CN103103260B - PCR (Polymerase China Reaction) primer and method for predicting aroma substance content of peach fruit - Google Patents
PCR (Polymerase China Reaction) primer and method for predicting aroma substance content of peach fruit Download PDFInfo
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Abstract
The invention discloses a PCR (Polymerase China Reaction) primer and a method for predicting aroma substance content of peach fruits. The primer comprises two pairs of primers, that is, the first pair of primers comprises: an upstream primer: 5'-AGTGGTTGAATCTATAATCCGA-3', and a downstream primer: 5'-TTCAACTCCTTTGTCAAGCC-3'; and the second pair of primers comprises: an upstream primer: 5'-TGTAAAACGACGGCCAGTTGCTGCTCTTTAGTCTTCTATGTT-3', and the downstream primer: 5'-ACATTCTTCAATGGCATCCTGT-3'. The method comprises the following steps of: extracting a genomic DNA (Deoxyribose Nucleic Acid) of peach tissue; using the genomic DNA as the template, respectively utilizing the two pairs of primers to carry out PCR amplification; analyzing an amplification product so as to obtain the gene type of PpACX1 and PTS1 genes; and predicting the aroma substance content of the tested type of the peach fruit.
Description
Technical field
The invention belongs to biology field, relate in particular to a kind of PCR primer and method of predicting peach Fruit Aroma content.
Background technology
Peach [Prunus persica (L.) Batsch] is Rosaceae cherry, originates in China western part, because of its strong adaptability, wide, the virgin phase of cultivation scope is short, mouthfeel good, liked by breeder and consumers in general.Over nearly 10 years, world's peach fruit is produced always in rising trend, and 2010 year outputs reach 20,274, and 287 tons, wherein China accounts for 52.8% (FAOSTAT 2012, http://faostat.fao.org/).Along with new variety are constantly introduced fruit market, and the requirement of world market to fruit fresh food quality and processing quality, fragrance, as a top important indicator of quality evaluation, more and more receives people's concern.
Fragrance is one of key factor of evaluating fruit interior quality.Peach fruit aroma is the complex character of controlled by multiple genes, and different peach kind aroma components and content difference are very large.More than 110 kind of volatile matter in peach fruit, detected, the route of synthesis of these volatile matter mainly comprises:
(1) in Fatty acid biosynthesis metabolism: lipoxygenase pathway 1); This approach is synthetic scent type material mainly, comprises C6 aldehydes, alcohols; 2) β-oxidation approach; This approach mainly synthesizes the lactone material in peach characteristic perfume, comprises and accounts for more than 95% γ of aroma component, δ-ten lactone; ACOD (ACX) participates in the first step reaction of β-oxidation approach, is rate-limiting enzyme.
Xi Wanpeng etc. clone and obtain 4 ACX genes (PpACX1-4) from two kinds of peach pulp (plain boiled pork peach ' lake scape honeydew ' and yellow meat peach ' beautiful yellow peach '), find that peach fruit PpACX1 expression amount and ACX enzymic activity in 20 ℃ and storage all can increase, the variation of lactone content is proportionate with the above two variation.Neat pure as jade grade obtains 5 ACX encoding genes (PpACX1-5) according to international peach genome sequence column information (www.phytozome.org/peach) clone, and it is specific expressed to carry out pulp organization, find in five gene members, PpACX1 expresses at most in the root of institute's research material, stem, leaf, pulp, is the gene member of most critical.
(2) terpene route of synthesis; Floral type composition phantol is mainly synthesized by monoterpene synthetic enzyme (PTS) the catalysis window ox acyl window ox base bisphosphate (GDP) in this approach.
In arabidopsis gene group, find that there is 20 terpene synthase genes and spending middle expression, At1g61680 gene coded protein has katalysis to synthetic (s)-phantol; 1 phantol synthase gene (LaLINS) that clone obtains from lavandula angustifolia can catalyze and synthesize aroma component (R)-(-)-phantol in a large number.
