CN105671161B - A method of containing high oleic acid according to BnaLCR78 genetic testing cabbage type rape - Google Patents
A method of containing high oleic acid according to BnaLCR78 genetic testing cabbage type rape Download PDFInfo
- Publication number
- CN105671161B CN105671161B CN201610118586.0A CN201610118586A CN105671161B CN 105671161 B CN105671161 B CN 105671161B CN 201610118586 A CN201610118586 A CN 201610118586A CN 105671161 B CN105671161 B CN 105671161B
- Authority
- CN
- China
- Prior art keywords
- oleic acid
- nucleotide sequence
- seq
- cdna
- bnalcr78
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/13—Plant traits
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Immunology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Botany (AREA)
- Mycology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Quickly, easily judge whether cabbage type rape belongs to the measuring method of the rape of high oleic acid content the present invention provides a kind of, the transcription by judging the BnaLCR78 gene of cabbage type rape is specifically disclosed with the presence or absence of variable sheer, i.e. the introne of BnaLCR78 gene judges whether rape belongs to high oleic acid rape with the presence or absence of the measuring method of variable sheer with this.
Description
Technical field: the present invention relates to oil crops fields, and in particular to a kind of to examine and whether judge cabbage type rape
Belong to the measuring method of high oleic acid rape.
Background technique
Cabbage type rape is the important kind in three big rapeseed germplasms, and in the typical oil of one kind that China is planted extensively
Expect crop, fatty acid composition is divided into saturated fatty acid and unsaturated fatty acid, health of the unsaturated fatty acid to human body, plant
It is degeneration-resistant and damage after reparation all play a significant role.According to Peng Qi Ph.D. Dissertation (Peng Qi, with Wild cabbage type in 2011
The clone of canola fatty acids component formation relative new gene, 2011, Ph.D. Dissertation, Agricultural University Of Hunan, middle National IP Network's number
According to library/CNKI；Hereinafter referred to as Peng Qi paper) in narration, T350078 gene is 35 days left sides of cabbage type rape " Hunan oil 15 " Post flowering
No. 78 EST cloned sequences in right seed specific expression library, DNA overall length 369bp, cDNA the overall length 237bp of gene, gene
Unknown Function；Meanwhile the article disclose the nucleotide sequence of the gene (paragraph 1 of page 54 of Peng Qi paper and the 2nd sections).It is logical
The comparison for crossing ncbi database finds a low molecular weight in the gene and arabidopsis, and is rich in the family of cysteine
Gene LCR23 homology is higher, therefore T350078 gene is named as BnaLCR78 again.By constructing corresponding expression vector
By T350078 genetic transformation arabidopsis, it is found that the gene may participate in the anabolism of oleic acid.
The prior art is obtained the coded sequence of BnaLCR78 gene with PCR amplification, is then utilized by design primer
DNAMAN6.0 software analyzes the sequence characteristic of BnaLCR78 gene, finds out and amplifies the sequence difference come with different templates；It is logical
Sequence difference is crossed, the oleic acid content of preliminary clear rape, for further increasing the correlative study of rape oleic acid in the future.In order to look for
To difference of the different materials in gene order, need to carry out gene magnification and examining order, work respectively with different templates
It is cumbersome.
On the other hand, at present canola fatty acids composition can also be measured to obtain correlated results by pertinent instruments, mainly had
Following methods: (one) near infrared spectroscopy: near infrared spectrometer (Near Infrared Spectrum Instrument,
NIRS) be between visible light (Vis) and in electromagnetic radiation as waves between infrared (MIR), U.S. material detects association (ASTM) will
Near infrared spectrum is defined as the region of 780-2526nm, is first non-visible light area that people have found in absorption spectrum.
