CN1958810A - Method for detecting CVM deleterious gene of oxen - Google Patents

Abstract

This invention provides a method for detecting genes related to milk cow complex vertebral malformation (CVM). The method comprises: (1) designing and synthesizing a pair of primers shown in SEQ ID No.1 and 2; (2) performing PCR on a bovine DNA sample with the primers, and annealing at 57 deg.C to obtain the nucleotide sequence shown in SEQ ID No.3; (3) performing single strand conformation polymorphism (SSCP) analysis, and determining according to the result of polyacrylamide gel electrophoresis. The method is simple, rapid ad accurate. According to the method, relevant test kit can be developed. This invention can be used for dividing and screening the genes related to milk cow CVM, and breeding milk cows.

Description

A kind of method that detects CVM deleterious gene of oxen

Technical field

The present invention relates to biological technical field, be specifically related to the detrimental molecular detecting method of a kind of ox spine malformation syndromes (CVM).

Background technology

Calendar year 2001 Denmark Agerholm etc. ^[1]Found to exist in the He Sitan cows recessive inheritance dcc gene, very fast death after can causing pregnant early abortion, stillborn foetus or calf to be born when isozygotying, suffer from that to raise notable attribute be syndromes such as hamstring tendon shortenings before the spinal curvature deformity, two, neck weak point, heart malformations, so be called " spine malformation syndromes " (ComplexVertebral Malformation is called for short CVM), be that (G → T) cause is recessive inheritance for single base mutation by SLC35A3 gene on the euchromosome ^[1,2]

The discovery of CVM hereditary defect has caused various countries cattle breeding association and breeding work person's attention at once, in the U.S. ^[3], Britain ^[4]And Japan ^[5]Reported in succession in country and in the He Sitan cows, to have had the CVM deleterious gene.A large amount of gene test and the pedigree analysises of the U.S., Canada and some countries of Europe found subsequently, this recessive inheritance mutator gene can be traced back to bull " Carlin-M Ivanhoe Bell " family that the U.S. is very famous, this bull may be the common ancestor of this hereditary defect gene, and this bull is extensive use of in the whole world.In preceding 100 the outstanding breeding oxen of U.S. TPI in 2004, the CVM carrier accounts for 17%, an outstanding breeding oxen can produce hundreds of thousands of agent even up to a million doses of frozen semens all one's life, because popularizing and widespread use of frozen semen and technology of artificial insemination, outstanding breeding oxen hereditation is conspicuous.As seen, what the CVM hereditary defect of finding in the He Sitan cows was brought is worldwide problem, and this causes in recent years the cattle breeding worker when paying attention to outstanding breeding oxen production performance performance, payes attention to outstanding breeding oxen more and whether carries recessive deleterious gene.

By gene test and molecule breeding technique, can on molecular level, solve the genetic improvement problem of milk cow, on the one hand can find that by technique of gene detection breeding oxen or seed cow carry recessive detrimental situation, and selected to reject, avoid propagating the risk of hereditary defect gene by technology of artificial insemination; Can position analysis to quantity character gene (QTL) by genetic marker gene or candidate gene on the other hand, realize early stage selection, can shorten the milk cow generation interval, accelerate genetic improvement speed the reserve breeding oxen.Milk industry such as America and Europe developed country is use molecule breeding technique in the seed selection of bullock and in eliminating.According to relevant expert prediction, along with milk cow and other main domestic animal variety and qualities with the quantitative character gene mapping is finished and the infiltration of molecule breeding technique, revolutionary change will take place in whole breeding system.

In recent years, at all corresponding molecular detecting method and the breeding system of setting up breeding oxen hereditary defect gene of the U.S., Canada, Japan and other countries, and on the breeding oxen pedigree, clearly mark the recessive detrimental situation of carrying, reasonably instruct the seed selection and selective pairing of breeding oxen, effectively reduced the detrimentally affect of recessive deleterious gene CVM.At present, the external detrimental molecular detecting method of CVM mainly contains AS-PCR (allele-specific polymerase chainreaction) method and PCR-PIRA (polymerase chain reaction-primer introducedrestriction analysis) method ^[6], the AS-PCR method has very high request and must just can reach certain accuracy by strict control reaction conditions archaeal dna polymerase; Though and the PCR-PIRA method improves to some extent than AS-PCR method, the PCR-PIRA method requires to set up positive control could guarantee accuracy, and is not suitable for the mass-producing detection, and cost is higher.So, set up a kind of accurate, quick, economic detrimental molecular method of detection CVM and be very important.

