CN117447567B - OsNramp5 mutant and related products and application thereof - Google Patents
OsNramp5 mutant and related products and application thereof Download PDFInfo
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Abstract
The invention discloses an OsNramp5 mutant and related products and applications thereof. The amino acid sequence of the OsNramp5 mutant comprises a sequence shown as SEQ ID NO.3 or SEQ ID NO. 4. The invention screens out 2 OsNramp5 mutants, the plant height, effective spike number, fruiting rate and thousand grain weight of rice containing the OsNramp5 mutants are superior to those of wild rice, the cadmium content in seeds is obviously reduced, and genetic germplasm resources can be provided for breeding rice varieties with low cadmium accumulation.
Description
Technical Field
The invention relates to the field of plant breeding, in particular to an OsNramp5 mutant, and a related product and application thereof.
Background
Cadmium (Cd) is an unnecessary heavy metal and is toxic to almost all organisms. Cadmium has a long biological half-life, ranging from 10 years to 30 years, and cadmium accumulated in the human body with the increase of age can pose serious health risks to the body even under low-level chronic contact. Continuous intake of Cd can lead to chronic renal toxicity, "pain disease," cancer, and other health problems. With the rapid development of industrialization, urbanization and economic globalization, heavy metal cadmium pollution has become a serious problem on a global scale. In China, sludge irrigation is a main source of cadmium in soil, and seriously affects the yield, quality and nutrition of crops. The main sources of cadmium in the air are the emission of industrial waste gas, vehicle waste gas and particulate matters, and the sedimentation of the atmosphere causes disturbance to the agricultural soil, which is more serious around industrial mining areas, highways and railways. Heavy metals including cadmium enter soil and the environment through drainage and rainfall due to improper metal mining and smelting waste treatment, thereby polluting the paddy field.
Rice is an important staple food for more than half of the world's population, and about 90% of the world's rice is produced in asia. Rice has the habit of enriching heavy metal cadmium, is grains with the strongest cadmium absorption capacity, and is comprehensively influenced by soil, air, water drainage and the like, so that rice cadmium pollution is serious. Investigation showed that the average concentration of Cd in rice grain samples from asia was higher than rice grain samples from europe, middle east and north america. Therefore, controlling cadmium accumulation in rice grains is of great importance to human food safety and health.
OsNramp5 is the main transporter involved in absorption of divalent metal cations by rice root cells, and mainly absorbs Mn 2+ 、Cd 2+ And Fe (Fe) 2+ And simultaneously, the transportation of the ions from the root to the overground part is responsible, and the expression is limited by the epidermis, the outer cortex and the tissues around the xylem of the root. Research shows that the defect transport protein coded by the mutant OsNramp5 obviously reduces the absorption of root system to cadmium, resulting in obvious reduction of cadmium content in straw and seeds, as CN 115961075A discloses rice OsNramp5 mutant, screening method and application thereof, which lacks 12 bases in chromosome 7, plants containing mutant 8612-12 are planted in soil with 0.217mg/kg cadmium content, and the cadmium content of roots, stems, leaves and mature seeds is foundLower than normal huahui; CN114014919B discloses a rice OsNramp5 mutant, a screening method and application thereof, which is characterized in that 6 bases are deleted in an intron of chromosome 7, the mutant is planted in soil with cadmium content of 0.98mg/kg, and the result shows that the cadmium content of mature seeds is reduced by more than 90% compared with that of wild Japanese sunny; CN110804676A discloses a rice OsNramp5-18 mutant gene, an identification method thereof, KASP typing primers for identification and application thereof, wherein 18 bp bases are deleted, and after 4 generations of backcrossing, the rice OsNramp5-18 mutant gene is planted in soil with cadmium content of 2mg/kg for 4 weeks, and the reduction of the cadmium content is found.
Therefore, creating rice germplasm resources with low cadmium accumulation and breeding rice varieties with low cadmium accumulation are the most fundamental and economical method for solving the problem of cadmium pollution and realizing safe production in rice fields.
Disclosure of Invention
In view of the above-mentioned shortcomings of the prior art, the present invention aims to provide OsNramp5 mutant and related products and applications thereof, to realize accurate identification and high throughput screening of OsNramp5 allelic variation in a mutagenesis population, and to obtain rice containing OsNramp5 mutant with plant height, effective spike number, fruiting rate and thousand grain weight superior to those of wild rice, and with significantly reduced cadmium content in seeds, which is beneficial to creating rice varieties with low cadmium accumulation.
To achieve the above and other related objects, the present invention is achieved by the following technical means.
It is an object of the present invention to provide an OsNramp5 mutant, wherein the nucleotide sequence of the OsNramp5 mutant comprises a sequence shown as SEQ ID NO.1 or SEQ ID NO. 2.
The second object of the present invention is to provide biological materials related to the OsNramp5 mutant as described, comprising any one of the following:
a) A recombinant expression vector comprising a nucleotide encoding an OsNramp5 mutant as described above;
b) A bioengineering bacterium containing the encoding nucleotide of the OsNramp5 mutant as described above, or a bioengineering bacterium containing the recombinant expression vector of a);
c) A nucleotide encoding an OsNramp5 mutant as described above.
It is a further object of the present invention to provide the use of an OsNramp5 mutant as described above or a biomaterial as described above for increasing rice tolerance to heavy metals or reducing rice heavy metal accumulation.
The fourth object of the present invention is to provide the use of an OsNramp5 mutant as described above or a biological material as described above for breeding rice with high heavy metal tolerance.
It is a fifth object of the present invention to provide a method for increasing rice tolerance to heavy metals or reducing rice heavy metal accumulation by introducing into rice a mutant as described above or a biological material as described above.
The sixth object of the present invention is to provide a method for cultivating transgenic rice, which comprises introducing the above-mentioned mutant or the above-mentioned biological material into rice, and cultivating to obtain the transgenic rice.
The seventh object of the invention is to provide a method for detecting an OsNramp5 mutant based on multiplex PCR, which comprises the following steps:
1) Extracting genome DNA of a rice sample; designing a primer according to a wild rice OsNramp5 gene;
2) Multiplex PCR amplification is carried out on the genome DNA by adopting the primer to obtain an amplified library;
3) And (3) carrying out identification analysis on the amplified library to identify whether the OsNramp5 mutant exists.
Compared with the prior art, the invention has the following beneficial effects:
according to the invention, the mutation rate of the genome of the plant is quickened by adopting radiation mutagenesis, the mutation of the OsNramp5 allele is detected from the radiation mutagenesis group by adopting a high-flux molecular screening technology in the seedling stage of the M2 generation of the mutagenized plant, and the efficiency and the accuracy rate of screening the OsNramp5 mutant are improved. According to the invention, 2 OsNramp5 mutants are screened, and compared with the OsNramp5 gene sequence of R498 rice, the OsNramp5 mutant gene of the first mutant comprises the following base deletion segments: a frame shift deletion mutation of [ G/- ]1 bp exists at the 8935306 position (RAP_Locus) of the 13 th exon of the gene, so that a stop codon is lost, and the stop codon mutation is caused to be related to a backward 216bp base, and 72 amino acids are transcribed. Compared with the OsNramp5 gene sequence of R498 rice, the second mutant OsNramp5 mutant gene comprises the following base deletion segments: the 8939534-8939541 position (RAP_Locus) of the 7 th exon of the gene has [ AGAAACTC/- ]8 bp frameshift deletion mutation, and compared with wild rice, the cadmium content in seeds of 2 OsNramp5 mutant plants is obviously reduced.
The rice OsNramp5 mutant provides genetic germplasm resources for breeding rice varieties with low cadmium accumulation.
Drawings
FIG. 1 is a schematic diagram showing a partial sequence alignment of the OsNramp5 gene and the OsNramp5 mutant gene of wild-type rice in example 2 of the present invention.
