CN108285927A - Molecular labeling Caps7 and its application in a kind of gene of rice Cd accumulation related gene OsHMA3 - Google Patents
Molecular labeling Caps7 and its application in a kind of gene of rice Cd accumulation related gene OsHMA3 Download PDFInfo
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Abstract
The invention belongs to technical field of molecular biology, provide molecular labeling Caps7 and its application in a kind of gene of rice Cd accumulation related gene OsHMA3, positive and negative two primers have been synthesized by design, paddy DNA is expanded in PCR system, then digestion is carried out to amplified production, determine that rice to be measured contains 2 allele of HMA3 1 or HMA3 finally by agarose electrophoresis detection, so that it is determined that rice Cd accumulation character and assisting the selection and breeding of the low Cd accumulation kind of long-grained nonglutinous rice.The present invention can effectively differentiate rice Cd accumulation character, and provide new technological means for the selection and breeding of long-grained nonglutinous rice low cadmium-accumulation kind, improve Breeding Efficiency.
Description
Technical field
The invention belongs to technical field of molecular biology, and in particular to a kind of rice Cd accumulation related gene OsHMA3's
Molecular labeling Caps7 and its application in gene.
Background technology
Rice cadmium is exceeded caused by rice field cadmium (Cadmium, Cd) pollution, seriously affects the food security and grain peace in China
Entirely.Therefore, the injury in order to avoid cadmium pollution to human body is badly in need of reducing the cadmium accumulated in rice grain.For rice varieties rice
The hereditary feature of rice Cd accumulation is the study found that rice variety often accumulates more cadmiums than japonica rice variety in seed.And at me
State south rice main producing region, single long-grained nonglutinous rice pattern of farming, the Soil Background of slant acidity and higher cadmium pollution degree aggravate
The exceeded risk of rice variety Cd concentration of brown rice.So, before thoroughly radical cure cadmium pollution, plantation rice low cadmium-accumulation
Rice variety is to avoid the most economical effective method of cadmium pollution risk at present.But since rice Cd accumulation character cannot be by breeding
Family is directly observed during actual breed breeding, therefore the selection and breeding of low cadmium rice varieties need to differentiate, utilize excellent base
Because of resource and corresponding molecule supplementary means (Marker Assisted Selection, MAS).It can effectively differentiate rice cadmium
The molecule supplementary means of character is accumulated still in the stage to be developed.
Invention content
For the above technical problem, the present invention provides molecules in a kind of gene of rice Cd accumulation related gene OsHMA3
Caps7 and its application are marked, can effectively differentiate rice varieties Cd accumulation character and accelerate the choosing of the low Cd accumulation kind of long-grained nonglutinous rice
Educate process.
Currently, for rice grain Cd accumulation character, scientific research personnel has identified a series of control rice Cd uptakes, has turned
Fortune and the Key number character site and related gene of distribution, wherein a control at No. seven the short arm of a chromosome of rice
The main effect QTL for influencing rice cadmium Transport Rate is cloned by separation, and OsHMA3 (Ueno et al, 2010) is named as.The gene
Mainly the cadmium that rice root vacuole absorbs root system is influenced by control to be isolated, reduce the cadmium transported to ground position,
To influence rice cadmium Transport Rate.Inventor studies rice Cd accumulation using linkage analysis and association analysis, finds base
Because OsHMA3 can influence accumulation of the rice from the cadmium of soil absorption in rice；Find that the gene is main in natural population simultaneously
There are two kinds of important allelotype HMA3-1 and HMA3-2, HMA3-2 nucleotide sequences are shown in SEQ ID NO:Shown in 1, this
Two kinds of allelotypes account for 85% or more in natural population, and the Cd concentration of brown rice of the rice varieties containing HMA3-1 is substantially less than
Rice varieties (P containing HMA3-2<0.01)；The further analysis of inventor finds HMA3-1 and HMA3-2, and there are apparent Xian-
The feature of round-grained rice differentiation, i.e. japonica rice variety often contain HMA3-1, and rice variety then largely containing HMA3-2 and small part contains
There is HMA3-1.
The genetic background of the present invention focuses on：1) Cd concentration of brown rice of most of rice variety is often in one
Higher level (under identical similar soil environment, long-grained nonglutinous rice often accumulates more), this reason is likely due to OsHMA3
Allelotype be high Cd accumulation genotype HMA3-2；It 2) can be by low cadmium-accumulation allelotype by molecule supplementary means
HMA3-1 imports high accumulation kind (the high accumulation allelotype HMA3-2 for replacing OsHMA3), to improve the cadmium product of rice variety
Tired feature provides important science of heredity means and related kind, strain.
