CN104593518B - With Fructus Cucumidis sativi trichome development gene M ict be divided into from molecular marker - Google Patents
With Fructus Cucumidis sativi trichome development gene M ict be divided into from molecular marker Download PDFInfo
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- CN104593518B CN104593518B CN201510088116.XA CN201510088116A CN104593518B CN 104593518 B CN104593518 B CN 104593518B CN 201510088116 A CN201510088116 A CN 201510088116A CN 104593518 B CN104593518 B CN 104593518B
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Abstract
The invention provides three kinds with Fructus Cucumidis sativi trichome development gene M ict be divided into from molecular marker, the first named Cosegregated SNP 01, the second named Cosegregated SNP 02, the third named Cosegregated SNP 03.The present invention is divided into Fructus Cucumidis sativi trichome development gene M ict from three molecular markers more helpful to the final clone of Fructus Cucumidis sativi trichome development gene and the foundation of Molecular Marker-Assisted Breeding of Cucumber system；The molecular marker of the present invention easy, quick, high-throughout can be applied to breed cucumber practice.
Description
Technical field
The present invention relates to technique for gene engineering, particularly to Fructus Cucumidis sativi trichome development gene M ict be divided into from point
Sub-labelling.
Technical background
Plant epidermal hair (Trichome) is organ biology of a kind of structure height specialization, procuticle cell send out
Educate, be from individual cell level study plant cell differentiation, including cell fate, cell cycle, cell polarity,
Cellular morphology such as builds up at the ideal model of aspect.Epidermal hair is widely present in most plant surface, is divided into slender
Born of the same parents or many cells, have body of gland or Non-gland body, has branch or without a few class of branch.Epidermal hair plant growth promoter and
The aspects such as the adaptation to environment, including biological or abiotic, play an important role.As increased skin layer thickness
To regulate body temperature, reduce transpiration moisture loss, reduce mechanical damage, reduce ultraviolet light harms, protect plant
From the infringement of insecticide, herbivore and pathogen, contribute to collection and the propagation etc. of pollen.
Fructus Cucumidis sativi (Cucumis sativus L.) belong to Magnoliatae (Magnoliopsida), Cucurbitaceae (Cucurbitaceae),
Cucumis (Cucumis), is 1 year herbaceous plant that overgrows, one of world's important vegetable, because its flower property type is many
Sample, by the model plant as flower property type research.Epidermal hair be distributed widely in the stem of cucumber plant, leaf, tendril,
Petal, sepal, ovary surface, cucumber fruits belongs to Peponidium, ovary and holder symbiotic developmental form, fruit surface
Hair form eggcase, is commonly referred to as " fruit thorn ".
Cao Chenxing found nothing in 1997 in the colony of North China type Fructus Cucumidis sativi local varieties " Folium Isatidis handle " self progeny
The natural mutant of hairs type, it shows as stem, leaf, tendril, petal, sepal, ovary surface all without epidermal hair,
This mutant is that Fructus Cucumidis sativi epidermal hair formation mechanism study provides preferable material, also provides for Fructus Cucumidis sativi genetic breeding
Stingless parent.The Fructus Cucumidis sativi of smooth in appearance pollutes few, easy to clean, is the preferable kind of pollution-free vegetable.Few without tumor
The Fructus Cucumidis sativi sarcocarp persticide residue of thorn is lower by 27% than spinosity Fructus Cucumidis sativi, and peel persticide residue is low by 18%.
Fructus Cucumidis sativi epidermal hair is different from arabidopsis and Cotton Gossypii, is that typical many cells are without branch epidermal hair.At present for planting
The research of thing epidermal hair focuses primarily upon the single cell type of arabidopsis, and the research to many cells epidermal hair is the deepest
Entering, the map based cloning of Fructus Cucumidis sativi epidermal hair related gene and regulatory mechanism research also have no report.Fructus Cucumidis sativi is without chalaza variant
Fruit is had no result tumor, and reproductive growth is in advance, and female flower calyx is loose, illustrate that Fructus Cucumidis sativi epidermal hair gene also take part in really tumor
Formation and other growth course.The present invention preliminary study to Fructus Cucumidis sativi epidermal hair related gene map based cloning, not only
Can reveal that the molecule mechanism of plant many cells trichome development, and can be the clone of more epidermal hair related gene
Research with function lays the foundation, and accelerates cucumber quality breeding process.
Map based cloning (Map-based cloning), also known as positional cloning (Positional cloning), can be at gene
In the case of product the unknown, utilize the genetic linkage analysis of segregating population or the exception of chromosome, genes of interest is fixed
Position, to a kind of particular location of chromosome, is found molecular marker closely linked with it, is constantly reduced candidate region and enter
And clone gene.The most frequently used mark screening method is that chorista is grouped Mixed method (Bulked segregant
Analysis, BSA), the method in there is for a pair the segregating population constructed by the parent of genes of interest phenotypic difference,
According to the phenotype of genes of interest, choose a number of individuality respectively, constitute two subgroups, by each subgroup
The DNA mixed in equal amounts of body, is formed " gene pool " (the Gene pool) of two relative characters, then with suitably
Two gene pools are analyzed by molecular marker, show molecular marker and the genes of interest seat of polymorphism between two ponds
Position is mutually chain, and recycling segregating population detects the linkage degree of gained molecular marker and genes of interest further, thus
Determine its position on known molecular collection of illustrative plates or chromosome.Group is specifically separated owing to structure gene pool employs
Body, and only genes of interest phenotype is selected when packet, this ensure that the genetic background of other character is basic
Identical, mainly should there are differences at genes of interest section in theory between two gene pools, eliminate environment and artificial
The impact of factor, makes result of study the most accurately and reliably.
