A rice OsNramp5 mutant M41, its genotype and its application

Abstract

The present invention discloses a rice OsNramp5 mutant M41, a genotype and an application thereof, and the technical field of rice mutagenesis breeding. Compared with the OsNramp5 gene sequence of Nipponbare rice, the nucleotide sequence of the OsNramp5 mutant M41 comprises the following base substitution: a base substitution G to A occurs at position 8877807 of chromosome 7. The present invention is of great significance in enriching the gene diversity related to low cadmium accumulation, balancing the positive and negative linkage effects of OsNramp5 on rice growth, development and yield, and exerting the socialization effect of low cadmium accumulation rice varieties.

Description

Technical Field

[0001] The invention relates to the technical field of rice mutation breeding, and in particular to a rice OsNramp5 mutant M41, a genotype, related biological materials and applications and screening methods thereof. Background Art

[0002] Excessive cadmium content in rice caused by soil cadmium pollution has become a key issue that needs to be urgently addressed in the rice industry. However, excessive cadmium content in rice has seriously affected the sustainable development of the agricultural economy and farmers' enthusiasm for rice planting, and has become a key issue that needs to be urgently addressed in the rice industry. Therefore, cultivating low-cadmium rice varieties is the most economical and effective way to increase the added value of early rice and drive new economic growth points in the rice industry. OsNRAMP5 is the main transporter gene that mediates cadmium absorption in rice. Its mutation can significantly reduce the cadmium content in rice plants, and the cadmium content in rice is reduced by more than 90%, providing an important target gene for the molecular breeding of low-cadmium accumulation rice. Therefore, it is of great significance to explore new and effective genetic loci related to grain cadmium accumulation and find new low-cadmium genes that can be directly used in hybrid breeding for the selection and breeding of low-cadmium rice.

[0003] At present, the use of technologies such as gene editing and transgenic technology to improve crop varieties is still relatively conservative, and physical and chemical mutagenesis have become irreplaceable methods for obtaining stable genetic germplasm innovation. The combination of radiation mutagenesis and chemical mutagenesis, and the large-scale processing of excellent rice germplasm resources, can increase the mutation rate in a short period of time and obtain better mutant gene types. The use of target gene liquid phase capture sequencing technology to detect gene markers in rice mutants can quickly obtain OsNramp5 site mutant plants, and is expected to create more new OsNramp5 gene mutation sites, providing new gene options for the breeding of low-cadmium rice varieties.

[0004] However, this mutation is random, and how to efficiently and accurately identify the mutation of the target gene related to cadmium accumulation is a difficulty in current mutagenesis breeding. At present, there are 6 OsNramp5 mutant patent applications (202111192651.1, 202111210318.9, 202111412187.2, 201911222439.8, CN201810525073, CN202211377945) that disclose the acquisition of OsNramp5 mutants, which are basically concentrated on heavy ion mutagenesis leading to multiple fragment deletions. However, different types of mutations have different effects on the content of metal ions in rice plants, rice growth and development, yield traits and rice quality. The greater the deletion, the greater the negative effect. Therefore, discovering effective single-base difference sites can better reduce this negative effect and provide favorable support for the efficient transformation of rice low-cadmium genes.

[0005] The present invention provides a rice OsNramp5 mutant M41, a genotype and an application thereof, so as to enrich the gene diversity related to low cadmium accumulation, balance the positive and negative linkage effects of OsNramp5 on rice growth, development and yield, and exert the socialization effect of low cadmium accumulation rice varieties. Summary of the invention

[0006] The primary purpose of the present invention is to provide a rice OsNramp5 mutant M41, a mutant genotype, and related biological materials to better guide the breeding of low cadmium accumulation rice varieties.

[0007] The secondary purpose of the present invention is to provide an application of related biological materials of rice OsNramp5 mutant M41 in rice breeding, so as to obtain more abundant rice varieties with low cadmium accumulation.

[0008] The third object of the present invention is to provide a screening method for accurately identifying and efficiently obtaining heritable rice OsNramp5 mutants.

[0009] The technical problem solved by the present invention is achieved by adopting the following technical solutions:

[0010] A rice OsNramp5 mutant M41, wherein the nucleotide sequence of the OsNramp5 mutant M41 comprises the following base substitutions compared with the OsNramp5 gene sequence of Nipponbare rice: a base substitution G to A occurs at position 8877807 of chromosome 7.

[0011] A rice OsNramp5 mutant M41 genotype, wherein the gene of the OsNramp5 mutant M41 comprises a nucleotide sequence as shown in SEQ ID NO.1, or comprises a sequence encoding a protein having the same function and having more than 95% homology with the nucleotide sequence as shown in SEQ ID NO.1, wherein the same function comprises reducing the cadmium content of rice.

[0012] The present invention provides biological materials related to the OsNramp5 mutant M41, including any one of the following:

[0013] A) a recombinant expression vector containing the nucleotide sequence of claim 2;

[0014] B) a bioengineering bacterium containing the nucleotide as claimed in claim 2, or a bioengineering bacterium containing the recombinant expression vector as claimed in A);

[0015] C) a transgenic plant cell containing the nucleotide sequence of claim 2, or a transgenic plant containing the recombinant expression vector of A);

[0016] D) A protein encoded by the nucleotide sequence of claim 2.

