CN104946642A

CN104946642A - Molecule specificity labeling primer for camellia oleifera improved variety changlin number eighteen and detection method

Info

Publication number: CN104946642A
Application number: CN201510407976.5A
Authority: CN
Inventors: 李海波; 魏海龙; 胡传久; 王丽玲; 徐梁
Original assignee: Zhejiang Academy of Forestry
Current assignee: Zhejiang Academy of Forestry
Priority date: 2015-07-10
Filing date: 2015-07-10
Publication date: 2015-09-30
Anticipated expiration: 2035-07-10
Also published as: CN104946642B

Abstract

The invention relates to a molecule specificity labeling primer for the camellia oleifera improved variety changlin number eighteen and a method capable of fast identifying the camellia oleifera improved variety changlin number eighteen. The primer has the following sequence of an upstream primer body 5'-CATTACACCATCCTCTTGTTGC-3' and a downstream primer body 5'-TGATCAATTACAGGGCTCGT-3'. The molecule specificity labeling primer can fast identify the camellia oleifera improved variety changlin number eighteen at the early stage, and the method is simple, fast and accurate and is an irreplaceable molecule means for identifying camellia oleifera improved variety through the apparent characteristics.

Description

The molecular specificity labeled primers of the long woods of oil tea breeding No. 18 and detection method

(1) technical field

The present invention relates to molecular specificity labeled primers and the authentication method thereof of the long woods of oil tea breeding No. 18.

(2) background technology

Oil tea is the woody oil tree species that China's planting area is maximum, distribution range is wider.Greatly develop camellia oleiferaindustry, to guarantee grain and oil safety, promote increasing peasant income, advance Integrated Development of The Mountainous Region and building a New Socialist Countryside, all tool is of great significance.China's camellia oleiferaindustry is in the ascendant, and development potentiality is huge.At present, China's camellia oleifera lam total area reaches more than 4,500 ten thousand mu, but output is very low, every per mu yield tea oil only about 4 kilograms.Its basic reason is, most camellia oleifera lam variety and quality difference or serious degradation, and a large amount of countries and the provincial improved seeds examining (recognizing) fixed are not applied.Meanwhile, some local seedling quality consciousness are thin, and operation control is extensive.For ensureing the good and fast development of camellia oleiferaindustry, the importance of oil tea breeding to development camellia oleiferaindustry be fully realized, the development of oil tea good seed must be accelerated and strengthen quality control.

China, at present to the supervision and management of oil tea seedling quality, mainly relies on every system that management layer is formulated, and not yet obtains key breakthrough at technological layer, particularly lacks the early stage quick identification technology system of oil tea breeding.12 the long woods kind series authorized by national high quality tree species are at present respectively long woods No. 3, long woods No. 4, long woods No. 18, long woods No. 21, long woods No. 23, long woods No. 26, long woods No. 27, long woods No. 40, long woods No. 53, long woods No. 55, long woods No. 56, long woods No. 166.These oil tea breedings have the early real feature such as high yield, stable yields, seed-producing rate is high, oleaginousness is high, strong resistance, wide adaptability.To put forth effort to solve the trouble waters of producing upper oil tea seedling at present to the supervision and management of oil tea seedling quality, and first from technological layer, discriminating protection be carried out to long woods oil tea breeding.

Discriminating Main Basis appearance features at present to camellia oleifera cultivar, but the cycle identified due to many morphological characterss is long, greatly affected by environment, and kind quantity is on the increase, and makes cultivar identification more and more difficult.Since this century, the molecular marking technique of some PCR-based is as RAPD (Random Amplified Polymorphic DNA, randomly amplified polymorphic DNA), ISSR (Inter-Simple Sequence Repea, Inter-simple sequence repeat) and SRAP (sequence-related amplified polymorphism, SRAP) in succession for the Genetic Diversity of Germplasm of oil tea, genetic distance is studied, but chain for molecule marker and oil tea proterties, molecular identificalion research between camellia oleifera cultivar seldom.Moreover, what these molecular marking techniques adopted is universal primer, its pcr amplification collection of illustrative plates is complicated, poor repeatability not only, and specificity is not high, therefore and be not suitable for Variety identification, only develop certain stable variety, special DNA fingerprint mark could really for the accurate Rapid identification of this kind.

(3) summary of the invention

The molecular specificity labeled primers of the long woods of oil tea breeding No. 18 that the object of the invention provides a pair specificity high, and a kind of method carrying out Rapid identification to the long woods of the long woods of oil tea breeding No. 18.

The technical solution used in the present invention is:

The molecular specificity labeled primers of the long woods of oil tea breeding No. 18, described primer sequence is as follows:

Upstream primer: 5 '-CATTACACCATCCTCTTGTTGC-3 ',

Downstream primer: 5 '-TGATCAATTACAGGGCTCGT-3 '.