The technical scheme is that：Provide molecular labeling in a kind of gene of rice Cd accumulation related gene OsHMA3
Caps7, gene OsHMA3 include allele HMA3-1 and allele HMA3-2, and the molecular labeling Caps7 can be distinguished
The sequence difference of position gene HMA3-1 and allele HMA3-2.
Further, the PCR amplification primer pair of the molecular labeling Caps7 is：
Forward primer F (SEQ ID NO:4)：CTCGTCAGCGGCCTCAAGG；
Reverse primer R (SEQ ID NO:5)：GCACGACGAGGAGGCACG；
The molecular labeling Caps7 is shown as：When carrying out PCR amplification using the primer pair, allele HMA3-1's
Amplified production has SEQ ID NO:The amplified production of nucleotide sequence shown in 2, allele HMA3-2 has SEQ ID NO:3
Shown nucleotide sequence；The allele HMA3-1 shows as low Cd accumulation, the nucleotides sequence of the allele HMA3-2
It is classified as SEQ ID NO:Shown in 1, high Cd accumulation is shown as.
The present invention also provides a kind of sides for distinguishing allele HMA3-1 and HMA3-2 using above-mentioned molecular labeling Caps7
Method carries out PCR amplification using primer pair noted earlier to oryza sativa genomic dna to be measured；To PCR product AscI restriction endonuclease enzymes
It cuts, agarose electrophoresis detects the amplified production after digestion, if agarose electrophoresis detection is shown as a band, rice tool to be measured
There is allele HMA3-1；If agarose electrophoresis detection is shown as two band, rice to be measured has allele HMA3-2,
If agarose electrophoresis detection is shown as three band, the gene OsHMA3 of rice to be measured is heterozygous state.
Further, if agarose electrophoresis detection is shown as a band, sequence length 337bp, then rice tool to be measured
There is allele HMA3-1, shows as low Cd accumulation；If agarose electrophoresis detection is shown as two band, sequence length is respectively
196bp, 141bp, then rice to be measured shows as high Cd accumulation with allele HMA3-2, if agarose electrophoresis detection display
For three band, sequence length is respectively 337bp, 196bp and 141bp, and the band that sequence length is 337bp is most bright, then waits for
The gene OsHMA3 for surveying rice is heterozygous state.
Further, the PCR system is：10~50ng of template DNA, forward primer and each 0.25um of reverse primer,
Taq archaeal dna polymerases 1U, four kinds of deoxyribonucleotide dATP, dTTP, dCTP, dGTP each 0.1mM, 2 × GC buffer I
10ul, rest part deionized water polishing to 20ul；
The PCR response procedures are：First in 94 DEG C of pre-degeneration 4min, enters PCR cycle later and expand, cyclic amplification packet
94 DEG C of denaturation 30sec, 60 DEG C or 62 DEG C annealing 30sec, 72 DEG C of extension 1min are included, totally three steps, circular flow 30~34 times, finally
After extending 10min at 72 DEG C, 25 DEG C of heat preservation 4min obtain amplified production；
The digestion system is 10ul systems, and following each component is mixed：In pcr amplification product 100~200ng, AscI
Enzyme cutting Buffer (10X) 1ul, AscI restriction endonuclease 2U, remaining uses deionized water polishing；Digestion program：The digestion body that will be mixed
System is positioned over 8~12h or more of reaction in 37 DEG C of insulating boxs and carries out digestion.
The present invention also provides molecular labeling Caps7 noted earlier and its PCR amplification primer pair in identification rice Cd accumulation
Purposes in character.
The present invention also provides molecular labeling Caps7 noted earlier and its PCR amplification primer pair in low Cd accumulation long-grained nonglutinous rice product
Purposes in kind selection and breeding.
The present invention also provides the methods of differentiation allele HMA3-1 and HMA3-2 noted earlier in low Cd accumulation long-grained nonglutinous rice
Purposes in breed breeding.
The present invention also provides a kind of methods of identification rice Cd accumulation character, using foregoing primer pair to be measured
The genomic DNA of rice carries out the PCR amplification of molecular labeling Caps7, and AscI endonuclease digestions, agarose electrophoresis detection is used in combination
The length of PCR product determines rice Cd accumulation character；
If agarose electrophoresis detection is shown as a band, sequence length 337bp, then rice to be measured is with allele
HMA3-1 shows as low Cd accumulation；If agarose electrophoresis detection is shown as two band, sequence length be respectively 196bp,
141bp, then rice to be measured shows as high Cd accumulation with allele HMA3-2, if agarose electrophoresis detection is shown as three
Band, sequence length is respectively 337bp, 196bp and 141bp, and the band that sequence length is 337bp is most bright, then rice to be measured
Gene OsHMA3 be heterozygous state.