[0017] The present invention provides the use of the OsNramp5 mutant M41 or the biological material in plant molecular assisted breeding.

[0018] The present invention provides the use of the OsNramp5 mutant M41 or the biological material in breeding and / or preparing low cadmium accumulation plants.

[0019] Furthermore, the invention relates to an application in breeding and / or preparing rice varieties with low cadmium accumulation.

[0020] Furthermore, the method for breeding low cadmium accumulation rice products is: using rice OsNramp5 mutant M41 as a donor and hybrid rice backbone parent as a recipient, backcrossing improvement is carried out, and conventional breeding combined with KASP molecular markers are used to breed low cadmium accumulation rice varieties with the OsNramp5 mutant M41 genotype.

[0021] The present invention also provides a method for screening a rice OsNramp5 mutant, comprising the following steps:

[0022] 1) Selecting induced rice seeds for planting, planting the M2 generation seedlings separately, taking leaves from multiple rice plants and mixing them in groups to obtain multiple groups of mixed samples; extracting mixed pool DNA from each group of mixed samples;

[0023] 2) Using KASP molecular markers to identify and type each group of mixed pool DNA, and screen for mixed pool samples containing rice M41 mutant genes;

[0024] 3) Extract DNA from individual rice plants corresponding to the mixed pool samples with the same genotype, further perform typing identification on the DNA extracted from the individual plants by detecting the KASP molecular markers corresponding to the differences, and screen out the individual plants with the rice M41 mutant gene, that is, the plants containing the rice M41 mutant gene.

[0025] In the screening method, the typing primer of the KASP molecular marker comprises the following sequence:

[0026] F1: GAAGGTGACCAAGTTCATGCTACAGTTTAATTGCGTGTCATTTAGTCAC

[0027] F2: GAAGGTCGGAGTCAACGGATTACAGTTTAATTGCGTGTCATTTAGTCAT

[0028] R:GCTAGCAGCTAATCTTGGAGTGGT.

[0029] Furthermore, in step 1), the rice variety is Xiangyue Si Miao.

[0030] Preferably, in step 1), physicochemical mutagenesis is performed, and the physicochemical mutagenesis method includes proton mutagenesis or EMS (ethyl methanesulfonate) mutagenesis.

[0031] Furthermore, in step 1), leaves from every 100-200 individual rice plants are grouped and mixed to obtain a mixed sample.

[0032] Furthermore, the number of generations for backcross improvement is 2-6 generations.

[0033] Beneficial effects of the present invention:

[0034] The rice M41 mutant of the present invention is a unique rice OsNramp5 genotype mutant screened out for the first time, and can be well applied to the breeding of low cadmium accumulation rice varieties.

[0035] The present invention can accurately screen out rice OsNramp5 mutants by mutagenizing the M1 generation through unique KASP molecular marker typing and identification. The operation is efficient, accurate and simple, and the cost is significantly reduced.

[0036] The rice M41 mutant described in the present invention has a simple cultivation and preparation method when breeding and creating low cadmium accumulation rice products, and does not require gene editing means. After the cultivated rice containing the M41 mutant genotype of the present invention is planted, the cadmium content in the roots, stems, leaves and mature seeds is significantly lower than that of wild rice of the same variety. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Schematic diagram of the comparison of base sequences of the mutation site and mutation type of the mutant gene in Example 1.

[0038] Figure 2 This is a scatter plot of the genotyping of the segregating population using KASP typing primers in Example 1.

[0039] Figure 3 This is a SNP variation analysis diagram of M41 associated with the low cadmium accumulation phenotype in Example 1.

[0040] Figure 4 These are plant type comparison photos of Xiangzao Low Cadmium No. 1 and Xiangzao No. 1 in Example 3. DETAILED DESCRIPTION

[0041] In order to make the technical means, creative features, objectives and effects of the present invention easy to understand,

[0042] The present invention is further described below in conjunction with specific embodiments.

[0043] Unless otherwise defined, all professional terms used hereinafter have the same meanings as those generally understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention. Unless otherwise specifically stated, the various raw materials, reagents, instruments and equipment used in the present invention can be purchased on the market or can be prepared by existing methods. The embodiments use the Xiangyue Si Miao variety as an example to illustrate the conception and experimental results achieved, and the actual application is not limited to Xiangyue Si Miao.

[0044] Example 1—Obtaining and verifying the rice cadmium low accumulation mutant M41

[0045] A rice M41 mutant gene having a nucleotide sequence as shown in SEQ ID NO.1. According to the test results of Huazhi Biotechnology Co., Ltd., the rice M41 mutant gene is a base substitution G to A at the splicing position 8877807 of the 4th exon and the 4th intron of the rice OsNramp5 gene, and the 4th intron in the translation process of the coding region is translated, resulting in the final mutant rice M41 mutant gene encoding the 6th-8th base of the 4th intron. The termination codon TAA appears in advance, causing its metabolic activity to be severely reduced, thereby achieving low accumulation of cadmium in rice grains. The rice M41 mutant gene of this embodiment has a mutant gene OsNRAMP5 nucleotide sequence as shown in SEQ ID No.1, and its encoded amino acid sequence is shown in SEQ ID No.2.

