CN104419752B - A kind of method detecting cattle FANCI gene delection and test kit - Google Patents
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Abstract
The invention discloses a kind of method for detecting cattle FANCI gene delection and test kit.Containing primer B being made up of by A and primer primer to group in test kit provided by the present invention, A is made up of by described primer two single stranded DNAs shown in sequence in sequence table 1 and sequence 2, and B is made up of by described primer two single stranded DNAs shown in the sequence 3 in sequence table and sequence 4.It is demonstrated experimentally that utilize primer provided by the present invention to group, cattle short spinal column syndrome (BS) genetic defect can be identified the most quickly, accurately and efficiently.The Molecular screening that the present invention is BS milch cow genetic defect gene provides reference and reference, provides technical support in a planned way reducing BS genetic defect gene frequency in breeding work from now on, and the seed selection and selective pairing carrying out science for cattle farm provides foundation.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of method for detecting cattle FANCI gene delection and test kit,
Particularly to a kind of for detecting the diagnostic kit whether relevant FANCI gene of spinal syndromes short to cattle lacks.
Background technology
Along with going deep into of genomics research, people gradually recognize that almost each animal can be carried some recessiveness and had
Evil gene, when they isozygoty, shows different types of illness symptom.In cattle breeding, due to progeny testing, genome
The application of the GENERALIZATION OF MODERN BREEDING TECHNIQUE such as selection, while drastically increasing selection accuracy, improves selection strong the most potentially
Degree；The most again because the application of the breeding biotechnology such as artificial insemination (AI), make indivedual outstanding breeding oxen be used widely, increase
The coefficient of inbreeding of Jia Liao colony, reduces effective population size.2010, Canada milch cow website (CDN) report Canada lotus
The average coefficient of inbreeding of Si Tanniu has reached 5.87%, and Jersey is 5.92%.In this case, what recessive deleterious alleles isozygotied is general
Rate increases, and genetic defect occurs the most therewith, and therefore every few years just has a kind of new genetic defect and occurs in some cattle breeds
In.As in He Sitan colony, citrullinemia (CN), bovine uridine monophosphate synthase deficiency disease since nineteen ninety, are the most in succession reported
(DUMPS) the milch cow genetic defect such as bovine leukocyte adhesion defects disease (BLAD), cattle deformity of spine syndrome (CVM).
2006, cattle short spinal column syndrome (Brachyspina syndrome, BS) was first in Denmark Holstein cow group
Secondary report, ill cattle clinical manifestation is that embryo's early abortion, hypoevolutism, extremity are elongated, cervical vertebra and thoracic vertebra serious curtailment, internal organs
The symptomes complices such as exception, and tentatively inferred that BS is probably emerging a kind of recessive inheritance's defect.This genetic defect gene
Find to cause cattle breeding association of various countries and the extensive concern of worker, subsequently, Italy in 2008, Germany in 2010,
Within 2010, Canada reports 2,1,1 ill stillborn fetus of BS the most in succession.7 the ill stillborn fetuses of BS found Europe altogether carry out pedigree
Analyze, find all to trace back to a common ancestor Sweat Haven Tradition(USAM1682485), demonstrate,prove the most further
Clear BS is not only present in the Holstein cow colony of Europe, also exists in the colony of North America, additionally in view of BS diseased individuals
The main breeding family related to, can be inferred that BS range of scatter and influence degree are the biggest.Until 2012, BS genetic defect
Just by the FANCI gene being successfully positioned at BTA21.
The molecular genetic mechanism of BS is the upper FANCI(Fanconi anemia of No. 21 chromosomes (BTA21) of cattle
Complementation-group I) deletion mutation of one section of 3329bp on gene, cause losing of 25,26,27 exons
Lose, there occurs that structure changes in 877 amino acids site: 451 amino acid whose peptide chains of C-terminal are by only containing 26 ammonia originally
The residual peptide of base acid is replaced.At present, BS Disease-causing gene can trace back to a famous U.S. breeding oxen Sweet Haven
Tradition(USAM1682485), it is born in 1974, but mainly by two son Bis-May Tradition
Cleitus(USAM1879085) and Rothrock Tradition Leadman(USAM1983348) spread.
