatacac atatatatat atatacacac acatatatat 1080

atatatatat agccatgtgt gtaataataa gaaataaatt tacatttgtt caattttgtt 1140

gtagatcagt ttatttatgg tccttaaaaa agaattacat tttctaatgt tgacttgtat 1200

gtgtctagaa atgtttaagt agagatacaa cgacatacgt aatgatctat gttaaattat 1260

atttacattt tacttccatg caagaaaaaa tatatatagt gtactgaaga acttggctct 1320

tcgatgtctg taaagatcaa gcaagtacgt tactgcatga ttttgacaca tcatgccgac 1380

gacgcaatac atgattaatc tctggtacta ctgcaagaca gaaaaatgat gcaggacgta 1440

catcttggtc gtcgttttca tatgtacgac gatcctctat atgcatggct ggaccatata 1500

tatacctgtc ctgcattggc ccctgctgtg ctataatact attgcatgag ctaggtagct 1560

aatgattcaa ttcaattcac tgcacatctc gatctatatt atgcacacac tgacctatag 1620

aacatggtgt cctgtagggg ttgcatgcat gcagggtgaa ggaccagcta gctagccaac 1680

tatcatcatt aatttatatt ctttaccaac aattattgaa actctaattt catgcatgga 1740

cagatagatg catatattta tgttacttgt acatttgctg ccaattgaga ttaagaaaca 1800

aactataatt aagcaatgca tatatatagt gtacgatctt acatagatag agttcttgtg 1860

gggccccatc ttgctcttac tgctgctgaa cttgagaaga gggatgagag caaacggcag 1920

ctcgaaggac agtatcatct gcatgcacac aacacgtacg gattaggaac aattaatcat 1980

atatatgtaa ttaagcaaac aagaactagc aatgatcaga tcactacttt gatttgattt 2040

aattatatat aattagtcct tacttattga gttggaagct agtctagcta gatggtacac 2100

tgtattatat atatgcatgg tgctatttta gatcagattg caaaaaaaga aaagatacct 2160

agctagtcag tgacattttg ctaatcagat cactactttg atttggcaat aatctaaaaa 2220

aagaagaaaa agtgcttttg ctctaatgac atatatatat gctcttagta ctatgtcact 2280

aggtacctag tggatacccc actactacct agctagctac ataaatccgc acgatcgact 2340

gcacacacgt gactgtttgg gcatgtgcaa aattaaaaat ttaaacgcat taaattttaa 2400

tcgatcggcg acacgacggc cggccggttg ctcgccggcg ggcgccgccg tatggcgcac 2460

gcagtttgct atagtaaaat agtaataata gtaaagattt gctatagtaa atagtaataa 2520

taaaaatgct gaccgaagcg atgatgatga ggcggccggc gcccctggag ccgatggaga 2580

cgatgaggct cggcgcgatg gcgatggttc ttgtcatcag gttccgaagc cacttcctca 2640

tcctgatgtc caagaaaccc tgcacataca tggatcatat catatatatc aacgcgacat 2700

gatgatgatt tatcatatac tccttccgtc attaaatatt tgacgccgtt gatttttcta 2760

aacatgttta accattcatc ttattcaata aatttaagta gttattaatt cttttcttat 2820

catttgattc attgttgtat atactactac tactctagtt ttttaaataa tattcataaa 2880

agtttttgca taagacgaac ggtcaaacat atttataaaa agtcaacgac gtcaaatatt 2940

tagagacgga gtgagtatat cctgaagatc agtcctttgg agtgaaaaaa taaatatata 3000

ttttcgggtc cgatttatcc cgtaactgaa atacgcttca tatcataaat agattattaa 3060

gaggaatgag actcgaacta gatcggttag cccgccaggt agattaggaa aactagttgt 3120

tctctttctc aacgttcact tcagaaagaa aattttacgg tgcgatggga tatcctattt 3180

tttttaatat aatttataga gtttcgttag catattttta ttaatcaatt ggttttctaa 3240