[0046] The rice M41 mutant gene of this embodiment is specifically obtained by screening and identifying by the following method:

[0047] 1) 200 g of seeds of the proprietary rice material "Xiang Yue Si Miao" were selected, and the dry seeds of "Xiang Yue Si Miao" were treated with proton beam radiation at a treatment dose of 200 Gy to obtain the induced M1 generation of "Xiang Yue Si Miao".

[0048] 2) Plant the seedlings of the M1 generation individually, use film isolation, strictly self-pollinate and harvest together to obtain M2 generation seeds.

[0049] 3) Planting the M2 seedlings individually, with 112 seedlings as a group, 14 rows per group, 8 plants per row, 96 groups, a total of 10,752 plants,

[0050] 4) Number each group; take each sample group as a unit, and when the seedlings grow to four leaves and one heart, take leaves with a 6 mm hole puncher to take equal amounts of leaves and mix them, and a total of 112 sample groups are obtained.

[0051] 5) A total of 112 DNA samples were extracted from each population, and each DNA sample was used as a sequencing sample. Targeted high-depth sequencing was performed on the exon region of the OsNramp5 gene (Os07g0257200), with a sequencing depth of >5000.

[0052] 6) The high-depth targeted sequencing data were compared with the Nipponbare sequence to obtain the SNP and Indel data of the OsNramp5 gene in 112 samples. After testing by Huazhi Biotechnology Co., Ltd., it was found that in sample No. 41, there was a [G / A] base substitution at the 8877807 site of the OsNramp5 gene, and the base substitution was located at the splicing position between the 4th exon and the 4th intron (see Figure 1 ).

[0053] 7) Then, KASP typing primers were designed based on the [G / A] base substitution site, and the primer sequences were:

[0054] >F1: GAAGGTGACCAAGTTCATGCTACAGTTTAATTGCGTGTCATTTAGTCAC

[0055] >F2:GAAGGTCGGAGTCAACGGATTACAGTTTAATTGCGTGTCATTTAGTCAT

[0056] >R:GCTAGCAGCTAATCTTGGAGTGGT

[0057] 8) Using the KASP typing primers for the deletion mutation site, 112 strains from population 41 were typed. After typing, it was found that the 4th strain (41-2-D1) in row 13 of population 41 contained a single base substitution genotype of [G / A] in exon 4 of the OsNramp5 gene (see Figure 2 ), and finally obtained an M2 generation single plant with target gene mutation, namely, OsNramp5 gene base substitution mutation heterozygous plant 41-2-D1. That is, there is a rice M41 mutant gene heterozygous plant of the present invention.

[0058] 9) The mixed seeds of 41-2-D1 were sown, and the KASP primers designed in step 8 were used to type the individual plants, and finally the homozygous mutant of the OsNramp5 mutant gene was obtained in the M3 population of 41-2-D1, that is, the rice M41 mutant gene of the present invention existed.

[0059] At the same time, according to the typing results, 20 plants were randomly selected from the three genotypes of G / A, G / G, and A / A and planted in fields with severe cadmium pollution (soil cadmium concentration of 1.5 mg / kg, pH of 6.5) and moderate cadmium pollution (soil cadmium concentration of 0.4 mg / kg, pH of 6.0). In order to accumulate as much cadmium as possible in rice, the cultivation management was carried out in the form of alternating dry and wet. After the rice was fully mature, the plants were harvested, threshed, and the brown rice was ground into powder. Referring to the national standard determination method (GB / T5009.15-2003), the cadmium content in brown rice was determined by ICP-MS. The results are shown in Table 1 below. The cadmium content of the grain of the homozygous genotype of the M41 mutant [A / A] was 0.056 mg / kg, which was significantly lower than that of other genotypes and lower than the national limit standard of 0.2 mg / kg.

[0060] Table 1 Phenotypic characteristics of cadmium content in grains of various mutant genotypes

[0061] serial number 8877807 site variation characteristics Average cadmium content in grains (mg / kg) M41-homozygous A / A 0.056 M41-heterozygous G / A 1.531 M41-Wild G / G 1.795

[0062] 10) In order to verify the effect of the mutation at the M41-8877807 site [G / A], an F2 population of M41 and wild-type Xiangyuesi seedling was constructed and planted in a heavily cadmium-contaminated field (soil cadmium concentration was 1.5 mg / kg, pH was 6.5). The cadmium content of the grains was detected using a high-precision X-ray fluorescence spectrometer from Jiapu Technology XR700. Ten F2 plants with high / low cadmium accumulation were selected to construct a DNA pool for sequencing and SNP chromosome distribution analysis was performed. The results showed that the variation at the 7.2M-9.1M position of chromosome 7 was very concentrated, and this region was preliminarily identified as the functional gene interval for low cadmium accumulation in rice (see Figure 3 ).

[0063] 11) Trizol (Invitrogen) was further used to extract RNA from the cadmium low accumulation mutant M41 and Xiangyuesima.

[0064] Reverse transcribe RNA into cDNA, reaction system: RNA 30ng, Oligo(dT)23VN (50μM): 1μL, HiScriptII Enzyme Mix: 2μL, 2×RT Mix: 10μL, RNase-free Water: to total volume 20μL.

[0065] Reaction program: 25°C for 5 min, 37°C for 45 min, 85°C for 10 s.

