CN102268429B

CN102268429B - Rapid cloning method of plant flowering related gene LFY/FLO homologous fragment and special primer thereof

Info

Publication number: CN102268429B
Application number: CN 201110217275
Authority: CN
Inventors: 鄢波; 于丽霞; 汤晓倩; 姚有林; 樊国盛
Original assignee: Southwest Forestry University
Current assignee: Southwest Forestry University
Priority date: 2011-08-01
Filing date: 2011-08-01
Publication date: 2013-01-30
Anticipated expiration: 2031-08-01
Also published as: CN102268429A

Abstract

The invention relates to a rapid cloning method of plant flowering related gene LFY/FLO homologous fragments and special primers thereof, and relates to the technical field of rapid isolation and cloning of plant homologous genes. The method is characterized in that the used special primer LYF has a nucleotide sequence of 5'-TAYATIAAYAARCCIAARATG-3', and LYR has a nucleotide sequence of 5'-ARIYKIGTIGGIACRTACCA-3'; the primer concentration is 25 muM, and the annealing temperature is 44 DEG C. The method of the invention can clone to obtain homologous fragments of LFY/FLO genes of plants from bryophytes to spermatophytes, and has a wide range of the kinds of applicable plants; the potential problem that a gene fragment is difficult to be amplified due to no expression or low expression of homologous fragments of the LFY/FLO gene when cDNA is used as a template is well avoided; and the method is rapid, simple, economical, and effective.

Description

Rapid clon method and the primer special thereof of the Blossoms Correlation Gene in Plants LFY/FLO homologous fragment

Technical field

The present invention relates to the homogenic sharp separation clone technology of plant field, be specifically related to from bryophyte to phanerogamous LFY/FLOThe fast separating process of dna homolog fragment and primer special thereof.

Background technology

The one-tenth flower process of plant is the common result who coordinates of external environment and inner gene, in the middle of the bloom controlling network, LFYGene is the hinge of GA signal and photoperiodic induction signal transduction, is the indispensable gene that determines that floral meristem forms, and has the vital role that activates the floral organ gene activity. LFY/ FLOHomologous gene all exists in flower and the higher plant without flower are arranged. LFY/FLOHomologous gene is the critical elements of decision from nourishing and growing and changing to generative growth phase, and is controlling flowering time, but it is not the single-minded colored related tissue that is expressed in into as the floral meristem characteristic gene. LFY/FLOThe homologous gene wide expression is in trophicity and the reproducibility tissue of higher plant, and may only have when its expression amount reaches certain level, could promote into colored. LFYGene is in the key position of bloom controlling network, not only regulates and control flowering time and becomes flower to change, and also play an important role in the growth of inflorescence and flower.

At present, be used for Cloning Plant Genes method commonly used the methods such as sequence clone method, functional cloning method, transposon tagging method, map based cloning method, subtractive hybridization method and Differential expression analysis are arranged.Although above several method has obtained certain application, ubiquity length consuming time, high in cost of production is not enough.

Summary of the invention

The objective of the invention is to set up a kind of all extensively applicable from the bryophyte to the spermatophyte LFY/ FLOThe dna homolog fragment is easy, cloning process fast, and its primer special is provided.

The objective of the invention is to be achieved through the following technical solutions:

1, a pair of the Blossoms Correlation Gene in Plants LFY/ FLOThe primer special of homologous fragment quick clone, described primer special is degenerated primer LYF and LYR, the nucleotide sequence of described LYF is 5 '-TAYATIAAYAARCCIAARATG-3 ', the nucleotide sequence of described LYR is 5 '-ARIYKIGTIGGIACRTACCA-3 '.

2, a kind of the Blossoms Correlation Gene in Plants LFY/FLOThe rapid clon method of homologous fragment, comprise the genomic dna that extracts plant sample, carry out pcr amplification with primer special, the steps such as the recovery of amplified production, Cloning and sequencing, described primer special is degenerated primer LYF and LYR, the nucleotide sequence of described primer LYF is 5 '-TAYATIAAYAARCCIAARATG-3 ', the nucleotide sequence of described primer LYR is 5 '-ARIYKIGTIGGIACRTACCA-3 '; The PCR reaction system of described pcr amplification is: in 25 μ l reaction systems, and 10 * Buffer, 2.5 μ l, dNTP Mixture 2 μ l, (respectively being 2.5mM), 25 μ M LYF, 1 μ l; 25 μ M LYR, 1 μ l, sample DNA 1 μ l, 5U/ μ l Taq enzyme 0.5 μ l, ddH ₂O 17 μ l; The PCR reaction conditions is: 94 ℃ of denaturation 2min; 94 ℃ of sex change 30s, 44 ℃ of annealing 30s, 72 ℃ are extended 30s, totally 35 circulations; 72 ℃ are extended 10min; Expection amplified fragments size is 236bp; Y represents t or c in the described primer special, and I represents inosine, and R represents g or a, and K represents g or t.