gtatatgtaa acagtaaaat cgctggttat tgatgagact gcaccgtgaa atggactaga 3300

ccaaaagagc ggagaaatat ggacgaaagt ggtaagtagt ggcagcaagg tagcgtctta 3360

attcgatctg actttaacaa aaacatatgg tgaaatccga cgaggatcgc cagattacct 3420

aattaataaa ggaatattat atcactaaat attcgtgatg taaacaggat taggtgtgca 3480

cccctacaat tcgtcagtta acctccataa ttgaacggat taacaaatta attatgtggc 3540

agctagccgt ttaattaatt atagttcaat catttaatta cgcgctaagc tagctggtga 3600

tgtacaggta caggtacagt taagttaatt aatttaatta attacctgca tgatgtactg 3660

tccagcgtat gtgccggtaa tagtggagct ctgcccagat gccaacagtg ccacgccgta 3720

cacgatcgca ctcgacttgc ccagcacgtt ctgaaccaca taaaaaaatt gacgtcacaa 3780

tttgaccata aattagttac tcctcctatt caaaaatata aacatatcta ataaagttat 3840

tatattttag gacggagaaa tcgtgtagac taattcggta ttattgtggt taattaaata 3900

ttgattaaat tgaacacata caagctagta ctacgaatct gaatagattt ctatctgtag 3960

tactaggata cgtctaatcc atttaaaaat ttttaatatt aaggagtagg aagtaataat 4020

gaatgaatga ccttgagaag gaaggaggag gtgtcgaggc tgaggttggc gcacttgtcg 4080

gcgtcctctt gggagaggtt ggcggaggag caggcggtgc cggagacgga gacgacggcg 4140

atgtttatca gcagcgccac gaacagcgcg aacccgctct cgtacaggaa gaacctgcac 4200

ccgtcctacg tggcaattca cacaccatat agtacatatt aattattagc ctggatcttt 4260

gttcggtttc tttccatgca tgaaaaaatt ctaagctgaa tccagtggta ctggagcccg 4320

aaattataga agattagtgc taaaataaaa cttagctaat aaagtattgg ttttctatga 4380

aatttacatg ttaatttaat aaaatttaaa caagatttaa gagacaggac tagatcgagt 4440

tcatgtccta gctaccctat acctatctcg atctgcccag gctgaatata gctaccttga 4500

ttcctctgac tgatgccggt gtcttcctcg atagcaccaa ggcagaatgc aagaacagat 4560

tgtggctgca tatatataaa tgcatgcagt agttaattaa ctgattagtt gtatgaatta 4620

attgattgat tgttgcgtat gtatgtatgt atgtaaaagc gtacggcatg acaagagctc 4680

cgaggagggc aatggcgtcg gcggtggcgc cgtcgccgtt gagcctgggg atgaagagcc 4740

ccttcatcac ctccttcgcc ggcggcttca cgatgctcag ctccccgaag aagcacgccg 4800

ccatcacgaa caccagcatc gatatcagaa actccagctt cctcacctgt tttttttcaa 4860

aaaaaattca tcaatcaatt ttcatgcagt tccacagtca gagaaaaaaa actgaataat 4920

gtacatataa tatgccattt gagtacttga caatcgatcc aactagctaa tttcatatac 4980

tattccattt ttataatcgt ttctttgagt gcatcctacg ctaatgcttg tgatactaaa 5040

caaaaacaac ttacaaaaaa taatttatac cccgtatttt tggaggccaa gaagcagtag 5100

agtgctggtg ccggtgatga ggacgccgac ccacaccgga atatggaaca atatgttgaa 5160

agcaaaggcc gtccctataa ctgcatccac caaaattaac aacatcaaac gacatgtaat 5220

taattaatta attataatca catcgatctg tacctttcta tctgttgatc gatctatcta 5280

tctatctata tatcaccttt gctatcatca tgtgaggtat cttccataac tagtttaatt 5340

tttttaaaaa agaacttgca tgcatgatca attaaaaagg caatttacaa cggttgaggt 5400