In the research of peach fragrance candidate gene, pass through Section Location, 4 terpene synthase genes (STC, STS, TS1, TC) are positioned at respectively to first and the 4th in linkage group of cherry (with reference to collection of illustrative plates (T × E)), but whether affect the synthetic and unclear of phantol.
Summary of the invention
The invention provides a kind of PCR primer of predicting peach Fruit Aroma content, utilize this PCR primer to detect the genotype of peach PpACX1 gene, PTS1 gene, and predict the content of aroma substance in peach fruit.
Predict a PCR primer for peach Fruit Aroma content, comprise two pairs of primers,
Pair of primers is:
Upstream primer: 5 '-AGTGGTTGAATCTATAATCCGA-3 ';
Downstream primer: 5 '-TTCAACTCCTTTGTCAAGCC-3 ';
Second pair of primer is:
Upstream primer: 5 '-TGTAAAACGACGGCCAGTTGCTGCTCTTTAGTCTTCTATGTT-3 ';
Downstream primer: 5 '-ACATTCTTCAATGGCATCCTGT-3 '.
The nucleotide sequence synthetic relevant to peach aroma component phantol analyzed to discovery, in PTS1 (GenBank accession number: DY639772) intron, contain a SSR fragment take (AT) as repeating, the sequence of this SSR fragment and both sides conservative fragments thereof is as shown in SEQ ID No.1.According to the sequences Design at these SSR fragment two ends above-mentioned pair of primers (PTS1-SSR), and add green fluorescence group at 5 ' end of PTS1-SSR upstream primer.
ACX synthetic gene PpACX1 (peach genome sequence ppa002510m) total length is analyzed to discovery, in this gene, have one section of SSR fragment take (CT) as repeating.The sequence of this SSR fragment and both sides conservative fragments thereof is as shown in SEQ ID No.2.According to the sequences Design at these SSR fragment two ends above-mentioned second pair of primer (ACX1), upstream sequence 5 ' the end of this primer contains a universal primer tail (M13 (21)-TGTAAAACGACGGCCAGT), so can utilize universal primer directly fluorophor to be added on product in PCR process.
Due to the repetition number difference of connecting in SSR fragment, and the sequence high conservative at fragment two ends, therefore, utilize the primer of special design can amplify the PCR product of different lengths, the clip size of amplified production has determined the genotype of sample.
The present invention also provides a kind of method of predicting peach Fruit Aroma content, comprising:
(1) genomic dna of extraction peach tissue;
Preferably adopt CTAB method to extract genomic dna; And because the secondary metabolite of plant has interference effect to nucleic acid extraction, but young tender cambium secondary metabolite is less, DNA content is high, be easy to fragmentation, and the tender cambium of described children can be selected young leaflet tablet.
(2), take described genomic dna as template, utilize respectively described PCR primer to carry out pcr amplification;
Wherein, the reaction system that adopts primer PTS1-SSR to carry out PCR is preferably 10 μ L, comprising: 10 × PCR buffer (Mg
2+) 2 μ L, 2mM dNTP 1 μ L, 5U rTaq DNApolymerase 0.1 μ L, forward primer 0.25 μ L, reverse primer 0.25 μ L, 20ng/ μ L DNA profiling 1 μ L, deionized water 4.5 μ L;
Response procedures is preferably: 94 ℃ of denaturation 5min; 94 ℃ of sex change 30s, 53 ℃ of annealing 40s, 72 ℃ are extended 40s, 35 circulations; Last 72 ℃ are extended 10min;
The reaction system that adopts primer ACX1 to carry out PCR is preferably 20 μ L, comprising: 10 × PCRbuffer (Mg
2+) 2 μ L, 2mM dNTP 0.2 μ L, 5U rTaq DNApolymerase 0.1 μ L, forward primer 0.1 μ L, reverse primer 0.5 μ L, the general tail 0.4 μ L of M13 (21) fluorescence, 20ng/ μ LDNA template 2 μ L, deionized water 14.7 μ L;
Response procedures is preferably: 94 ℃ of denaturation 5min; 94 ℃ of sex change 30s, 60 ℃ of annealing 30s, 72 ℃ are extended 30s, 30 circulations; 94 ℃ of sex change 30s, 53 ℃ of annealing 30s, 72 ℃ are extended 30s, 10 circulations; Last 72 ℃ are extended 10min;
Wherein, the pcr amplification of DNA fragmentation is mainly carried out in first round PCR reaction, and second takes turns PCR increases the general tail of M13 (21) fluorescence at the PCR product end obtaining that increases;
(3) amplified production is analyzed, obtained the genotype of PpACX1 gene and PTS1 gene, predict examined kind peach Fruit Aroma content;
Described peach Fruit Aroma is ten lactones and phantol.