Summary of the invention
It is an object of the invention to provide a kind of primer for detecting the short spinal syndromes of cattle to group.
Primer for detecting the short spinal syndromes of cattle provided by the present invention to group, specifically by primer to A and primer to B
Composition, A is made up of by described primer two single stranded DNAs shown in sequence in sequence table 1 and sequence 2, described primer to B by sequence
Sequence 3 in table and two single stranded DNA compositions shown in sequence 4.
In the present invention, the described detection short spinal syndromes of cattle is particularly as follows: detect FANCI gene in the genome of cattle to be measured
(the 9701-74362 position of GenBank:AC_000178.1, concrete network address is as follows: http: //
www.ncbi.nlm.nih.gov/nuccore/AC_000178.1?report=genbank&from=21124257&to=
21208316) whether there is the large fragment deletion of 3329bp.According to the described primer nucleotide sequence to A amplification products therefrom
As shown in sequence 6 in sequence table, in the genome of the most described cattle to be measured there is the large fragment deletion of 3329bp in FANCI gene, this
Illustrate that described cattle to be measured is cattle short spinal syndromes recessiveness carrier or cattle short spinal syndromes homozygote.The short spinal syndromes of cattle
Homozygote is most lethal embryo, seldom occurs in cows, and BS homozygote phenotype exists deformity, is not required to any inspection
Survey means can be identified.The purpose of the present invention is exactly examination cows short-and-medium spinal syndromes recessiveness carriers.
Described primer is 1:1:1:1 to the mol ratio in PCR reaction system of four single stranded DNAs in group.
In one embodiment of the invention, described primer to every single stranded DNA in group in multi-PRC reaction system
Final concentration be 240pM.
Wherein, sequence 1 is made up of 25 nucleotide；Sequence 2 is made up of 27 nucleotide；Sequence 3 is 20 nucleotide groups
Become；Sequence 4 is made up of 20 nucleotide；Sequence 5 is made up of 3738 nucleotide；Sequence 6 is made up of 409 nucleotide.
Containing described primer, the test kit of group is fallen within protection scope of the present invention.
It is also another object of the present invention to provide a kind of prepare described primer to group method.
Provided by the present invention prepare described primer to group method, specifically include described primer four lists in group
The step that chain DNA is individually packed.
The present invention a further object is a kind of method preparing described test kit of offer.
The method preparing described test kit provided by the present invention, specifically includes following steps: by described primer in group
Four single stranded DNAs individually pack after, be packaged in same reagent box with at least one in following substances: PCR reacts
Buffer, archaeal dna polymerase (such as Taq archaeal dna polymerase, specifically produce such as TaKaRa biological engineering (Dalian) company limited
R001B) and 4 kinds of dNTP TaKaRa Taq polymerase, its article No. is:.
Described primer is to group, or whether described test kit FANCI gene in preparation detects the genome of cattle to be measured lacks
Product in application fall within protection scope of the present invention.
It is a further object to provide the side that in a kind of genome detecting cattle to be measured, whether FANCI gene lacks
Method.
The method that in the genome of detection provided by the present invention cattle to be measured, whether FANCI gene lacks, specifically can include
Following steps:
(a1) with the genomic DNA of cattle to be measured as template, with described primer, group is carried out multiplexed PCR amplification, it is thus achieved that PCR
Product；
(a2) size of detecting step (a1) gained PCR primer, determines the genome of described cattle to be measured as follows
Whether middle FANCI gene lacks: in PCR primer, containing the DNA fragmentation that size is 269bp, (explanation PCR reaction itself is not asked
Topic, credible result) on the premise of, if containing size in described PCR primer is the DNA fragmentation of 409bp, the base of the most described cattle to be measured
Because in group, FANCI gene exists disappearance, show that described cattle to be measured is that cattle short spinal syndromes recessiveness carrier or the short vertebra of cattle are combined
Simulator sickness homozygote；If described PCR primer not containing the DNA fragmentation that size is 409bp, in the genome of the most described cattle to be measured
FANCI gene does not exist disappearance, shows that described cattle to be measured is normal individual, not cattle short spinal syndromes recessiveness carrier or
Cattle short spinal syndromes homozygote.