Summary of the invention
The purpose of the present invention, it is simply that in order to provide three kinds with Fructus Cucumidis sativi trichome development gene M ict be divided into from molecule
Labelling.
In order to realize the purpose of the present invention, present invention employs techniques below scheme:
A kind of be divided into Fructus Cucumidis sativi trichome development gene M ict from molecular marker, named
Cosegregated-SNP-01, by the nucleotide fragments shown in SEQ ID NO.1 in sequence table and SEQ ID NO.2
Shown nucleotide fragments composition, wherein shown in SEQ ID NO.1, fragment is divided into trichome development gene M ict
From, fragment shown in SEQ ID NO.2 and hairless gene mict be divided into from.
Above-mentioned be divided into Fructus Cucumidis sativi trichome development gene M ict from molecular marker, wherein, described molecular marker
Cosegregated-SNP-01 is drawn by forward primer shown in SEQ ID NO.7 and the downstream shown in SEQ ID NO.8
Thing amplification obtains.
A kind of be divided into Fructus Cucumidis sativi trichome development gene M ict from molecular marker, named
Cosegregated-SNP-02, shown in the nucleotide fragments shown in SEQ ID NO.3 and SEQ ID NO.4
Nucleotide fragments forms, and wherein fragment shown in SEQ ID NO.3 and trichome development gene M ict are divided into from, SEQ
Fragment shown in ID NO.4 and hairless gene mict be divided into from.
Above-mentioned be divided into Fructus Cucumidis sativi trichome development gene M ict from molecular marker, wherein, described molecular marker
Cosegregated-SNP-02 is drawn by forward primer shown in SEQ ID NO.9 and the downstream shown in SEQ ID NO.10
Thing amplification obtains.
A kind of be divided into Fructus Cucumidis sativi trichome development gene M ict from molecular marker, named
Cosegregated-SNP-03, shown in the nucleotide fragments shown in SEQ ID NO.5 and SEQ ID NO.6
Nucleotide fragments forms, and wherein fragment shown in SEQ ID NO.5 and trichome development gene M ict are divided into from, SEQ
Fragment shown in ID NO.6 and hairless gene mict be divided into from.
Above-mentioned be divided into Fructus Cucumidis sativi trichome development gene M ict from molecular marker, wherein, described molecular marker
Cosegregated-SNP-03 is by forward primer shown in SEQ ID NO.11 and the downstream shown in SEQ ID NO.12
Primer amplification obtains.
The present invention is the method by map based cloning, utilizes Fructus Cucumidis sativi without chalaza variant (mict) and hairiness (Mict)
Wild type (S77) preparing hybrid combines, and F1 generation selfing generation F2 is for colony, and totally 7936 strains are individual, and identify
Blade hairiness and without hair phenotype, wherein individual 5904 strains of hairiness, without hair individuality 2032 strains, every strain takes a piece of tender leaf
Extract genomic DNA, in conjunction with BSA method screening with Mict gene be divided into from molecular marker, through dideoxy-chain
Cessation method order-checking obtains.
The present invention is divided into Fructus Cucumidis sativi trichome development gene M ict from three molecular markers to Fructus Cucumidis sativi epidermis hair
The foundation of finally clone and the Molecular Marker-Assisted Breeding of Cucumber system of educating gene is more helpful；The molecule mark of the present invention
Note easy, quick, high-throughout can be applied to breed cucumber practice.
Specific implementation method
F2Structure for segregating population
Fructus Cucumidis sativi is utilized to combine without chalaza variant (mict) and hairiness (Mict) wild type (S77) preparing hybrid,
F1F is produced for selfing2For colony, totally 7936 strains are individual, and identify blade hairiness and without hair phenotype, wherein hairiness
Individual 5904 strains, without individual 2032 strains of hair, chi-square analysis method checking segregation ratio.Because of when plant rough leaf
Expansion just can carry out phenotype judgement period, and segregating population is planted in hole tray.