First utilize phenotype and the genotype of population genetics analytical procedure to peach kind Aromatic Matter Contents to carry out correlation analysis; Take the correlation analysis result that obtains as foundation, the pcr amplification product of testing sample to be analyzed, the genotype of record take clip size as standard, predicts the content of ten lactones and phantol in the fruit of examined peach kind.
If the genotype of PpACX1 gene is 209/213 or 213/213,, compared with other genotype, in this genotypic peach fruit, the content of ten lactones is higher; If the genotype of PTS1 gene is 183/187,, compared with other genotype, in this genotypic peach fruit, the content of phantol is higher.
Compared with prior art, beneficial effect of the present invention is:
(1) utilize primer of the present invention can identify accurately and efficiently the Aromatic Matter Contents genotype of peach kind, prediction peach Fruit Aroma content, and then peach flavouring essence quality is carried out to preliminary evaluation;
(2) utilize primer of the present invention to peach leaf sheet, to detect in seedling stage, greatly improved the screening efficiency of high flavouring essence quality peach kind, accelerated the process of peach fruit aroma breeding, shortened breeding cycle.
Accompanying drawing explanation
Fig. 1 is PpACX1 genotype and phenotype correlation analysis result figure;
Fig. 2 is PTS1 genotype and phenotype correlation analysis result figure.
Embodiment
The extraction of 1 genomic dna
The genomic dna that extracts respectively 345 peach kinds by CTAB method, concrete grammar is as follows:
(1) preparation damping fluid
CTAB damping fluid: 2%CTAB, 0.1M Tris, 20mM EDTA, 1.4M NaCl, pH8.0;
DNA dissolves damping fluid TE:10mM Tris; 1mM EDTA; PH 8.0.
(2) extract genomic dna
1. with balance, take rapidly about 1.0g and be stored in-80 ℃ of peach young leaflet tablet tissues in refrigerator, with liquid nitrogen fully grinding in mortar, add the anti-oxidation of a little PVP, powder is proceeded to fast in the 10mL centrifuge tube that fills 4mLCTAB solution and 80 μ L beta-mercaptoethanols (65 ℃ of preheatings) to 65 ℃ of water-bath 1h;
2. add 4mL chloroform/primary isoamyl alcohol (24: 1), mix, the centrifugal 10min of 12,000rpm, gets supernatant liquor in new 10mL pipe, again adds 4mL chloroform/primary isoamyl alcohol (24: 1), mixes the centrifugal 10min of 12,000rpm;
3. by step 2. the supernatant liquor of centrifugal rear gained proceed to new 10mL pipe, and add the Virahol of equal-volume-20 ℃ precooling, mix gently, at-20 ℃, place 30min to DNA and precipitate.The centrifugal 2min of 10,000rpm, removes supernatant liquor;
4. use 3. DNA throw out 2 times of 75% ethanol cleaning step;
5. the DNA after 4. step being washed dries and is dissolved in the TE damping fluid of 400 μ L, adds 2 μ LRNAase enzymes (10mg/mL) to remove RNA;
6. in the DNA crude extract 5. obtaining in step, add phenol/chloroform/primary isoamyl alcohol (25: 24: 1) of 400 μ L to mix, the centrifugal 10min of 12,000rpm.Draw supernatant liquor, centrifugal under similarity condition after adding 400 μ L chloroform/primary isoamyl alcohol (24: 1) to mix;
7. draw the supernatant liquor of step in 6., by method precipitation 3., 4. of step, after cleaning DNA, add the TE damping fluid of 200 μ L to dissolve;
DNA detection adopts ultraviolet spectrophotometer (Beckman coulter, USA), determines its concentration and quality, gets 1-2 μ L simultaneously and detects on 1.0% sepharose.DNA stoste is stored in-20 ℃, and working fluid dilution is that 20ng/ μ L is standby.
