CN101629206A

CN101629206A - Quick detection method for FAD2 gene expression of different sources in brassica napus seeds

Info

Publication number: CN101629206A
Application number: CN200810144001A
Authority: CN
Inventors: 刘春林; 阮颖; 彭琦
Original assignee: Individual
Current assignee: Individual
Priority date: 2008-07-19
Filing date: 2008-07-19
Publication date: 2010-01-20

Abstract

The invention belongs to the technical field of biology, and discloses a quick detection method for FAD2 gene expression of different sources in brassica napus seeds. The method comprises the following steps: by comparing DNA sequences of brassica FAD2 genes of different sources, screening four single nucleotide polymorphism (SNP) sites which can differentiate FAD2 genes of an A genome and a C genome; then designing specific SNP primers according to the four SNP sites; and combining RT-PCR to identify FAD2 gene types expressed in the seeds. The method has positive meanings for further researching a molecular regulation mechanism formed by oleic acid of rape seeds and quickening the pace of selecting and breeding high oleic acid rape variety.

Description

The method for quick of FAD 2 gene expression of different sources in brassica napus seeds

Technical field

The present invention relates to utilize single nucleotide polymorphism (SNP) technology to set up to detect in the swede type rape seed from effective detection method of A genome and C genome FAD2 genetic expression, belong to biological technical field.

Background technology

Rape is a third place in the world large economy crop, and rapeseed oil has become second largest vegetables oil.More than 100,000,000 mu of China's rapeseed cultivation area ranks first place, the whole world.At present, one of major objective of rapeseed breeding is the high oleic acid kind of seed selection on two low bases.On function, saturated fatty acid is lower than 3%, oleic acid content is a kind of highly beneficial healthy eating oil near 90% oil.High oleic oil has many good qualities, and is difficult for smoldering during as high temperature; Oleic acid can reduce the low density lipoprotein cholesterol and suppress the low density lipoprotein oxidation, the effect of prevention coronary artery disease; Oleic acid has the effect of the mammary cancer of killing, and oleic acid is more even more important than linolic acid aspect health care.In today of energy dilemma, oleic acid (18: 1) is 18 carbochains, and is suitable with the diesel oil carbon chain lengths, is the ideal source of biofuel.

Swede type rape is the main cultivation rape in the whole world, says in the heredity that it is allotrtraploid (AACC).The height of oleic acid content depends on the active height of fatty acid desaturation enzyme 2 (FAD2) in the existing experiment confirm rapeseed oil, and the active height of FAD2 is directly controlled by FAD2 expression of gene amount.The existing method that detects the FAD2 expression of gene is special primer RT-PCR and specific probe Northern analytical procedure.It is from the A genome or from the C genome that but existing special primer RT-PCR can not distinguish the FAD2 gene of expression, and wherein the A genome is Chinese cabbage (Brassica campestris, AA, genome 2n=20); The C genome is wild cabbage (Brassica oleracea, CC, genome 2n=18); The FAD2 gene that the Northern analytical procedure not only can not be distinguished expression is from the A genome or from the C genome, and because method itself can't be used for extensive sieve in breeding process.For the molecular regulation mechanism that further research Semen Brassicae campestris oleic acid forms, accelerate the paces of high oleic acid rape variety seed selection, the FAD2 gene that is a long felt need for the expression of a kind of energy service area branch is from A genome or the genomic detection method of C.

Summary of the invention

The objective of the invention is to set up that a kind of energy is quick, easy, effectively to distinguish the FAD2 gene of expressing in the seed be from A genome or the genomic detection method of C.

First goal of the invention of the present invention is achieved through the following technical solutions, and comprises the steps:

1.FAD2 the clone of gene and order-checking

According to the full length cDNA sequence of Arabidopis thaliana FAD2 gene of having delivered and rape FAD2 gene,, design the degenerated primer of a pair of FAD2 full length gene cDNA by bioinformatic analysis.Because the coding region of FAD2 gene does not have intron, so utilize degenerated primer directly the genomic dna of swede type rape (AACC) to be carried out the high-fidelity pcr amplification.Amplified production separates through 1.2% agarose gel electrophoresis, and glue reclaims, and obtains the target DNA fragment of purifying.Purpose fragment tailing rear clone to the T-carrier, will be had the segmental carrier of purpose then and forwards in the competent bacillus coli DH 5 alpha, and the bacterium liquid after will transforming again is taped against on the screening culture medium of kantlex and filters out resistance clone.Random choose 30 clones check order.To remove the compare of analysis of the target dna order-checking of carrier sequence, determine that these 30 sequences all are from the FAD2 gene by the Blast on the NCBI.