[0066] Using cDNA as template, PCR amplification was performed using the full-length primers of OsNramp5.

[0067] The primer sequences are:

[0068] >TF:ATGGAGATTGAGAGAGAGAGCAGT

[0069] >TR:CTACCTTGGGAGCGGGATG

[0070] 12) The PCR products were subjected to Sanger sequencing. After the sequencing results were obtained, the OsNramp5 transcription products of the low cadmium accumulation mutant M41 and Xiangyuesima were compared (see Figure 3 ), it was found that the base substitution G at the splicing position 8877807 between the 4th exon and the 4th intron of the mutant M41 was changed to A, and the 4th intron in the translation process of the coding region was translated, and its transcription expression sequence is shown in SEQ ID No. 3. As a result, the mutant gene of the final mutant rice M41 was encoded with the termination codon TAA in advance at the 6th to 8th bases of the 4th intron, and its amino acid sequence is shown in SEQ ID No. 2.

[0071] Example 2--- Phenotypic characteristics of low cadmium accumulation in different genotypes of rice OsNramp5 mutants

[0072] The cadmium accumulation phenotypic effect analysis of rice OsNramp5 mutants with different genotypes, with the donors being mutants lcd1, mutant R001, mutant lcd100 and other multiple mutants with different sites obtained by the present invention, specifically comprises the following steps:

[0073] 1) The donor mutation site information described in this example is as follows:

[0074] Table 2 Site variation characteristics of mutants with different genotypes

[0075]

[0076] 2) 30 mutants with different differential sites were extracted and planted in a moderately cadmium-contaminated field (soil cadmium concentration of 0.4 mg / kg, pH of 6.0). In order to make rice accumulate as much cadmium as possible, the cultivation management was carried out in the form of alternating dry and wet. After the rice was fully mature, the individual plants were harvested, threshed, and brown rice was ground. With reference to the national standard determination method (GB / T5009.15-2003), the cadmium content in brown rice was determined by ICP-MS. The results showed that the different differential site mutants showed significant differences in the accumulation of cadmium in rice grains. Among them, the mutant M41 of the three stable genetic mutants obtained in this embodiment had the best inhibitory effect on the accumulation of cadmium in the grains. Although the cadmium content of the mutant M54 was also lower than the national standard of 0.2 mg / kg, it was impossible to achieve a low cadmium accumulation phenotype in high cadmium soil. At the same time, although M78 also caused changes in the amino acids of OsNramp5, its grain cadmium content was consistent with the cadmium content of the wild-type Xiangyue silk seedlings. Based on this, the OsNramp5 gene mutant M41 obtained in this experiment is a mutant with a stable genetic phenotype of low cadmium accumulation (Table 3).

[0077] Table 3 Phenotypic characteristics of cadmium content in grains at different genotype mutant loci

[0078] Mutant name Average cadmium content in grains mg / kg lcd1 0.097 R001 0.040 lcd100 0.051 M41 0.047 M78 0.345 M54 0.159 Xiangyue Silk 0.308

[0079] Example 3 - Application of rice OsNramp5 mutant M41 in creating new low-cadmium rice varieties

[0080] In order to verify the application of rice OsNramp5 mutant M41 in creating new low-cadmium rice varieties, the following steps are specifically included:

[0081] 1) Using the mutant M41 obtained in Example 1 as the donor parent and the early rice core material Xiangzao No. 1 as the recipient parent, hybridization and breeding were performed to obtain F1 seeds;

[0082] 2) Selecting a single plant from the above sowing F1, using the tested early rice as the recipient parent, and backcrossing to obtain BC1F1 seeds;

[0083] 3) Sowing the above BC1F1, performing typing detection using KASP markers, obtaining plants containing the M41 heterozygous genotype locus, performing background selection, using the tested early rice as the recipient parent, and backcrossing to obtain BC2F1 seeds;

[0084] 4) Sowing the above BC2F1, performing typing detection using KASP markers, obtaining plants containing the M41 heterozygous genotype locus, performing background selection, using the tested early rice as the recipient parent, and backcrossing to obtain BC3F1 seeds;

[0085] 5) Sowing the above BC3F1, performing typing detection using KASP markers, obtaining single plants containing the M41 homozygous genotype locus, performing background selection, and self-pollinating to obtain BC3F2 seeds;

[0086] 6) The above-mentioned BC3F2 was sown and typing was performed using the KASP marker to obtain a single plant containing the M41 homozygous genotype locus, and background selection was performed. Single plants with the same genetic background as the rice Xiangzao No. 1 were selected for harvest to obtain Xiangzao Low Cadmium No. 1.

[0087] 7) The Xiangzao Low Cadmium No. 1 obtained in this example was planted simultaneously with Xiangzao No. 1 in soil with excessive cadmium content (0.45 mg / kg), and the yield, plant height, grain cadmium content and other indicators were tested after maturity. The results showed (Table 4) that the cadmium content of Xiangzao Low Cadmium No. 1 seeds was significantly lower than that of Xiangyuesi seedlings, indicating that the low cadmium phenotype of mutant M41 has a genetic effect and can be effectively applied to low cadmium rice molecular breeding.