The uptake zone one of the sum of fundamental frequencies of hydric group (O-H, N-H, C-H) vibration and frequency multiplication at different levels near infrared spectrum and organic molecule
It causes, by scanning the near infrared spectrum of sample, the characteristic information of organic molecule hydric group, disadvantage in available sample
Are as follows: 1. need sample known to a large amount of representative and chemical scores to establish model.In this way, using the analysis of small lot sample close red
Just seem not practical outside.2. model needs to constantly update, since instrument state change or standard sample change, model
To change therewith.3. model is not general, the model of every instrument is different from, and increases the limitation used.4. modeling capital
Height, test expenditure are big.Therefore, it is unreliable that the conclusion that this method obtains is used alone.(2) gas chromatography: use gas as shifting
The chromatography of dynamic phase.Disadvantage: when directly carrying out qualitative analysis to component, it is necessary to use known substance or given data and corresponding color
Spectral peak compares, or is combined with other methods (such as mass spectrum, spectrum), could obtain result directly certainly.In quantitative analysis
When, it often needs to be corrected the signal exported after detection with known substance pure sample, therefore analyze the reliability very great Cheng of result
The selection that known substance is depended on degree, it is bad to be used alone the result reliability that this method obtains；(3) liquid chromatography: liquid is used
Chromatography of the body as mobile phase.Disadvantage: liquid chromatogram cannot be directly given the qualitative results of unknown material by chromatogram, and necessary
It is compared by known standard qualitative.When no pure material compares, Qualitative Identification with regard to highly difficult, at this moment need to by mass spectrum, it is infrared and
The cooperation such as chemical method, therefore the selection of known standard is whether result is reliable crucial, and the result that this method obtains is used alone
Reliability is bad, and in addition most metals salt and the substance of thermal stability difference can't be analyzed.Although the disadvantage can efficient liquid
Phase chromatography overcomes, but high-efficient liquid phase chromatogram technique analysis is at high cost, expensive equipment and maintenance cost is high, analysis time compared with
It is long.
Existing research emphasis cultivates kind often by means such as conventional breeding and molecular breedings, measures in rape
Oleic acid content, as certain material whether be high-oleic acid material direct basis, and very to the sequence characteristic of corresponding controlling gene
The sequence characteristic of gene is studied in few research, and targeted screening material carries out relevant downstream work, can be to selection, cultivation
More good high-oleic acid material establishes important basis.
Summary of the invention
The present invention provide can a kind of measuring method of the cabbage type rape containing high oleic acid, contain following steps:
(1) transcription of the BnaLCR78 gene of cabbage type rape is measured there are variable sheer, then the cabbage type rape
Oleic acid content is high；Or variable sheer is not present in the transcription of the BnaLCR78 gene of measurement cabbage type rape, then the Wild cabbage type is oily
Dish is not belonging to high oleic acid rape.
Since gene order generally has exon and introne simultaneously, formed active mRNA coded sequence it
Before, it is processed, shears introne, exon is connected in a particular manner.It is obtained by template amplification of cDNA
Gene be mostly only retain exon code area a sequence.Present invention discover that the BnaLCR78 base of the rape of high oleic acid content
Because having the form of variable sheer, rather than the BnaLCR78 gene of high oleic acid content rape does not have the form of variable sheer.This
Invention provides a kind of measuring method, for prejudging the height of cole crop oleic acid content, examines and whether anticipation belongs to high oil
The rape of acid.