2 design of primers
(1)PTS1-SSR
1. the search nucleotide sequence 4 (GenBank accession number: DY639772, DY647590, DY646265, DY640744) synthetic relevant to peach aroma component " phantol " from GenBank, design respectively primer 4 right, synthetic by Shanghai Ying Weijie base trade Co., Ltd;
2. take peach genomic dna as template, carry out pcr amplification, PCR product first reclaims purification kit purifying (gene of direct Sequencing does not need to carry out the following steps) through QiaquickGel Extraction Kit (Qiagen, Germany);
3. purified product is connected on pGEM-T (Promega, Madison, Wis.) carrier, proceeds to DH5 α competent cell (Takara Biotechnology, Dalian, China);
4. 37 ℃ of cultivation 12-16h left and right on the LB substratum that is added with IPTG, glycerine, peace benzyl and X-gal will be inoculated in, carry out subsequently bacterial plaque screening, select 6-8 of uniform size white recombinant monoclonal bacterial plaque and shake after bacterium through spending the night, respectively get 1.0 μ L and serve the order-checking of Hai Yingweijie base trade Co., Ltd;
5. with DNAstar, sequencing result is carried out to sequence comparative analysis, in discovery PTS1 (GenBank accession number: DY639772) intron, contain a SSR fragment take (AT) as repeating;
⑥Kua SSR district redesigns primer pair PTS1-SSR, and upstream primer 5 ' end adds fluorophor HEX, entrusts Shanghai Ying Weijie base trade Co., Ltd synthetic rear standby.
The base sequence of PTS1-SSR primer in Table the nucleotide sequence of 1, PTS1 gene SSR fragment and two ends conservative fragments as shown in SEQ ID No.1.
(2)ACX1
From peach genomic dna is resurveyed the data of order, ACX synthetic gene PpACX1 (peach genome sequence ppa002510m) total length is copied, analyze with DNAstar, find this gene exist one section take (CT) the SSR fragment for repetition;
At this SSR fragment left and right design primer ACX1,5 ' of upstream primer is held the universal primer tail (M13 (21)-TGTAAAACGACGGCCAGT) that contains 18bp, entrusts Shanghai Ying Weijie base trade Co., Ltd synthetic.While carrying out PCR, in reaction system, need to add the universal primer tail with fluorophor.The base sequence of each fragment in Table the nucleotide sequence of 1, PpACX1 gene SSR fragment and two ends conservative fragments as shown in SEQ ID No.2.
Table 1 primer PTS1-SSR and ACX1 relevant information
(3) pcr amplification
Take the genomic dna in step (1) as template, utilize respectively primer PTS1-SSR and primer ACX1, carry out pcr amplification, 345 peach kinds are carried out to the genotype detection relevant to Aromatic Matter Contents.
If without specified otherwise, the aroma substance in the present invention refers to phantol and ten lactones.
1) PCR of primer PTS1-SSR reaction
1. reaction system: totally 10 μ L, comprising: 10 × PCR buffer (Mg
2+) 2 μ L, 2mMdNTP 1 μ L, 5U rTaq DNA polymerase 0.1 μ L, forward primer 0.25 μ L, reverse primer 0.25 μ L, 20ng/ μ L DNA profiling 1 μ L, deionized water 4.5 μ L.
2. response procedures: 94 ℃ of denaturation 5min, 94 ℃ of sex change 30s, 53 ℃ of annealing 40s, 72 ℃ are extended 40s, 35 circulations, last 72 ℃ are extended 10min.
3. product electrophoresis detection on 1% sepharose, observes and photographic recording result under ultraviolet lamp.