2.SNP the screening in site

Utilize DNAStar software, the dna sequence dna of the FAD2 gene that will oneself record from us carries out sequence alignment analysis, the position of searching mononucleotide difference SNP together with the cDNA sequence of having announced from the FAD2 gene of Chinese cabbage (AA), wild cabbage (CC) and swede type rape (AACC) on the net.By relatively, filtered out four SNP sites: the initiator codon ATG on the cDNA sequence of FAD2 gene begins counting, 1) at 700bp between the 760bp, genomic from A

The 4th site on the fragment and the thymidylic acid T in the 8th site (italic mark) are with genomic from C

The 4th site on the fragment and the deoxycytidylic acid C in the 8th site (italic mark) form two SNP sites; 2) at 920bp between the 1000bp, genomic from A

The guanine deoxyribonucleoside in deoxycytidylic acid C in the 1st site (italic mark) and the 4th site acid G (italic mark) is with genomic from C on the fragment

The deoxycytidylic acid C (italic mark) in thymidylic acid T in the 1st site (italic mark) and the 4th site forms two SNP sites on the fragment.Red arrow indication among Fig. 1 is specifically seen in these four SNP sites.

3.SNP the design that primer is right

Layout strategy design SNP primer according to primer extension is right, and primer-design software is Primer Priemer5.Wherein primer designs a SNP special primer with the 3 ' end of one of the Nucleotide in above-mentioned four SNP sites as this primer; Another primer design is that the upstream and downstream in selected SNP site finds the common sequence from A genome and C genome FAD2 gene, at general primer of common sequences design.It is right to form a SNP primer by a general primer and SNP special primer.A SNP primer has amplified production to making from the genomic FAD2 gene of A, does not have amplified production from the genomic FAD2 gene of C; Another SNP primer is right, and can only make from the genomic FAD2 gene of C has amplified production, does not have amplified production from the genomic FAD2 gene of A.The molecular weight of the right pcr amplification product of these two groups of SNP primers is varied in size.

4. the separation of the total RNA of seed and reverse transcription

Utilize total RNA separating kit from Semen Brassicae campestris, to separate total RNA.Be template with total RNA then, polythymidylic acid dT (Polydeoxythymidine acid) is a primer, according to the explanation of reverse transcription test kit, in the reverse transcription damping fluid, by the synthetic cDNA of reversed transcriptive enzyme.Be template with cDNA again, carry out SNP special primer pcr amplification, the validity in check SNP site.

5.SNP the checking of site validity

Isolation of RNA from Chinese cabbage (AA), wild cabbage (CC) and 40 days seed (Time Calculation is to pick up counting after blooming) of swede type rape (AACC) growth, by reverse transcription, obtaining cDNA separately, is template with separately cDNA again, carries out SNP special primer pcr amplification.Amplified production separates and the ethidium bromide staining fluorometric analysis by agarose gel electrophoresis.If can not effectively separate with agarose gel electrophoresis with the amplified fragments size of the genomic FAD2 gene of C from the A genome, the amplified reaction of then distinguishing from A genome and the genomic FAD2 genetic expression of C carries out in different PCR pipes respectively.On the contrary, if can effectively separate with agarose gel electrophoresis with the amplified fragments size of the genomic FAD2 gene of C from the A genome, the amplified reaction of then distinguishing from A genome and C genome FAD2 genetic expression can carry out in same PCR pipe.The SNP primer of determining design according to the size that has or not amplified band and amplified band is from the A genome or from the C genome to whether distinguishing the FAD2 gene.Right to two SNP primers can distinguishing A genome and C genome FAD2 gene, the experimental material of increase Chinese cabbage, wild cabbage and swede type rape is further checked the right validity of SNP primer.It is from A genome or right from the genomic SNP primer of C that final acquisition can be distinguished the FAD2 gene.

In one embodiment, if the amplified fragments size of electrophoretic separation is about 430bp, illustrate that the FAD2 gene of expressing is from the A genome;

In another embodiment, if the amplified fragments size of electrophoretic separation is about 630bp, illustrate that the FAD2 gene of expressing is from the C genome;

In another embodiment, if the amplified fragments of electrophoretic separation has about 430bp and two amplified fragments of 630bp simultaneously, illustrate that the FAD2 gene of expressing is respectively from A genome and C genome.

