CN105543219B - Molecular labeling with controlling cucumber epidermal hair/fruit thorniness initial gene Tril close linkages - Google Patents
Molecular labeling with controlling cucumber epidermal hair/fruit thorniness initial gene Tril close linkages Download PDFInfo
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- CN105543219B CN105543219B CN201610043932.3A CN201610043932A CN105543219B CN 105543219 B CN105543219 B CN 105543219B CN 201610043932 A CN201610043932 A CN 201610043932A CN 105543219 B CN105543219 B CN 105543219B
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Abstract
The invention provides two molecular labelings with controlling cucumber epidermal hair/fruit thorniness initial gene Tril close linkages.FlankT 01, FlankT 02, FlankT 01 is respectively designated as to be made up of the fragment of 293 nucleotides shown in the fragment and SEQ ID NO.2 of 290 nucleotides shown in SEQ ID NO.1.Wherein, SEQ ID NO.1 are chain with there is epidermal hair/fruit thorniness gene Tril, SEQ ID NO.2 with it is chain without epidermal hair/fruit thorniness gene tril.FlankT 02 is made up of 197 nucleotides shown in 187 nucleotide fragments and SEQ ID NO.4 in sequence table shown in SEQ ID NO.3.Wherein, SEQ ID NO.3 are chain with there is epidermal hair/fruit thorniness gene Tril, SEQ ID NO.4 with it is chain without epidermal hair/fruit thorniness gene tril.Two molecular labelings of the present invention by the Tril assignments of genes gene mapping in 37.4Kb sections, and all have higher stability respectively in the both sides of Tril genes, can simply and quickly be applied to the molecular mark screening system that cucumber whether there is epidermal hair/fruit thorniness type.
Description
Technical field
The present invention relates to the molecular labeling of gene engineering technology field, and more particularly to two with controlling cucumber epidermal hair/fruit
Pierce the molecular labeling of initial gene Tril close linkages.
Background technology
Cucumber (Cucumis sativus L.) is that Curcurbitaceae (Cucurbitaceae) Cucumis (Cucumis) 1 year is climing
Raw herbaceous plant, cucumber is as one of big important vegetable crop in the world ten, and one of main cultivation vegetable crop in China.Cucumber
Plant is coated to epidermal hair, and epidermal hair can effectively prevent the infringement gnawed, resist pathogen of herbivore and insect, mitigate sunlight
The injury of direct projection (including ultraviolet light), the machinery damage for slowing down transpiration, the moisture for adjusting plant absorption and nutrient, reducing surface
Wound and surface temperature etc., make plant more Adaptable growth environment.The fruit of cucumber belongs to Peponidium, is the most important portion of economic characters
Point, formed by ovary and holder symbiotic developmental.Fruit thorniness is form eggcase of the epidermal hair on fruit, and important fruit
Character.Research on cucumber fruit thorniness is reported, can be related earliest by 1997, North China type cucumber local varieties " smalt handle " are certainly
The microtriche natural mutant of offspring is handed over to be found, it shows as the smooth no fruit thorniness of blade, passes through high power electron microscopic observation stem, leaf, calyx
Piece, fruit surface cover the difficult microtriche discovered of naked eyes, and are named as " glabrous (gl) ".Found by genetic analysis,
The microtriche gene is recessive mutation, and gene is then further cloned and Molecular.It is subsequently found European greenhouse cucumber type
Natural mutant, the mutant shows as the smooth no epidermal hair in stem, branch, leaf, tendril, petal, sepal and ovary surface, fruit
Real stingless no knurl and there is wax gloss, observed by electron-microscope scanning and find epidermal hair of the plant surface without any form, named
For " Trichome-less (Tril) ", the key gene of control epidermal hair/fruit thorniness starting is named as Tril.Pass through heredity point
Analysis, it is dominant to find to have the wild characters (Tril) of epidermal hair/fruit thorniness, and the mutant character (tril) of no epidermal hair/fruit thorniness is
It is recessive.By the transcriptome analysis to tril mutant plants, obtain coding and participate in many cells trichome development and fruit thorniness development machine
The key transcription factor of system, but the crucial candidate gene not whether there is to control epidermal hair and fruit thorniness is further positioned
And analysis.