Further, the present invention provides specific measuring methods comprising the steps of:
(1) mRNA of the BnaLCR78 gene of cabbage type rape is extracted, reverse transcription obtains the cDNA of the mRNA, reverse transcription
The nucleotide sequence of primer is as shown in the nucleotide sequence of sequence table SEQ ID NO.1；
(2) PCR amplification, the primer nucleotide sequences of PCR amplification such as sequence table SEQ are carried out to the cDNA that step (1) obtains
Shown in the nucleotide sequence of ID NO.2 and SEQ ID NO.3；
(3) the cDNA amplified production that step (2) is prepared is sequenced, obtains the cDNA nucleotide sequence；
(4) nucleotide sequence comparison for the cDNA nucleotide sequence and BnaLCR78 gene for obtaining step (3) sequencing, step
Suddenly sequencing result described in (3) had both included the cDNA nucleotide sequence containing BnaLCR78 gene intron, described in step (3)
Sequencing result also includes the cDNA nucleotide sequence without BnaLCR78 gene intron, then the cabbage type rape contains for high oleic acid
The cabbage type rape of amount；The nucleotide sequence of the BnaLCR78 gene is as shown in the nucleotide sequence of SEQ ID NO.4；Or it will
The nucleotide sequence comparison of the nucleotide sequence of the cDNA that step (3) measures and BnaLCR78 gene, sequencing described in step (3)
Result is the cDNA nucleotide sequence without BnaLCR78 gene intron, then the cabbage type rape is the sweet of low oleic acid content
Blue type rape；The nucleotide sequence of the BnaLCR78 gene is as shown in the nucleotide sequence of SEQ ID NO.4.
Meanwhile the nucleotide sequence of BnaLCR78 gene described in step (4), it can be replaced cabbage type rape to be measured
The nucleotide sequence of BnaLCR78 gene.
High oleic acid of the present invention is that the oleic acid content of rape is greater than or equal to 60%, and the low oleic acid is the oil of rape
Acid content is less than 60%.
Further, measuring method of the present invention comprises the steps of:
The method of reverse transcription:
(1) reagent (operate in and carry out on ice) being added in following table in sterile centrifuge tube；
Table 1-1RNA reverse transcription operating procedure one
(2) it is uniformly mixed, 65 DEG C of 5min, rapidly as at least 1min on ice；
(3) following reagent (operate in and carry out on ice) is added into centrifuge tube again；
Table 1-2RNA reverse transcription operating procedure two
4) it mixes and is placed on 50 DEG C, 60min；
5) 70 DEG C, 15min terminates reaction
6) reverse transcription product can be placed at one 20 DEG C and save
7) PCR amplification: the PCR instrument T100 of Bio-Rad is used；
Response procedures are as follows: 94 DEG C of 4min at, 94 DEG C of 40s at, 58 DEG C of 40s at, 72 DEG C of for of 30s at
35cycles；and 7min at 72℃
Reaction system are as follows: 2 μ L of cDNA template, each 0.8 μ L, ES Taq DNA of 0.5 μ L, 10mM dNTPS of upstream and downstream primer
3 μ L of Polymerase (5U/ μ L) 0.8 μ L, 10 × Polymerase Buffer, sterile water volume complement to 30 μ L
8) the cDNA product obtained to step 7) amplification is sequenced, and obtains the nucleotide sequence of sequencing result cDNA；
9) sequence compares, and obtains sequence comparison result.It can be by sequencing result cDNA nucleotide sequence that step 8) obtains
With the nucleotide sequence comparison of BnaLCR78 gene, sequencing result described in step 8) had both included including containing BnaLCR78 gene
The cDNA nucleotide sequence of son, sequencing result described in step 8) also include the cDNA nucleosides without BnaLCR78 gene intron
Acid sequence, then the cabbage type rape is the cabbage type rape of high oleic acid content；The nucleotide sequence of the BnaLCR78 gene is such as
Shown in the nucleotide sequence of SEQ ID NO.4；
Or the nucleotide sequence ratio of the nucleotide sequence for the sequencing result cDNA for measuring step 8) and BnaLCR78 gene
Compared with sequencing result described in step 8) is the cDNA nucleotide sequence without BnaLCR78 gene intron, then the Wild cabbage type
Rape is the cabbage type rape of low oleic acid content；The nucleosides of the nucleotide sequence of the BnaLCR78 gene such as SEQ ID NO.4
Shown in acid sequence.