FIG. 2 is a second schematic diagram showing a partial sequence alignment of the OsNramp5 gene and the OsNramp5 mutant gene of wild-type rice in example 2 of the present invention.
FIG. 3 is a photograph showing an OsNramp5 mutant rice and a wild type rice of sample group number K8 in example 2 of the present invention.
FIG. 4 is a photograph showing an OsNramp5 mutant rice and a wild type rice of sample group No. A44 in example 2 of the present invention.
FIG. 5 shows a bar graph of cadmium content for OsNramp5 mutant rice having sample group number K8, osNramp5 mutant rice having sample group number A44, and wild type rice in example 2 of the present invention.
Detailed Description
The inventor carries out radiation mutagenesis on dry rice seeds, and adopts a multiplex PCR method to amplify and construct a sequencing library, and obtains 2 mutants through sequencing analysis and radiation mutagenesis; after the mutant is planted, the plant height, the effective spike number, the fruiting rate and the thousand grain weight of 2 mutant rice are superior to those of wild rice, and the cadmium content in the grains is obviously lower than that of the wild rice. The present invention has been completed on the basis of this finding.
The first aspect of the invention protects an OsNramp5 mutant, wherein the amino acid sequence of the OsNramp5 mutant comprises a sequence shown as SEQ ID NO.3 or SEQ ID NO. 4.
After the rice containing the OsNramp5 mutant is planted in a field with the effective cadmium content of 1.25mg/Kg until the rice is mature, the cadmium content of seeds is lower than 0.03mg/Kg, and the cadmium content of seeds of wild rice exceeds 0.2mg/Kg, and the reduction rate exceeds 90%.
MEIERESSERGSISWRASAAHDQDAKKLDADDQLLMKEPAWKRFLAHVGPGFMVSLAYLDPGNLETDLQAGANHRYELLWVILIGLIFALIIQSLAANLGVVTGRHLAEICKSEYPKFVKIFLWLLAELAVIAADIPEVIGTAFAFNILFHIPVWVGVLITGTSTLLLLGLQKYGVRKLEFLISMLVFVMAACFFGELSIVKPPAKEVMKGLFIPRLNGDGATADAIALLGALVMPHNLFLHSALVLSRKTPASVRGIKDGCRFFLYESGFALFVALLINIAVVSVSGTACSSANLSQEDADKCANLSLDTSSFLLKNVLGKSSAIVYGVALLASGQSSTITGTYAGQYIMQGFLDIRMRKWLRNLMTRTIAIAPSLIVSIIGGSRGAGRLIIIASMILSFELPFALIPLLKFSSSKSKMGPHKNSIYIIVFSWFLGLLIIGINMYFLSTSFVGWLIHNDLPKYANVLVGAAVFPFMLVYIVAVVYLTIRKDSVVTFVADSSLAAVVDAEKADAGDLAVDDDEPLPYRDDLADIPLQGREEEDRHAYVWYSVCIRTYIRVHCEYTYLRTYMCILYGCHVRASEPFVYICAVQIARRSIDRQGCAINI(SEQ ID NO.3)
MEIERESSERGSISWRASAAHDQDAKKLDADDQLLMKEPAWKRFLAHVGPGFMVSLAYLDPGNLETDLQAGANHRYELLWVILIGLIFALIIQSLAANLGVVTGRHLAEICKSEYPKFVKIFLWLLAELAVIAADIPEVIGTAFAFNILFHIPVWVGVLITGTSTLLLLGLQKYGVRKLDIDAGVRDGGVLLRGAEHREAAGEGGDEGALHPQAQRRRRHRRRHCPPRSSCHAPQSVLAFCLGAIEEDTGISQRNQGRVQVLPVRERVRAVRGAADKHRRRLRLRHRLLLRQPLPRGRRQVRQPQPRHLLLPSQERAGQVECDRVRRGTVGIWAELHYYRHIRWTVHHAGFLGHQDEEVASEPDDKNHRHRAEPHRLHHRRLQGRRPPHHHRFDDTVLRAAVCSHPSSQVQQQ(SEQ ID NO.4)
The second aspect of the invention protects biological material associated with an OsNramp5 mutant as described above, comprising any one of the following:
a) A recombinant expression vector comprising a nucleotide encoding an OsNramp5 mutant as described above;
b) A bioengineering bacterium containing the encoding nucleotide of the OsNramp5 mutant as described above, or a bioengineering bacterium containing the recombinant expression vector of a);
c) A nucleotide encoding an OsNramp5 mutant as described above.
In a second aspect of the invention, the recombinant expression vector of the nucleotide is a recombinant expression vector in which the nucleotide sequence encoding the mutant is inserted into an expression vector. The term "expression vector" refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses or other vectors well known in the art. In general, any plasmid or vector can be used as long as it replicates and is stable in the host. An important feature of expression vectors is that they generally contain an origin of replication, a promoter, a marker gene and translational control elements. Methods well known to those skilled in the art can be used to construct expression vectors containing the nucleotide sequence encoding the stress-resistant protein and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.
