CN104152448A - Rye chromosome oligonucleotide probe and preparation method thereof - Google Patents

Abstract

The invention relates to the field of biotechnology, specifically a rye chromosome oligonucleotide probe and a preparation method thereof. The probe sequence is shown as SEQ ID NO:1. The acquisition method includes: (1) sequencing rye genome DNA by a Slaf-seq method to obtain a large amount of reads in the length of 120bp; (2) finding highly repetitive sequences in the measured sequences by a bioinformatic method, and selecting oligonucleotide in the length of less than 60bp from the highly repetitive sequences used for probe synthesis; (3) sending the preliminarily selected oligonucleotide sequences to a company for probe synthesis; and (4) after probe synthesis, conducting a FISH test probe by probe to validate the probe functions; and (5) determining the right oligonucleotide probes. The probe sequence obtained by the invention can play due functions, allows easy experiments for identification of rye chromosome in wheat background, and greatly reduces the experimental cost.

Description

Chromosomes of rye oligonucleotide probe and preparation method thereof

Technical field

The present invention relates to biological technical field, is a kind of body oligonucleotide probe for detection of rye dyeing and the preparation method of this probe specifically.

Background technology

In wheat breeding process, rye is that the sibling species of important improvement wheat belongs to.Rye beneficial gene can be imported to wheat genetic background, thereby reach the object of improvement wheat.Most typical example is the long-armed generation transposition of rye 1R the short arm of a chromosome and wheat 1B karyomit(e) and the 1BL.1RS translocation chromosome that forms, and the application that succeeds in wheat breeding of this translocation chromosome, obtains world wheat output and increase substantially.In addition, on other karyomit(e) of rye also with the gene that is conducive to wheat improvement.Distant hybirdization and chromosome engineering method are these beneficial genes to be imported to the important channel of wheat.And in chromosome engineering method, genomic in situ hybridization technology (GISH) and fluorescence in situ hybridization technique (FISH) are indispensable.By GISH and FISH technology, whether integrity, clip size and wheat and the chromosomes of rye that chromosomes of rye in Wheat Background can be detected there is transposition, wheat-rye small segment transposition (Fu et al.2013a can also be detected; Fu et al.2013b; Hao et al.2013; Chaudhary et al.2014; Yang Manyu etc., 2014).In GISH technology, mainly utilize rye genome DNA to analyze (S á nchez-Mor á n et al.1999 as probe; Fu et al.2013a; Fu et al.2013b; Hao et al.2013; Chaudhary et al.2014; Yang Manyu etc., 2014), in FISH technology, mainly utilize the special tumor-necrosis factor glycoproteins of rye to analyze (Ko et al.2002 as probe; Tomita et al.2008; Liu et al.2008; Jia et al.2009; Tomita et al.2009a; Tomita et al.2009b).Yet utilizing at present the special tumor-necrosis factor glycoproteins of rye genomic dna and rye to make probe relatively wastes time and energy.

GISH technology: utilize at present rye genome DNA to make probe, mainly extract DNA by own laboratory, then carry out mark (S á nchez-Mor á n et al.1999; Fu et al.2013a; Fu et al.2013b; Hao et al.2013; Chaudhary et al.2014; Yang Manyu etc., 2014), its step and scheme are as follows: in probe preparation, first will from the histoorgans such as the blade of rye plant or root, extract rye genome DNA; Then genomic dna is carried out to purifying, measure concentration, with probe mark test kit, carry out mark, whether mark is successful for detection probes; Finally with the successful probe of mark, test materials is carried out to GISH analysis.This method is deposited problem both ways: (1) step is various, easily causes mark failure, and from above-mentioned probe mark flow process, existing probe mark technological step is various, and wherein mistake appears in any one link, all can cause the mark failure of probe; (2) expensive, in existing probe mark technology, probe mark test kit (Nick translation) is the most expensive, according to different wave length fluorescein, its unit price is between 3000～5000 yuans, and a common test kit, containing the dosage of 25 μ l, can be marked into 1000 μ l probes, each sample is calculated by 0.2 μ l consumption, and the multipotency of test kit is made 5000 samples.That is on prior art basis, make 5000 samples and at least need to spend 3000～5000 yuans, and the expense of other enzyme of using in probe mark process and relevant reagent also do not count, as the factor of mark failure is taken into account, its cost is higher.