taactaatcc cgatcagcaa agcttcgcgc agctccgttt tgcaactgct acaccactga 5460

tctctcttcc atgttgctag atagatagat atttagatat tgttgttatt gtttaaacac 5520

acatggatgt atacaggaca ggacaaatgt gaaatgacaa gagggtgaca aaatagttgt 5580

ttactaatct gcgtaagatg atgaacgtac tcacccagtg tttaaaatct aatcttgatg 5640

tgcatcattg acacttttat gggaagctac tgtggacgtg gtcactcctc taaacagcta 5700

aaacgcgcat ttgcttatgt ttgaaacaga ttattcaaca gatatattat tggtacatat 5760

tatctacctt attatatgga tgaattaaat atttttttaa aaaatggctt agaacttgta 5820

ttctatgcaa tatcgcagaa tatatttttt ttagaaaaac atatttacta gctagagaca 5880

tgaaagaaat aatatgtgag aataatctgg tgagtagctg agaagaacgt ggcttgatgg 5940

tgattggtga gatattaatt tgcctatgtt tgtggattag gaattccagt atgtaggccc 6000

gtacggttgg agctacatat gctagccaga tcaacttgtt ttcttctact gtcagagagc 6060

ttaaactgaa ttaattaatt agctcaaagt caaagatggt gacactgtta aacacaagta 6120

gtttaataaa ttaattattc aggctggacc gtgtcaagtt gttactaata agtatatacc 6180

ttctgggata tctgcagcga tgacggccaa ctctgccagc agccatagga aaatcttgac 6240

gaacttgggg tactcactct tgcagatctc agccagatgc ctccctagga tgtaaattaa 6300

tgccggccat atattagcac tttatcaagt tggtaattac agtttaattg cgtgtcattt 6360

agtcacctgt aaccactcca agattagctg ctagcgactg tatgataagt gcgaagatga 6420

gtccaatcag aatcacccag agcagctgca aattgaaaca acacaatgga cagaaaagca 6480

catagaaatc aattaatggt gcagattatt cacttttctc ttttctctag atgatgtgag 6540

agtgagatac atatgcactg gggattaaaa gtggttctga aaagtttgtc aatttggaga 6600

atttgatgga gctagcagat gaattaatgg agtgtgagtc tgcgagggac aaggaaaggg 6660

acgaacacct agaaagcatg aaagttactc atgctttcag tcattcagtg cgtaaacagt 6720

aaaataaagt cgactgtacg tacctcatat ctgtggttgg ctccggcttg cagatcggtt 6780

tccactgttt cattttgtga caaaattaac agttacaatc cagagtttgt caaaataaaa 6840

gtttcagttg agagagagag agagagagag agagagagag agagagagag agagagagag 6900

agagattctc acaattgcca ggatccaagt aggctaaaga caccatgaat ccaggaccaa 6960

catgggcaag gaaccttttc catgcaggct cctgtacaca atttaccaaa actgagaaac 7020

taacaaaaag aaacacagac ataattgagg atccaatgtt ccatcagtcc ttaattacca 7080

catacacaca cacacatgaa cattcatgag caatgaatgc catgccagga agtactctca 7140

agatctatct acattaagct ctgctcttgc ttctgcagtt acagtgaaca actgtccatg 7200

gtgacagtgg agtgcttaca agaagacaac tttgtgataa ttaaggtagc aactgacctt 7260

cattagcagc tgatcatctg cgtcgagctt cttggcatct tgatcatgtg ccgcactagc 7320

tctccagctg atgctccctc tctcactgct ctctctctca atctccattg ttgcttcctc 7380

tcttagcttc ttcaggctaa gctagagcta agctagagct caggctagct ggaagtagac 7440

gaagaagaga atggtggtag tgac 7464

<210> 3

<211> 7470

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atccaatcca ccaacttgca ctaattatta attattgaca gagatcaaac tactgctccg 60