In a second aspect of the present invention, the bioengineering bacterium containing nucleotides or the bioengineering bacterium containing a) the recombinant expression vector means that the bioengineering bacterium contains the recombinant expression vector or the nucleotides as described above integrated in the genome. Bioengineering bacteria to enable expression of proteins. The bioengineered bacteria may be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as plant cells. Representative examples are: coli, streptomyces and agrobacterium; fungal cells such as yeast; plant cells, and the like.
In a second aspect of the present invention, the nucleotide sequence comprises a sequence as shown in SEQ ID NO.1 or SEQ ID NO. 2.
TCCAATCCACCAACTTGCACTAATTATTAATTATTGACAGAGATCAAACTACTGCTCCGAAGTCTATACAGTACACTACCGTACGTGTCTCTAGTTTCTGGAAAAAAAAGGTACAGCTATAGTACTACTCTTCCAATACTAATATATTAATCTAATTGCTTAATTAATTAATTAATTAATAGTAGTTGATTCATCACAGAAAATTGAAGGACGTTGGCTCTGCCCTGAATTATGATAAATCATATCGTACATATAAATTACAAGGATACATGAGCCACCTCCCCTCAAATGCTTATATATTAATTGCACACCCTTGTCGATCGATCGATCTGCGAGCGATCTGGACCGCACAAATATAAACGAACGGCTCCGACGCACGCACGTGGCATCCATAGAGGATGCACATATACGTACGTAAGTACGTATATTCACAATGTACGCGTACATACGTACGTATACATACGCTATACCATACGTATGCATGTCGATCTTCTTCTTCTCTACCTTGGAGCGGGATGTCGGCCAGGTCGTCGCGGTACGGCAAGGGCTCGTCGTCGTCGACGGCGAGGTCGCCGGCGTCGGCCTTCTCGGCGTCGACGACGGCGGCGAGGGAGGAGTCGGCGACGAAGGTGACGACGGAGTCCTTCCTGATGGTGAGGTAGACGACGGCGACGATGTAGACGAGCATGAACGGGAAGACGGCGGCGCCGACGAGCACGTTGGCGTACTTGGGGAGGTCGTTGTGGATGAGCCAGCCGACGAAGCTCGTGCTCAGGAAGTACATGTTGATGCCGATGATGAGCAGCCCCAGGAACCACGAGAACACTATTATCTGCATTCAATTCAATTCAATTTATTTTTTGACATACATGACGCTAATTTATTCATTGATAGCGTCACTAAATAACCAGTCATCTTTTACGGGTGGTAACTTATATATCTTTTTAACGGGTATCGACTGATGAGCATGCGCAATTTTTATAGGTAGTATATTAATATATATATATATAGCCATGTGTGTAATAATAAGAAATAAATTTACATTTGTTCAATTTTGTTGTAGATCAGTTTATTTATGGTCCTTAAAAAAGAATTACATTTTCTAATGTTGACTTGTATGTGTCTAGAAATGTTTAAGTAGAGATACAACGACATACGTAATGATCTATGTTAAATTATATTTACATTTTACTTCCATGCAAGAAAAAATATATATAGTGTACTGAAGAACTTGGCTCTTCGATGTCTGTAAAGATCAAGCAAGTACGTTAATGCATGATTTTGACACATCATGCCGACGACGCAATACATGATTAATCTCTGGTACTACTGCAAGACAGAAAAATGATGCAGGACGTACATCTTGGTCGTCGTTTTCATATGTACGACGATCCTCTATATGCATGGCTGGACCATATATATACCTGTCCTGCATTGGCCCCTGCTGTGCTATATTACTATTGCATGAGCTAGGTAGCTAATGATTCAATTCAATTCACTGCACATCTCGATCTACATTATGCACACACTGACCTATAGAACATGGTGTCCTGCAGGGGTTGCATGCATGCAGGGTGAAGGACCAGCTAGCTAGCCAACTATCATCATTAATTTATATTCTTTACCAACAATTATTGAAA
CTCTAATTTCATGCATGGACAGATAGATGCATATATTTATGTTACTTGTACATTTGCTGC
CATTTGAGATTAAGAAACAAACTATAATTAAGCAATGCATATATATAGTGTACGATCTT
ACATAGATAGAGTTCTTGTGGGGCCCCATCTTGCTCTTACTGCTGCTGAACTTGAGAAG
AGGGATGAGAGCAAACGGCAGCTCGAAGGACAGTATCATCTGCATGCACACAACACG
TACGGATTAGGAACAAATTAATCATATATATGTAATTAAGCAAACAAGAACTAGCAAT
GATCAGATCACTACTTTGATTTGATTTAATTATATATAATTAGTCCTTACTTATTGAGTT
GGAAGCTAGTCTAGCTAGATGGTACACTGTATTATATATATGCATGGTGCTATTTTAGA
TCAGATTGCAAAAAAAGAAAAGATACCTAGCTAGTCAGTGACATTTTGCTAATCAGAT
CACTACTTTGATTTGGCAATAATCTAAAAAAAGAAGAAAAAGTGCTTTTGCTCTAATG
ACATATATATATGCTCTTAGTACTATGTCACTAGGTACCTAGTGGATACCCCACTACTA
CCTAGCTAGCTACATAAATCCGCACGATCGACTGCACACACGTGACTGTTTGGGCATGT
GCAAAATTAAAAATTTAAACGCATTAAATTTTAATCGATCGGCGACACGACGGCCGGC
CGGTTGCTCGCCGGCGGGCGCCGCCGTATGGCGCACGCAGTTTGCTATAGTAAAATAG
TAATAATAGTAAAGATTTGCTATAGTAAATAGTAATAATAAAAATGCTGACCGAAGCG
ATGATGATGAGGCGGCCGGCGCCCCTGGAGCCGCCGATGATGGAGACGATGAGGCTC
GGCGCGATGGCGATGGTTCTTGTCATCAGGTTCCGAAGCCACTTCCTCATCCTGATGTC
CAAGAAACCCTGCACATACATGGATCATATCATATATATCAACGCGACATCATGATGA
TTTATCATATACTCCCTCCGTCATTAAATATTTAAACATATTTAACCATTCATCTTATTC
AATAAATTTAAGTAGTTATTAATTCTTTTCTTATCATTTGATTCATTGTTGTATATACTA
CTACTACTCAAGTTTTTTAAATAATATTCACAAAAGTTTTTGCATAAGACGAACGGTCA
AACATATTTATAAAAAATCAACGACGTCAAATATTTAGAGACGGAGTGAGTATATCCT
GAAGATCAGTCCTTTGGAGTGAAAAAATAAATATATATTTTCGGGTCCGATTTATCCCG
TAACTGAAATACGCTTCATATCATAAATAGATTATTAAGAGGAATGAGACTCGAACTA
GATCGGTTAGCCCGCCAGGTAGATTAGGAAAACTAGTTGTTCTCTTTCTCAACGTTCAC
TTCAGAAAGAAAATTTTACGGTGCGATGGGATATCCTATTTTTTTTAATATAATTTATA
GAGTTTCATTAGCATATTTTTATTAATCAATTGGTTTTCTAAGTATATGTAAACAGTAA
AATCGCTGGTTATTGATGAGACTGCACCGTGAAATGGACTAGACCAAAAGAGCGGAGA
AATATGGACGAAAGTGGTAAGTAGTGGCAGCAAGGTAGCGTCTTAATTCGATCTGACT
TTAACAAAAACATATGGTGAAATCCGACGAGGATCGCCAGATTACCTAATTAATAAAG
GAATATTATATCACTAAATATCCGTGATGTAAACAGGATTAGGTGTGCACCCCTACAAT
TCGTCAGTTAACCTCCATAATTGAACGGATTAACAAATTAATTATGTGGCAGCTAGCCG
TTTAATTAATTATAGTTCAATCATTTAATTACGCGCTAAGCTAGCTGGTGATGTACAGG
TACAGGTACAGTTAAGTTAATTAATTTAATTAATTACCTGCATGATGTACTGTCCAGCG
TATGTGCCGGTAATAGTGGAGCTCTGCCCAGATGCCAACAGTGCCACGCCATACACGA
TCGCACTCGACTTGCCCAGCACGTTCTGAACCACATAAAAAATTGACGTCACAATTTGA
CCATAAATTAGTTACTCCTCCTATTCAAAAATATAAACATATCTAATAAAGTTATTCTA
CATTAGGACAGAGAAATCGTGTAGACTAATTCGGTGTTATTGTGGTTAATTAAATATTG