In actual applications, it is judged that when whether PCR primer contains target DNA fragment, can be by PCR primer be carried out fine jade
Sepharose electrophoresis, sees and whether contains purpose band on electrophoresis pattern: containing purpose band on electrophoresis pattern, then in PCR primer
Containing target DNA fragment；Otherwise, then PCR primer does not contains target DNA fragment.
In above-mentioned application or method, described disappearance specially lacks the 231-3559 of sequence 5 in described FANCI gene
Position.
Further, described size is that the DNA fragmentation of 409bp is specially the DNA fragmentation shown in sequence 6 in sequence table；Described
Size is that the DNA fragmentation of 269bp is specially the DNA fragmentation shown in sequence 7 in sequence table.
In the above-mentioned methods, with described primer, group carried out the annealing temperature of multiplexed PCR amplification and be specially 60 DEG C.
More specific, the reaction condition of described multiplex PCR is: 94 DEG C of denaturations 5min；94 DEG C of degeneration 30s, 60 DEG C are moved back
Fire 45s, 72 DEG C extend 45s, carry out 35 circulations；72 DEG C extend 10min.
In the present invention, above all of described cattle to be measured is milch cow, and specially Holstein cow is more specific such as Beijing
The Holstein sire in area and holstein cows.
It is demonstrated experimentally that utilize primer provided by the present invention to group, use cheap common archaeal dna polymerase (such as Taq
Archaeal dna polymerase) cattle short spinal column syndrome (BS) genetic defect can be identified quickly, accurately and efficiently.The present invention is that BS milch cow is lost
The Molecular screening passing dcc gene provides reference and reference, in a planned way reducing BS genetic defect in breeding work from now on
Gene frequency provides technical support, and the seed selection and selective pairing carrying out science for cattle farm provides foundation.
Accompanying drawing explanation
Fig. 1 is the test kit utilizing embodiment 1 to prepare, and by multiplexed PCR amplification, whether detection cattle FANCI gene lacks
Agarose gel electrophoretogram.Wherein, swimming lane M is pBR322DNA/MSPI ladder Marker；Swimming lane 1,3,4 and 5 is
Normal cattle (non-BS genetic defect cattle)；Swimming lane 2 is BS genetic defect carrier.
Fig. 2 is BY-F/BY-R primer (i.e. the primer described in literary composition is to A) substance PCR based on LA-Taq polymerase detection
The agarose gel electrophoretogram whether cattle FANCI gene lacks.Wherein, swimming lane M1 is pBR322DNA/Mspl；Swimming lane M2 is
1kb DNA Ladder；Swimming lane AA is normal cattle (non-BS genetic defect cattle)；Swimming lane AB is BS genetic defect carrier.
Detailed description of the invention
Experimental technique used in following embodiment if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, the most commercially obtain.
Embodiment 1, for detecting the preparation of test kit of the short spinal syndromes of cattle
Provided in the present embodiment for detecting the test kit of the short spinal syndromes of cattle, for multiple PCR detection kit,
Containing being used for the primer detecting cattle FANCI gene delection, B, PCR reaction buffer, Taq DNA are polymerized by A and internal reference primer
Enzyme, and 4 kinds of dNTP.Wherein, specific as follows to group for detecting the primer of cattle FANCI gene:
1, BY-F/BY-R primer
According on GenBank cattle FANCI gene order (the 9701-74362 position of GenBank:AC_000178.1,
The concrete network address of GenBank:AC_000178.1 sees as follows: http://www.ncbi.nlm.nih.gov/nuccore/AC_
000178.1?Report=genbank&from=21124257&to=21208316), and with reference to Carole et al(2012) pin
Primer sequence to FANCI gene design, it is determined that the specific primer BY of detection BS genetic defect cattle:
Forward primer BY-F(sequence 1): 5 '-GCTCAAGTAGTTAGTTGCTCCACTG-3 ' (the 1-25 position of sequence 5)；
Downstream primer BY-R(sequence 2): 5 '-the ATAAATAAATAAAGCAGGATGCTGAAA-3 ' (3712-of sequence 5
The reverse complementary sequence of 3738).