aagtctatac agtacactac cgtacgtgtc tctagtttct ggaaaaaaag gtacagctat 120

agtactactc ttccaatact aatatattaa tctaattgct taattaatta attaattaat 180

agtagttgat tcatcacaga aaattgaagg acgttggctc tgccctgaat tatgataaat 240

catatcgtac atataaatta caaggataca tgagccacct cccctcaaat gcttatatat 300

taattgcaca cccttgtcga tcgatcgatc tgcgagcgat ctggaccgca caaatataaa 360

cgaacggctc cgacgcacgc acgtggcatc catagaggat gcacatatac gtacgtaagt 420

acgtatattc acaatgtacg cgtatatacg tacgtataca tacgctatac catacgtatg 480

catgtcgatc ttcttcttct ctaccttggg agcgggatgt cggccaggtc gtcgcggtac 540

ggcaagggct cgtcgtcgtc gacggcgagg tcgccggcgt cggccttctc ggcgtcgacg 600

acggcggcga gggaggagtc ggcgacgaag gtgacgacgg agtccttcct gatggtgagg 660

tagacgacgg cgacgatgta gacgagcatg aacgggaaga cggcggcgcc gacgagcacg 720

ttggcgtact tggggaggtc gttgtggatg agccagccga cgaagctcgt gctcaggaag 780

tacatgttga tgccgatgat gagcagaccc aggaaccacg agaacactat tatctgcatt 840

caattcaatt caatttattt tttgacatac atgacgctaa ttaattcatt gatagcgtca 900

ctaaataacc agtcatcttt gacgggtggt aacttatata tctttttaac gggtatcgac 960

tgatgagcat gcgcaatttt tataggtagt atatatatat atatatacat atatatatat 1020

atatatatat acacatatat atatatacac atatatatat atatacacac acatatatat 1080

atatatatat agccatgtgt gtaataataa gaaataaatt tacatttgtt caattttgtt 1140

gtagatcagt ttatttatgg tccttaaaaa agaattacat tttctaatgt tgacttgtat 1200

gtgtctagaa atgtttaagt agagatacaa cgacatacgt aatgatctat gttaaattat 1260

atttacattt tacttccatg caagaaaaaa tatatatagt gtactgaaga acttggctct 1320

tcgatgtctg taaagatcaa gcaagtacgt tactgcatga ttttgacaca tcatgccgac 1380

gacgcaatac atgattaatc tctggtacta ctgcaagaca gaaaaatgat gcaggacgta 1440

catcttggtc gtcgttttca tatgtacgac gatcctctat atgcatggct ggaccatata 1500

tatacctgtc ctgcattggc ccctgctgtg ctataatact attgcatgag ctaggtagct 1560

aatgattcaa ttcaattcac tgcacatctc gatctatatt atgcacacac tgacctatag 1620

aacatggtgt cctgtagggg ttgcatgcat gcagggtgaa ggaccagcta gctagccaac 1680

tatcatcatt aatttatatt ctttaccaac aattattgaa actctaattt catgcatgga 1740

cagatagatg catatattta tgttacttgt acatttgctg ccaattgaga ttaagaaaca 1800

aactataatt aagcaatgca tatatatagt gtacgatctt acatagatag agttcttgtg 1860

gggccccatc ttgctcttac tgctgctgaa cttgagaaga gggatgagag caaacggcag 1920

ctcgaaggac agtatcatct gcatgcacac aacacgtacg gattaggaac aattaatcat 1980

atatatgtaa ttaagcaaac aagaactagc aatgatcaga tcactacttt gatttgattt 2040

aattatatat aattagtcct tacttattga gttggaagct agtctagcta gatggtacac 2100

tgtattatat atatgcatgg tgctatttta gatcagattg caaaaaaaga aaagatacct 2160

agctagtcag tgacattttg ctaatcagat cactactttg atttggcaat aatctaaaaa 2220

aagaagaaaa agtgcttttg ctctaatgac atatatatat gctcttagta ctatgtcact 2280

aggtacctag tggatacccc actactacct agctagctac ataaatccgc acgatcgact 2340

gcacacacgt gactgtttgg gcatgtgcaa aattaaaaat ttaaacgcat taaattttaa 2400

tcgatcggcg acacgacggc cggccggttg ctcgccggcg ggcgccgccg tatggcgcac 2460

gcagtttgct atagtaaaat agtaataata gtaaagattt gctatagtaa atagtaataa 2520

taaaaatgct gaccgaagcg atgatgatga ggcggccggc gcccctggag ccgccgatga 2580