This primer pair adopts round pcr, after a large amount of shaker test obtains the DNA fragment specific of the long woods of oil tea breeding No. 18, by this fragment cloning and sequencing, Auele Specific Primer is designed based on the DNA sequence dna obtained, pcr amplification is carried out with this primer pair oil tea breeding, only long woods No. 18 Absorbable organic halogens obtain the specific fragment of 983bp size, and other camellia oleifera cultivar all can not obtain this specific fragment.It should be noted that, molecular specificity labeled primers of the present invention is only limitted to the discriminating (differentiating whether it is long woods No. 18) of oil tea breeding, and namely testing sample is only limitted to oil tea.

The invention still further relates to and a kind ofly utilize described molecular specificity labeled primers to the long woods of oil tea breeding No. 18 method for distinguishing that reflect fast, described method is: extract the genomic dna of camellia oleifera cultivar blade to be measured as template, using described molecular specificity labeled primers as amplimer, carry out pcr amplification, electrophoresis detection is carried out to amplified production, if the DNA band of 983bp size appears in electrophoresis result, then camellia oleifera cultivar to be measured is the long woods of oil tea breeding No. 18, otherwise then no; Described molecular specificity labeled primers sequence is:

Upstream primer: 5 '-CATTACACCATCCTCTTGTTGC-3 ',

Downstream primer: 5 '-TGATCAATTACAGGGCTCGT-3 '.

The inventive method key is the selection of amplimer, and DNA extraction, PCR reaction system and reaction conditions are determined, and electrophoresis detection, all can carry out according to this area ordinary method.

Preferably, 20 μ L PCR reaction systems of the present invention are composed as follows:

Described pcr amplification condition is as follows: 94 DEG C of denaturation 7min; 94 DEG C of sex change 45min, 63 DEG C of annealing 45s, 72 DEG C of extensions 2min, totally 30 circulations; Finally fill 7min in 72 DEG C, final temperature is 4 DEG C.

Concrete, described method is as follows:

(1) get oil tea blade to be measured, add liquid nitrogen and grind, extract the genomic dna of oil tea to be measured;

Described extracting genome DNA can utilize novel quick-speed plant extracting genome DNA box

(DP3111, BioTeke, Beijing hundred Tyke Bioisystech Co., Ltd) carries out;

(2) genomic dna extracted with step (1) for template, with described molecular specificity mark

Note primer, as amplimer, carries out pcr amplification:

Every 20 μ L are composed as follows for PCR reaction system:

PCR reaction conditions is as follows:

94 DEG C of denaturation 7min; 94 DEG C of sex change 45min, 63 DEG C of annealing 45s, 72 DEG C of extensions 2min, totally 30 circulations; Finally fill 7min in 72 DEG C, final temperature is 4 DEG C.

(3) step (2) amplified production 5 μ L is got, mix with 1 μ L0.25% bromjophenol blue damping fluid, point sample is on the sepharose of 1.5%, electrophoresis in 1 × TAE damping fluid, under 5V/cm voltage, electrophoresis terminates rear EB and dyes, and takes a picture on automatic gel image analysis instrument, if the DNA band of 983bp appears in electrophoresis result, then camellia oleifera cultivar to be measured is long woods No. 18, otherwise then no.

Beneficial effect of the present invention is mainly reflected in: molecular specificity labeled primers of the present invention can carry out Early Identification fast to the long woods of oil tea breeding No. 18, and method is simple, quick, accurate, is that appearance features distinguishes the irreplaceable Molecular tools of oil tea breeding.

(4) accompanying drawing explanation

Fig. 1 is the result of oil tea breeding being carried out to pcr amplification; M is DNA molecular amount standard; Numbering 3 is the long woods of oil tea breeding No. 18, has amplified the specific DNA band of a 983bp size; All the other are numbered other long woods oil tea breeding, produce there are no the much little specific DNA bands of 900bp.

(5) embodiment

Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in this:

Embodiment 1:

(1) extraction of oil tea fine breed gene group DNA:

Get oil tea breeding young leaflet tablet 0.03g to be measured, add liquid nitrogen thoroughly to grind, extraction and application novel quick-speed plant extracting genome DNA box (DP3111, the BioTeke of genomic dna, Beijing hundred Tyke Bioisystech Co., Ltd), extract the genomic dna crude extract obtaining oil tea breeding.DNA crude extract detects integrity, purity and concentration by the agarose gel electrophoresis of 1.5% and DNA/RNA ultraviolet spectrophotometer (Nanodrop Technologies, USA).OD ₂₆₀/ OD ₂₈₀the DNA sample of >1.8 is used for subsequent PCR amplification.DNA extraction thing is for subsequent use in-20 DEG C of refrigerator storages.

(2) design specific PCR amplification primer, the sequence of primer pair is:

Upstream primer: 5 '-CATTACACCATCCTCTTGTTGC-3 ' and downstream primer: 5 '-TGATCAATTACAGGGCTCGT-3 ', synthesized by Shanghai biotechnology company limited.