When cattle to be measured is healthy cattle (non-BS genetic defect cattle), use with as above primer carrying out PCR amplification, in theory
Target product is 3738bp(sequence 5), but owing to target product is excessive, use common archaeal dna polymerase (such as Taq archaeal dna polymerase)
Carry out PCR amplification, it is impossible to realize amplification, i.e. without amplified production.When cattle to be measured is BS carrier, use as above primer to carrying out
PCR expands, and can obtain the target DNA fragment (sequence 6) that size is 409bp.
2, mtATP-F/mtATP-R primer
According to the Mitochondrial gene sequence (NO:HQ184035.1) of cattle, devise internal standard upstream and downstream primer:
Forward primer mtATP-F(sequence 3): 5 '-TAAGTTAGAGATTGAGAGCC-3 ' (the 1-20 position of sequence 7)；
Downstream primer mtATP-R(sequence 4): 5 '-GATAAGGGTTACGAGAGGGA-3 ' (the 250-269 position of sequence 7
Reverse complementary sequence).
With the genomic DNA of cattle to be measured as template, using above primer to carrying out PCR amplification, its expanding fragment length is
269bp, its nucleotide sequence is as shown in sequence 7 in sequence table.
Embodiment 2, the test kit detection short spinal syndromes of cattle prepared by embodiment 1
One, the multi-PRC reaction system of test kit of embodiment 1 preparation and the optimization of response procedures
1, the extraction of cattle STb gene
The Holstein sire selecting Beijing's Breeding bull station (includes potential BS genetic defect carrier, i.e. by U.S. lotus
This smooth association website carries out the offspring of the BS carrier of pedigree verification) it is experiment material, extract total from fresh in vitro blood
DNA, expands template as PCR.Certainly STb gene is possible not only to extract from blood (such as, fresh or freezing), it is also possible to from
Seminal fluid (such as, fresh or freezing), tissue sample (such as ear tissue etc.) or containing hair follicle ox hair sample in extracting and developing and
Purification.
2, BY-F/BY-R primer substance PCR amplification
The test kit that the present inventor uses embodiment 1 to prepare carries out BY-F/BY-R primer substance PCR(TaqDNA
Polymerase), and response procedures is optimized.
Reaction system (25 μ l): dNTP mixture (4 × 2.5mmol/L) 2.0 μ l；10 × amplification buffer is (containing Mg2+) 2.5
μl；Forward primer BY-F(20pmol/L) 0.3 μ l；Downstream primer BY-R(20pmol/L) 0.3 μ l；Taq archaeal dna polymerase (3U/ μ
L) 0.5 μ l；Template DNA (30ng/ μ l) 1.0 μ l；ddH2O complements to 25 μ l.
Response procedures: 94 DEG C of denaturations 5min；94 DEG C of degeneration 30s, 55 ± 10 DEG C of annealing 30s, 72 DEG C extend 30s, carry out
35 circulations；72 DEG C extend 7min, then 4 DEG C of preservations.
Result shows: use the test kit of embodiment 1 preparation to carry out BY-F/BY-R primer substance PCR(Taq DNA polymerization
Enzyme) annealing temperature be advisable with 58 DEG C.
3, mtATP-F/mtATP-R primer substance PCR amplification
The test kit that the present inventor uses embodiment 1 to prepare carries out mtATP-F/mtATP-R primer substance PCR
(Taq archaeal dna polymerase), and response procedures is optimized.
Reaction system (25 μ l): dNTP mixture (4 × 2.5mmol/L) 2.0 μ l；10 × amplification buffer is (containing Mg2+) 2.5
μl；Forward primer BY-F(20pmol/L) 0.3 μ l；Downstream primer BY-R(20pmol/L) 0.3 μ l；Taq archaeal dna polymerase (3U/ μ
L) 0.5 μ l；Template DNA (30ng/ μ l) 1.0 μ l；ddH2O complements to 25 μ l.
Response procedures: 94 DEG C of denaturations 5min；94 DEG C of degeneration 30s, 55 ± 8 DEG C of annealing 30s, 72 DEG C extend 30s, carry out 35
Individual circulation；72 DEG C extend 7min, then 4 DEG C of preservations.
Result shows: use the test kit of embodiment 1 preparation to carry out mtATP-F/mtATP-R primer substance PCR(Taq
Archaeal dna polymerase) annealing temperature be advisable with 56 DEG C.