ATTAAATTGAACACATACAAGTACTACGAATGTGAATAAATTTCTATATCTGTAGTACT
AGGATACGTCTAATCCATTTAAAAACTTTTAATATTAAAGAGTAGGAAGTAATAATGA
ATGAATGACCTTGAGAAGGAAGGAGGAGGTGTCGAGGCTGAGGTTGGCGCACTTGTCG
GCGTCCTCTTGGGAGAGGTTGGCGGAGGAGCAGGCGGTGCCGGAGACGGAGACGACG
GCGATGTTTATCAGCAGCGCCACGAACAGCGCGAACCCGCTCTCGTACAGGAAGAACC
TGCACCCGTCCTACGTGGCAATTCACACACCATATAGTACATATTAATTATTAGCCTGG
ATCTTTGTTCAGTTTCTTTCCATGCATGAAAAAATTCTTAGCTGAATCCAGTGGTACTG
GAGCCCGAAATTATAGAAGATTAGTGCTAAAATAAAACTTAGCTAATAAAGTATTGGT
TTTCTATGAAATTTACATGTTAATTTAATAAAATTTAAACAAGATTTAAGAGACAGGAC
TAGATCGAGTTCATGTCCTAGCTACCCTATACCTATCTCGATCTGCCCAGGCTGAAGAT
AGCTACCTTGATTCCTCTGACTGATGCCGGTGTCTTCCTCGATAGCACCAAGGCAGAAT
GCAAGAACAGATTGTGGCTGCATATATATAAATGCATGCAGTAGTTAATTAACTGATT
AGTTGTATGAATTAATTGATTGATTGTTGCGTATGTATGTATGTATGTATGTAAAAGCG
TACGGCATGACAAGAGCTCCGAGGAGGGCAATGGCGTCGGCGGTGGCGCCGTCGCCGT
TGAGCCTGGGGATGAAGAGCCCCTTCATCACCTCCTTCGCCGGCGGCTTCACGATGCTC
AGCTCCCCGAAGAAGCACGCCGCCATCACGAACACCAGCATCGATATCAGAAACTCCA
GCTTCCTCACCTGTTTTTTTTCAAAAAAAATTCATCAATCAATTTTCATGCAGTTCCACA
GTCAGAGAAAAAAAAACTGAATAATGTACATATAATATGCCATTTGAGTACTTGACAA
TCGATCCAACTAGCTAATTTCATATACTATTCCATTTTTATAATCGTTTCTTTGAGTGCA
TCCTACGCTAATGCTTGTGATACTAAACAAAAACAACTTACAAAAAATAATTTATACCC
CGTATTTTTGGAGGCCAAGAAGCAGTAGAGTGCTGGTGCCGGTGATGAGGACGCCGAC
CCACACCGGAATATGGAACAATATGTTGAAAGCAAAGGCCGTCCCTATAACTGCATCC
ACATCAAACGACATGTAATTAATTAATTAATTATAATCACATCGATCTGTACCTTTCTA
TCTGTTGATCGATCTATCTATCTATCTATATATCACCTTTGCTATCATCATGTGAGGTAT
CTTCCATAACTAGTTTAATTTTTTTAAAAAAGAACTTGCATGCATGATCAATTTAAAAG
GCAATTTACAACGGTTGAGGTTAACTAATCCCGATCAGCAAAGCTTCGCGCAGCTCCG
TTTTGCAACTGCTACACCACTGATCTCTCTTCCATGTTGCTAGATAGATAGATATTTAG
ATATTGTTGTTATTGTTTAAACACACATGGATGTATACAGGACAGGACAAATGTGAAA
TGACAAGAGGGTGACAAAATAGTTGTTTACTAATCTGCGTAAGATGATGAACGTACTC
ACCCAGTGTTTAAAATCTAATCTTGATGTGCATCATTGACACTTTTATGGGAAGCTACT
GTGGACGTGGTCACTACTCTAAACAGCTAAAACGCGCATTTGCTTATGTTTGAAACAG
ATTATTCAACAGATATATTATTGGTACATATTATCTACCTTATTATATGGATGAATTAA
ATATTTTTTTAAAAAATGGCTTAGAACTTGTAATCTATGCAATATCGCAGAATATATTT
TTTTTAGAAAAACATATTTACTAGCTAGAGACATGAAAGAAATAATATGTGAGAATAA
TCTGGTGAGTAGCTGAGAAGAACGTGGCTTGATGGTGATTGGTGAGATATTAATTTGC
CTATGTTTGTGGATTAGGAATTCCAGTATGTAGGCCCGTACGGTTGGAGCTACATATGC
TAGCCAGATCAACTTGTTTTCTTCTACTGTCAGAGAGCTTAAACTGAATTAATTAATTA
GCTCAAAGTCAAAGATGGTGACACTGTTAAACACAAGTAGTTTAATAAATTAATTATT
CAGGCTGGACCGTGTCAAGTTGTTGCTAATAAGTATATACCTTCTGGGATATCTGCAGC
GATGACGGCCAACTCTGCCAGCAGCCATAGGAAAATCTTGACGAACTTGGGGTACTCA
CTCTTGCAGATCTCAGCCAGATGCCTCCCTAGGATGTAAATTAATGCCGGCCATATATT
AGCACTTTATCAAGTTGGTAATTACAGTTTAATTGCGTGTCATTTAGTCACCTGTAACC
ACTCCAAGATTAGCTGCTAGCGACTGTATGATAAGTGCGAAGATGAGTCCAATCAGAA
TCACCCAGAGCAGCTGCAAATTGAAACAACACAATGGACAGAAAAGCACATAGAAAT
CAATTAATGGTGCAGATTATTCACTTTTCTCTTTTCTCTAGATGATGTGAGAGTGAGAT
ACATATGCACTGGGGATTAAAAGTGGTTCTGAAAAGTTTGTCAATTTGGAGAATTTGAT
GGAGCTAGCAGATGAATTAATGGAGTGTGAGTCTGCGAGGGACAAGGAAAGGGACGA
ACACCTAGAAAGCATGAAAGTTACTCATGCTTTCAGTCATTCAGTGCGTAAACAGTAA
AATAAAGTCGACTGTACGTACCTCATATCTGTGGTTGGCTCCGGCTTGCAGATCGGTTT
CCACTGTTTCATTTTGTGACAAAATTAACAGTTACAATCCAGAGTTTGTCAAAATAAAA
GTTTCAGTTGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGATTC
AGAGATTCTCACAATTGCCAGGATCCAAGTAGGCTAAAGACACCATGAATCCAGGACC
AACATGGGCAAGGAACCTTTTCCATGCAGGCTCCTGTACACAATTTACCAAAACTGAG
AAACTAACAAAAAGAAACACAGACATAATTGAGGATCCAATGTTCCATCAGTCCTTAA
TTACCACATACACACACACACATGAACATTCATGAGCAATGAATGCCATGCCAGGAAG
TACTCTCAAGATCTATCTACATTAAGCTCTGCTCTTGCTTCTGCAGTTACAGTGAACAA
CTGTCCATGGTGACAGTGGAGTGCTTACAAGAAGACAACTTTGTGATAATTAAGGTAG
CAACTGACCTTCATTAGCAGCTGATCATCTGCGTCGAGCTTCTTGGCATCTTGATCATG
TGCCGCACTAGCTCTCCAGCTGATGCTCCCTCTCTCACTGCTCTCTCTCTCAATCTCCAT
TGTTGCTTCCTCTCTTAGCTTCTTCAGGCTAAGCTAGAGCTAAGCTAGACTCAGGCTAG
CTGGAAGTAGACGAAGAAGAGAATGGTGGTAGTGACGAACTCTCAAGCCACACCCTCC
TGTCCTACTTATACACAAACCATGCAAGTATGCAGCCAAATTAAC(SEQ ID NO.1)
TCCAATCCACCAACTTGCACTAATTATTAATTATTGACAGAGATCAAACTACTGCTCCG
AAGTCTATACAGTACACTACCGTACGTGTCTCTAGTTTCTGGAAAAAAAAGGTACAGC
TATAGTACTACTCTTCCAATACTAATATATTAATCTAATTGCTTAATTAATTAATTAATT
AATAGTAGTTGATTCATCACAGAAAATTGAAGGACGTTGGCTCTGCCCTGAATTATGAT
AAATCATATCGTACATATAAATTACAAGGATACATGAGCCACCTCCCCTCAAATGCTTA
TATATTAATTGCACACCCTTGTCGATCGATCGATCTGCGAGCGATCTGGACCGCACAAA
TATAAACGAACGGCTCCGACGCACGCACGTGGCATCCATAGAGGATGCACATATACGT
ACGTAAGTACGTATATTCACAATGTACGCGTACATACGTACGTATACATACGCTATACC
ATACGTATGCATGTCGATCTTCTTCTTCTCTACCTTGGGAGCGGGATGTCGGCCAGGTC
GTCGCGGTACGGCAAGGGCTCGTCGTCGTCGACGGCGAGGTCGCCGGCGTCGGCCTTC
TCGGCGTCGACGACGGCGGCGAGGGAGGAGTCGGCGACGAAGGTGACGACGGAGTCC