Map based cloning (Map-based cloning), the something lost of segregating population can be utilized in the case where gene outcome is unknown
The exception of linkage analysis or chromosome is passed, target gene is navigated to a particular location of chromosome, finds and closely connects with it
The molecular labeling of lock, constantly reduce candidate region and then clone gene.The most frequently used mark screening method is that chorista packet is mixed
Analytic approach (Bulked segregant analysis, BSA) is closed, this method has the parent of target gene phenotypic difference at a pair
In segregating population constructed by this, according to the phenotype of target gene, the individual of identical quantity is chosen respectively, forms two subgroups,
By DNA mixed in equal amounts individual in each subgroup, " gene pool " of two relativity of formation, then with suitable molecular labeling
Two gene pools are analyzed, it is mutually chain then sharp that molecular labeling and the target gene seat of polymorphism are shown between two ponds
The linkage degree of gained molecular labeling and target gene is further detected with segregating population, so that it is determined that it is in known molecular collection of illustrative plates
Or the position on chromosome.Because structure gene pool has used specific segregating population, and in packet only to target gene table
Type is selected, this ensure that the genetic background of other characters is essentially identical, should mainly be existed in theory between two gene pools
Target gene section has differences, and eliminates the influence of environment and human factor, makes result of study more accurately and reliably.And purpose
The molecular labeling that gene has the advantages that close linkage have efficiently, it is quick, do not limited by environmental condition, can be selected in seedling stage
Select, accelerate the process of breeding.
As the improvement of people's living standards, cucumber quality breeding has been brought into schedule.Cucumber fruit thorniness character belongs to fruit sense
See quality category.The cucumber of European greenhouse is the pericarp without fruit thorniness, is called Fruit Cucumber, and its market price is common yellow
2-3 times of melon.Cucumber fruits smooth in appearance without fruit thorniness, Mechanical wound are few, residual contamination is few, easy to clean, sanitary edible, are
The preferable kind of pollution-free vegetable.Detection indicate that：Without fruit thorniness cucumber pulp persticide residue lower than spinosity cucumber 27%, pericarp
Persticide residue low 18%.The presence or absence of cucumber fruit thorniness gene Tril control fruit thorniness, its research will promote cucumber quality breeding
Process.It is highly effective that assisted Selection, which is marked, in cucumber genetic breeding using the molecular labeling with fruit thorniness close linkage
Method.The mutant tril of stable heredity provides preferable material for cucumber fruit thorniness formation mechanism study, itself and spinosity parent
F1Colony shows as few thorn, pierced less for cultivating cucumber improved Varieties provide stably without fruit thorniness parent.
The content of the invention
The purpose of the present invention, it is to provide two and control cucumber epidermal hair/fruit thorniness initial gene Tril close linkages
Molecular labeling.
The present invention is achieved by the following technical solutions：
Molecular labeling with controlling cucumber epidermal hair/fruit thorniness initial gene Tril close linkages, is named as FlankT-01,
It is made up of the DNA fragmentation shown in SEQ ID NO.1 and the DNA fragmentation shown in SEQ ID NO.2；Wherein, shown in SEQ ID NO.1
DNA fragmentation with there is epidermal hair/fruit thorniness gene Tril chain, DNA fragmentation shown in SEQ ID NO.2 with without epidermal hair/fruit thorniness
Gene tril is chain.
The FlankT-01 is expanded as the sense primer shown in SEQ ID NO.5 and the anti-sense primer shown in SEQ ID NO.6
Increasing obtains, and its amplification program is：94 DEG C 5 minutes；94 DEG C 30 seconds, 57 DEG C 30 seconds, 72 DEG C 30 seconds, 35 circulation；72 DEG C 5 minutes.
Molecular labeling with controlling cucumber epidermal hair/fruit thorniness initial gene Tril close linkages, is named as FlankT-02,
It is made up of the DNA fragmentation in sequence table shown in SEQ ID NO.3 and the DNA fragmentation shown in SEQ ID NO.4；Wherein, SEQ ID
DNA fragmentation shown in NO.3 with there is epidermal hair/fruit thorniness gene Tril chain, DNA fragmentation shown in SEQ ID NO.4 with without epidermis
Hair/fruit thorniness gene tril is chain.
The FlankT-02 is expanded as the sense primer shown in SEQ ID NO.7 and the anti-sense primer shown in SEQ ID NO.8
Increasing obtains, and its amplification program is：94 DEG C 5 minutes；94 DEG C 30 seconds, 57 DEG C 30 seconds, 72 DEG C 30 seconds, 35 circulation；72 DEG C 5 minutes.
Above-mentioned primer is synthesized by Shanghai life work.