Second goal of the invention of the present invention provides the SNP site that is used to detect different sources FAD2 gene in the swede type rape seed, wherein, initiator codon ATG on the cDNA sequence of FAD2 gene begins counting, 1) at 700bp between the 760bp, genomic from A

The 3rd goal of the invention of the present invention provides the special primer group for preparing by first goal of the invention, and this primer sets can be used for detecting the SNP of different sources FAD2 gene in the swede type rape seed.Wherein, primer sets is made up of with the reverse primer of two different SNP that are used to increase identical forward primer.

In a specific embodiments, in the described primer sets, common forward (forward) primer (FFP) sequence SEQ ID NO:1 is: 5 '-CCACGCCTTCAGCGACCACCAG-3 '; In the described primer sets, the sequence SEQ IDNO:2 of reverse (reverse) primers F RAP430 that is used for detecting the SNP of A genome is: 5 '-CATCGAGGCAACTCCTTGGACAGCA-3 ', and the sequence SEQ ID NO:3 of reverse (reverse) primers F RCP630 that is used for detecting the SNP of C genome is: 5 '-TGCGCCACGTGCGTGTCGGTA-3.

Advantage of the present invention: utilize SNP special primer group to carry out PCR, can FAD2 gene easy, check expression apace be from the A genome or from the C genome, or this two class FAD2 gene expresses simultaneously, solved current to a bottleneck problem in Canola oil acid metabolic study on regulation and the high oleic acid breed breeding process.

Description of drawings

Four SNP sites of Fig. 1 sign picture, the wherein site of SNP shown in the arrow.

Fig. 2 wild cabbage, Chinese cabbage and swede type rape SNP special primer pcr amplification product gel electrophoresis figure.

Embodiment

Below in conjunction with accompanying drawing and example the present invention is further described, but is not intended to limit the scope of the invention.

Embodiment:

1) design of primers.Choose long the fragment 5 '-CCACGCCTTCAGCGACCACCAG-3 ' of one section 22nt total identical on A genome and C genome as forward (forward) primers F FP (SEQ ID NO:1) at the 5 ' end in these four known SNP sites.At 700bp between the 760bp, at from reverse (reverse) primers F RAP430 (SEQ ID NO:2): the 5 '-CATCGAGGCAACTCCTTGGACAGCA-3 ' of the design of two thymidylic acid T on the genomic 5 '-CGCTGCT-3 ' fragment of A; At 920bp between the 1000bp, at genomic from C

Thymidylic acid T (italic) on the fragment and a deoxycytidylic acid C (italic) design be (reverse) primers F RCP630 (SEQ IDNO:3): 5 '-TGCGCCACGTGCGTGTCGGTA-3 ' oppositely.

2) PCR reaction system: cumulative volume 30 μ l, three kinds of primers add in the PCR reaction tubes simultaneously, and concrete concentration is composed as follows:

10x?Buffer： 3.0μl

dNTPs 10μM

Taq 2.5U

FFP 0.1μM

FRAP430 0.1μM

FRCP630 0.1μM

Template DNA 80ng

DdH2O adds to 30 μ l

3) PCR thermal cycling program:

The pre-sex change of the first step: 94 ℃ 3 minutes

Second the step sex change: 94 ℃ 40 seconds

Three-step annealing and extension: 72 ℃ 1 minute 20 seconds

Circulate 35 times

Extend the back: 72 ℃ 7 minutes

4) agarose gel electrophoresis detects

Pcr amplification product is 1.5% in concentration, and is added with on the sepharose of ethidium bromide and carries out electrophoretic separation, takes pictures by the gel analysis system then and analyzes (Fig. 2).Wherein 1-3 electrophoresis road template cDNA is from 3 different Chinese cabbage seeds, and amplification obtains the fragment that size is about 430bp, illustrates that the FAD2 gene of expressing is from the A genome; 4-6 electrophoresis road template cDNA is from 3 different cabbage seeds, and amplification obtains the fragment that size is about 630bp, illustrates that the FAD2 gene of expressing is from the C genome; 7-9 electrophoresis road template cDNA is from 3 different swede type rape seeds, and amplification obtains size and is about 430bp and these two fragments of 630bp, illustrates from A genome and the genomic FAD2 gene of C and expresses simultaneously; 10 electrophoresis roads are blank.The result clearly demonstrates, and She Ji one group of 3 primer can effectively be distinguished wild cabbage (CC), Chinese cabbage (AA) and the genomic FAD2 gene of swede type rape (AACC) like this.