Specific sequence alignment method, using 6.0 software of DNAMAN, version 6.0.3.99, operating procedure includes: choosing
Trim vegetables for cooking simple sequence (S), selects Multiple Sequence Alignment (M) below in comparison (A), clicks in the window " sequence and file " of pop-up
" file " button selects suitable sequential file, is nucleotide sequence or protein sequence according to sequence, selects DNA either
Protein, selection in next step, in " mode " window, if it is comparison dna sequence, make hook before " attempting to use double-strand ", point
It hits in next step, if it is protein sequence is compared, this step be can be omitted, it is clicked in " Multiple Sequence Alignment " window in next step,
Remaining parameter is set according to system default value, and " completion " button is clicked in " multiple alignment " window, can enter " multisequencing ratio
It is right " result interface
The present invention also provides can pass through the BnaLCR78 gene of measurement cabbage type rape with the presence or absence of variable sheer form
Method, to examine and prejudge the purposes whether rape belongs to high oleic acid content rape, i.e., measurement BnaLCR78 gene exist can
Become the method for shearing, examine and judges that cabbage type rape belongs to the purposes of high oleic acid rape.
In addition, the present invention also provides a kind of methods of genotype for detecting cabbage type rape BnaLCR78 gene, specifically
Provide a kind of PCR method of genotype for detecting cabbage type rape BnaLCR78 gene, comprising:
(1) reverse transcription: extracting the mRNA of detected materials, on ice, extraction is added in sterile centrifuge tube
MRNA10pg-5 μ g, 1 μ L of primer 1 μ L, 10mM dNTP (mixture of four kinds of deoxynucleotides) mixture, is then added by coke
Diethyl carbonate processing and pure water through autoclave sterilization filtering is to 13 μ L；Such as sequence table of the nucleotide sequence of the primer
Shown in the nucleotide sequence of SEQ ID NO.1；
(2) it is uniformly mixed, 65 DEG C are kept for 5 minutes, then rapidly as holding 1 minute on ice；
(3) on ice, the RNA degrading enzyme of 5 times of 4 μ L, 40u/ μ L of First-Strand buffer is added into centrifuge tube
The reverse transcriptase III SuperScript III of 1 μ L, the 200u/ μ L of dithiothreitol (DTT) DTT of RNase inhibitor 1 μ L, 0.1mol
1 μ L of Reverse Transcriptase, until 20 μ L of total volume；
(4) it mixes, is placed in 50 DEG C, kept for 60 minutes；
(5) 70 DEG C are placed in be kept for 15 minutes, reaction is terminated, obtains reverse transcription product cDNA；
(6) reverse transcription product prepared by step (5) is placed in subzero 20 DEG C of preservations；
(7) the PCR instrument T100 of Bio-Rad, response procedures PCR amplification: are used are as follows: 94 DEG C are kept for 4 minutes, and 94 DEG C keep 40
Second, 58 DEG C are kept for 40 seconds, and 72 DEG C are kept for 30 seconds, are carried out 35 circulations, are kept for 7 minutes in 72 DEG C；Reaction system are as follows: cDNA template
2 μ L, the ES Taq of each 0.8 μ L, 5U/ μ L of 0.5 μ L, 10mM dNTPS (mixture of four kinds of deoxynucleotides) of upstream and downstream primer
Then archaeal dna polymerase 0.8 μ L, 10 times of 3 μ L of polymerase chain reaction buffer complement to 30 μ L with sterile water volume；
(8) the cDNA product obtained to step (7) amplification is sequenced, and obtains the nucleotide sequence of sequencing result cDNA；
(9) sequence alignment: the nucleosides of sequencing result cDNA nucleotide sequence and BnaLCR78 gene that step (8) is obtained
Acid sequence compares, and sequencing result described in step (8) had both included the cDNA nucleotide sequence containing BnaLCR78 gene intron,
Sequencing result described in step (8) also includes the cDNA nucleotide sequence without BnaLCR78 gene intron, then the Wild cabbage type
Rape is the cabbage type rape of high oleic acid content；The nucleosides of the nucleotide sequence of the BnaLCR78 gene such as SEQ ID NO.4
Shown in acid sequence；
Or the nucleotide sequence ratio of the nucleotide sequence for the sequencing result cDNA for measuring step (8) and BnaLCR78 gene
Compared with sequencing result described in step (8) is the cDNA nucleotide sequence without BnaLCR78 gene intron, then the Wild cabbage type
Rape is the cabbage type rape of low oleic acid content；The nucleosides of the nucleotide sequence of the BnaLCR78 gene such as SEQ ID NO.4
Shown in acid sequence.