tggagacgat gaggctcggc gcgatggcga tggttcttgt catcaggttc cgaagccact 2640

tcctcatcct gatgtccaag aaaccctgca catacatgga tcatatcata tatatcaacg 2700

cgacatgatg atgatttatc atatactcct tccgtcatta aatatttgac gccgttgatt 2760

tttctaaaca tgtttaacca ttcatcttat tcaataaatt taagtagtta ttaattcttt 2820

tcttatcatt tgattcattg ttgtatatac tactactact ctagtttttt aaataatatt 2880

cataaaagtt tttgcataag acgaacggtc aaacatattt ataaaaagtc aacgacgtca 2940

aatatttaga gacggagtga gtatatcctg aagatcagtc ctttggagtg aaaaaataaa 3000

tatatatttt cgggtccgat ttatcccgta actgaaatac gcttcatatc ataaatagat 3060

tattaagagg aatgagactc gaactagatc ggttagcccg ccaggtagat taggaaaact 3120

agttgttctc tttctcaacg ttcacttcag aaagaaaatt ttacggtgcg atgggatatc 3180

ctattttttt taatataatt tatagagttt cgttagcata tttttattaa tcaattggtt 3240

ttctaagtat atgtaaacag taaaatcgct ggttattgat gagactgcac cgtgaaatgg 3300

actagaccaa aagagcggag aaatatggac gaaagtggta agtagtggca gcaaggtagc 3360

gtcttaattc gatctgactt taacaaaaac atatggtgaa atccgacgag gatcgccaga 3420

ttacctaatt aataaaggaa tattatatca ctaaatattc gtgatgtaaa caggattagg 3480

tgtgcacccc tacaattcgt cagttaacct ccataattga acggattaac aaattaatta 3540

tgtggcagct agccgtttaa ttaattatag ttcaatcatt taattacgcg ctaagctagc 3600

tggtgatgta caggtacagg tacagttaag ttaattaatt taattaatta cctgcatgat 3660

gtactgtcca gcgtatgtgc cggtaatagt ggagctctgc ccagatgcca acagtgccac 3720

gccgtacacg atcgcactcg acttgcccag cacgttctga accacataaa aaaattgacg 3780

tcacaatttg accataaatt agttactcct cctattcaaa aatataaaca tatctaataa 3840

agttattata ttttaggacg gagaaatcgt gtagactaat tcggtattat tgtggttaat 3900

taaatattga ttaaattgaa cacatacaag ctagtactac gaatctgaat agatttctat 3960

ctgtagtact aggatacgtc taatccattt aaaaattttt aatattaagg agtaggaagt 4020

aataatgaat gaatgacctt gagaaggaag gaggaggtgt cgaggctgag gttggcgcac 4080

ttgtcggcgt cctcttggga gaggttggcg gaggagcagg cggtgccgga gacggagacg 4140

acggcgatgt ttatcagcag cgccacgaac agcgcgaacc cgctctcgta caggaagaac 4200

ctgcacccgt cctacgtggc aattcacaca ccatatagta catattaatt attagcctgg 4260

atctttgttc ggtttctttc catgcatgaa aaaattctaa gctgaatcca gtggtactgg 4320

agcccgaaat tatagaagat tagtgctaaa ataaaactta gctaataaag tattggtttt 4380

ctatgaaatt tacatgttaa tttaataaaa tttaaacaag atttaagaga caggactaga 4440

tcgagttcat gtcctagcta ccctatacct atctcgatct gcccaggctg aatatagcta 4500

ccttgattcc tctgactgat gccggtgtct tcctcgatag caccaaggca gaatgcaaga 4560

acagattgtg gctgcatata tataaatgca tgcagtagtt aattaactga ttagttgtat 4620

gaattaattg attgattgtt gcgtatgtat gtatgtatgt aaaagcgtac ggcatgacaa 4680

gagctccgag gagggcaatg gcgtcggcgg tggcgccgtc gccgttgagc ctggggatga 4740

agagcccctt catcacctcc ttcgccggcg gcttcacgat gctcagctcc ccgaagaagc 4800

acgccgccat cacgaacacc agcatcgata tcagaaactc cagcttcctc acctgttttt 4860

tttcaaaaaa aattcatcaa tcaattttca tgcagttcca cagtcagaga aaaaaaactg 4920

aataatgtac atataatatg ccatttgagt acttgacaat cgatccaact agctaatttc 4980

atatactatt ccatttttat aatcgtttct ttgagtgcat cctacgctaa tgcttgtgat 5040

actaaacaaa aacaacttac aaaaaataat ttataccccg tatttttgga ggccaagaag 5100