[0088] Table 4 Comparison results of related traits between Xiangzao Low Cadmium 1 and Xiangzao 1 (WT)

[0089]

[0090] The above shows and describes the basic principles, main features and advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

[0091] Sequence Listing

[0092] SEQ ID NO.1:

[0093] ATGGAGATTGAGAGAGAGAGCAGTGAGAGAGGGAGCATCAGCTGGAGAGCTAGTGCG

[0094] GCACATGATCAAGATGCCAAGAAGCTCGACGCAGATGATCAGCTGCTAATGAAGGTCA

[0095] GTTGCTACCTTAATTATCACAAAGTTGTCTTCTTGTAAGCACTCCACTGTCACCATGGA

[0096] CAGTTGTTCACTGTAACTGCAGAAGCAAGAGCAGAGCTTAATGTAGATAGATCTTGAG

[0097] AGTACTTCCTGGCATGGCATTCATTGCTCATGAATGTTCATGTGTGTGTGTGTGTATGTG

[0098] GTAATTAAGGACTGATGGAACATTGGATCCTCAATTATGTCTGTGTTTCTTTTTGTTAGT

[0099] TTCTCAGTTTTGGTAAATTGTGTACAGGAGCCTGCATGGAAAAGGTTCCTTGCCCATGT

[0100] TGGTCCTGGATTCATGGTGTCTTTAGCCTACTTGGATCCTGGCAATTGTGAGAATCTCTG

[0101] AATCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCCAACTGAAACTTTTATTTTGACAAACTCT