4, multiplexed PCR amplification condition optimizing
The test kit that the present inventor uses embodiment 1 to prepare carries out primer, and to the multiplex PCR of group, (Taq DNA gathers
Synthase), and reaction system and response procedures are optimized, are finally defined below reaction system and response procedures:
Multi-PRC reaction system (25 μ l): dNTP mixture (4 × 2.5mmol/L) 2.0 μ l；10 × amplification buffer (contains
Mg2+) 2.5 μ l；Primer BY-F(20pmol/L) 0.3 μ l；Primer BY-R(20pmol/L) 0.3 μ l；Primer mtATP-F(20pmol/
L) 0.3 μ l；Primer mtATPR(20pmol/L) 0.3 μ l；Taq archaeal dna polymerase (3U/ μ l) 0.5 μ l；Template DNA (30ng/ μ l)
1.0μl；ddH2O complements to 25 μ l.
Multi-PRC reaction condition is: 94 DEG C of denaturations 5min；94 DEG C of degeneration 30s, 60 DEG C of annealing 45s, 72 DEG C extend 45s,
Carry out 35 circulations；72 DEG C extend 10min, then 4 DEG C of preservations.
Two, the multi-PRC reaction amplification of the test kit of embodiment 1 preparation judges
In the multiple PCR detection kit of embodiment 1 preparation, mtATP-F/mtATP-R primer is as interior label primer, right
Quality Control effect is played itself in PCR reaction, is 269bp(sequence 7 when amplification obtains size) DNA fragmentation time, reliable experiment result.
Therefore, the test kit using embodiment 1 preparation being carried out the primer multiplex PCR (Taq archaeal dna polymerase) to group, result judges mark
Accurate as follows: if PCR expands obtains DNA fragmentation (sequence 6) and the 269bp(sequence 7 that size is 409bp) DNA fragmentation, the most to be measured
Cattle is BS genetic defect gene carrier, (i.e. the 231-3559 position of FANCI gene delection sequence 5 in genome)；If amplification
Do not obtain the DNA fragmentation (sequence 6) that size is 409bp, but there is 269bp(sequence 7) DNA fragmentation time, cattle the most to be measured is
Normal cattle (i.e. in genome, FANCI gene does not lacks).
In short, cow genome type to be measured judges as follows:
Healthy cattle (non-BS genetic defect cattle): genotype is AA type, 269bp internal standard electrophoresis band only occurs；
, i.e. there is marker tape and 409bp electrophoresis band in 269bp in BS recessiveness carrier: genotype is AB type.
BS homozygote is most lethal embryo, seldom occurs, examination recessive inheritance's defect base of the present invention in cows
Because the purpose of BS finds out BS recessiveness carrier exactly.
Three, with the actual application of the test kit detection short spinal syndromes of cattle of embodiment 1 preparation
Cattle to be measured: 206 Holstein sire of Beijing area and 136 holstein cows.
1, the multiplex PCR detection of the test kit of embodiment 1 preparation
Extract the genomic DNA of cattle to be measured, with it as template, utilize test kit prepared by embodiment 1, according to step one 4
Optimization after reaction system (archaeal dna polymerase be TaKaRa biological engineering (Dalian) company limited produce TaKaRaTaq polymerization
R001B) and response procedures carries out multiplexed PCR amplification enzyme, is a kind of general T aq enzyme, and its article No. is:.After reaction terminates, take 5 μ l
PCR primer detects by 2% sepharose electrophoresis, and judges result according to standard described in step 2.
Result shows: have 10 bulls and 3 cows to obtain size through multiplexed PCR amplification is 269bp's and 409bp simultaneously
Two purpose bands, it is determined that these 13 cattle are BS recessiveness carrier, calculate the BS carrying rate of bull and cow be respectively 4.85%,
2.21%.The PCR amplification of part cattle to be measured is as shown in Figure 1.The purpose band of obtained 269bp and 409bp is entered respectively
Row sequencing, the nucleotide sequence of the purpose band of result 409bp as shown in sequence 6, the nucleoside of the purpose band of 269bp
Acid sequence is as shown in sequence 7.
2, pedigree checking
Being followed the trail of by pedigree and find, the common ancestor of 10 bulls being accredited as BS recessiveness carrier through step 1 is Sweet
Haven Tradition(USAM1682485) (and son Cleitus(USA1879085), with the common ancestral reported in the world
The most consistent, more confirm the accuracy of institute's method for building up.