The present invention also provides a kind of low Cd accumulation long-grained nonglutinous rice auxiliary breeding means, the method includes：By noted earlier
Method, detection molecules mark Caps7, with determination long-grained nonglutinous rice Cd accumulation character to be measured.
Compared with prior art, beneficial effects of the present invention are：
1, the present invention is studied by influencing the natural variation of the main effect QTL gene OsHMA3 of rice Cd accumulation on control, is opened
It sends out in the gene that one can effectively distinguish low Cd accumulation allelotype HMA3-1 and high Cd accumulation allelotype HMA3-2
Molecular labeling Caps7.
2, molecular labeling Caps7 can effectively differentiate that rice is Cd accumulation character in gene of the invention, and be selection and breeding
Low Cd accumulation rice variety provides new technological means, accelerates Breeding Process.
3, the near isogene based material NIL (93-11) that the embodiment of the present invention 3 obtains is that low Cd accumulation Indica Rice Varieties carry
Good donor material is supplied.
Description of the drawings
Natural variation and the haplotype analysis figure that Fig. 1 is OsHMA3, the natural variation analysis and list times that wherein a is OsHMA3
Type analysis, Oxford gray show long-grained nonglutinous rice genotype, and grayish to be shown to be japonica rice genotype, the number in the grid of right side is
The kind number that the genotype occurs in natural population；B is HMA3-1 and HMA3-2 rice cadmiums in 105 parts of rice varieties
The comparison of accumulation, P<0.01 level shows that difference is extremely notable；C is that histogram frequency distribution diagram shows that HMA3-1 and HMA3-2 exist
The distribution of 300 parts of rice varieties, black are shown to be HMA3-2 allelotypes, and light gray is HMA3-1 allelotypes；D is
The difference of HMA3-1 and HMA3-2 Cd concentration of brown rice in 300 parts of materials, P<0.01 level shows that difference is extremely notable；
Fig. 2 is the weak function alleles analysis figure that HMA3-2 is OsHMA3 genes, wherein a is OsHMA3 allele
Differential expressions of the HMA3-1 and HMA3-2 in yeast, Glucose are glucose, as the carbon source for inhibiting expression, galactose
For galactolipin, as the carbon source of induced expression, for yeast growth on SC-Ura (uracil) auxotrophy culture medium, Cd stress is dense
Degree is 10uM；B is growth curve of yeast under Cd stress, and culture medium is SC-Ura (uracil) fluid nutrient medium；C is GFP-HMA3
The yeast cell to express of fusion protein positions, HMA3CKKIndicate the nonfunctional allelotype of OsHMA3；
Fig. 3 is that developed OsHMA3 gene internal labeling Caps7 is utilized to identify electrophoresis to the PCR of HMA3-1 and HMA3-2
Figure, a concentration of the 2% of Ago-Gel；9311 and Nip is check variety, wherein 9311 contain HMA3-2, and Nip contains
HMA3-1.HMA3-1/HMA3-2 is expressed as heterozygous state；
Fig. 4 is Caps7 pairs of two kinds of near isogenic lines NIL (HMA3-1) and the PCR identification glue figures of NIL (HMA3-2), agar
A concentration of the 3% of sugared gel；Wherein NIL (HMA3-1) contains HMA3-1, and glue figure is a single band, and NIL (HMA3-
2) contain HMA3-2, glue figure is two bands.
Specific implementation mode
Technical solution of the present invention is further described below with reference to drawings and examples.
Embodiment 1：Excavations of the HMA3 in rice long-grained nonglutinous rice subgroup and japonica rice subgroup allelic function difference
(1) natural variation of gene HMA3
For rice Cd accumulation correlation QTL gene OsHMA3, to OsHMA3 in 105 parts of Germplasms (collection of this room)
The sequence of gene has carried out sequence analysis.Haplotype point has been carried out by MEGA sequence analysis softwares and STRUCTURE softwares
Analysis, it is found that the gene co-exists in 9 kinds of genotype in nature, be denoted as HMA3-1~HMA3-9 successively；Wherein HMA3-2~HMA3-
6 uniformly belong to long-grained nonglutinous rice genotype, and HMA3-1, HMA3-7~HMA3-9 then belong to japonica rice genotype, see Fig. 1 a.HMA3-1 and
HMA3-2 is two kinds of main allele genes, they account for the 83.8% of all material altogether；Further analysis finds,
HMA3-1 largely comes across japonica rice variety small part and comes across rice variety, and HMA3-2 then mainly appears on rice variety,
The haplotype analysis of OsHMA3 finds that the gene has more apparent Xian-round-grained rice differentiating phenomenon.In conjunction with Cd concentration of brown rice phenotype, hair
The rice cadmium that rice now containing HMA3-1 genotype averagely accumulates is substantially less than the rice varieties of the allelotype containing HMA3-2,
See Fig. 1 b.In rice variety, OsHMA3 genes equally exist the distribution between HMA3-1 and HMA3-2, but in rice variety
Kind number shared by middle HMA3-1 is less, sees Fig. 1 c, inside long-grained nonglutinous rice, it is again seen that the rice containing HMA3-1 genotype is flat
The rice cadmium accumulated is substantially less than the rice varieties of the allelotype containing HMA3-2, sees Fig. 1 d.