The relevant term of the present invention:
Double-low rapeseed: while there is the rape of low erucic acid and low glucosinolate characteristic
PCR: polymerase chain reaction, it is a kind of under specific primer, polymerase and four kinds of nucleotide participations, by becoming in advance
Property, denaturation, annealing, extension, reach the technology of rapid amplifying specific DNA fragment
High-oleic acid material: pass through the physics perhaps high rape material of the oleic acid content of chemical mutagenesis or self-assembling formation, oil
Acid content is general > and 60%, it is indicated with G in figure.
The holding that high-oleic acid material of the present invention can be selected from cabbage type rape is the seed of 100B, or is also possible to
The high rape material of the oleic acid content of self-assembling formation.There are extremely significant negative correlation between the erucic acid and oleic acid of cabbage type rape
Relationship (Zhou Yongming, the heredity of several primary fat acid contents, Acta Agronomica Sinica [J], 1987 in cabbage type rape seed), thus it is low
Erucic acid rape is considered high oleic acid rape.The industry mark publicized and implemented according to The Ministry of Agriculture of the People's Republic of China, MOA 2000
It is quasi- --- the regulation of Canola Oil NY416-2000, the content of canola oleic acid is between 50%-66%, according to another dish
The regulation of seed oil GB1536-2004, the oleic acid content of Canola Oil is between 51%-70%, but high oleic acid rape
The national standard of content has no always announcement, according to the document delivered at present, academia be generally acknowledged that the oleic acid content of rape >
60% ability is referred to as high oleic acid rape (Shi Dongqiao, Zhou Yihua, Zhang Lihua etc., the canola fatty acids regulation of mediated by agriculture bacillus
Genetic engineering research, high-tech communicate [J], and 2001, (2): 1-7) (Guan Chunyun, radioactive breeding obtain rape high oleic acid material, make
Object journal [J], 2006,32 (11): 1626-1629).In conjunction with the regulation of above-mentioned document and national standard, height of the present invention
Oleic acid content > 60% of oleic acid rape is considered high oleic acid rape.
The control material of high-oleic acid material: the original material of any processing is not done, is indicated in figure with CK
Low oleic acid material: oleic acid content < 40% is mostly the material of high erucic acid, is indicated in figure with D
Introne: the noncoding region of split gene can be transcribed, but be cut away in mRNA process
Exon: the coded sequence in split gene.Exon is a part of eukaryotic gene, it after montage still
It can be saved, and protein can be expressed as during Protein synthesis.
Variable sheer: the mRNA precursor that most of eukaryotic transcriptions generate be montage in one way produce it is a kind of at
Ripe mRNA molecule, thus only translate into a kind of protein.But a mRNA precursor of some genes passes through different montage modes
(selecting different splice sites) generates different mRNA splicing isomers, this process is known as alternative splicing.
Type has following several: it is generally believed that alternative splicing has 5 kinds of citation forms: 1. introne retains；2. variable 5 '
End；3. 3 ' variable ends；4. exon box；5. mutual exclusion exon (only selects one in one group of exon).Also have and be divided into 7 kinds of forms
, in addition variable starting or terminal exon, and both forms are more likely alternative promoter, the variable site polyA
Caused by (Li Zhifeng, eukaryotic gene alternative splicing status and prospectives, bioinformatics [J], 2004,2 (2), 35-38)
(Roberts GC,Smith CW.Alternative splicing:combinatorial output from the
genome.Curr Opin Chem Biol[J],2002,6(3):375-383)(Moderk B,Lee C.A genomic
view of alternative splicing.Nature Genet[J],2002,30:13-19)。
Using cloning, being sequenced to a relatively simple gene, sequence compares the present invention, can be right within a short period of time
The oleic acid content of rape makes quickly preliminary judgement.Advantage is as follows: (1) RNA extract and reverse transcription technical operation all very
Maturation, time-consuming short, failure rate is extremely low；(2) PCR reaction system and response procedures are all convenient for operating；(3) full length gene less than
400bp is easy clone, and the result time being sequenced is short；(4) DNAMAN6.0 software is easy to operate, and display interface is clear, right
The fast response time of order can complete the comparison work of sequence in a very short period of time, obtain accurate conclusion.