2) PCR of primer ACX1 reaction
1. reaction system: totally 20 μ L, comprising: 10 × PCR buffer (Mg
2+) 2 μ L, 2mM dNTP0.2 μ L, 5U rTaq DNA polymerase 0.1 μ L, forward primer 0.1 μ L, reverse primer 0.5 μ L, M13 (21) fluorescence tail 0.4 μ L, 20ng/ μ L DNA profiling 2 μ L, deionized water 14.7 μ L.
2. response procedures: 94 ℃ of denaturation 5min, 94 ℃ of sex change 30s, 60 ℃ of annealing 30s, 72 ℃ are extended 30s, 30 circulations; 94 ℃ of sex change 30s, 53 ℃ of annealing 30s, 72 ℃ are extended 30s, 10 circulations; Last 72 ℃ are extended 10min.
3. product electrophoresis detection on 1% sepharose, observes and photographic recording result under ultraviolet lamp.
(4) fragment analysis
Get 4 μ L PCR product ddH
2o is diluted to 25 μ L, absorption diluent joins and is added with in 12 μ LFormamide and the upper model of the interior target 96 hole ABI3130 of 0.3 μ L LIZ500 (75-500bp) fluorescence, 95 ℃ of sex change 5min, on ABI3130 sequenator, carry out capillary electrophoresis analysis, utilize genemapper 4.0 softwares to carry out clip size reading.
The PpACX1 gene of 345 parts of peach kinds and the genotype of PTS1 gene are as shown in table 2.
The genotype data of table 2.345 part peach kind PpACX1 gene and PTS1 gene
Note:-/-for not obtaining amplified production
From table 2, in Hakuho.pop (Bai Feng colony), landraces (local variety), Yu Lu.pop (Yu Lu colony), the oligogene type of PpACX1 gene is respectively 209/215,209/213,213/213, and wherein 209/213 is topmost genotype; The oligogene type of PTS1 gene is respectively 177/177,183/187,177/183, and wherein 183/187 is topmost genotype.
22 peach kinds (being numbered 324-345) of known phenotype data and corresponding genotype are carried out to association analysis, and analytical results is shown in Fig. 1 and Fig. 2.Shown in Fig. 1 is associated between genotype and the ten lactone absolute contents of PpACX1 gene.Shown in Fig. 2 is associated between the genotype of PTS1 gene and phantol absolute content.
As seen from Figure 1, in the genotype of PpACX1 gene, genotype is that the content of 209/213 and 213/213 peach kind ten lactones is the highest, and flavouring essence quality is better; As seen from Figure 2, in the genotype of PTS1 gene, the peach kind phantol content that genotype is 183/187 is the highest, and flavouring essence quality is better.
Claims (3)
1. a PCR primer of predicting peach Fruit Aroma content, is characterized in that, comprises two pairs of primers,
Pair of primers is:
Upstream primer: 5 '-AGTGGTTGAATCTATAATCCGA-3 ';
Downstream primer: 5 '-TTCAACTCCTTTGTCAAGCC-3 ';
Second pair of primer is:
Upstream primer: 5 '-TGTAAAACGACGGCCAGTTGCTGCTCTTTAGTCTTCTATGTT-3 ';
Downstream primer: 5 '-ACATTCTTCAATGGCATCCTGT-3 '.
2. a method of predicting peach Fruit Aroma content, comprising:
(1) genomic dna of extraction peach tissue;
(2) take described genomic dna as template, utilize respectively PCR primer as claimed in claim 1, carry out pcr amplification;
(3) amplified production is analyzed, obtained the genotype of PpACX1 gene and PTS1-SSR gene, predict examined kind peach Fruit Aroma content;
Described peach Fruit Aroma is ten lactones and phantol;
In step (1), described peach is organized as peach young leaflet tablet;
In step (2), while adopting second pair of primer to carry out pcr amplification, carry out two-wheeled PCR reaction.
3. method as claimed in claim 2, is characterized in that, adopts CTAB method to extract genomic dna.
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