(2) HMA3-2 is the weak function allelotype of OsHMA3
Function difference analysis is carried out using wild-type yeast BY4741.Under 10uMCdSO4 coercing cultivations, contain HMA3-1
Yeast strain with HMA3-2 is compared to empty carrier and nonfunctional allelotype HMA3CKKIt grows less, contains simultaneously
The yeast strain that HMA3-2 yeast strain Mortality Ratios contain HMA3-1 is more, sees Fig. 2 b, while yeast growth is experiments have shown that contain
The yeast growth rate of HMA3-2 is substantially less than the yeast strain containing HMA3-1, sees Fig. 2 b；Pass through the yeast of GFP fusion proteins
Subcellular localization, find no matter HMA3-1 or HMA3-2 be primarily located on tonoplast (other plasma membranes also have on a small quantity divides
Cloth), see Fig. 2 c.The above results illustrate HMA3-2 and carry the work(being transported to the cadmium in the yeast external world in yeast as HMA3-1
Energy；The cadmium in cell body is retained in the ability in vacuole since HMA3 has, and HMA3-1 is powerful etc. as OsHMA3
Position gene, into vacuole turn-over capacity be relatively better than HMA3-2, therefore the yeast strain containing HMA3-2 is in Cd stress environment
Growth to be weaker than the yeast strain containing HMA3-1, therefore HMA3-2 is the weak function allele of OsHMA3.
Embodiment 2：The exploitation of molecular labeling Caps7 and genotype identification method
(1) exploitation of molecular labeling Caps7
The molecular labeling Caps7 of the present invention, marks the principle of design to be：Utilize bioinformatics website NCBI
(https://www.ncbi.nlm.nih.gov/) in BLAST tools sequence alignment is carried out to the sequence of HMA3-1 and HMA3-2,
Difference site is found, which forms two kinds of genotype, wherein cannot be limited from japonica rice genotype HMA3-1
Property restriction endonuclease AscI cutting, after the AscI enzymic digestions after PCR amplification, single band is detected as by agarose electrophoresis, PCR
Primer size is 337bp；Long-grained nonglutinous rice genotype HMA3-2 can then be cut by restriction enzyme A scI, be examined by agarose electrophoresis
It is two bands to survey, and PCR primer sizes are 196bp and 141bp, see Fig. 3.
(2) genotype identification method
PCR programs are referring to Pehanorm Brooker etc., and 2002, Molecular Cloning:A Laboratory guide, the third edition, golden winter wild goose etc. (translating), section
The method that publishing house is introduced is learned, and according to the condition of the Institute of Subtropical Agriculture, The Chinese Academy of Sciences and requirement, is modified.
Forward primer (F)：CTCGTCAGCGGCCTCAAGG；
Reverse primer (R)：GCACGACGAGGAGGCACG.
The specific steps of genotype identification：
1) genomic DNA of rice plant is extracted；
2) PCR amplification, using the rice plant genomic DNA of step S1 as template, by above-mentioned forward primer (F) and reversely
Primer (R) is added in PCR reaction systems, is expanded to obtain amplified production to the rice plant genomic DNA；
The PCR system is the system of 20ul：10~50ng of genomic DNA of rice plant, forward primer (F) and anti-
To primer (R) each 0.25um, Taq archaeal dna polymerases 1U, four kinds of deoxyribonucleotides dATP, dTTP, dCTP, dGTP is each
0.1mM, 2 × GC buffer I 10ul, rest part deionized water polishing to 20ul.2 × GC buffer I by
TAKARA treasured bioengineering Co., Ltd produces；
The response procedures of the PCR are：First in 94 DEG C of pre-degeneration 4min, enters PCR cycle later and expand, cyclic amplification
Including 94 DEG C of denaturation 30sec, 60 DEG C or 62 DEG C annealing 30sec, 72 DEG C extend 1min, totally three steps, circular flow 30~34 times, most
After extending 10min at 72 DEG C afterwards, 25 DEG C of heat preservation 4min obtain amplified production；
3) digestions of PCR product, digestion system are 10ul, and following each component is mixed：Pcr amplification product 100~
200ng, AscI restriction endonuclease Buffer (10X) 1ul, AscI restriction endonuclease 2U, remaining uses deionized water polishing；The digestion that will be mixed
System is positioned over 8~12h or more of reaction in 37 DEG C of insulating boxs；
4) 2% agarose gel electrophoresis detects, after point sample, in 250V DC voltage 15~30min of electrophoresis, ethidium bromide
Dyeing 1h is placed under ultraviolet lamp, reads the PCR banding patterns of each sample；According to PCR banding patterns to judge the equipotential base of OsHMA3
Because of type, it is HMA3-2 to have two bands, and containing a band then be HMA3-1；When rice Cd accumulation related gene OsHMA3 is
When heterozygous state, three bands will be generated after being identified by gene internal labeling Caps7 in agar sugar detection, PCR product is big
Small is respectively 337bp, 196bp and 141bp, and 337bp banding patterns are most bright, see Fig. 3.9311 and Nip is check variety, wherein
9311 contain HMA3-2, and Nip contains HMA3-1.HMA3-1/HMA3-2 is expressed as heterozygous state.