Cucumber germplasm DNA extraction
Parent and F is extracted by CTAB method2Genomic DNA for segregating population blade.Take just launched a piece of
True leaf is in 1.5mL centrifuge tube and add liquid nitrogen, and tissue abrasion is powdered, adds 500 μ L CTAB cracking
Buffer, acutely vibration 30 seconds, 60 DEG C of water-baths 30 minutes, period turns upside down fully mixing.To above-mentioned homogenate
Adding 500 μ L chloroform isoamyl alcohol (24:1, v/v) in lysate, turn upside down fully mixing, 13200rpm 4 DEG C
Centrifugal 15 minutes.Aspirate supernatant 500 μ L, in new centrifuge tube, adds equal-volume isopropanol, turns upside down
Fully mixing, stands 30 minutes in-20 DEG C, centrifugal 15 minutes of 13200rpm 4 DEG C.Abandon supernatant, along centrifugal
Tube wall adds 1mL 75% ethanol, and turn upside down washing centrifuge tube tube wall, and 13200rpm 4 DEG C is after centrifugal 5 minutes
Discard ethanol.Drying at room temperature precipitates 30 minutes, adds 100 μ L RNase TE buffer (7:993, v/v)
Dissolving, 37 DEG C of water-baths 30 minutes, nucleic acid instrument dilutes final concentration of 30 with TE buffer after measuring DNA concentration
Ng/ μ L, standby in-20 DEG C.
Mark scan F2Segregating population
The three kinds of molecular markers utilizing the present invention to develop scan above-mentioned F2Segregating population.PCR system: 1 × Taq
Buffer, 1.5mM MgCl2, 200 μMs of dNTPs, each 0.2 μM of primer, 30ng template DNA, 0.5U Taq
DNA Polymerase, overall reaction system is 10 μ L.PCR program: 94 DEG C 5 minutes；35 circulations, 94 DEG C
30 seconds, 55 DEG C 30 seconds, 72 DEG C 30 seconds；72 DEG C 5 minutes.BioTeke SYBR is added in PCR primer
Green I nucleic acid dye 3 μ L, 4 DEG C of standings make dyestuff be combined with DNA in 15 minutes, use 1.5% agarose gel
Electrophoretic separation, cuts purpose fragment under uviol lamp, uses TaKaRa MiniBEST Agarose Gel DNA
Extraction Kit reclaims purifying DNA fragment, concrete steps reference reagent box description, production code member 9762.
Being sent by purified fragments and checked order by Sani bio tech ltd, Shanghai, sequencing result passes through Chromas 2.3 software
Analyze.
Above 3 molecular markers are through F2Segregating population is verified, be all divided into Mict gene loci from, and special
Property PCR has high stable feature.Utilize these molecular markers to build high density heredity and physical map, will help
Final clone in Fructus Cucumidis sativi trichome development gene.
Claims (6)
1. be divided into Fructus Cucumidis sativi trichome development gene M ict from a molecular marker, named
Cosegregated-SNP-01, by the nucleotide fragments shown in SEQ ID NO.1 in sequence table and SEQ ID NO.2
Shown nucleotide fragments composition, wherein shown in SEQ ID NO.1, fragment is divided into trichome development gene M ict
From, fragment shown in SEQ ID NO.2 and hairless gene mict be divided into from.
2. as claimed in claim 1 and Fructus Cucumidis sativi trichome development gene M ict be divided into from molecular marker, its feature
Being, described molecular marker Cosegregated-SNP-01 is by forward primer and SEQ ID shown in SEQ ID NO.7
Downstream primer amplification shown in NO.8 obtains.
3. be divided into Fructus Cucumidis sativi trichome development gene M ict from a molecular marker, named
Cosegregated-SNP-02, shown in the nucleotide fragments shown in SEQ ID NO.3 and SEQ ID NO.4
Nucleotide fragments forms, and wherein fragment shown in SEQ ID NO.3 and trichome development gene M ict are divided into from, SEQ
Fragment shown in ID NO.4 and hairless gene mict be divided into from.
4. as claimed in claim 3 and Fructus Cucumidis sativi trichome development gene M ict be divided into from molecular marker, its feature
Being, described molecular marker Cosegregated-SNP-02 is by forward primer and SEQ ID shown in SEQ ID NO.9
Downstream primer amplification shown in NO.10 obtains.
5. be divided into Fructus Cucumidis sativi trichome development gene M ict from a molecular marker, named
Cosegregated-SNP-03, shown in the nucleotide fragments shown in SEQ ID NO.5 and SEQ ID NO.6
Nucleotide fragments forms, and wherein fragment shown in SEQ ID NO.5 and trichome development gene M ict are divided into from, SEQ
Fragment shown in ID NO.6 and hairless gene mict be divided into from.
6. as claimed in claim 5 and Fructus Cucumidis sativi trichome development gene M ict be divided into from molecular marker, its feature
Being, described molecular marker Cosegregated-SNP-03 is by forward primer and SEQ ID shown in SEQ ID NO.11
Downstream primer amplification shown in NO.12 obtains.
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Inventor after: Pan Jian Inventor after: Zhao Junlong Inventor after: Cai Run Inventor after: Pan Junsong Inventor after: He Huanle Inventor after: Wang Yunli Inventor after: Zhu Wenying Inventor after: Peng Jialin Inventor after: Chen Long Inventor before: Zhao Junlong Inventor before: Cai Run Inventor before: Pan Junsong Inventor before: He Huanle Inventor before: Wang Yunli Inventor before: Zhu Wenying Inventor before: Peng Jialin Inventor before: Chen Long |
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