FISH technology: utilize at present the special tumor-necrosis factor glycoproteins of rye to make probe, mainly carry out mark (Ko et al.2002 by own laboratory; Tomita et al.2008; Liu et al.2008; Jia et al.2009; Tomita et al.2009a; Tomita et al.2009b), its markers step and scheme are as follows: in probe preparation, first to from rye genome, obtain by DNA clone technology the sequence fragment of these probes, connect in cloning vector, carrier is transformed into and in Bacillus coli cells, carries out amount reproduction, then from Bacillus coli cells, extract a large amount of cloned sequences, or from rye genomic dna, amplify these sequences by the method for PCR, with glue, reclaim test kit and reclaim sequence, with purification kit, carry out purifying, the sequence obtaining by these methods could be used as probe mark; Its less important purchase probe mark test kit carries out probe mark to the sequence of these acquisitions in own laboratory; Then whether mark is successful for detection probes; Finally with the successful probe of mark, test materials is carried out to FISH test.The probe mark of FISH technology is very numerous and diverse at present, equally easily runs into the situation of probe mark failure.The shortcoming existing is: (1), from above-mentioned probe mark flow process, existing probe mark technological step is various, and wherein mistake appears in any one link, all can cause the mark failure of probe; (2) expensive: in existing probe mark technology, probe mark test kit (Nick translation) is the most expensive.According to different wave length fluorescein, its unit price is between 3000～5000 yuans.A common test kit, containing the dosage of 25 μ l, can be marked into 1000 μ l probes, and each sample is calculated by 0.2 μ l consumption, and the multipotency of test kit is made 5000 samples.That is on prior art basis, make 5000 samples and at least need to spend 3000～5000 yuans, and the expense of other enzyme of using in probe mark process and relevant reagent also do not count, as the factor of mark failure is taken into account, its cost is higher.

Summary of the invention

The object of the invention is to solve current GISH and loaded down with trivial details, the expensive problem of FISH technology middle probe mark.Be intended to find some oligonucleotide sequences, by synthetic way, obtain probe (because oligonucleotide sequence is easily synthesized, and low price), and can replace black wheat genomic dna and the function of the special tumor-necrosis factor glycoproteins of rye according to the synthetic probe of these oligonucleotide sequences, identify the chromosomes of rye in wheat genetic background.So just save the complicated processes of existing probe mark technology, eliminated the situation of probe mark failure.In addition, synthesising probing needle price is not high at present, compares with existing probe mark technology, and its experimental cost can reduce greatly.Therefore, the problem to be solved in the present invention is: find the oligonucleotide sequence of energy replace black wheat genomic dna and the special tumor-necrosis factor glycoproteins of rye as probe, when carrying out hybridization in situ experiment, remove probe mark process, raise the efficiency, reduce difficulty, reduce costs.

For achieving the above object, the technical solution used in the present invention is: a kind of oligonucleotide probe that checks chromosomes of rye, described probe nucleotide sequence is SEQ ID NO:1.

The invention provides a kind of method that obtains chromosomes of rye oligonucleotide probe, comprise the steps:

(1) by Slaf-seq method, rye genomic dna is checked order, obtain the reads that a large amount of length is 120bp;

(2) utilize Perl language editor, the sequence recording is analyzed and cluster, the group of low depth and high depth is filtered, the group of screening certain depth scope is defined as SLAF, therefrom finds multiplicity at more than 75% highly repetitive sequence; By Blast method, highly repetitive sequence and known wheat cdna group sequence are compared again, reject with wheat sequence similarity degree in more than 80% sequence, obtain the highly repetitive sequence of rye genome specific; In NCBI website, utilize blastn instrument again, the highly repetitive sequence that the rye of acquisition is special is compared with the rye distinguished sequence of having announced, obtain similarity and reach more than 80% highly repetitive sequence; The sequence highly repeating from these, choose the oligonucleotide of length below 60bp, synthetic as probe;

(3) oligonucleotide sequence preliminary screening being gone out is delivered to company, and to carry out probe synthetic;

(4) after probe synthesizes, carry out one by one FISH test, the function of checking probe;

(5) determine correct oligonucleotide probe, nucleotide sequence is as SEQ ID NO:1.

Useful technique effect of the present invention is:

(1) oligonucleotide sequence can play due function: oligonucleotide sequence Oligo1162 makes probe, can replace the special tumor-necrosis factor glycoproteins of rye genomic dna and rye, identifies the chromosomes of rye in wheat genetic background.

(2) experiment that makes to identify the chromosomes of rye in Wheat Background becomes simple and easy to do: the acquisition of oligonucleotide sequence Oligo1162, saved the labeling process of probe, after synthesizing by company, just can directly use, avoid the situation of own laboratory probe mark failure.