(3) pcr amplification:

PCR reaction solution forms: 2 × Power Taq PCR Master Mix (PR1700, BioTeke, Beijing hundred Tyke Bioisystech Co., Ltd) 10 μ L, 10 μMs of upstream and downstream special primers are to each 1 μ L, 20ng/ μ L template DNA 3 μ L, ddH ₂o 5 μ L.

Amplified reaction carries out on Life ECO type amplification instrument (Bioer, Hangzhou BIOER Technology Co., Ltd).

Amplification condition:

94 DEG C of denaturation 7min;

94 DEG C of sex change 45min, 63 DEG C of annealing 45s, 72 DEG C of extensions 2min, totally 30 circulations;

Finally fill 7min in 72 DEG C, final temperature is 4 DEG C.

(4) electrophoresis detection: get step (3) pcr amplification product 5 μ L, mix with 1 μ L 0.25% bromjophenol blue damping fluid, point sample on the sepharose of 1.5%, in 1 × TAE damping fluid, electrophoresis under 5V/cm voltage, after electrophoresis terminates, dye 30 minutes, then at the automatic gel image analysis instrument of U.S. Bole (Chemi Doc XRS imaging system in containing the aqueous solution of 0.5 μ g/mL EB, Bio-Rad, Hercules, CA, USA) upper photograph.

According to the method described above, respectively electrophoresis detection is carried out to the specific primer PCR AFLP system of multiple oil tea breeding (numbering 1 ~ 12 represents oil tea breeding and is followed successively by: 1: long woods No. 3,2: long woods No. 4,3: long woods No. 18,4: long woods No. 21,5: long woods No. 23,6: long woods No. 26,7: long woods No. 27,8: long woods No. 40,9: long woods No. 53,10: long woods No. 55,11: long woods No. 56,12: long woods No. 166), the results are shown in Figure 1.

In figure, arrow indication is from the long woods of oil tea breeding No. 18 being numbered 3, only amplified clear bright, a stable molecular weight be about the much little specific DNA bands of 900bp respectively, and the long woods oil tea breeding of all the other numberings, produce there are no the much little special DNA bands of 900bp, other non-object band is not had to produce yet, visible the present invention opens the discriminating of molecular specificity marker for the long woods of oil tea breeding No. 18, and its stability, specificity are very high.

Claims

1. the molecular specificity labeled primers of the long woods of oil tea breeding No. 18, described primer sequence is as follows:

Upstream primer: 5 '-CATTACACCATCCTCTTGTTGC-3 ',

Downstream primer: 5 '-TGATCAATTACAGGGCTCGT-3 '.

2. one kind utilizes the molecular specificity labeled primers described in claim 1 to carry out the method for Rapid identification to the long woods of oil tea breeding No. 18, described method is: extract the genomic dna of camellia oleifera cultivar blade to be measured as template, using described molecular specificity labeled primers as amplimer, carry out pcr amplification, electrophoresis detection is carried out to amplified production, if the specific DNA band of 983bp appears in electrophoresis result, then camellia oleifera cultivar to be measured is the long woods of oil tea breeding No. 18, otherwise then no; Described molecular specificity labeled primers sequence is:

Upstream primer: 5 '-CATTACACCATCCTCTTGTTGC-3 ',

Downstream primer: 5 '-TGATCAATTACAGGGCTCGT-3 '.

3. method as claimed in claim 2, is characterized in that described pcr amplification condition is as follows: 94 DEG C of denaturation 7min; 94 DEG C of sex change 45min, 63 DEG C of annealing 45s, 72 DEG C of extensions 2min, totally 30 circulations; Finally fill 7min in 72 DEG C, final temperature is 4 DEG C.

4. method as claimed in claim 2, is characterized in that described method is as follows:

(1) get oil tea blade to be measured, add liquid nitrogen and grind, extract the genomic dna of oil tea blade to be measured;

(2) genomic dna extracted with step (1), for template, using described molecular specificity labeled primers as amplimer, carries out pcr amplification:

Every 20 μ L are composed as follows for PCR reaction system:

2×Power Taq PCR Master Mix 10μL

10 μMs of each 1 μ L of upstream and downstream primer

20ng/ μ L template DNA 3 μ L

DdH ₂o complements to 20 μ L;

PCR reaction conditions is as follows:

94 DEG C of denaturation 7min; 94 DEG C of sex change 45min, 63 DEG C of annealing 45s, 72 DEG C of extensions 2min, totally 30 circulations; Finally fill 7min in 72 DEG C, final temperature is 4 DEG C;

(3) step (2) amplified production 5 μ L is got, mix with 1 μ L 0.25% bromjophenol blue damping fluid, point sample is on the sepharose of 1.5%, electrophoresis in 1 × TAE damping fluid, under 5V/cm voltage, electrophoresis terminates rear EB and dyes, and takes a picture on automatic gel image analysis instrument, if the DNA band of 983bp appears in electrophoresis result, then camellia oleifera cultivar to be measured is long woods No. 18, otherwise then no.