Obviously, above content according to the present invention is not being departed from according to the ordinary technical knowledge and customary means of this field
Under the premise of the above-mentioned basic fundamental thought of the present invention, the modification, replacement or change of other diversified forms can also be made.
Specific implementation method in the form of embodiment below is doing further specifically above content of the invention
It is bright, but should not be construed as following embodiments for limiting the scope of protection of the present invention.
Detailed description of the invention
Fig. 1, Peng Qi paper BnaLCR78 gene order and the BnaLCR78 gene order of high-oleic acid material, high-oleic acid material
BnaLCR78 gene reservation introne cDNA gene order comparison result figure.
In Fig. 1, black intermittent line indicates G-BnaLCR78genome DNA and G-BnaLCR78-2 ratio
The nucleotide that BnaLCR78genome DNA has more, the continuous solid line of black indicate that G-BnaLCR78-2 remains introne sequence
Column；
Wherein, BnaLCR78 gene order disclosed in DNA: Peng Qi paper of BnaLCR78genome；
G-BnaLCR78genome DNA: the gene order obtained using the DNA of high oleic acid rape as template amplification；
G-BnaLCR78-2: the cDNA obtained using the mRNA reverse transcription of high oleic acid rape BnaLCR78 gene is template amplification
Obtain gene order.
The BnaLCR78 gene of the cDNA sequence and high-oleic acid material of BnaLCR78 gene disclosed in Fig. 2 a Peng Qi paper
The control material of cDNA sequence, high-oleic acid material without introne and low oleic acid material
The comparison result figure of the cDNA sequence of BnaLCR78 gene.
The BnaLCR78 gene of the cDNA sequence and high-oleic acid material of BnaLCR78 gene disclosed in Fig. 2 b Peng Qi paper
The control material of cDNA sequence, high-oleic acid material without introne and low oleic acid material
The cDNA sequence of BnaLCR78 gene respectively corresponds the comparison result figure of the protein sequence translated.
The cDNA sequence for the gene that BnaLCR78: Peng Qi paper is announced；
G-BnaLCR78: the cDNA sequence of the BnaLCR78 gene in high-oleic acid material；
CK-BnaLCR78: the cDNA sequence of BnaLCR78 gene in the control material of high-oleic acid material；
D-BnaLCR78: the cDNA sequence of BnaLCR78 gene in low oleic acid material.
The schematic diagram for the fracture albumen that the nucleotide sequence of the BnaLCR78 gene of Fig. 3 high-oleic acid material is translated, is shown in
Embodiment one.
The schematic diagram for the fracture albumen that Fig. 4 Peng Qi paper BnaLCR78 gene nucleotide series in 2011 are translated, is shown in
Embodiment one.
The following are specific embodiments
Embodiment
Embodiment one
(1) take high-oleic acid material cabbage type rape holding be 100B seed, it is compound by Flight Mutagenesis+iterating sodium
Processing, and the offspring for stablizing heredity is cultivated through excessively generation, oleic acid content 62.60% is indicated with G.Also self-assembling formation can be used
High-oleic acid material (oleic acid content > 60%) seed substitution it is oily by Flight Mutagenesis+iterating sodium combined processing Wild cabbage type
The holding of dish is the seed of 100B, to carry out this experiment.