[0102] GGATTGTAACTGTTAATTTTGTCACAAAATGAAACAGTGGAAACCGATCTGCAAGCCG

[0103] GAGCCAACCACAGATATGAGGTACGTACAGTCGACTTTATTTTACTGTTTACGCACTGA

[0104] ATGACTGAAAGCATGAGTAACTTTCATGCTTTCTAGGTGTTCGTCCCTTTCTCTGTCCCT

[0105] CGCAGACTCACACTCCATTAATTCATCTGCTAGCTCCATCAAATTCTCCAAATTGACAA

[0106] ACTTTTCAGAACCACTTTTAATCCCCAGTGCATATGTATCTCACTCTCACATCATCTAGA

[0107] GAAAAGAGAAAAGTGAATAATCTGCACCATTAATTGATTTCTATGTGCTTTTCTGTCCAT

[0108] TGTGTTGTTTCAATTTGCAGCTGCTCTGGGGTGATTCTGATTGGACTCATCTTCGCACTTA

[0109] TCATACAGTCGCTAGCAGCTAATCTTGGAGTGGTTACAGATGACTAAATGACACGCAAT

[0110] TAAACTGTAATTACCAACTTGATAAAGTGCTAATATATGGCCGGCATTAATTTACATCCTA

[0111] GGGAGGCATCTGGCTGAGATCTGCAAGAGTGAGTACCCCAAGTTCGTCAAGATTTTCC

[0112] TATGGCTGCTGGCAGAGTTGGCCGTCCATCGCTGCAGATATCCAGAAGGTATATACTTAT

[0113] TAGAACATTGACACGGTCCAGCCTGAATAATTATTATTAAACTACTTGTGTTTAAC

[0114] AGTGTCACCATCTTTGACTTTGAGCTAATTAATTAATTCAGTTTAAGCTCTCTGACAGTA

[0115] GAAGAAAACAAGTTGATCTGGCTAGCATATGTAGCTCCAACCGTACGGCCTACATACT

[0116] GGAATTCCTAATCCCACAAACATAGGCAAATTAATATCTCACCAATCACCATCAAGCCAC

[0117] GTTCTTTCTCAGCTACTCACCAGATTATTCTCACATATTATTCTTTCATGTCTCTAGCTAG

[0118] TAAATATGTTTTTCTAAAAAAAATATATTCTGCGATATTGCATAGATTACAAGTTCTAAGC

[0119] CATTTTTTTAAAAAATATTTAATTCATCCATATAATAAGGTAGATAATATGTACCAATAATA

[0120] TATCTGTTGAATAATCTGTTTCAAACATAAGCAAATGCGCGTTTTAGCTGTTTAGAGTAG

[0121] TGACCACGTCCACAGTAGCTTCCCATAAAATTGTCAATGATGCACATCAAGATTAGATT

[0122] TTAAACACTGGGTGAGTACGTTCATCATCTTACGCAGATTAGTAAACAACTATTTTGTC

[0123] ACCCTCTTGTCATTTCACATTTGTCCTGTCCTGTATACATCCATGTGTGTTTAAACAATA

[0124] ACAACAATATCTAAATATCTATCTATCTAGCAACATGGAAGATAGATCAGTGGTGTAGCA

[0125] GTTGCAAAACGGAGCTGCGCGAAGCTTTGCTGATCGGGATTAGTTAACCTCAACCGTT

[0126] GTAATTGCCTTTTTAATTGATCATGCATGCAAGTTCTTTTTTAAAAAAATTAAACTAGT

[0127] TATGGAAGATACCTCACATGATGATAGCAAAGGTGATATATAGATAGATAGATAGATCGA

[0128] TCAACAGATAGAAAGGTACAGATCGATGTGATTATAATTAATTAATTAATTACATGTCGT

[0129] TTGATGTTGTTAATTTTGGTGGATGCAGTTATAGGGACGGCCTTTGCTTTCAACATATTG

[0130] TTCCATATTCCGGTGTGGGTCGGCGTCCTCATCACCGGCACCAGCACTCTACTGCTTCT

[0131] TGGCCTCCAAAAATACGGGGTATAAATTATTTTTTGTAAGTTGTTTTTGTTTAGTATCAC

[0132] AAGCATTAGCGTAGGATGCACTCAAAGAAACGATTATAAAAATGGAATAGTATATGAAA

[0133] TTAGCTAGTTGGATCGATTGTCAAGTACTCAAATGGCATATTATATGTACATTATTCAGTT

[0134] TTTTTTTCTCTGACTGTGGAACTGCATGAAAATTGATTGATGAATTTTTTTTGAAAAAA

[0135] AACAGGTGAGGAAGCTGGAGTTTCTGATATCGATGCTGGTGTTCGTGATGGCGGCGTG

[0136] CTTCTTCGGGGAGCTGAGCATCGTGAAGCCGCCGGCGAAGGAGGTGATGAAGGGGCT

[0137] CTTCATCCCCAGGCTCAACGGCGACGGCGCCACCGCCGACGCCATTGCCCTCCTCGGA

[0138] GCTCTTGTCATGCCGTACGCTTTTACATACATACATACATACATACGCAACAATCAATCA

[0139] ATTAATTCATACAACTAATCAGTTAATTAACTACTGCATGCATTTATATATATGCAGCCAC

[0140] AATCTGTTCTTGCATTCTGCCTTGGTGCTATCGAGGAAGACACCGGCATCAGTCAGAGG

[0141] AATCAAGGTAGCTATCTTCAGCCTGGGCAGATCGAGATAGGTATAGGGTAGCTAGGACA

[0142] TGAACTCGATCTAGTCCTGTCTCTTAAATCTTGTTTAAATTTTATTAATTAATTAACATGTAAA

[0143] TTTCATAGAAAACCAATACTTTATTAGCTAAGTTTTATTTTAGCACTAATCTCTCTATAATT

[0144] TCGGGCTCCCAGTACCACTGGATTCAGCTAAGAATTTTTTCATGCATGGAAAGAAACTG

[0145] AACAAAGATCCAGGCTAATAATTAATATGTACTATATGGTGTGTGAATTGCCACGTAGGA

[0146] CGGGTGCAGGTTCTTCCTGTACGAGAGCGGGTTCGCGCTGTTCGTGGCGCTGCTGATA

[0147] AACATCGCCGTCGTCTCCGTCTCCGGCACCGCCTGCTCCTCCGCCAACCTCTCCCAAG

[0148] AGGACGCCGACAAGTGCGCCAACCTCAGCCTCGACACCTCCCTCCTTCCTTCTCAAGGT

[0149] CATTCATTCATTATTACTTCCTACTCTTTAATATTAAAAGTTTTTAAATGGATTAGACGTAT

[0150] CCTAATACTACAGATATAGGAATTTATTCAGATTCGTAGTACTAGCTTGTATGTGTTCAAT

[0151] TTAATCAATATTTAATTAACCACAATAACACCGAATTAGTCTACACGATTTCTCCGTCCT

[0152] AAAATATAATAACTTTATTAGATATGTTTATATTTTTGAATAGGAGGAGTAACTAATTTAT

[0153] GGTCAAATTGTGACGTCAATTTTTTATGTGGTTCAGAACGTGCTGGGCAAGTCGAGTG

[0154] CGATCGTGTACGGCGTGGCACTGTTGGCATCTGGGCAGAGCTCCACTATTACCGGCAC