Compared with prior art, the invention has the beneficial effects as follows:

1, by the designed primer special of the present invention (being degenerated primer LYF and LYR), the optimization of PCR reaction conditions can be cloned acquisition from the bryophyte to the spermatophyte LFY/ FLOThe homologous fragment of gene, applicable floristics is extensive.

2, the exon that used primer special is based on after the intron 2 among the present invention partly carries out design of primers, can be directly used in the amplification of genomic dna as template, in the time of avoiding preferably with cDNA as template, may exist because LFY/ FLOThe homologous fragment of gene is not expressed or the expression amount former thereby problem that causes this gene fragment to be difficult to increase such as low.

3, the inventive method is quick, easy, economical and effective.

Description of drawings

Fig. 1 is the result that the PCR temperature of reaction is optimized, and each mark represents among the figure: 1. 36 ℃; 2. 39 ℃; 3. 44 ℃; 4. 49 ℃; 5. 55 ℃; M. DNA Marker.

Fig. 2 uses degenerated primer LYF of the present invention and LYR 14 kind of plant are carried out electrophoresis detection result behind the pcr amplification, wherein:

M is Marker,

Swimming lane 1 is Podocarpus macrophyllus; Swimming lane 2 is liver mosses; Swimming lane 3 is Chinese hollys; Swimming lane 4 is Flower of Lesser Bougainvillea; Swimming lane 5 is cuckoos; Swimming lane 6 is sweet osmanthus; Swimming lane 7 is Canna generalis Baileys; Swimming lane 8 is epipremnum aureums; Swimming lane 9 is dove trees; Swimming lane 10 is Qinggang; Swimming lane 11 is winter jasmines; Swimming lane 12 is Japan winter cherries; Swimming lane 13 is bamboos; Swimming lane 14 is to lead a cow.

Fig. 3 clones from 14 kind of plant LFY/ FLOThe homology analysis result of the aminoacid sequence of dna homolog fragment and the paddy rice of having reported and this gene fragment of corn, percentage ratio represents corresponding two kind of plant among the figure LFY/ FLOThe homology of dna homolog fragment aminoacid sequence.

Embodiment

Below in conjunction with embodiment the present invention is described further, be convenient to understand better the present invention, but be not construed as limiting the invention, experimental technique is ordinary method among the lower routine embodiment, agents useful for same, consumptive material, all can buy from reagent company, embodiment 1 usefulness the inventive method has been carried out the Blossoms Correlation Gene in Plants to following 14 kinds of plants from bryophyte to phanerogamous not equal genus LFY/ FLOThe quick clone of homologous fragment and Genetic homology of carbapenem-resistant thereof.

Podocarpus macrophyllus ( Podocarpus macrophyllus), Podocarpaceae, Podocarpus;

Liver moss ( Bryophyta);

Chinese holly ( Ilex cornuta), Aquifoliaceae, Ilex;

Flower of Lesser Bougainvillea ( Bougainvillea spectabilis), Nyctaginaceae, bougainvillea;

Cuckoo ( Rhododendron hybrida), Ericaceae, Genus Rhododendron;

Sweet osmanthus ( Osmanthus fragrans), Oleaceae, Osmanthus;

Canna generalis Bailey ( Canna indica), Cannaceae, Canna generalis Bailey belongs to;

Epipremnum aureum ( Epipremnum aureum), Araeceae, the kylin leaf belongs to;

Dove tree ( Davidia involucrata), Nyssaceae, dove tree belongs to;

Qinggang ( Cyclobalanopsis glauca), Fagaceae, Cyclobalanopsis;

Winter jasmine ( Jasminum nudiflorum), Oleaceae, gelsemium;

Japan winter cherry ( Prunus subhirtella(Miq.) Sok.), the Rosaceae, Prunus;

Cizu ( Sinocalmus affinis), Gramineae, Dendrocalamus;

Lead a cow ( Pharbifis nil), convolvulaceae, ipomoea.

Embodiment 1

(1) primer special is degenerated primer LYF and LYR design and synthetic

According to what reported LFY/ FLODegenerated primer LYF and the LYR of homologous gene sequences Design, the nucleotide sequence of primer LYF is 5 '-TAYATIAAYAARCCIAARATG-3 ' (namely shown in SEQ ID NO:1), the nucleotide sequence of primer LYR is 5 '-ARIYKIGTIGGIACRTACCA-3 ' (namely shown in SEQ ID NO:2), the target product of expection amplification is 236bp.