In China's breeding oxen seed selection, do not carry out recessive detrimental detection of CVM and molecule breeding technique as yet, the detrimental situation of carrying of CVM in breeding oxen and the milk cow colony be it be unclear that.Actively utilize molecular genetics and molecular biotechnology means to set up recessive detrimental detection method of breeding oxen and molecule breeding technique, dwindle gap with milk industry developed country, to cultivating Chinese elite reserve breeding oxen voluntarily, it is crucial improving the breeding oxen breeding of method.The present invention will introduce detrimental fast, method-SSCP (Single-Strand Conformation Polymorphism, the single strand conformation polymorphism) method accurately of a kind of brand-new detection CVM.

SSCP method ultimate principle: pcr amplification target DNA, then with special pcr amplification product sex change, snapback then, make it to become single strand dna with certain space structure, the single stranded DNA fragment is complicated space folded conformation, this three-dimensional arrangement is mainly kept by interaction force in its inner base pairing equimolecular, when a base changes, can influence its space conformation more or less, conformation is changed, and the discrepant single strand dna of space conformation is varied in size by exclusion power in polyacrylamide gel.Therefore, by native polyacrylamide gel electrophoresis (PAGE), can very observantly discrepant molecular separation on the conformation be opened.

The PCR-SSCP technology ^[7,8]Be to detect dna mutation one of experimental technique means the most intuitively and accurately, the sensitivity that the dna fragmentation sudden change between 100-300bp detects to length of this method can reach 100%, binding sequence is analyzed, can accomplish " full scan " analysis to the target DNA fragment of batch samples, not only cost is low, and level of automation is higher, therefore, carrying out gene diagnosis and polymorphic analysis, comprise in the detection of mononucleotide polymorphic (SNP) PCR-SSCP technology a kind of accurate, quick, easy, economic technique means of can yet be regarded as.

Summary of the invention

The object of the present invention is to provide the detrimental molecular detecting method of a kind of ox spine malformation syndromes (CVM).

The present invention goes up ox SLC35A3 gene order (NO:AY160683) design according to GenBank and detects the detrimental specific PCR primer of CVM, the length of PCR product is all about 150-300bp, be fit to adopt SSCP to detect, by screening and optimization to the PCR primer, the Auele Specific Primer that has found suitable SSCP to detect, thereby created a cover and detected the detrimental method fast and accurately of CVM, accuracy rate can reach more than 99%.

The primer that the present invention design, screening obtain by this primer, carries out pcr amplification to total DNA of ox shown in sequence table SEQ ID NO:1 and 2, the amplified production sequence is carried out sscp analysis to amplified production shown in sequence table SEQ ID NO.3.Ox spine malformation syndromes (CVM) carrier compare with normal ox, because the sudden change of G → T, make to have 4 strands in the amplified production, and normal ox has only 2 strand (see figure 3)s.Check order filtering out different genotype, determine that 5 ' end the 73rd bit base from SEQ ID NO.3 is G or N, further verify the reliability of SSCP.Experimental result shows that accuracy rate can reach more than 99%.

In the present invention, the 73rd bit base among the described SEQ ID NO.3 is the result that N is meant order-checking, and what show is NF base, and in fact these site two kinds of situations of existence are G and T in sample, and sequencing result is shown as N.The present invention also is illustrated in the base in this site with N.

The present invention has also further optimized the pcr amplification condition, and the amplification condition of optimization is: 94 ℃ of pre-sex change 5min; 94 ℃ of sex change 30s, 57 ℃ of annealing 30s, 72 ℃ are extended 30s, carry out 35 circulations; 72 ℃ are extended 7min.

Need to prove that the present invention is not a kind of detection method of disease.For ox spine malformation syndromes, when the 73rd bit base that is to say SEQ ID NO.3 is isozygotying of T, very fast death after can causing pregnant early abortion, stillborn foetus or calf to be born, suffer from that to raise notable attribute be syndromes such as hamstring tendon shortenings before the spinal curvature deformity, two, neck weak point, heart malformations, these are remarkable symptoms, do not need special detection.And of the present invention to liking the normal ox of sign, by the detection CVM deleterious gene of oxen, thereby the carrier of eliminating CVM gets rid of the hidden danger in the ox breeding.

The present invention has set up a kind of method by PCR-SSCP analyzing and testing CVM deleterious gene of oxen carrier.Method of the present invention is simple, quick, accurate, and can further develop detection kit by method of the present invention.The present invention provides new method and thinking for the detrimental somatotype of milk cow CVM and screening, for the molecule seed selection of China milk cow provides reliable theoretical foundation.

Description of drawings

Fig. 1 is the gel electrophoresis figure of PCR product, and the annealing temperature of swimming lane 1 is 65 ℃, and swimming lane 12 temperature are 45 ℃, swimming lane 1～12 lapse of temperature, and M is 100bp ladder Mark;

Fig. 2 is the gel electrophoresis figure of PCR product, and swimming lane 1～6 is the PCR product, and M is the 100bp molecular weight standard, and annealing temperature is 57 ℃;

Fig. 3 is PCR-SSCP analyzing and testing figure, and wherein the AA type is normal ox, and the AB type is the recessive carrier of CVM;

Fig. 4 is the order-checking peak figure of PCR product, and part shown in the arrow is the 73rd bit base of SEQ ID NO.3, is the AA type above, is the BB type below.