[0155] ATACGCTGGACAGTACATCATGCAGGTAATTAATTAAATTAATTAACTTAACTGTACCTG

[0156] TACCTGTACATCACCAGCTAGCTTAGCGCGTAATTAAATGATTGAACTATAATTAATTAA

[0157] ACGGCTAGCTGCCACATAATTAATTTGTTAATCCGTTCAATTATGGAGGTTAACTGACGA

[0158] ATTGTAGGGGTGCACACCTAATCCTGTTTACATCACGAATATTTAGTGATATAATATTCCT

[0159] TTATTAATTAGGTAATCTGGCGATCCTCGTCGGATTTCACCATATGTTTTTGTTAAAGTAA

[0160] GATCGAATTAAGACGCTACCTTGCTGCCACTACTTACCACTTTCGTCCATATTTCTCCGC

[0161] TCTTTTGGTCTAGTCCATTTCACGGTGCAGTCTCATCAATAACCAGCGATTTTACTGTTT

[0162] ACATATACTTAGAAAACCAATTGATTAATAAAAATATGCTAACGAAACTCTATAAATTAT

[0163] ATTAAAAAAAATAGGATATCCCATCGCACCGTAAAATTTTCTTTCTGAAGTGAACGTTG

[0164] AGAAAGAGAACAACTAGTTTTCCTAATCTACCTGGCGGGCTAACCGATCTAGTTCGAG

[0165] TCTCATTCCTCTTAATAATCTATTTGATATGAAGTGTATTTCAGTTACGGGATAAATCG

[0166] GACCCGAAAATATATTTATTTTCACTCCAAAGGACTGATCTTCAGGATATACTCAC

[0167] TCCGTCTCTAAATTTGACGTCGTTGATTTTTTATAATATGTTTGACCGTTCGTCTTAT

[0168] GCAAAAACTTTTGTGAATATTTTAAAAAACTTGAGTAGTAGTAGTATATACAACAATG

[0169] AATTAATAATAAAAAGAATTAATAACTACTTAATTTGAATAAGATGAATGGTT

[0170] AAAATGTTTAGAAATCAATGGTGTCAAAATATTTAATGACGGAGGGAGTATATGATAA

[0171] ATCATCATCATGTCGCGTTGATATATATGATATGATCCATGTATGTGCAGGGTTTCTTGGA

[0172] CATCAGGATGAGGAAGTGGCTTCGGAACCTGATGACAAGAACCATCGCCATCGCGCCCG

[0173] AGCCTCATCGTCTCCATCATCGGCGGCTCCAGGGGCGCCGGCCGCCTCATCATCATCGC

[0174] TTCGGTCAGCATTTTTTATTACTATTTACTATAGCAAATCTTTACTATTTACTATTTTA

[0175] CTATATCAAACTGCGTGCGCCATACGGCGGCGCCCGCCGGCGAGCCAACCGGCCGGCCG

[0176] TCGTGTCGCCGATCGATTAAAATTTAATGCGTTTAAATTTTTAATTTTGCACATGCCCAA

[0177] ACAGTCACGTGTGTGCAGTCGATCGTGCGGATTTATGTAGCTAGCTAGGTAGTAGTGGG

[0178] GTATCCACTAGGTACCTAGTGACATAGTACTAAGAGCATATATATATGTCATTAGAGCAA

[0179] AAGCACTTTTTCTTCTTTTTTTAGATTATTGCCAAATCAAAGTAGTGATCTGATTAGCAA

[0180] AATGTCACTGACTAGCTAGGTATCTTTTCTTTTTTTGCAATCTGATCTAAAATAGCACCA

[0181] TGCATATATATAATACAGTGTACCATCTAGCTAGACTAGCTTCCAACTCAATAAGTAAGG

[0182] ACTAATTATATATAATTAAATCAAATCAAAGTAGTGATCTGATCATTGCTAGTTCTTGTTT

[0183] GCTTAATTACATATATATGATTAATTTGTTCCTAATCCGTACGTGTTGTGTGCATGCAGAT

[0184] GATACTGTCCTTCGAGCTGCCGTTTGCTCTCATCCCTCTTCTCAAGTTCAGCAGCAGCAGTA

[0185] AGAGCAAGATGGGGCCCCACAAGAACTCTATCTATGTAAGATCGTACACTATATATATGC

[0186] ATTGCTTAATTATAGTTTGTTTCTTAATCTCAAATGGCAGCAAATGTACAAGTAACATAA

[0187] ATATATGCATCTATCTGTCCATGCATGAATTAGAGTTTCAATAATTGTTGGTAAAGAATA

[0188] TAAATTAATGATGATAGTTGGCTAGCTAGCTGGTCCTTCACCCTGCATGCATGCAACCCC

[0189] TGCAGGACACCATGTTCTATAGGTCAGTGTGTGCATAATGTAGATCGAGATGTGCAGTG

[0190] AATTGAATTGAATCATTAGCTACCTAGCTCATGCAATAGTAATATAGCACAGCAGGGGCC

[0191] AATGCAGGACAGGTATATATATGGTCCAGCCATGCATATAGAGGATCGTCGTACATATGA

[0192] AAACGACGACCAAGATGTACGTCCTGCATCATTTTTCTGTCTTGCAGTAGTACCAGAGA

[0193] TTAATCATGTATTGCGTCGTCGGCATGATGTGTCAAAATCATGCAGTAACGTACTTGCTT

[0194] GATCTTTACAGACATCGAAGAGCCAAGTTCTTCAGTACACTATATATATTTTTTCTTGCA

[0195] TGGAAGTAAATGTAAATATAATTTAACATAGATAATTACGTATATCGTTGTACTCTACT

[0196] TAAACATTTCTAGACACATACAAGTCAACATTAGAAAATGTAATTCTTTTTTAAGGACC

[0197] ATAAATAAACTGATCTACAACAAAATTGAGCAAATGTAAATTTATTTCTTATTATTACAC

[0198] ACATGGCTATATATATATATATATATATATATATATATATATATATATACACATACTACCTATA

[0199] AAAATTGCGCATGCTCATCAGTCGATACCCGTTAAAAAGATATATAAGTTACCACCC

[0200] GTCAAAGATGACTGGTTATTTAGTGACGCTATCAATGAATAAATCAGCGTCATGTATG

[0201] TCAAAAAATAAATTGAATTGAATTGAATGCAGATAATAGTGTTCTCGTGGTTCCTGGG

[0202] GCTGCTCATCATCGGCATCAACATGTACTTCCTGAGCACGAGCTTCGTCGGCTGGCTC

[0203] ATCCACAACGACCTCCCCAAGTACGCCAACGTGCTCGTCGGCGCCGCCGTCTTCCCGT

[0204] TCATGCTCGTCTACATCGTCGCCGTCGTCTACCTCACCATCAGGAAGGACTCCGTCGT

[0205] CACCTTCGTCGCCGACTCCTCCCTCGCCGCCGTCGTCGACGCCGAGAAGGCCGACGCC

[0206] GGCGACCTCGCCGTCGACGACGACGAGCCCTTGCCGTACCGCGACGACCTGGCCGAC

[0207] ATCCCGCTCCCAAGGTAG

[0208] SEQ ID NO.2:

[0209] MEIERESSERGSISWRASAAHDQDAKKLDADDQLLMKEPAWKRFLAHVGPGFMVSLAY

[0210] LDPGNLETDLQAGANHRYELLWVILIGLIFALIIQSLAANLGVVTDD

[0211] SEQ ID No.3

[0212] ATGGAGATTGAGAGAGAGAGCAGTGAGAGAGGGAGCATCAGCTGGAGAGCTAGTGCG

[0213] GCACATGATCAAGATGCCAAGAAGCTCGACGCAGATGATCAGCTGCTAATGAAGGAGC

[0214] CTGCATGGAAAAGGTTCCTTGCCCATGTTGGTCCTGGATTCATGGTGTCTTTAGCCTAC

[0215] TTGGATCCTGGCAATTTGGAAACCGATCTGCAAGCCGGAGCCAACCACAGATATGAGC

[0216] TGCTCTGGGTGATTCTGATTGGACTCATCTTCGCACTTATCATACAGTCGCTAGCAGCTA

[0217] ATCTTGGAGTGGTTACAGATGACTAAATGACACGCAATTAAACTGTAATTACCAACTTG

[0218] ATAAAGTGCTAATATATGGCCGGCATTAATTTACATCCTAGGGAGGCATCTGGCTGAGAT

[0219] CTGCAAGAGTGAGTACCCCAAGTTCGTCAAGATTTTCCTATGGCTGCTGGCAGAGTTG

[0220] GCCGTCATCGCTGCAGATATCCCAGAAGTTATAGGGACGGCCTTTGCTTTCAACATATT

[0221] GTTCCATATTCCGGTGTGGGTCGGCGTCCTCATCACCGGCACCAGCACTCTACTGCTTC

[0222] TTGGCCTCCAAAAATACGGGGTGAGGAAGCTGGAGTTTCTGATATCGATGCTGGTGTT

[0223] CGTGATGGCGGCGTGCTTCTTCGGGGAGCTGAGCATCGTGAAGCCGCCGGCGAAGGA

[0224] GGTGATGAAGGGGCTCTTCATCCCCAGGCTCAACGGCGACGGCGCCACCGCCGACGC

[0225] CATTGCCCTCCTCGGAGCTCTTGTCATGCCCCACAATCTGTTCTTGCATTCTGCCTTGGT

[0226] GCTATCGAGGAAGACACCGGCATCAGTCAGAGGAATCAAGGACGGGTGCAGGTTCTT

[0227] CCTGTACGAGAGCGGGTTCGCGCTGTTCGTGGCGCTGCTGATAAACATCGCCGTCGTC

[0228] TCCGTCTCCGGCACCGCCTGCTCCTCCGCCAACCTCTCCCAAGAGGACGCCGACAAGT

[0229] GCGCCAACCTCAGCCTCGACACCTCCTCCTTCCTTCTCAAGAACGTGCTGGGCAAGTC

[0230] GAGTGCGATCGTGTACGGCGTGGCACTGTTGGCATCTGGGCAGAGCTCCACTATTACC

[0231] GGCACATACGCTGGACAGTACATCATGCAGGGTTTCTTGGACATCAGGATGAGGAAGT

[0232] GGCTTCGGAACCTGATGACAAGAACCATCGCCATCGCGCCGAGCCTCATCGTCTCCAT

[0233] CATCGGCGGCTCCAGGGGCGCCGGCCGCCTCATCATCATCGCTTCGATGATACTGTCCT

[0234] TCGAGCTGCCGTTTGCTCTCATCCCTCTTCTCAAGTTCAGCAGCAGTAAGAGCAAGAT

[0235] GGGGCCCCACAAGAACTCTATCTATATAATAGTGTTCTCGTGGTTCCTGGGGCTGCTCA

[0236] TCATCGGCATCAACATGTACTTCCTGAGCACGAGCTTCGTCGGCTGGCTCATCCACAA

[0237] CGACCTCCCCAAGTACGCCAACGTGCTCGTCGGCGCCGCCGTCTTCCCGTTCATGCTC

[0238] GTCTACATCGTCGCCGTCGTCTACCTCACCATCAGGAAGGACTCCGTCGTCACCTTCG

[0239] TCGCCGACTCCTCCCTCGCCGCCGTCGTCGACGCCGAGAAGGCCGACGCCGGCGACC

[0240] TCGCCGTCGACGACGACGAGCCCTTGCCGTACCGCGACGACCTGGCCGACATCCCGC

[0241] TCCCAAGGTAG

Claims

1. A rice OsNramp5 gene mutant, characterized in that: The nucleotide sequence of the mutant is shown in SEQ ID NO.

1.

2. A biomaterial, characterized in that: The biological material is any of the following: A) a recombinant expression vector containing the gene mutant according to claim 1; B) Bioengineered bacteria containing the recombinant expression vector described in A).

3. Use of the gene mutant according to claim 1 or the biological material according to claim 2 in breeding rice with low cadmium accumulation.

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