Claims

1. the method for quick of a FAD 2 gene expression of different sources in brassica napus seeds is characterized in that:

1) carry out the sequence alignment analysis according to the sequence of the FAD2 gene of brassica plant wild cabbage, Chinese cabbage and swede type rape, the result filters out four SNP sites.Initiator codon ATG on the cDNA sequence of FAD2 gene begins counting, to between the 760bp, form two SNP sites at 700bp from the thymidylic acid T (italic mark) in the 4th site on the genomic 5 '-CGCTGCT-3 ' fragment of A and the 8th site and deoxycytidylic acid C (italic mark) from the 4th site on the genomic 5 '-CGCCGCC-3 ' fragment of C and the 8th site; At 920bp between the 1000bp, from the guanine deoxyribonucleoside acid G (italic mark) in the deoxycytidylic acid C (italic mark) in the 1st site on the genomic 5 '-CACGGAC-3 ' fragment of A and the 4th site with from the thymidylic acid T (italic mark) in the 1st site on the genomic 5 '-TACCGAC-3 ' fragment of C and two SNP sites of deoxycytidylic acid C (italic mark) composition in the 4th site.

2) based on above-mentioned four SNP sites, choose two SNP special primers of one or two SNP site design arbitrarily, article one, be the special primer of A genome FAD2 gene, another is the special primer of C genome FAD2 gene, chooses one section total primer of sequence identical segments conduct on A genome and the genomic FAD2 gene of C at 5 ' or the 3 ' end in the SNP site of selecting for use simultaneously;

3) from the growth seed of swede type rape, separate total RNA, after the reverse transcription, carry out SNP special primer PCR reaction respectively;

4) the PCR product separates with agarose gel electrophoresis, and ethidium bromide is a dyestuff, carries out image analysis with the gel analysis system behind the electrophoresis, according to the having or not or vary in size of SNP primer specificity and electrophoresis band, determines which genome the FAD2 gene of expressing is from.

2. the method described in the claim 1, wherein in step 4):

If can not effectively separate with agarose gel electrophoresis with the amplified fragments size of the genomic FAD2 gene of C from the A genome, the amplified reaction of then distinguishing from A genome and the genomic FAD2 genetic expression of C carries out in different PCR pipes respectively; If can effectively separate with agarose gel electrophoresis with the amplified fragments size of the genomic FAD2 gene of C from the A genome, the amplified reaction of then distinguishing from A genome and C genome FAD2 genetic expression can carry out in same PCR pipe.

3. the method described in the claim 1 or 2, wherein in step 4):

If the amplified fragments size of electrophoretic separation is about 430bp, illustrate that the FAD2 gene of expressing is from the A genome;

If the amplified fragments size of electrophoretic separation is about 630bp, illustrate that the FAD2 gene of expressing is from the C genome;

If the amplified fragments of electrophoretic separation has about 430bp and two amplified fragments of 630bp simultaneously, illustrate that the FAD2 gene of expressing is respectively from A genome and C genome.

4. any described SNP site that is used to detect different sources FAD2 gene in the swede type rape seed among the claim 1-3.

5. the SNP site described in the claim 4, it is characterized in that the initiator codon ATG on the cDNA sequence of FAD2 gene begins counting, 1) at 700bp between the 760bp, form two SNP sites from the thymidylic acid T (italic mark) in the 4th site on the genomic 5 '-CGCTGCT-3 ' fragment of A and the 7th site and deoxycytidylic acid C (italic mark) from the 4th site on the genomic 5 '-CGCCGCC-3 ' fragment of C and the 8th site; 2) at 920bp between the 1000bp, from the guanine deoxyribonucleoside acid G (italic mark) in the deoxycytidylic acid C (italic mark) in the 1st site on the genomic 5 '-CACGGAC-3 ' fragment of A and the 4th site with from the thymidylic acid T (italic mark) in the 1st site on the genomic 5 '-TACCGAC-3 ' fragment of C and two SNP sites of deoxycytidylic acid C (italic mark) composition in the 4th site.

6. described method of claim 1-3 or the described SNP of claim 4-5 site are used to detect the purposes of FAD 2 gene expression of different sources in brassica napus seeds, and infer that the genome type in the unknown material is band A genome or C genome.

7. the described purposes of claim 6 wherein utilizes above-mentioned SNP site as the SNP molecule marker, is used for the assignment of genes gene mapping and molecular mark.