Embodiment 3:
(1) identify, screen 300 parts of rice long-grained nonglutinous rice materials rice Cd accumulation related gene OsHMA3 allelotype and
Its efficiency assay
Using ensemble average value as the standard for distinguishing Cd concentration of brown rice height, reflected using the molecular labeling Caps7 of the present invention
The allelotype of the OsHMA3 of fixed 300 parts of long-grained nonglutinous rice materials.
The rice Cd accumulation phenotype of 300 parts of materials is collected in pollution field experiment, and field planting mode is with alternation of wetting and drying
Mode carries out, make utmostly to accumulate in rice grain cadmium element (crop field cadmium concentration is serious pollution, a concentration of 1.9mg/kg,
5.4) pH value is.Its genomic DNA is extracted from 300 parts of long-grained nonglutinous rice materials of table 1, utilizes rice Cd accumulation related gene OsHMA3
Gene internal labeling Caps7 (its PCR amplification primer be SEQ ID NO:4 and SEQ ID NO:Shown in 5), according in embodiment 2
Shown method carries out allelotype identification.
Table 1 the result shows that, have 49 parts of rice long-grained nonglutinous rice materials qualification result be 337bp, show the allele of OsHMA3
Type is HMA3-1, has 45 parts of its Cd concentration of brown rice of material lower than ensemble average value level in this 49 parts of materials, accounts for 91.8%；Have
The qualification result of 251 parts of materials is 196bp and 141bp, shows to carry HMA3-2 allele, has 151 in this 251 parts of materials
Its Cd concentration of brown rice of part material is higher than ensemble average value, accounts for 60.2%.Illustrate that the molecular labeling Caps7 of the present invention is to differentiate rice
The molecular labeling of Cd accumulation kind can be used for the prediction and screening of rice low cadmium-accumulation kind.
The OsHMA3 genotype and rice Cd content phenotypes of 1 300 parts of long-grained nonglutinous rice materials of table
(2) selection and breeding of low Cd accumulation rice variety
According to screening in (1) as a result, inventor selects rice variety 93-11 as improving the breed (containing HMA3-2 etc.
Position genotype), the kind Cd concentration of brown rice is higher, and seed Cd concentration of brown rice is in 3.6~4.5mg/kg.Choose japonica rice simultaneously
Kind Nipponbare improves the rice of 93-11 as donor kind (containing HMA3-1 allelotypes) in the way of back cross breeding
The high Cd accumulation characteristic of rice.
After hybridization and subsequent three generations backcrossing, obtain under 93-11 genetic backgrounds, OsHMA3 allelotypes are
Then the F1 offsprings of HMA3-1/HMA3-2 heterozygous states are detached after F1 selfings.
The identification of molecular labeling Caps7 through the invention, obtain respectively in 93-11 genetic background HMA3-1 homozygosis and
Two kinds of near isogenic lines familys of HMA3-2 homozygosis, are denoted as NIL (HMA3-1) and NIL (HMA3-2), two kinds of near isogenic lines
Caps7 Marker Identification glue figures are shown in that Fig. 4, NIL (HMA3-1) contain HMA3-1, and glue figure is a single band, and NIL
(HMA3-2) contain HMA3-2, glue figure is two bands.Yield is carried out to both materials and the investigation of rice Cd accumulation phenotype is shown in
Table 2 finds that the Cd concentration of brown rice of NIL (HMA3-1) is substantially less than NIL (HMA3-2), about 30.7% or so (P<0.01)；In rice
Without significant difference (P on meter Xin, manganese accumulation>0.05)；Without significant difference (P in plant height, the economical characters such as single plant yield>
0.05)。
The above results illustrate that molecular labeling Caps7 is in the gene of the rice Cd accumulation related gene OsHMA3 of the present invention
Effective molecular labeling, and can be used for the selection and breeding of low Cd accumulation rice variety.