The control material of high-oleic acid material is taken, i.e., is 100B without the holding of the cabbage type rape of any processing, in field
Between grow under natural conditions, the seed harvested after mature, oleic acid content 56.57%；It is indicated with CK.
The seed of oil 821 in low oleic acid material is taken, oleic acid content < 40% is 25.17%, is mostly the material of high erucic acid, is used
D is indicated.
(2) according to Peng Qi paper in 2011, about the narration of BnaLCR78 gene, (BnaLCR78 gene nucleotide series are such as
Shown in sequence table SEQ ID NO.4), pass through software DNAMAN6.0 design primer, primer sequence such as sequence table SEQ ID NO.2
Shown in nucleotide sequence with SEQ ID NO.3.
(3) cDNA obtained respectively using genomic DNA and reverse transcription is template, the nucleotide sequence of reverse transcription primer such as sequence
Shown in list SEQ ID NO.1.The complete gene order of the gene and coded sequence are obtained by PCR amplification.PCR response procedures
Are as follows: 94 degree 4 minutes, 94 degree 40 seconds, 58 degree 40 seconds, 72 degree 30 seconds, 35 circulation, 72 degree heat preservation 7 minutes；
The specific method of reverse transcription:
(1) reagent (operate in and carry out on ice) being added in following table in sterile centrifuge tube；
Table 1-1RNA reverse transcription operating procedure one
(2) it is uniformly mixed, 65 DEG C of 5min, rapidly as at least 1min on ice；
(3) following reagent (operate in and carry out on ice) is added into centrifuge tube again；
Table 1-2RNA reverse transcription operating procedure two
4) it mixes and is placed on 50 DEG C, 60min；
5) 70 DEG C, 15min terminates reaction
6) reverse transcription product can be placed at one 20 DEG C and save
7) method of PCR amplification:
PCR amplification uses the PCR instrument T100 of Bio-Rad
Response procedures are as follows: 94 DEG C of 4min at, 94 DEG C of 40s at, 58 DEG C of 40s at, 72 DEG C of for of 30s at
35cycles；and7min at 72℃
Reaction system are as follows: 2 μ L of cDNA template, each 0.8 μ L, ES TaqDNA of 0.5 μ L, 10mM dNTPS of upstream and downstream primer
3 μ L of Polymerase (5U/ μ L) 0.8 μ L, 10 × Polymerase Buffer, sterile water volume complement to 30 μ L
8) the cDNA product obtained to step 7) amplification is sequenced, and obtains the nucleotide sequence of sequencing result cDNA；
Nucleotide sequence comparison/comparison can be used sequence alignment program commonly used in the art and carry out sequence comparison, such as can benefit
With 6.0 software of DNAMAN, version 6.0.3.99 selects menu sequence (S), selects Multiple Sequence Alignment (M) below in comparison (A),
" file " button is clicked in the window " sequence and file " of pop-up, selects suitable sequential file, is nucleotide according to sequence
Sequence or protein sequence select DNA either protein, selection next step, in " mode " window, if it is comparison dna
Sequence is made hook before " attempting to use double-strand ", is clicked in next step, and if it is protein sequence is compared, this step be can be omitted,
It is clicked in next step in " Multiple Sequence Alignment " window, remaining parameter is set according to system default value, at " multiple alignment " window midpoint
" completion " button is hit, the result interface of " Multiple Sequence Alignment " can be entered
The method of amino acid sequence prediction can be used amino acid sequence forecasting software commonly used in the art and carry out, can such as adopt
With DNAMAN6.0 software.Concrete operation method are as follows: open DNAMAN6.0 software, open " file " life under File menu
It enables, selection suitably containing the file of nucleotide sequence, clicks open button, selects " Quan Xuan " life under Edit menu
It enables, selects " from selected project " subcommand under " being loaded into sequence " order under " sequence " menu, click "Yes" button, selection
" translation " order under " protein " menu, in " translation parameter " dialog box, " reading frame " column selects " reading frame 1 ",
The column " display DNA " selects "None", remaining parameter is set according to system default value, clicks " determination " button, that is, the coding occurred
The prediction result of albumen occurs with the format of sequential file
The comparison that gene order is carried out using the function that the multiple sequence in DNAMAN6.0 software compares, finds different materials
The difference of gene order in material, as the preliminary foundation for quickly judging rape oleic acid content.