1058 plant Fs of the present invention using microtriche mutant gl and without epidermal hair/fruit thorniness mutant tril hybridization2Separate group
Body, it is non-allelic genes to be determined cucumber epidermal hair/fruit thorniness whether there is character and microtriche character by phenotypic analysis, and has epidermis
Hair/fruit thorniness character belongs to the dominant character of Dominant gene.Take F respectively afterwards2Every plant of tender leaf extraction genomic DNA of segregating population,
With reference to the screening of BSA methods and the molecular labeling of Tril gene close linkages.It is final to provide two using high density collection of illustrative plates molecular labeling
With the mark of cucumber epidermal hair/fruit thorniness gene Tril close linkages, Tril genes both sides, two mark genetic distances are located in respectively
Only 37.4Kb, and the physical map between two molecular labelings is advantageous to the final clone of Tril genes, be easy to whether there is epidermal hair/
The molecular mark Establishing of fruit thorniness.The molecular labeling of the present invention can easy, the application and breeding of quick and high-flux
In practice.
Brief description of the drawings
Fig. 1 shows that SSR marker FlankT-01 and FlankT-02 and control epidermal hair/fruit thorniness initial gene Tril closely connect
Lock；
Fig. 2 shows SSR marker FlankT-01 F2Colony's PCR amplified bands；
Fig. 3 shows SSR marker FlankT-02 F2Colony's PCR amplified bands.
Embodiment
First, F2The structure of segregating population
Build F2Stingless mutant parent used in colony is European greenhouse mutant tril, and microtriche mutant parent is
North China type cucumber mutant gl.The present embodiment prepares F using the two parents1Generation, F1Generation selfing produces F2For colony.More
Kind F2Colony's identification whether there is epidermal hair/fruit thorniness phenotype, ultimate analysis F1Phenotype and F2Segregation ratio, chi-square analysis method is verified, is obtained
Going out cucumber has the character of epidermal hair and fruit thorniness to belong to same gene control, and is the dominant character of Dominant gene.
2nd, Cucumber germplasm DNA extraction
Parent and F are extracted with CTAB methods2The genome DNA of segregating population blade.Take a piece of true leaf just deployed extremely
In 1.5mL centrifuge tubes and add liquid nitrogen, tissue abrasion adds 700 μ L CTAB lysis buffers, acutely vibrates 30 into powdered
Second, 60 DEG C of water-baths 30 minutes, abundant mixing of during which turning upside down.700 μ L chloroform isoamyl alcohols are added into above-mentioned homogenate lysate
(24:1, v/v), turn upside down abundant mixing, 4 DEG C of 13000r/min is centrifuged 15 minutes.The μ L of Aspirate supernatant 400 to it is new from
In heart pipe, add isometric isopropanol, abundant mixing of turning upside down, 30 minutes stood in -20 DEG C, 4 DEG C of 12000r/min from
The heart 10 minutes.Supernatant is abandoned, adds the ethanol of 200 μ L 75% along centrifugation tube wall, turn upside down washing centrifuge tube tube wall, 12000r/
Ethanol is discarded after 4 DEG C of min centrifugations 1 minute.Drying at room temperature precipitates 30 minutes, adds 100 μ L RNase TE buffer solutions (7:993,
V/v) dissolve, 37 DEG C of water-baths 30 minutes, dilute final concentration of 30ng/ μ L with TE buffer solutions after nucleic acid instrument measure DNA concentration, in-
20 DEG C standby.
3rd, SSR marker scanning F2Segregating population
Two molecular labelings developed using the present invention scan above-mentioned F2Segregating population, find marking type and trait expression type
Difference individual plant, obtain mark and exchange individual plant with Tril genes.PCR system：1 × Taq Buffer, 1.5mmol/LMgCl2,
200 μm of ol/L dNTPs, each 0.2 μm of ol/L of primer, 30ng template DNAs, 0.5U Taq DNA Polymerase, overall reaction body
It is for 10 μ L.PCR programs：94 DEG C 5 minutes；35 circulation, 94 DEG C 30 seconds, 57 DEG C 30 seconds, 72 DEG C 30 seconds；72 DEG C 5 minutes.10μL
PCR primer in add 1.5 μ L 6 × loading buffer, be well mixed the polyacrylamide gel electrophoresis after 8%,
Silver staining is dyed afterwards, and band banding pattern is analyzed on white light platform.
Using parent tril and gl genomic DNA as template, 50 μ L PCR systems are done, PCR reaction conditions are same as above.50 μ L are produced
The μ L of BioTeke SYBR Green I nucleic acid dyes 7 are added in thing, being stored at room temperature 10 minutes makes dyestuff be combined with DNA, with 1.5%
Agarose gel electrophoresis separates, and cuts purpose fragment under uviol lamp, uses TaKaRa MiniBEST Agarose Gel DNA
Extraction Kit recovery purifying DNA fragmentations, specific steps are with reference to kit specification, production code member 9762.Piece will be purified
Section is sent to be sequenced by Shanghai Sani bio tech ltd, and sequencing result passes through the software analysis of Chromas 2.3.