TTCCTGATGGTGAGGTAGACGACGGCGACGATGTAGACGAGCATGAACGGGAAGACG
GCGGCGCCGACGAGCACGTTGGCGTACTTGGGGAGGTCGTTGTGGATGAGCCAGCCGA
CGAAGCTCGTGCTCAGGAAGTACATGTTGATGCCGATGATGAGCAGCCCCAGGAACCA
CGAGAACACTATTATCTGCATTCAATTCAATTCAATTTATTTTTTGACATACATGACGCT
AATTTATTCATTGATAGCGTCACTAAATAACCAGTCATCTTTTACGGGTGGTAACTTAT
ATATCTTTTTAACGGGTATCGACTGATGAGCATGCGCAATTTTTATAGGTAGTATATTA
ATATATATATATATAGCCATGTGTGTAATAATAAGAAATAAATTTACATTTGTTCAATT
TTGTTGTAGATCAGTTTATTTATGGTCCTTAAAAAAGAATTACATTTTCTAATGTTGACT
TGTATGTGTCTAGAAATGTTTAAGTAGAGATACAACGACATACGTAATGATCTATGTTA
AATTATATTTACATTTTACTTCCATGCAAGAAAAAATATATATAGTGTACTGAAGAACT
TGGCTCTTCGATGTCTGTAAAGATCAAGCAAGTACGTTAATGCATGATTTTGACACATC
ATGCCGACGACGCAATACATGATTAATCTCTGGTACTACTGCAAGACAGAAAAATGAT
GCAGGACGTACATCTTGGTCGTCGTTTTCATATGTACGACGATCCTCTATATGCATGGC
TGGACCATATATATACCTGTCCTGCATTGGCCCCTGCTGTGCTATATTACTATTGCATG
AGCTAGGTAGCTAATGATTCAATTCAATTCACTGCACATCTCGATCTACATTATGCACA
CACTGACCTATAGAACATGGTGTCCTGCAGGGGTTGCATGCATGCAGGGTGAAGGACC
AGCTAGCTAGCCAACTATCATCATTAATTTATATTCTTTACCAACAATTATTGAAACTC
TAATTTCATGCATGGACAGATAGATGCATATATTTATGTTACTTGTACATTTGCTGCCA
TTTGAGATTAAGAAACAAACTATAATTAAGCAATGCATATATATAGTGTACGATCTTAC
ATAGATAGAGTTCTTGTGGGGCCCCATCTTGCTCTTACTGCTGCTGAACTTGAGAAGAG
GGATGAGAGCAAACGGCAGCTCGAAGGACAGTATCATCTGCATGCACACAACACGTA
CGGATTAGGAACAAATTAATCATATATATGTAATTAAGCAAACAAGAACTAGCAATGA
TCAGATCACTACTTTGATTTGATTTAATTATATATAATTAGTCCTTACTTATTGAGTTGG
AAGCTAGTCTAGCTAGATGGTACACTGTATTATATATATGCATGGTGCTATTTTAGATC
AGATTGCAAAAAAAGAAAAGATACCTAGCTAGTCAGTGACATTTTGCTAATCAGATCA
CTACTTTGATTTGGCAATAATCTAAAAAAAGAAGAAAAAGTGCTTTTGCTCTAATGAC
ATATATATATGCTCTTAGTACTATGTCACTAGGTACCTAGTGGATACCCCACTACTACC
TAGCTAGCTACATAAATCCGCACGATCGACTGCACACACGTGACTGTTTGGGCATGTG
CAAAATTAAAAATTTAAACGCATTAAATTTTAATCGATCGGCGACACGACGGCCGGCC
GGTTGCTCGCCGGCGGGCGCCGCCGTATGGCGCACGCAGTTTGCTATAGTAAAATAGT
AATAATAGTAAAGATTTGCTATAGTAAATAGTAATAATAAAAATGCTGACCGAAGCGA
TGATGATGAGGCGGCCGGCGCCCCTGGAGCCGCCGATGATGGAGACGATGAGGCTCG
GCGCGATGGCGATGGTTCTTGTCATCAGGTTCCGAAGCCACTTCCTCATCCTGATGTCC
AAGAAACCCTGCACATACATGGATCATATCATATATATCAACGCGACATCATGATGAT
TTATCATATACTCCCTCCGTCATTAAATATTTAAACATATTTAACCATTCATCTTATTCA
ATAAATTTAAGTAGTTATTAATTCTTTTCTTATCATTTGATTCATTGTTGTATATACTAC
TACTACTCAAGTTTTTTAAATAATATTCACAAAAGTTTTTGCATAAGACGAACGGTCAA
ACATATTTATAAAAAATCAACGACGTCAAATATTTAGAGACGGAGTGAGTATATCCTG
AAGATCAGTCCTTTGGAGTGAAAAAATAAATATATATTTTCGGGTCCGATTTATCCCGT
AACTGAAATACGCTTCATATCATAAATAGATTATTAAGAGGAATGAGACTCGAACTAG
ATCGGTTAGCCCGCCAGGTAGATTAGGAAAACTAGTTGTTCTCTTTCTCAACGTTCACT
TCAGAAAGAAAATTTTACGGTGCGATGGGATATCCTATTTTTTTTAATATAATTTATAG
AGTTTCATTAGCATATTTTTATTAATCAATTGGTTTTCTAAGTATATGTAAACAGTAAA
ATCGCTGGTTATTGATGAGACTGCACCGTGAAATGGACTAGACCAAAAGAGCGGAGAA
ATATGGACGAAAGTGGTAAGTAGTGGCAGCAAGGTAGCGTCTTAATTCGATCTGACTT
TAACAAAAACATATGGTGAAATCCGACGAGGATCGCCAGATTACCTAATTAATAAAGG
AATATTATATCACTAAATATCCGTGATGTAAACAGGATTAGGTGTGCACCCCTACAATT
CGTCAGTTAACCTCCATAATTGAACGGATTAACAAATTAATTATGTGGCAGCTAGCCGT
TTAATTAATTATAGTTCAATCATTTAATTACGCGCTAAGCTAGCTGGTGATGTACAGGT
ACAGGTACAGTTAAGTTAATTAATTTAATTAATTACCTGCATGATGTACTGTCCAGCGT
ATGTGCCGGTAATAGTGGAGCTCTGCCCAGATGCCAACAGTGCCACGCCATACACGAT
CGCACTCGACTTGCCCAGCACGTTCTGAACCACATAAAAAATTGACGTCACAATTTGA
CCATAAATTAGTTACTCCTCCTATTCAAAAATATAAACATATCTAATAAAGTTATTCTA
CATTAGGACAGAGAAATCGTGTAGACTAATTCGGTGTTATTGTGGTTAATTAAATATTG
ATTAAATTGAACACATACAAGTACTACGAATGTGAATAAATTTCTATATCTGTAGTACT
AGGATACGTCTAATCCATTTAAAAACTTTTAATATTAAAGAGTAGGAAGTAATAATGA
ATGAATGACCTTGAGAAGGAAGGAGGAGGTGTCGAGGCTGAGGTTGGCGCACTTGTCG
GCGTCCTCTTGGGAGAGGTTGGCGGAGGAGCAGGCGGTGCCGGAGACGGAGACGACG
GCGATGTTTATCAGCAGCGCCACGAACAGCGCGAACCCGCTCTCGTACAGGAAGAACC
TGCACCCGTCCTACGTGGCAATTCACACACCATATAGTACATATTAATTATTAGCCTGG
ATCTTTGTTCAGTTTCTTTCCATGCATGAAAAAATTCTTAGCTGAATCCAGTGGTACTG
GAGCCCGAAATTATAGAAGATTAGTGCTAAAATAAAACTTAGCTAATAAAGTATTGGT
TTTCTATGAAATTTACATGTTAATTTAATAAAATTTAAACAAGATTTAAGAGACAGGAC
TAGATCGAGTTCATGTCCTAGCTACCCTATACCTATCTCGATCTGCCCAGGCTGAAGAT
AGCTACCTTGATTCCTCTGACTGATGCCGGTGTCTTCCTCGATAGCACCAAGGCAGAAT
GCAAGAACAGATTGTGGCTGCATATATATAAATGCATGCAGTAGTTAATTAACTGATT
AGTTGTATGAATTAATTGATTGATTGTTGCGTATGTATGTATGTATGTATGTAAAAGCG
TACGGCATGACAAGAGCTCCGAGGAGGGCAATGGCGTCGGCGGTGGCGCCGTCGCCGT
TGAGCCTGGGGATGAAGAGCCCCTTCATCACCTCCTTCGCCGGCGGCTTCACGATGCTC
AGCTCCCCGAAGAAGCACGCCGCCATCACGAACACCAGCATCGATATCCAGCTTCCTC
ACCTGTTTTTTTTCAAAAAAAATTCATCAATCAATTTTCATGCAGTTCCACAGTCAGAG
AAAAAAAAACTGAATAATGTACATATAATATGCCATTTGAGTACTTGACAATCGATCC
AACTAGCTAATTTCATATACTATTCCATTTTTATAATCGTTTCTTTGAGTGCATCCTACG