2 NIL of table (HMA3-1) and NIL (HMA3-2) in rice Cd accumulation and other elements (manganese, zinc), economical character
Compare
* P is indicated respectively with * *<0.05, P<0.01
The above content is a further detailed description of the present invention in conjunction with specific preferred embodiments, and it cannot be said that
The specific implementation of the present invention is confined to these explanations.For those of ordinary skill in the art to which the present invention belongs, exist
Under the premise of not departing from present inventive concept, a number of simple deductions or replacements can also be made, all shall be regarded as belonging to the present invention's
Protection domain.
SEQUENCE LISTING
<110>The Institute of Subtropical Agriculture, The Chinese Academy of Sciences
<120>Molecular labeling Caps7 and its application in a kind of gene of rice Cd accumulation related gene OsHMA3
<130> 2018
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 3015
<212> DNA
<213>Rice (Oryza sativa)
<400> 1
atggccggaa aggatgaggc ggaagggctc gaggcgaggc tgctgctgct gccgcctgag 60
gcggcggcgg aggagccgac gaggtgtggc ggcggcgacg gcggcggcgg cgggaggaag 120
cggaagaaga cgtaccttga tgtccttggc gtgtgctgct ccgcggaggt cgcgctggtg 180
gagcggctgc tcgcgccgct cgacggcgtc cgcgtggtgt ccgtcgtcgt ggcgtcccgc 240
accgtcgtcg tcgagcacga ccccgccgcc gccccggagt ccgccatcgt gaaggcgctg 300
aacaaggccg gcctcgaggc gtcggtgcga gcctacggca gcagcggcgt ggtcagccgg 360
tggccgagcc cgtacatcgt cgcctccggc gtcctgctca cggcgtcctt cttcgagtgg 420
ctcttccctc ccctgcagtg cctggccgtg gcggccgtcg tcgccggcgc gccgccgatg 480
gtgcgccgtg ggttcgccgc ggcgagccgg ctgtcgctcg acatcaacgt cctcatgctc 540
atcgccgtcg ccggcgcgct ctgcctcggc gactacacgg aggccggcgc catcgtcttc 600
ctcttcacca ccgcagaatg gctcgagacg ctggcctgca caaaggcgag cgccgggatg 660
tcgtcgttga tgggcatgct gccggtgaag gcggtcatcg cgacgacggg cgaggttgtc 720
agcgtgcgcg acgtccgcgt gggcgacgtc gtcgcggtca gggccggcga gatcgtcccc 780
gtcgacggcg tggtggtcga cggccagagc gaggtcgacg agaggagcct caccggcgag 840
tccttcccgg tgccgaagca gccgcactcc gaggtctggg ccggcacaat gaacttagac 900
ggttacattg ctgtgagaac tacggctctc gccgagaact cgacggtggc gaagatggag 960
aggctggtgg aggcggcgca gaacagcagg tcgaagacgc agcggctgat cgattcgtgc 1020
gcaaagtact acacgccggc cgtggtggtt gttgcagcag gagtggccct gatcccggct 1080
ctgctcggag cagatggcct tgagcaatgg tggaagctgg ctctggtgat gcttgtgagc 1140
gcgtgcccct gcgcattagt gctgtcgaca ccggtggcat ccttctgcgc aatgctgcgc 1200
gctgcgagga tggggatctt catcaagggt ggagatgttc ttgaatcact tggggagatc 1260
agggccgtcg cgttcgacaa gaccgggacg atcaccagag gagagttcag catcgactcg 1320
ttccatctgg ttggggatca caaggttgag atggatcatc ttctttactg gattgcaagc 1380
attgagagca agtcaagcca cccaatggca gctgcacttg tggagtatgc tcaatccaaa 1440
tccatccaac caaacccgga aaatgtcggc gattttcgga tatatcccgg ggaggggatc 1500
tatggagaga tccatggaaa gcacatctac attgggaaca gaaggacatt ggcaagagcg 1560
tcatcacctc agtcaactca agaaatgggt gagatgatca agggcgtgtc gatcggctac 1620
gtgatctgcg acggcgagct cgccggcatc ttctcgctct ccgacgactg ccggaccggc 1680