(3) experimental cost reduces greatly: synthesising probing needle cheap.The unit price of synthetic Oligo1162 probe (is determined according to different colours mark) between 350～1500 yuans.A synthetic probe, containing 5 OD values, can be diluted to the working fluid of 2500 μ l, and each sample is calculated by 0.5 μ l consumption, and synthetic Oligo1162 probe can be made 5000 samples equally; That is do the situ Analysis of 5000 samples, probe cost is at 350～1500 yuans; If own laboratory label probe, making 5000 samples at least needs to spend 3000～5000 yuans.So, by result of the present invention, carrying out In situ hybridization, cost reduces greatly.

Figure of description

Fig. 1 is that probe of the present invention is for the identification of the chromosomes of rye result figure in wheat-rye double diploid.

Embodiment

Below in conjunction with the embodiment of the present invention, the technical scheme in the embodiment of the present invention is clearly and completely described, obviously, described embodiment is only the present invention's part embodiment, rather than whole embodiment.Embodiment based in the present invention, those of ordinary skills, not making the every other embodiment obtaining under creative work prerequisite, belong to the scope of protection of the invention.

Embodiment 1

S101: extract rye genomic dna, and purifying, whether qualifiedly detect genomic dna quality.

S102: by the method for Slaf-Seq, rye genomic dna is checked order, obtain the reads that magnanimity length is 120bp.

S103: utilize Perl language to programme, by editor's program, the sequence obtaining is analyzed to cluster, find multiplicity at more than 75 highly repetitive sequences.

S104: utilize Blast method that highly repetitive sequence and known wheat cdna group sequence are compared, reject with wheat sequence similarity degree in more than 80% sequence, obtain the highly repetitive sequence of rye genome specific.

S105: in NCBI website, utilize blastn instrument again, the highly repetitive sequence that the rye of acquisition is special is compared with the rye distinguished sequence of having announced, obtain similarity and reach more than 80% highly repetitive sequence.

S106: the sequence highly repeating from these, choose the oligonucleotide of length below 60bp, synthetic as probe.

S107: after probe is synthetic, carry out one by one FISH test, the function of checking probe, verifies whether synthetic oligonucleotide sequence can play the effect of the evaluation chromosomes of rye of rye genomic dna and the special tumor-necrosis factor glycoproteins of rye; Obtain oligonucleotide probe.

The active oligonucleotide sequence obtaining at step S107 is as follows:

5’-tgtggcttta?tgttgttttg?gtatctttct?tttggatctt?cacccgtagt?cgggttgt-3’

The oligonucleotide that the present invention obtains can play the effect of the evaluation chromosomes of rye of rye genomic dna and the special tumor-necrosis factor glycoproteins of rye.

Embodiment 2

By above-mentioned sequence, make probe (Oligo1162), can identify the chromosomes of rye in wheat-rye partial amphidiploid.

5 ' end base of oligonucleotide Oligo1162 sequence is carried out to mark with 6-FAM, as probe, the tip of a root medium cell of wheat-rye partial amphidiploid is carried out to fish analysis, result shows, probe Oligo1162 has significantly very strong green hybridization signal (as shown in Fig. 1 arrow) on chromosomes of rye, and with the hybridization signal of chromosome of wheat not obvious or amixia signal almost.This explanation oligonucleotide Oligo1162 makes probe and can replace rye genomic dna and the special tumor-necrosis factor glycoproteins of rye to identify the chromosomes of rye in Wheat Background.

The foregoing is only preferred embodiment of the present invention, in order to limit the present invention, within the spirit and principles in the present invention not all, any modification of doing, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.

Claims (2)

1. an oligonucleotide probe that checks chromosomes of rye, is characterized in that, described probe nucleotide sequence is SEQ ID NO:1.

2. a method of preparing the oligonucleotide probe that detects chromosomes of rye, is characterized in that, comprises the steps:

(1) by Slaf-seq method, rye genomic dna is checked order, obtain the reads that a large amount of length is 120bp;

(2) utilize Perl language editor, the sequence recording is analyzed and cluster, the group of low depth and high depth is filtered, the group of screening certain depth scope is defined as SLAF, therefrom finds multiplicity at more than 75% highly repetitive sequence; By Blast method, highly repetitive sequence and known wheat cdna group sequence are compared again, reject with wheat sequence similarity degree in more than 80% sequence, obtain the highly repetitive sequence of rye genome specific; In NCBI website, utilize blastn instrument again, the highly repetitive sequence that the rye of acquisition is special is compared with the rye distinguished sequence of having announced, obtain similarity and reach more than 80% highly repetitive sequence; The sequence highly repeating from these, choose the oligonucleotide of length below 60bp, synthetic as probe;

(3) oligonucleotide sequence preliminary screening being gone out is delivered to company, and to carry out probe synthetic;

(4) after probe synthesizes, carry out one by one FISH test, the function of checking probe;

(5) determine correct oligonucleotide probe, nucleotide sequence is as SEQ ID NO:1.