CTAATGCTTGTGATACTAAACAAAAACAACTTACAAAAAATAATTTATACCCCGTATTT
TTGGAGGCCAAGAAGCAGTAGAGTGCTGGTGCCGGTGATGAGGACGCCGACCCACAC
CGGAATATGGAACAATATGTTGAAAGCAAAGGCCGTCCCTATAACTGCATCCACATCA
AACGACATGTAATTAATTAATTAATTATAATCACATCGATCTGTACCTTTCTATCTGTTG
ATCGATCTATCTATCTATCTATATATCACCTTTGCTATCATCATGTGAGGTATCTTCCAT
AACTAGTTTAATTTTTTTAAAAAAGAACTTGCATGCATGATCAATTTAAAAGGCAATTT
ACAACGGTTGAGGTTAACTAATCCCGATCAGCAAAGCTTCGCGCAGCTCCGTTTTGCA
ACTGCTACACCACTGATCTCTCTTCCATGTTGCTAGATAGATAGATATTTAGATATTGTT
GTTATTGTTTAAACACACATGGATGTATACAGGACAGGACAAATGTGAAATGACAAGA
GGGTGACAAAATAGTTGTTTACTAATCTGCGTAAGATGATGAACGTACTCACCCAGTG
TTTAAAATCTAATCTTGATGTGCATCATTGACACTTTTATGGGAAGCTACTGTGGACGT
GGTCACTACTCTAAACAGCTAAAACGCGCATTTGCTTATGTTTGAAACAGATTATTCAA
CAGATATATTATTGGTACATATTATCTACCTTATTATATGGATGAATTAAATATTTTTTT
AAAAAATGGCTTAGAACTTGTAATCTATGCAATATCGCAGAATATATTTTTTTTAGAAA
AACATATTTACTAGCTAGAGACATGAAAGAAATAATATGTGAGAATAATCTGGTGAGT
AGCTGAGAAGAACGTGGCTTGATGGTGATTGGTGAGATATTAATTTGCCTATGTTTGTG
GATTAGGAATTCCAGTATGTAGGCCCGTACGGTTGGAGCTACATATGCTAGCCAGATC
AACTTGTTTTCTTCTACTGTCAGAGAGCTTAAACTGAATTAATTAATTAGCTCAAAGTC
AAAGATGGTGACACTGTTAAACACAAGTAGTTTAATAAATTAATTATTCAGGCTGGAC
CGTGTCAAGTTGTTGCTAATAAGTATATACCTTCTGGGATATCTGCAGCGATGACGGCC
AACTCTGCCAGCAGCCATAGGAAAATCTTGACGAACTTGGGGTACTCACTCTTGCAGA
TCTCAGCCAGATGCCTCCCTAGGATGTAAATTAATGCCGGCCATATATTAGCACTTTAT
CAAGTTGGTAATTACAGTTTAATTGCGTGTCATTTAGTCACCTGTAACCACTCCAAGAT
TAGCTGCTAGCGACTGTATGATAAGTGCGAAGATGAGTCCAATCAGAATCACCCAGAG
CAGCTGCAAATTGAAACAACACAATGGACAGAAAAGCACATAGAAATCAATTAATGG
TGCAGATTATTCACTTTTCTCTTTTCTCTAGATGATGTGAGAGTGAGATACATATGCACT
GGGGATTAAAAGTGGTTCTGAAAAGTTTGTCAATTTGGAGAATTTGATGGAGCTAGCA
GATGAATTAATGGAGTGTGAGTCTGCGAGGGACAAGGAAAGGGACGAACACCTAGAA
AGCATGAAAGTTACTCATGCTTTCAGTCATTCAGTGCGTAAACAGTAAAATAAAGTCG
ACTGTACGTACCTCATATCTGTGGTTGGCTCCGGCTTGCAGATCGGTTTCCACTGTTTCA
TTTTGTGACAAAATTAACAGTTACAATCCAGAGTTTGTCAAAATAAAAGTTTCAGTTGA
GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGATTCAGAGATTCTCA
CAATTGCCAGGATCCAAGTAGGCTAAAGACACCATGAATCCAGGACCAACATGGGCA
AGGAACCTTTTCCATGCAGGCTCCTGTACACAATTTACCAAAACTGAGAAACTAACAA
AAAGAAACACAGACATAATTGAGGATCCAATGTTCCATCAGTCCTTAATTACCACATA
CACACACACACATGAACATTCATGAGCAATGAATGCCATGCCAGGAAGTACTCTCAAG
ATCTATCTACATTAAGCTCTGCTCTTGCTTCTGCAGTTACAGTGAACAACTGTCCATGG
TGACAGTGGAGTGCTTACAAGAAGACAACTTTGTGATAATTAAGGTAGCAACTGACCT
TCATTAGCAGCTGATCATCTGCGTCGAGCTTCTTGGCATCTTGATCATGTGCCGCACTA
GCTCTCCAGCTGATGCTCCCTCTCTCACTGCTCTCTCTCTCAATCTCCATTGTTGCTTCCT
CTCTTAGCTTCTTCAGGCTAAGCTAGAGCTAAGCTAGACTCAGGCTAGCTGGAAGTAG
ACGAAGAAGAGAATGGTGGTAGTGACGAACTCTCAAGCCACACCCTCCTGTCCTACTTATACACAAACCATGCAAGTATGCAGCCAAATTAAC(SEQ ID NO.2)
The third aspect of the invention protects the use of an OsNramp5 mutant as described above or a biomaterial as described above for increasing rice tolerance to heavy metals or reducing rice heavy metal accumulation. Preferably comprising increasing one or more of rice plant height, effective spike number and seed setting rate. The heavy metal is selected from one or more of cadmium, chromium, manganese and iron; mainly cadmium.
The fourth aspect of the present invention protects the use of an OsNramp5 mutant as described above or a biomaterial as described above for breeding high heavy metal tolerant rice. The heavy metal is selected from one or more of cadmium, chromium, manganese and iron; mainly cadmium.
In a fifth aspect the invention provides a method of increasing rice tolerance to heavy metals or reducing rice heavy metal accumulation by introducing into rice a mutant as described above or a biological material as described above.
A sixth aspect of the present invention provides a method for breeding transgenic rice, comprising introducing the mutant as described above or the biological material as described above into rice, and breeding the rice to obtain the transgenic rice.
The seventh aspect of the present invention provides a method for detecting an OsNramp5 mutation based on multiplex PCR, comprising the steps of:
1) Extracting genome DNA of a rice sample; designing a primer according to a wild rice OsNramp5 gene;
2) Multiplex PCR amplification is carried out on the genome DNA by adopting the primer to obtain an amplified library;
3) And (3) carrying out identification analysis on the amplified library to identify whether the OsNramp5 mutant exists.
In a seventh aspect of the present invention, in 1), the primer comprises a sequence represented by SEQ ID NO.5 to SEQ ID NO. 42.