gcggcggagg ctatccggga gcttggatcg ctgggcatca agtcggttat gctgaccggg 1740
gacagcagcg cggcggcgac gcacgcgcag ggccagctcg gcggcgtcat ggaggagctc 1800
cactccgagc tcctcccgga ggacaaggtc cggctcgtcg gcggcctcaa ggcgaggttc 1860
ggcccgacga tgatggttgg cgacgggatg aacgacgcgg cggcgctggc ggcggcggac 1920
gtgggcgtgt ccatgggcat ctccgggtcg gcggcggcga tggagaccag ccacgcgacg 1980
ctcatgtcga gcgacgtgct cagggtcccc gaggccgtca ggctcggccg gcgcgcccgc 2040
cggaccatcg ccgtcaacgt ggccggctcg gtggccgtga aggccgccgt gctcgcgctg 2100
gccgcggcgt ggcgccccgt gctctgggcc gccgtgctcg ccgacgtcgg gacgtgcctc 2160
ctcgtcgtgc tcaacagcat gacgctgctc agggaggagt ggaagggcgg cgccaaggag 2220
gacggcgcgt gccgcgccac ggcgaggtcg ctggcgatga ggtcccagct cgccgccgac 2280
tcccaagcac ccaacgccgc agacgctggc gccgccggcc gtgagcaaac gaacggctgc 2340
cgttgctgtc cgaagccggg catgtcgccc gagcactcgg ttgtcatcga catccgagcc 2400
gacggcgagc gtcaagaaga gcggccggcc gaggcggccg tcgttgccaa atgttgcggc 2460
ggaggcggcg gcgagggcat ccgctgcgga gcctccaaga agccaactgc aacggtcgtt 2520
gttgccaaat gttgcggggg cggcggcggc ggcgagggca ccaggtgcgg cgcgtccaag 2580
aatccggcaa cggcggccgt cgttgccaaa tgttgcagcg gaggcggcgg cgagggcatc 2640
ggctgcggag cctccaagaa gccaactgca acggccgttg ttgccaaatg ttgcggcggc 2700
ggcggcgagg gcaccaggtg cgccgcgtcc aagaagccgg caacggcggc cgtcgttgcc 2760
aaatgttgcg gtggcgatgg cggcgagggc accggatgtg gtgcctccaa gaggtcgccg 2820
ccggctgagg gaagctgcag cggcggtgaa ggcggtacca atggtgttgg tcgttgctgc 2880
acgagcgtga agaggccaac ctgttgcgac atgggagcgg cggaggtgtc cgattcttcg 2940
ccggagacgg cgaaagactg cagaaatggg aggtgttgcg cgaagacgat gaactccggt 3000
gaagtgaaag gatga 3015
<210> 2
<211> 337
<212> DNA
<213>Rice (Oryza sativa)
<400> 2
ctcgtcagcg gcctcaaggc gaggttcggc ccgacgatga tggtcgggga cgggatgaac 60
gacgcggcgg cgctggcggc ggcggacgtg ggcgtgtcca tgggcatctc cgggtcggcg 120
gcggcgatgg agaccagcca cgcgacgctc atgtcgagcg acgtgctcag ggtccccgag 180
gccgtcaggc tcggccggtg cgcccgccgg accatcgccg tcaacgtggc cggctcggtg 240
gccgtgaagg ccgccgtgct cgcgctggcc gcggcgtggc gccccgtgct ctgggccgcc 300
gtgctcgccg acgtcgggac gtgcctcctc gtcgtgc 337
<210> 3
<211> 337
<212> DNA
<213>Rice (Oryza sativa)
<400> 3
ctcgtcggcg gcctcaaggc gaggttcggc ccgacgatga tggttggcga cgggatgaac 60
gacgcggcgg cgctggcggc ggcggacgtg ggcgtgtcca tgggcatctc cgggtcggcg 120
gcggcgatgg agaccagcca cgcgacgctc atgtcgagcg acgtgctcag ggtccccgag 180
gccgtcaggc tcggccggcg cgcccgccgg accatcgccg tcaacgtggc cggctcggtg 240
gccgtgaagg ccgccgtgct cgcgctggcc gcggcgtggc gccccgtgct ctgggccgcc 300
gtgctcgccg acgtcgggac gtgcctcctc gtcgtgc 337
<210> 4
<211> 19
<212> DNA
<213>Rice (Oryza sativa)
<400> 4
ctcgtcagcg gcctcaagg 19
<210> 5
<211> 18
<212> DNA
<213>Rice (Oryza sativa)
<400> 5
gcacgacgag gaggcacg 18
Claims (10)
1. molecular labeling Caps7 in a kind of gene of rice Cd accumulation related gene OsHMA3, it is characterised in that：Gene OsHMA3
Including allele HMA3-1 and allele HMA3-2, the molecular labeling Caps7 can distinguish allele HMA3-1 and wait
The sequence difference of position gene HMA3-2.