cagtagagtg ctggtgccgg tgatgaggac gccgacccac accggaatat ggaacaatat 5160

gttgaaagca aaggccgtcc ctataactgc atccaccaaa attaacaaca tcaaacgaca 5220

tgtaattaat taattaatta taatcacatc gatctgtacc tttctatctg ttgatcgatc 5280

tatctatcta tctatatatc acctttgcta tcatcatgtg aggtatcttc cataactagt 5340

ttaatttttt taaaaaagaa cttgcatgca tgatcaatta aaaaggcaat ttacaacggt 5400

tgaggttaac taatcccgat cagcaaagct tcgcgcagct ccgttttgca actgctacac 5460

cactgatctc tcttccatgt tgctagatag atagatattt agatattgtt gttattgttt 5520

aaacacacat ggatgtatac aggacaggac aaatgtgaaa tgacaagagg gtgacaaaat 5580

agttgtttac taatctgcgt aagatgatga acgtactcac ccagtgttta aaatctaatc 5640

ttgatgtgca tcattgacac ttttatggga agctactgtg gacgtggtca ctcctctaaa 5700

cagctaaaac gcgcatttgc ttatgtttga aacagattat tcaacagata tattattggt 5760

acatattatc taccttatta tatggatgaa ttaaatattt ttttaaaaaa tggcttagaa 5820

cttgtattct atgcaatatc gcagaatata ttttttttag aaaaacatat ttactagcta 5880

gagacatgaa agaaataata tgtgagaata atctggtgag tagctgagaa gaacgtggct 5940

tgatggtgat tggtgagata ttaatttgcc tatgtttgtg gattaggaat tccagtatgt 6000

aggcccgtac ggttggagct acatatgcta gccagatcaa cttgttttct tctactgtca 6060

gagagcttaa actgaattaa ttaattagct caaagtcaaa gatggtgaca ctgttaaaca 6120

caagtagttt aataaattaa ttattcaggc tggaccgtgt caagttgtta ctaataagta 6180

tataccttct gggatatctg cagcgatgac ggccaactct gccagcagcc ataggaaaat 6240

cttgacgaac ttggggtact cactcttgca gatctcagcc agatgcctcc ctaggatgta 6300

aattaatgcc ggccatatat tagcacttta tcaagttggt aattacagtt taattgcgtg 6360

tcatttagtc acctgtaacc actccaagat tagctgctag cgactgtatg ataagtgcga 6420

agatgagtcc aatcagaatc acccagagca gctgcaaatt gaaacaacac aatggacaga 6480

aaagcacata gaaatcaatt aatggtgcag attattcact tttctctttt ctctagatga 6540

tgtgagagtg agatacatat gcactgggga ttaaaagtgg ttctgaaaag tttgtcaatt 6600

tggagaattt gatggagcta gcagatgaat taatggagtg tgagtctgcg agggacaagg 6660

aaagggacga acacctagaa agcatgaaag ttactcatgc tttcagtcat tcagtgcgta 6720

aacagtaaaa taaagtcgac tgtacgtacc tcatatctgt ggttggctcc ggcttgcaga 6780

tcggtttcca ctgtttcatt ttgtgacaaa attaacagtt acaatccaga gtttgtcaaa 6840

ataaaagttt cagttgagag agagagagag agagagagag agagagagag agagagagag 6900

agagagagag attctcacaa ttgccaggat ccaagtaggc taaagacacc atgaatccag 6960

gaccaacatg ggcaaggaac cttttccatg caggctcctg tacacaattt accaaaactg 7020

agaaactaac aaaaagaaac acagacataa ttgaggatcc aatgttccat cagtccttaa 7080

ttaccacata cacacacaca catgaacatt catgagcaat gaatgccatg ccaggaagta 7140

ctctcaagat ctatctacat taagctctgc tcttgcttct gcagttacag tgaacaactg 7200

tccatggtga cagtggagtg cttacaagaa gacaactttg tgataattaa ggtagcaact 7260

gaccttcatt agcagctgat catctgcgtc gagcttcttg gcatcttgat catgtgccgc 7320

actagctctc cagctgatgc tccctctctc actgctctct ctctcaatct ccattgttgc 7380

ttcctctctt agcttcttca ggctaagcta gagctaagct agagctcagg ctagctggaa 7440

gtagacgaag aagagaatgg tggtagtgac 7470

<210> 4

<211> 120

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gtcactacca ccattctctt cttcgtctac ttccagctag cctgagctct agcttagctc 60