In a seventh aspect of the present invention, in 1), the rice sample is a transgenic rice obtained by the cultivation method of the sixth aspect. In one embodiment, the transgenic rice is prepared by subjecting rice to radiation mutagenesis. Radiation mutagenesis includes gamma-ray mutagenesis and heavy ion mutagenesis. Preferably, the source of mutagenesis of the gamma-ray mutagenesis is selected from 60 Co-gamma rays, said 60 The dosage of Co-gamma rays is 200-500 Gy, such as 80-150 Gy. In one specific embodiment, 300Gy. The said 60 The dosage rate of Co-gamma rays is 2-16 Gy/min, such as 8-12 Gy/min. In one embodiment, 8Gy/min. The mutagenesis source of the heavy ion mutagenesis is selected from 12C+6 heavy ions. Preferably, the dosage of heavy ion mutagenesis is 80-150 Gy, such as 100-140 Gy. The dosage rate of the heavy ion mutagenesis is 0.8-2.2 Gy/Min, such as 1.2-1.7 Gy/Min. More specifically, the method comprises the following steps: a) Carrying out radiation mutagenesis on seeds of rice, 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 the leaves on each planted single plant, and mixing the leaves of each single plant; d) Extracting the genome DNA from the mixed leaves. In the step b), after the M1 generation seeds are planted singly, each spike seed of each single plant is harvested according to a spike harvesting method so as to form the M2 generation seeds. In the step c), the M2 generation seeds are planted singly, each 100-1000 plants are one sample group, each sample group is numbered, and the rice leaves in each sample group are taken and mixed to extract DNA. In the step c), when the leaves on each planted single plant are taken, the leaves on different parts of the same single plant are selected for equal amount mixing; equal amounts of different individuals were mixed.
The invention can accelerate the plant gene mutation by nuclear radiation mutagenesis, and adopts a high-flux molecular typing identification technology to screen mutation, thereby creating the low-cadmium gene OsNramp5 mutant. The molecular marker assisted selection and the traditional hybridization technology are utilized to realize the gene transfer of breeding materials, and the rice variety with excellent agronomic comprehensive properties and low cadmium accumulation is cultivated; in addition, the method has great significance for realizing the safe production of rice varieties in paddy fields with severe pollution of cadmium.
Further advantages and effects of the present invention will become apparent to those skilled in the art from the disclosure of the present invention, which is described by the following specific examples.
Before the embodiments of the invention are explained in further detail, it is to be understood that the invention is not limited in its scope to the particular embodiments described below; it is also to be understood that the terminology used in the examples of the invention is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention. The test methods in the following examples, in which specific conditions are not noted, are generally conducted under conventional conditions or under conditions recommended by the respective manufacturers.
Where numerical ranges are provided in the examples, it is understood that unless otherwise stated herein, both endpoints of each numerical range and any number between the two endpoints are significant both in the numerical range. 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, materials used in the embodiments, any methods, devices, and materials of the prior art similar or equivalent to those described in the embodiments of the present invention may be used to practice the present invention according to the knowledge of one skilled in the art and the description of the present invention.
Example 1
In this example, the wild rice OsNramp5 Gene was found in the Gene bank database, specific primers within the synthetic Gene were designed using published sequence information, and primers were designed using BatchPrimer3 (http:// probes. Pw. Usda. Gov/batch Primer3 /). Primers were commissioned for Invitrogen corporation synthesis.
The primer is designed according to the wild rice OsNramp5 gene, and the sequence of the primer comprises sequences shown in SEQ ID NO. 5-SEQ ID NO.42, and the details are shown in Table 1.
TABLE 1
Example 2
The OsNramp5 mutant was selected in this example 2, comprising the following:
1. subjecting the plant to radiation mutagenesis treatment comprising the following steps:
1) The types of the Xiangfu 1822 and Fuxiang 116 rice dry seeds are 1000 grains each,using 60 Co-gamma ray radiation mutagenesis treatment, the dosage is 300Gy, the dosage rate is 1.2Gy/Min, and M1 generation seeds are obtained.
2) The method comprises the steps of planting the single plants of the seed seedling raising of each variety M1 generation, carrying out the strict selfing and setting of the M1 generation, selecting 2 spikes on each single plant according to the diagonal line according to the spike harvesting method, and harvesting to obtain the M2 generation seeds.
3) Dividing the plants of the seed seedling raising of the M2 generation into single plants, planting about 20000 plants in each variety, 100 plants in each group, numbering each group; taking each sample group as a unit, taking equal amount of leaves from each single plant by adopting a puncher with the diameter of 6mm to mix when seedlings grow to two leaves and one heart, and mixing different plants of the same variety in equal amount, wherein each variety has 200 sample groups.
2. Extraction of DNA from a sample population by CTAB method
And (3) numbering 200 sample groups of each variety obtained in the step (1), wherein the variety Xiangfu 1822 is sequentially numbered as A1-A200, the variety Fuxiang 116 is sequentially numbered as K1-K200, and each individual plant in each sample group is also numbered, and the sample group A1 is taken as an example, and is sequentially numbered as A1-1 to A1-100.
After extracting DNA from each sample group by adopting CTAB, quantifying by adopting Qubit fluorescence, wherein the concentration is more than or equal to 100 ng/. Mu.L, and the total amount is more than or equal to 1 mu g; sample purity: OD 260/280=1.7-2.0, OD260/230 is not less than 1.8; agarose gel electrophoresis is used to detect genomic DNA integrity, requiring that the major band of electrophoresis be greater than 2000bp. In this example, the DNA actually detected below 2000bp is less than 50%. As a DNA template for subsequent library construction.
3. Sequencing library construction
The construction of the sequencing library was performed by Huazhi biotechnology limited company, and the specific steps are as follows:
3.1, DNA Targeted amplification
1) Sterile PCR centrifuge tubes were prepared, an amplification system was formed according to Table 2, and were blow-mixed. The 2 XPCR enzyme mixture contains Taq DNA polymerase, dNTPs, mgCl2, and PCR buffer.
TABLE 2DNA targeting amplification System
2) The amplification system of Table 2 was subjected to PCR amplification according to the procedure of Table 3, to obtain amplified products.
TABLE 3PCR reaction conditions
3.2 sequencing adapter ligation
1) Preparing a sterile PCR centrifuge tube, forming a joint connection system according to the table 4, and blowing and uniformly mixing, so that the amplification product obtained in the step 3.1) is connected with a sequencing joint.
TABLE 4 sequencing plug ligation System
2) The adaptor-ligated system of Table 4 was subjected to PCR reaction according to the procedure of Table 5 to obtain PCR products.
Table 5 reaction procedure
3.3 recovery of PCR products
After the PCR reaction of step 3.2 was completed, the DNA fragment was purified using Agencourt AMPure XP magnetic beads, as follows:
1) Taking out the magnetic bead solution from the refrigerator at 4 ℃ for 30min in advance, placing the magnetic bead solution at room temperature for incubation for 30min, adding the magnetic bead solution into a PCR centrifuge tube where the PCR product obtained in the step 3.2 is located, and uniformly mixing the magnetic bead solution.
2) 50 mu L of magnetic beads (1X) are added into a PCR reaction tube, and the mixture is blown and sucked for 7 to 8 times, so that the magnetic beads are uniformly distributed in the solution, and the reaction is carried out for 5 minutes at room temperature, and DNA fragments are adsorbed on the magnetic beads.
3) The PCR tube was placed on a magnetic rack for about 7min, and after the beads had aggregated and the solution had clarified, the supernatant was discarded.
4) The tubes were kept on the magnet rack during ethanol wash twice with 80% ethanol, 200 μl each for 30s, and then the supernatant was discarded.
5) Taking down the tube, centrifuging instantaneously, placing on a magnetic rack again, sucking residual liquid in the tube, opening the tube cover, and drying at normal temperature for 3min.
6) Aspirate 30 μl of DNA eluate: adding from the side with the magnetic beads, and flushing the magnetic beads into the bottom of the centrifuge tube; and then repeatedly blowing or vibrating the mixture to mix the magnetic beads uniformly with the PCR amplification product at a low speed, and reacting for 5min at room temperature.
7) The EP tube was inserted into the tube hole of the magnetic rack, and after the magnetic beads adsorbed the complete PCR amplification product and the solution became clear, 28. Mu.L was pipetted into a new EP tube (note that the magnetic beads could not be attracted) with a pipette to obtain a DNA second generation sequencing library.
4. Sequencing library on-machine quality inspection
The library concentration detection adopts Qubit or Real-time PCR system, the library quality detection adopts Agilent 2100/2200Bioanalyzer, and after the library quality is qualified, the sample detection and the depth analysis are carried out by adopting an Illumina PE150/MGIPE150 high-throughput sequencing platform.