Above-mentioned primer special is given birth to worker's biotechnology company limited by Shanghai and is synthesized.

(2) with described primer special to above-mentioned 14 kind of plant LFY/ FLOHomologous fragment carries out pcr amplification, and its step is as follows:

1. the collection of sample

Described 14 kind of plant samples are taken from each park, Kunming, Yunnan Province.

2. the extraction of DNA

Use fast-type plant genome DNA test kit (being purchased from sky, Beijing root company) and from plant tissue, extract DNA, the operation instruction operation that operation steps provides according to sky root company.

3. the optimization of PCR reaction conditions

The Taq that this experiment provides with precious biotechnology (Dalian) company limited Finish the PCR reaction.

Utilize different annealing temperatures to carry out grads PCR, the result shows, under the condition of 36 ℃ and 39 ℃ and 49 ℃, institute's amplified band a little less than, do not have the amplification of purpose band in the time of 55 ℃, in 5 kinds of different annealing temperature, 44 ℃ of amplifications better, therefore, finally determining 44 ℃ is the optimum annealing temperature (Fig. 1) of this research.

Because primer LYF and LYR have higher degeneracy, have designed different primer working concentrations, without the purpose band, when primer concentration is brought up to 25 μ M, can obtain the purpose band when finding Application standard concentration (10 μ M).

4. PCR reaction

The PCR reaction system:

The PCR reaction conditions

94℃ 2min

72℃ 10min

5. electrophoresis detection

Behind the PCR reaction terminating, get 5 μ lPCR products, 1.5% agarose electrophoresis detects amplification.The fragment that can amplify about 236bp is LFY/ FLOHomologous fragment is such as Fig. 2.

6. Cloning and sequencing

Reclaim test kit with sky root glue and reclaim the purifying pcr amplification product, pMD18-T carrier (be purchased from precious biotechnology company limited) connects, transform and clone identification routinely working method carry out, and serve the sea and give birth to worker's biotechnology Services Co., Ltd and finish order-checking.

7. sequence alignment

Utilize Cluxtal X with 14 kind of plant that obtain LFY/ FLOThe dna homolog fragment carries out with the paddy rice reported and this gene fragment of corn that aminoacid sequence is compared and homology analysis, and by the online comparison of NCBI BLAST (http://www.ncbi.nlm.nih.gov/BALST/), all show higher homology, as shown in Figure 3, illustrate that utilizing the inventive method successfully to clone has obtained from 14 kind of plant LFY/FLOThe homologous fragment of gene.

Sequence table

＜110〉southwestern Forestry University

＜120〉rapid clon method of the Blossoms Correlation Gene in Plants LFY/FLO homologous fragment and primer special thereof

<130> -

<160> 2

<170> PatentIn version 3.3

<210> 1

<211> 21

<212> DNA

＜213〉artificial sequence

<220>

＜223〉i represents inosine

<400> 1

tayatiaaya arcciaarat g 21

<210> 2

<211> 20

<212> DNA

＜213〉artificial sequence

<220>

＜223〉i represents inosine

<400> 2

ariykigtig giacrtacca 20

Claims

1. a pair of the Blossoms Correlation Gene in Plants LFY/ FLOThe primer special of homologous fragment quick clone, described primer special is degenerated primer LYF and LYR, the nucleotide sequence of described LYF is 5 '-TAYATIAAYAARCCIAARATG-3 ', the nucleotide sequence of described LYR is 5 '-ARIYKIGTIGGIACRTACCA-3 '.

2. the Blossoms Correlation Gene in Plants LFY/FLOThe rapid clon method of homologous fragment, comprise the genomic dna that extracts plant sample, carry out pcr amplification with primer special, the recovery of amplified production, Cloning and sequencing, it is characterized in that: described primer special is degenerated primer LYF and LYR, the nucleotide sequence of described primer LYF is 5 '-TAYATIAAYAARCCIAARATG-3 ', the nucleotide sequence of described primer LYR is 5 '-ARIYKIGTIGGIACRTACCA-3 '; The PCR reaction system of described pcr amplification is: in 25 μ l reaction systems, and 10 * Buffer, 2.5 μ l, dNTP Mixture 2 μ l, 25 μ M LYF, 1 μ l; 25 μ M LYR, 1 μ l, sample DNA 1 μ l, 5U/ μ l Taq enzyme 0.5 μ l, ddH ₂O 17 μ l; The PCR reaction conditions is: 94 ℃ of denaturation 2min; 94 ℃ of sex change 30s, 44 ℃ of annealing 30s, 72 ℃ are extended 30s, totally 35 circulations; 72 ℃ are extended 10min; Expection amplified fragments size is 236bp.