Embodiment

The present invention will further describe in the following embodiments, and wherein, molecular biological routine operation adopts method well known to those skilled in the art and technology to carry out.Ox DNA sample among the embodiment is from the He Sitan breeding oxen at Beijing breeding oxen station, genomic dna can be from the blood of ox (for example, fresh or refrigerated), seminal fluid (for example, fresh or refrigerated), tissue sample (as ear tissue etc.) or contain extraction in the ox hair sample of hair follicle, separation and purifying.

The optimization of embodiment 1 primer design and amplification condition

Go up ox SLC35A3 gene order (NO:AY160683) according to GenBank, detect the detrimental specific PCR primer of CVM with the Oligo6.0 design, the length of PCR product is all about 150-300bp, be fit to adopt SSCP to detect, by to PCR primer design, screening and optimization, finally determine following primer sequence:

CVMF (upstream): 5-TGGCCCTCAGATTCTCAAGAG-3 '

CVMR (downstream): 5-CCAAGTTGAATGTTTCTTATC-3 '

PCR primer optimal conditions is: 94 ℃ of pre-sex change 5min; 94 ℃ of sex change 30s, 55 ± 10 ℃ of annealing 30s, 72 ℃ are extended 30s, carry out 35 circulations; 72 ℃ are extended 7min.The agarose electrophoresis detected result of PCR condition optimizing as shown in Figure 1, according to the detected result of PCR condition optimizing, the annealing temperature of determining this primer is 57 ℃.

Reaction system is: primer is 25pmol, template DNA 50ng, and Taq enzyme 0.5U, dNTP concentration is 100umol/L, and the total reaction system is 25ul, and PCR product length is 173bp.

With above-mentioned primer and reaction system, total DNA carries out pcr amplification to ox, get the 3ulPCR product and detect with 2% agarose electrophoresis, the pcr amplification result as shown in Figure 2, amplified band is single, concentration is higher, can be used for SSCP and detects.

Embodiment 2 12% non-denaturing polyacrylamide gels are made and electrophoresis

1. the cleaning sheet glass is dried the slit between 0.8% agarose closed glass plate and adhesive tape;

2. third rare acid amides glue working fluid (30% acrylamide 12.0ml, 50% glycerine 3ml, 10 * TBE 3ml, 10% ammonium persulphate, 200 μ l, TEMED12 μ l, pure water 12ml) of preparation 30ml12% mixes back encapsulating rapidly;

3. stop encapsulating when watering extremely from sheet glass upper edge 0.1cm, insert comb, polymerized at room temperature is about half an hour, and unnecessary acrylamide is preserved down for 4 ℃.And observe the gel polymerisation situation at any time, add acrylamide;

4. after the gel polymerisation, in electrophoresis chamber, add 1 * TBE, use the irrigation with syringe well;

5. prerunning 20min prepares to go up sample simultaneously;

6. get 1.5 μ l PCR products and place the PCR pipe, add 6 μ l sex change Buffer, 98 ℃ of sex change 10min place rapidly on ice, ice bath 5min, point sample;

7.120V, electrophoresis 14-16h.

The PCR-SSCP of embodiment 3 CVM analyzes

Utilize that selected specific PCR primer CVMF/CVMR carries out pcr amplification in the DNA sample, embodiment 1 of breeding oxen, this amplified fragments comprises the G → T base mutation that causes CVM, get 1.5 μ l PCR products and place the PCR pipe, add 6 μ l sex change Buffer, 98 ℃ of sex change 10min, place immediately on ice until electrophoresis, adopt the method among the embodiment 2 to make 12% acr: bis is that 29: 1 polyacrylamide gel carries out electrophoresis, 120V, 14-16h, after gel electrophoresis finishes, the silver staining method dyeing of (1991) such as employing Bassam ^[9], find to have 2 kinds of different genotype: AA type and AB type, the gene type result as shown in Figure 3.Find that through order-checking the 73rd bit base of SLC35A3 gene the 4th exon (exon4) of AA type (identical with SEQ ID NO.3 sequence) is G, this genotype belongs to normal individual, with " TV " expression; And the 4th exon (exon4) the 73rd bit base of AB type is N, and this genotypic individuality is the CVM carrier, with " CV " expression.DNA sample to 54 breeding oxens has carried out sscp analysis, and the result shows, 47 of AA genotype, and 7 of AB genotype that is to say, in 54 breeding oxens that detected, have 7 to be the CVM carrier.