5. Bioinformatic analysis
In order to ensure the quality of information analysis, the method uses Fastp to control the quality of Raw Data (Chen et al 2018) to obtain Clean Data, and the subsequent analysis is based on the Clean Data.
The main filtering conditions for data quality control are as follows:
1) Removing the sequencing linker (Adapter) in the Reads;
2) Excising bases with mass values below 3 starting from the beginning (5') of Reads;
3) Excising bases with mass values below 3 starting from the end of Reads (3');
4) Removing Reads with mass values below 15 and base numbers exceeding 40% of the length of the Read;
5) Removing Reads with the number of "N" bases exceeding 5;
6) Searching an overlapping region of each pair of Reads, and correcting unmatched base pairs in the overlapping region;
7) The Reads with a length of less than 36 were removed after filtration.
And (3) comparing the BWA software with a rice reference genome (wild OsNramp 5), and analyzing mutation sites of a sequencing result by using Freebayes software. Variation was checked using IGV in comparison to the rice reference genome. Table 6 shows the detected genetic variation.
TABLE 6 Gene variation
As is clear from Table 6, the sample group genes of accession numbers K8 and A44 were found to differ from the wild-type OsNramp5 gene sequence, respectively, the sample group gene of accession number K8 had a deletion of 1 base G at position 8835306 (RAP_Locus), and the sample group gene of accession number A44 had a deletion of 8 bases AGAAACTC at position 8939534-8939541 (RAP_Locus).
6. According to the sequencing data result, the sequence of the gene with the difference site is designed by utilizing Primer Premier 5.0 software, and the genome DNA of 100M 2 generation single plant samples in the sample groups K8 and A44 are respectively subjected to PCR amplification, and the PCR amplification products are directly sequenced.
Molecular primers were designed based on the gene of the mutant numbered 24 in the sample group numbered POOL No. k 8:
forward primer: 5'-TTGTCGATCGATCGATCTGC-3' (SEQ ID NO. 43)
Reverse primer: 5'-TCATGCTCGTCTACATCGTC-3' (SEQ ID NO. 44)
Molecular primers were designed based on the gene of the mutant numbered 92 in the sample population numbered POOL No. a 44:
forward primer: 5'-AAGCGTACGGCATGACAAGAG-3' (SEQ ID NO. 45)
Reverse primer: 5'-TCTCTGACTGTGGAACTGCATG-3' (SEQ ID NO. 46)
PCR amplification was performed according to Table 7.
TABLE 7 PCR amplification System
The nucleotide sequence of the OsNramp5 mutant obtained by the K8 sample group is shown in figure 2; the nucleotide sequence of the OsNramp5 mutant obtained by the A44 sample group is shown in figure 3.
FIG. 1 is a schematic diagram of a partial sequence of an OsNramp5 mutant gene.
As can be seen from FIG. 1, there was a1 base G deletion of the OsNramp5 gene from the No. 24 plant in the sample group numbered K8, as compared with the wild-type OsNramp 5.
FIG. 2 is a schematic diagram of a partial sequence of an OsNramp5 mutant gene.
As can be seen from FIG. 2, there was an 8-base AGAAACTC deletion in the OsNramp5 gene of plant No. 92 in the sample group No. A44, as compared with the wild-type OsNramp 5.
7. Planting the single plants with the difference of the genome sequences in the step 6 into a field, carrying out strict selfing propagation on offspring, and taking leaves respectively in a seedling stage for mutation verification again. Finally, the mutant which is stable in heredity, excellent in comprehensive properties and meets the breeding target properties is selected.
The content of effective cadmium in the field is detected to be 1.25mg/Kg by adopting an atomic absorption spectrophotometry, and seeds are collected after rice is ripe to determine the cadmium content. Wherein the content of cadmium is measured according to GB5009.15-2014, and the result is shown in figure 5.
The mutant rice plants and the wild rice plants were photographed, and the plant height, the effective spike number, the seed setting rate, and the thousand seed weight were counted, and the results are shown in fig. 3, 4, and tables 8 and 9.
FIG. 3 is a photograph of an OsNramp5 mutant rice and a wild type rice of sample group number K8.
FIG. 4 is a photograph of an OsNramp5 mutant rice and a wild type rice of sample group number A44.
FIG. 5 is a bar graph showing cadmium content of OsNramp5 mutant rice designated K8 and OsNramp5 mutant rice designated A44, respectively, and wild type rice.
TABLE 8
| Classification | Name of the name | Height of plant (cm) | Effective spike number | Spike length (cm) | Set percentage (%) | Thousand grain weight |
| Wild rice | Hunan spoke 1822 | 114.8±2.3 | 8.2±2.1 | 27.3±2.6 | 88.2±3.2 | 19.9±1.3 |
| Mutant rice | A44-92 | 115.6±3.5 | 9.3±1.9 | 26.8±1.6 | 88.5±2.8 | 20.1±2.6 |
TABLE 9
| Classification | Name of the name | Height of plant (cm) | Effective spike number | Spike length (cm) | Set percentage (%) | Thousand grain weight |
| Wild rice | Fuxiang 116 | 132.3±3.1 | 13.5±3.2 | 26.3±2.2 | 85.6±1.3 | 25.6±2.7 |
| Mutant rice | K8-24 | 133.4±2.8 | 15.8±2.1 | 27.2±3.4 | 86.5±2.1 | 26.2±1.6 |
From FIGS. 3 and 4 and tables 8 and 9, it is understood that OsNramp5 mutant rice has a higher plant height, effective spike number, seed setting rate and thousand kernel weight than wild type rice.
As can be seen from FIG. 5, the OsNramp5 mutant rice has significantly reduced cadmium content in the grain and significantly different levels compared with the wild type rice.
The above embodiments are merely illustrative of the principles of the present invention and its effectiveness, and are not intended to limit the invention. Modifications and variations may be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the invention. Accordingly, it is intended that all equivalent modifications and variations of the invention be covered by the claims, which are within the ordinary skill of the art, be within the spirit and scope of the present disclosure.
Claims (6)
- Application of an OsNramp5 mutant or biological material related to the OsNramp5 mutant in improving heavy metal tolerance of rice or reducing heavy metal accumulation of rice, wherein the amino acid sequence of the OsNramp5 mutant is shown as SEQ ID NO.3 or SEQ ID NO.4, and the biological material is any one of the following:a) A recombinant expression vector containing the encoding nucleotide of the OsNramp5 mutant;b) Bioengineering bacteria containing the encoding nucleotide of the OsNramp5 mutant or bioengineering bacteria containing the recombinant expression vector of a);c) A nucleotide encoding the OsNramp5 mutant.
- Application of an OsNramp5 mutant or biological material related to the OsNramp5 mutant in cultivation of rice with high heavy metal tolerance, wherein the amino acid sequence of the OsNramp5 mutant is shown as SEQ ID NO.3 or SEQ ID NO.4, and the biological material is any one of the following:a) A recombinant expression vector containing the encoding nucleotide of the OsNramp5 mutant;b) Bioengineering bacteria containing the encoding nucleotide of the OsNramp5 mutant or bioengineering bacteria containing the recombinant expression vector of a);c) A nucleotide encoding the OsNramp5 mutant.
- 3. The use according to any one of claims 1-2, further comprising increasing one or more of rice plant height, effective spike number, and seed setting rate;and/or the heavy metal is selected from cadmium.
- 4. The use according to any one of claims 1 to 2, wherein the nucleotide sequence is as set forth in SEQ ID No.1 or as set forth in SEQ ID No. 2.
- 5. A method for increasing rice tolerance to heavy metals or reducing rice heavy metal accumulation, characterized by introducing the mutant of claim 1 or the biological material into rice.
- 6. A method for cultivating transgenic rice, comprising introducing the mutant or the biological material according to claim 1 into rice, and cultivating to obtain said transgenic rice.
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