tagcttagcc tgaagaagct aagagaggaa gcaacaatgg agattgagag agagagcagt 120

<210> 5

<211> 120

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gagagaggga gcatcagctg gagagctagt gcggcacatg atcaagatgc caagaagctc 60

gacgcagatg atcagctgct aatgaaggtc agttgctacc ttaattatca caaagttgtc 120

<210> 6

<211> 120

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gttagtttct cagttttggt aaattgtgta caggagcctg catggaaaag gttccttgcc 60

catgttggtc ctggattcat ggtgtcttta gcctacttgg atcctggcaa ttgtgagaat 120

<210> 7

<211> 120

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

tgtcacaaaa tgaaacagtg gaaaccgatc tgcaagccgg agccaaccac agatatgagg 60

tacgtacagt cgactttatt ttactgttta cgcactgaat gactgaaagc atgagtaact 120

<210> 8

<211> 120

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

agctgctctg ggtgattctg attggactca tcttcgcact tatcatacag tcgctagcag 60

ctaatcttgg agtggttaca ggtgactaaa tgacacgcaa ttaaactgta attaccaact 120

<210> 9

<211> 120

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

tcctagggag gcatctggct gagatctgca agagtgagta ccccaagttc gtcaagattt 60

tcctatggct gctggcagag ttggccgtca tcgctgcaga tatcccagaa ggtatatact 120

<210> 10

<211> 120

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

tgcagttata gggacggcct ttgctttcaa catattgttc catattccgg tgtgggtcgg 60

cgtcctcatc accggcacca gcactctact gcttcttggc ctccaaaaat acggggtata 120

<210> 11

<211> 120

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

tttctgatat cgatgctggt gttcgtgatg gcggcgtgct tcttcgggga gctgagcatc 60

gtgaagcctt tggcgaagga ggtgatgaag gggctcttca tccccaggct caacggcgac 120

<210> 12

<211> 120

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

acgcaacaat caatcaatta attcatacaa ctaatcagtt aattaactac tgcatgcatt 60

tatatatatg cagccacaat ctgttcttgc attctgcctt ggtgctatcg aggaagacac 120

<210> 13

<211> 120

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

agccacaatc tgttcttgca ttctgccttg gtgctatcga ggaagacacc ggcatcagtc 60

agaggaatca aggtagctat attcagcctg ggcagatcga gataggtata gggtagctag 120

<210> 14

<211> 120

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

atatatgcag ccacaatctg ttcttgcatt ctgccttggt gctatcgagg aagacaccgg 60

catcagtcag aggaatcaag gtagctatat tcagcctggg cagatcgaga taggtatagg 120

<210> 15

<211> 120

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

atccaggcta ataattaata tgtactatat ggtgtgtgaa ttgccacgta ggacgggtgc 60

aggttcttcc tgtacgagag cgggttcgcg ctgttcgtgg cgctgctgat aaacatcgcc 120

<210> 16

<211> 120

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

agaggacgcc gacaagtgcg ccaacctcag cctcgacacc tcctccttcc ttctcaaggt 60

cattcattca ttattacttc ctactcctta atattaaaaa tttttaaatg gattagacgt 120

<210> 17

<211> 120

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

tcagaacgtg ctgggcaagt cgagtgcgat cgtgtacggc gtggcactgt tggcatctgg 60

gcagagctcc actattaccg gcacatacgc tggacagtac atcatgcagg taattaatta 120

<210> 18

<211> 120

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

gtgcatgcag atgatactgt ccttcgagct gccgtttgct ctcatccctc ttctcaagtt 60

cagcagcagt aagagcaaga tggggcccca caagaactct atctatgtaa gatcgtacac 120

<210> 19

<211> 120

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

gataatagtg ttctcgtggt tcctgggtct gctcatcatc ggcatcaaca tgtacttcct 60

gagcacgagc ttcgtcggct ggctcatcca caacgacctc cccaagtacg ccaacgtgct 120

<210> 20

<211> 120

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

ctcgtctaca tcgtcgctgt tgtttacctc accatcagga aggactccgt cgtcaccttc 60

gtcgccgact tctctctctg ctgctgttgt tgacgccgag aaggccgacg ccggcgacct 120

<210> 21

<211> 120

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

agcccttgcc gtaccgcgac gacctggccg acatcccgct cccaaggtag agaagaagaa 60

gatcgacatg catacgtatg gtatagcgta tgtatacgta cgtatatacg cgtacattgt 120

<210> 22

<211> 120

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

tgtatacgta cgtatatacg cgtacattgt gaatatacgt acttacgtac gtatatgtgc 60

atcctctatg gatgccacgt gcgtgcgtcg gagccgttcg tttatatttg tgcggtccag 120