2 molecular labelings of the above pass through F2Segregating population is verified, with Tril gene loci close linkages, and is located respectively
In Tril genes both sides.SSR molecular marker has high stable feature, using these molecular labelings build dense genetic map and
Physical map, it will help the final clone of control cucumber epidermal hair/fruit thorniness initial gene.
Fig. 1 shows that SSR marker FlankT-01 and FlankT-02 and control epidermal hair/fruit thorniness initial gene Tril closely connect
Lock, and Tirl genes both sides are respectively seated at, it is respectively present a single cross-over event.Each cross represents a single-swap thing
Part.
Fig. 2 shows SSR marker FlankT-01 F2Colony's PCR amplified bands.M represents Marker 1, F2Without fruit thorniness colony and
F2There is fruit thorniness colony to be illustrated respectively in F210 plants selected at random in colony have fruit thorniness plant without fruit thorniness and 10 plants.
Fig. 3 shows SSR marker FlankT-02 F2Colony's PCR amplified bands.M represents Marker 1, F2Without fruit thorniness colony and
F2There is fruit thorniness colony to be illustrated respectively in F210 plants selected at random in colony have fruit thorniness plant without fruit thorniness and 10 plants.
Claims (4)
1. the molecular labeling with control cucumber epidermal hair/fruit thorniness initial gene Tril close linkages, is named as FlankT-01, by
DNA fragmentation composition shown in DNA fragmentation and SEQ ID NO.2 shown in SEQ ID NO.1；Wherein, shown in SEQ ID NO.1
DNA fragmentation is chain with there is epidermal hair/fruit thorniness gene Tril, the DNA fragmentation shown in SEQ ID NO.2 with without epidermal hair/fruit thorniness base
Because tril is chain.
2. the molecular labeling according to claim 1 with control cucumber epidermal hair/fruit thorniness initial gene Tril close linkages,
It is characterized in that：The FlankT-01 is drawn as the sense primer shown in SEQ ID NO.5 and the downstream shown in SEQ ID NO.6
Thing expands to obtain, and its amplification program is：94 DEG C 5 minutes；94 DEG C 30 seconds, 57 DEG C 30 seconds, 72 DEG C 30 seconds, 35 circulation；72 DEG C 5 points
Clock.
3. the molecular labeling with control cucumber epidermal hair/fruit thorniness initial gene Tril close linkages, is named as FlankT-02, by
DNA fragmentation composition shown in DNA fragmentation and SEQ ID NO.4 in sequence table shown in SEQ ID NO.3；Wherein, SEQ ID
DNA fragmentation shown in NO.3 with there is epidermal hair/fruit thorniness gene Tril chain, DNA fragmentation shown in SEQ ID NO.4 with without epidermis
Hair/fruit thorniness gene tril is chain.
4. the molecular labeling according to claim 3 with control cucumber epidermal hair/fruit thorniness initial gene Tril close linkages,
It is characterized in that：The FlankT-02 is drawn as the sense primer shown in SEQ ID NO.7 and the downstream shown in SEQ ID NO.8
Thing expands to obtain, and its amplification program is：94 DEG C 5 minutes；94 DEG C 30 seconds, 57 DEG C 30 seconds, 72 DEG C 30 seconds, 35 circulation；72 DEG C 5 points
Clock.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101250534A (en) * | 2008-03-20 | 2008-08-27 | 上海交通大学 | Protein coded sequence of cucumber TTG1-like gene |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101250534A (en) * | 2008-03-20 | 2008-08-27 | 上海交通大学 | Protein coded sequence of cucumber TTG1-like gene |
Non-Patent Citations (3)
| Title |
|---|
| Genome-wide characterization of simple sequence repeats in cucumber (Cucumis sativus L.);Pablo F Cavagnaro 等;《BMC Genomics》;20101231;第11卷(第569期);第1-18页 * |
| Identiication and mapping of Tril, a homeodomain‑leucine zipper gene involved in multicellular trichome initiation in Cucumis sativus;yun li wang 等;《theor appl genet》;20151030(第129期);第305-316页 * |
| 光滑少刺型黄瓜新品种水果黄瓜1号的选育;张守才 等;《中国蔬菜》;20071231(第11期);第25-26页 * |
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