The purifying and the sequencing of embodiment 4 pcr amplification products

Pcr amplification product with glass milk elution method recovery CVM uses ABI PRISM377DNA sequenator directly to carry out forward and reverse order-checking, further verifies gene type result among the embodiment 2.Sequencing result shows that the gene type result among the embodiment 2 is correct, and rate of accuracy reached illustrates that the detrimental method of setting up of detection CVM is feasible more than 99%.The CVM deleterious gene sequence that pcr amplification product obtains by checking order is shown in SEQ ID NO.3, and the 4th exon G73T missense mutation of SLC35A3 gene is the base mutation that causes CVM.The sequencing result of different genotype as shown in Figure 4.

Utilize the molecular detecting method of above-mentioned foundation, 54 breeding oxens at breeding oxen station have been carried out the detection that the recessive deleterious gene of CVM carries situation, detected result shows that it is the recessive deleterious gene carrier of CVM that 7 breeding oxens are arranged, and carrying rate is 13%.

Reference

1.Agerholm，J.S.，Bendixen，C.，Andersen，O.and?Arnbjerg，J.2001.Complexvertebral?malformation?in?Holstein?calves.J.Vet.Diagn.Invest.13：283-289.

2.Thomsen，B.，Horn，P.，Panitz，F.2002.A?missense?mutation?in?SLC35A3，encodingan?UDP-N-acetylglucosamine?transporter，causes?vertebral?malformation?in?cattle.GenBank?accession?number：AY160683.

3.Duncan，R.B.Jr.，Carrig，C.B.，Agerholm，J.S.and?Bendixen，C.2001.Complexvertebral?malformation?in?a?Holstein?calf：report?of?a?case?in?the?USA.J.Vet.Diagn.Invest.13：333-336.

4.Revell，S.2001.Complex?vertebral?malformation?in?a?Holstein?calfin?the?UK.Vet.Rec.149：659-660.

5.Nagahata?H，Oota?H，Nitanai?A，et?al.2002.Complex?Vertebral?Malformation?in?aStillborn?Holstein?Calf?in?Japan.J.Vet.Med.Sci.64(12)：1107-1112.

6.Kanae?Y，Endoh?D，Nagahata?H2?et?al.?2005.A?method?for?detecting?complexvertebral?malformation?in?Holstein?calves?using?polymerase?chain?reaction-primerintroduced?restriction?analysis.J?Vet?Diagn?Invest.17(3)：258-262.

7.Orita?M，Iwahana?H，Kanazawa?H.1989.Detection?of?polymorphisms?of?humanDNA?by?gel?electrophoresis?as?single-strand?conformational?polymorphisms.Proc?NatlAcad?Sci，USA.86：2766-2770.

8.Orita?M，Suzuki?Y，Sekiya?T，et?al.1989.A?rapid?and?sensitive?detetion?of?pointmutation?and?genetic?polymorphism?using?polymerase?chain?reaction.Genomics.5：874-879.

9.Bassam?BJ，Caetano-Anolles?G，Gresshoff?PM.1991.Fast?and?sensitive?silverstaining?of?DNA?in?polyacrylamide?gels.Anal?Biochem.196：80-83.

Sequence table

＜110〉Beijing Milk Cow Center

＜120〉a kind of method that detects CVM deleterious gene of oxen

<130>

<160>3

<170>PatentIn?version?3.3

<210>1

<211>21

<212>DNA

＜213〉artificial sequence

<400>1

tggccctcag?attctcaaga?g 21

<210>2

<211>21

<212>DNA

＜213〉artificial sequence

<400>2

ccaagttgaa?tgtttcttat?c 21

<210>3

<211>173

<212>DNA

＜213〉ox Bos taurus

<220>

<221>misc_feature

<222>(73)..(73)

＜223〉" n " expression " g " or " t "

<400>3

tggccctcag?attctcaaga?gcttaattct?aaggaacttt?cagctggctc?acaatttgta 60

ggtctcatgg?canttctcac?agcatgtttt?tccagtggct?ttgctggggt?ttactttgag 120

aaaatcttaa?aagaaaccaa?acaatcagtg?tggataagaa?acattcaact?tgg 173

Claims (4)

1, a kind of specificity amplification primer, it has the sequence shown in SEQ ID NO.1 and 2.

2, a kind of method that detects CVM deleterious gene of oxen is characterized in that using the described primer of claim 1, ox DNA sample is carried out PCR-SSCP analyze, according to the polyacrylamide gel electrophoresis genotype of judgement sample as a result.

3, method as claimed in claim 2, wherein the reaction conditions of PCR is: 94 ℃ of pre-sex change 5min; 94 ℃ of sex change 30s, 57 ℃ of annealing 30s, 72 ℃ are extended 30s, carry out 35 circulations; 72 ℃ are extended 7min.

4, the detection kit that contains the described primer of claim 1.

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