<210> 23

<211> 120

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

atcgctcgca gatcgatcga tcgacaaggg tgtgcaatta atatataagc atttgagggg 60

aggtggctca tgtatccttg taatttatat gtacgatatg atttatcata attcagggca 120

<210> 24

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

atgatccatg tatgtgcag 19

<210> 25

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

actactacct agctagctac 20

<210> 26

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

atgctgaccg aagcgatgat g 21

<210> 27

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

atgacaagaa ccatcgccat c 21

Claims (9)

1. The OsNramp5 mutant is characterized in that the nucleotide sequence for coding the OsNramp5 mutant is shown as SEQ ID NO.1 or SEQ ID NO. 2.
2. 2. Biomaterial related to an OsNramp5 mutant according to claim 1, comprising any of the following:

   A) a recombinant expression vector comprising the nucleotide of claim 1;

   B) a bioengineered bacterium comprising the nucleotide of claim 1 or the recombinant expression vector of a).
3. 3. Use of the OsNramp5 mutant according to claim 1 or the biomaterial according to claim 2 in molecular assisted breeding of rice.
4. 4. Use of the OsNramp5 mutant according to claim 1 or the biomaterial according to claim 2 for breeding and/or producing low cadmium accumulation rice.
5. 5. The use of claim 4, wherein said method of selective breeding comprises the steps of:

   1) after radiation mutagenesis is carried out on rice, genome DNA is extracted, and a DNA library is constructed according to the genome DNA;

   2) designing a probe according to the OsNramp5 gene of the wild rice; hybridizing the probe to the DNA library;

   3) enriching the hybridized product by using magnetic beads to construct an enriched library;

   4) and (4) performing identification analysis on an enrichment library to identify whether the OsNramp5 mutant exists.
6. 6. The use of claim 5, wherein said radiation mutagenesis comprises gamma ray mutagenesis and heavy ion mutagenesis;

   when radiation-induced to gamma-ray mutagenesis, one or more of the following are included:

   the mutagenesis source of the gamma ray mutagenesis is selected from⁶⁰Co-gamma rays;

   and/or the dosage of the gamma ray is 200-500 Gy;

   and/or the dose rate of the gamma rays is 2-16 Gy/min;

   when the radiation is induced to heavy ion mutagenesis, one or more of the following are included:

   and/or the mutagenesis source of the heavy ion mutagenesis is selected from¹²C⁺⁶Heavy ions;

   and/or the dosage of heavy ion mutagenesis is 80-150 Gy;

   and/or the dosage rate of heavy ion mutagenesis is 0.8-2.2 Gy/min.
7. 7. The use of claim 5, wherein the probe has a sequence as set forth in SEQ ID No.4 to SEQ ID No. 23;

   and/or the probe is a biotin-labeled probe;

   and/or the magnetic beads are streptavidin-labeled magnetic beads.
8. 8. Use according to claim 5, wherein the method in step 1) is as follows:

   a) carrying out radiation mutagenesis on rice seeds to obtain M1 generation seeds;

   b) planting the M1 generation seeds to obtain M2 generation seeds;

   c) planting the M2 generation seeds, taking leaves on each planted individual plant, and mixing the leaves of each individual plant;

   d) extracting the genomic DNA from the mixed leaves;

   e) and (2) fragmenting the genome DNA, adding A tail to the fragmented DNA fragment, connecting a sequencing adaptor, and amplifying to obtain the DNA library.
9. 9. The use according to claim 8, wherein in step c), the M2 seed is planted in single plants, each plant is divided into 100-1000 plants, each sample is numbered, and the leaves of each sample are mixed to extract DNA;

   and/or, in the step c), when taking the leaves on each planted single plant, selecting the leaves at different parts of the same single plant for equal mixing; equal mixing of different individuals.

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