3, BY-F/BY-R primer substance PCR based on LA-Taq polymerase detection
In order to be further characterized by multi-PCR detection method provided by the present invention accurately and reliably, the present inventor
206 Holstein sire and 136 holstein cows as cattle to be measured have been carried out based on LA-Taq polymerase the most simultaneously
BY-F/BY-R primer substance PCR detects.Wherein, LA-Taq polymerase is purchased from TaKaRa biological engineering (Dalian) company limited, goods
Number it is: RR02MA, for a kind of archaeal dna polymerase expanding long segment, uses this archaeal dna polymerase to carry out PCR amplification, if cattle to be measured is
Purpose band (the 1-of sequence 5 of the amplifiable 3738bp obtaining being consistent with theoretical size of healthy cattle (non-BS genetic defect cattle)
3738), if cattle to be measured is BS recessiveness carrier, expand the band (sequence 6) obtaining 409bp mesh.
Reaction system (25 μ l): dNTP mixture (4 × 2.5mmol/L) 4.0 μ l；10 × LA PCR buffer is (containing Mg2+)
2.5μl；Forward primer BY-F(20pmol/L) 0.5 μ l；Downstream primer BY-R(20pmol/L) 0.5 μ l；LA-Taq DNA is polymerized
Enzyme (5U/ μ l) 0.3 μ l；Template DNA (30ng/ μ l) 1.0 μ l；ddH2O complements to 25 μ l.
Response procedures: 94 DEG C of denaturations 5min；94 DEG C of degeneration 30s, 58 DEG C of annealing 1min, 72 DEG C extend 2.5min, carry out
35 circulations；72 DEG C extend 10min, then 4 DEG C of preservations.
Result shows: be accredited as 10 bulls of BS recessiveness carrier and 3 cows are expanded through above substance PCR through step 1
Increasing and all obtained the purpose band that size is 409bp, remaining cattle the most only obtains the purpose band that size is 3738bp.Part is treated
Survey the PCR amplification of cattle as shown in Figure 2.
The result of three aspects in comprehensive above 1-3, it is seen that the test kit that the present invention utilizes embodiment 1 to prepare uses multiple
PCR detects whether cattle to be measured is BS genetic defect carrier, and it tests reliable results, and is not necessarily dependent on special DNA polymerization
Enzyme TaKaRa LA-Taq polymerase.It addition, the present invention has carried out the molecule of BS genetic defect gene to China's Holstein cow
Diagnosis, and confirm that BS genetic defect exists certain proportion in China Holstein cow group, it is therefore necessary to China's Holstein cow
Group carries out large-scale examination, identifies or eliminate BS genetic defect gene carrier, and it is potential that the reduction new genetic defect of BS is brought
Harm.
Claims (7)
1., for detecting the primer of the short spinal syndromes of cattle to group, primer, B is made up of by A and primer, described primer to A by sequence
Sequence 1 and two single stranded DNAs compositions shown in sequence 2 in list, described primer to B by the sequence 3 in sequence table and sequence 4 institute
Two the single stranded DNA compositions shown；
Described primer is 1:1:1:1 to the mol ratio of four single stranded DNAs in group.
2. contain the test kit to group of the primer described in claim 1.
3. the preparation method of test kit described in claim 2, comprises the steps: described primer four strands in group
After DNA individually packs, it is packaged in same reagent box with at least one in following substances: PCR reaction buffer, DNA
Polymerase and 4 kinds of dNTP.
4. primer described in claim 1 is to the group product that whether FANCI gene lacks in preparation detects the genome of cattle to be measured
In application.
Application the most according to claim 4, it is characterised in that: described disappearance is the 231-3559 position of deletion sequence 5.
6. in the product that whether FANCI gene lacks in preparation detects the genome of cattle to be measured of test kit described in claim 2
Application.
Application the most according to claim 6, it is characterised in that: described disappearance is the 231-3559 position of deletion sequence 5.
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| 《特别报道——一种新的荷斯坦牛遗传缺陷短脊椎综合征》;马春生;《中国奶牛》;20090430(第4期);26-28 * |
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