2. molecular labeling Caps7 according to claim 1, which is characterized in that the PCR amplification of the molecular labeling Caps7
Primer pair is：
Forward primer F (SEQ ID NO:4)：CTCGTCAGCGGCCTCAAGG；
Reverse primer R (SEQ ID NO:5)：GCACGACGAGGAGGCACG；
The molecular labeling Caps7 is shown as：When carrying out PCR amplification using the primer pair, the amplification of allele HMA3-1
Product has SEQ ID NO:The amplified production of nucleotide sequence shown in 2, allele HMA3-2 has SEQ ID NO:Shown in 3
Nucleotide sequence；The allele HMA3-1 shows as low Cd accumulation, and the nucleotides sequence of the allele HMA3-2 is classified as
SEQ ID NO:Shown in 1, high Cd accumulation is shown as.
3. a kind of method that molecular labeling Caps7 using described in claim 2 distinguishes allele HMA3-1 and HMA3-2,
It is characterized in that：PCR amplification is carried out to oryza sativa genomic dna to be measured using primer pair described in claim 2, PCR product is used
AscI endonuclease digestions, agarose electrophoresis detect the amplified production after digestion, if agarose electrophoresis detection is shown as a band,
Then rice to be measured has allele HMA3-1；If agarose electrophoresis, which detects, is shown as two band, rice to be measured has etc.
Position gene HMA3-2, if agarose electrophoresis detection is shown as three band, the gene OsHMA3 of rice to be measured is heterozygous state.
4. the method according to claim 3 for distinguishing allele HMA3-1 and HMA3-2, it is characterised in that：If agarose
Electrophoresis detection is shown as a band, sequence length 337bp, then rice to be measured has allele HMA3-1, shows as low
Cd accumulation；If agarose electrophoresis detection is shown as two band, sequence length is respectively 196bp, 141bp, then rice tool to be measured
There is allele HMA3-2, show as high Cd accumulation, if agarose electrophoresis detection is shown as three band, sequence length is respectively
337bp, 196bp and 141bp, and the band that sequence length is 337bp is most bright, then the gene OsHMA3 of rice to be measured is heterozygosis shape
State.
5. the method according to claim 3 for distinguishing allele HMA3-1 and HMA3-2, which is characterized in that the PCR
System is：10~50ng of template DNA, forward primer and each 0.25um of reverse primer, Taq archaeal dna polymerases 1U, four kinds of deoxidation cores
Ribotide dATP, dTTP, dCTP, dGTP each 0.1mM, 2 × GC buffer I 10ul, rest part deionized water polishing
To 20ul；
The PCR response procedures are：First in 94 DEG C of pre-degeneration 4min, enters PCR cycle later and expand, cyclic amplification includes 94
DEG C denaturation 30sec, 60 DEG C or 62 DEG C annealing 30sec, 72 DEG C of extension 1min, totally three step, circular flow 30~34 times, finally 72
DEG C extend 10min after, 25 DEG C heat preservation 4min obtain amplified production；
The digestion system is 10ul systems, and following each component is mixed：Pcr amplification product 100~200ng, AscI restriction endonuclease
Buffer (10X) 1ul, AscI restriction endonuclease 2U, remaining uses deionized water polishing；Digestion program：The digestion system mixed is put
It is placed in 8~12h or more of reaction in 37 DEG C of insulating boxs and carries out digestion.
6. the use of molecular labeling Caps7 and its PCR amplification primer pair described in claim 2 in identifying rice Cd accumulation character
On the way.
7. the use of molecular labeling Caps7 and its PCR amplification primer pair in low Cd accumulation Indica Rice Varieties described in claim 2
On the way.
8. any one of claim 3 or claim 4 or claim 5 side for distinguishing allele HMA3-1 and HMA3-2
Purposes of the method in low Cd accumulation Indica Rice Varieties.
9. a kind of method of identification rice Cd accumulation character, it is characterised in that：Using the primer pair described in claim 2 to be measured
The genomic DNA of rice carries out the PCR amplification of molecular labeling Caps7 described in claim 2, and AscI endonuclease digestions, fine jade is used in combination
The length of lipolysaccharide electrophoresis detection PCR product determines rice Cd accumulation character；
If agarose electrophoresis detection is shown as a band, sequence length 337bp, then rice to be measured is with allele
HMA3-1 shows as low Cd accumulation；If agarose electrophoresis detection is shown as two band, sequence length be respectively 196bp,
141bp, then rice to be measured shows as high Cd accumulation with allele HMA3-2, if agarose electrophoresis detection is shown as three
Band, sequence length is respectively 337bp, 196bp and 141bp, and the band that sequence length is 337bp is most bright, then rice to be measured
Gene OsHMA3 be heterozygous state.
10. a kind of low Cd accumulation long-grained nonglutinous rice auxiliary breeding means, which is characterized in that the method includes：Pass through claim 3,4,5
Or claim 9 any one of them method, test right requires the molecular labeling Caps7 described in 2, with determination long-grained nonglutinous rice cadmium to be measured
Accumulate character.
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