Experiment as a result, the result of sequence alignment:
CDNA, Peng Qi paper of the BnaLCR78 gene of high-oleic acid material, the BnaLCR78 gene of high-oleic acid material
The control experiment result that BnaLCR78 gene order compares is shown in Figure of description 1.
Fig. 1 the experimental results showed that, in the rape of high oleic acid content, using its cDNA as template, still through PCR amplification
To be the full-length gene order for remaining the BnaLCR78 of introne.Show the genome of high oleic acid cabbage type rape
BnaLCR78 gene contains introne, and the cDNA of the genome BnaLCR78 for the high-oleic acid material that reverse transcription obtains also remains interior
Containing son.
The nucleotide sequence institute of the nucleotide sequence of the BnaLCR78 gene of high-oleic acid material such as sequence table SEQ ID NO.5
Show, the albumen of the gene translation stops in advance, include fracture protein sequence SEQ ID NO.7, SEQ ID NO.18,
SEQ ID NO.19、SEQ ID NO.20、SEQ ID NO.21、SEQ ID NO.22、SEQ ID NO.23、SEQ ID
NO.24, SEQ ID NO.25, SEQ ID NO.26, SEQ ID NO.27 totally ten one fracture protein/polypeptide sequence；Fracture
The concrete form of albumen is as shown in attached drawing 3.
The nucleotide of the nucleotide sequence of the cDNA of the BnaLCR78 gene of high-oleic acid material such as sequence table SEQ ID NO.6
Shown in sequence, the amino acid sequence of the albumen of translation is as shown in the amino acid sequence of sequence table SEQ ID NO.8, SEQ ID
NO.8 translation is complete.
Peng Qi paper BnaLCR78 gene (BnaLCR78 gene nucleotide series such as sequence table SEQ ID NO.4 in 2011
It is shown) albumen that translates stops in advance, and it include fracture protein sequence SEQ ID NO.9, SEQ ID NO.28, SEQ
ID NO.29 totally three fracture protein/polypeptide sequence；The concrete form of albumen is broken as shown in attached drawing 4.
It is carried out in parallel at another group, the cDNA sequence of BnaLCR78 gene disclosed in Peng Qi paper, with high-oleic acid material
BnaLCR78 gene cDNA sequence, the BnaLCR78 gene of the control material of high-oleic acid material and low oleic acid material
CDNA sequence control experiment result is shown in the control experiment knot of Figure of description 2a and the protein amino acid sequence translated respectively
Fruit is as shown in Fig. 2 b.
Fig. 2 the experimental results showed that, the BnaLCR78 gene obtained using the cDNA of high oleic acid content rape as template amplification
Another form be exactly the introne being normally sheared, with other two type (high oleic acid control material, low oleic acid material
Material) result that expands in rape is consistent.Fig. 1 experimental result is shown, is expanded using the cDNA of high oleic acid content rape as template
Another form for increasing obtained BnaLCR78 gene is the form containing introne.The variable sheer of introne, only in height
It is found in oleic acid rape.
The nucleotide sequence of the cDNA sequence of the BnaLCR78 gene of high-oleic acid material is as such as sequence table SEQ ID NO.10
Nucleotide sequence shown in；The amino acid sequence institute of the amino acid sequence such as sequence table SEQ ID NO.14 of its albumen translated
Show, SEQ ID NO.14 translation is complete.
The nucleotide sequence of the cDNA sequence of the control material of high-oleic acid material is as such as sequence table SEQ ID NO.11
Shown in nucleotide sequence；Its translate albumen amino acid sequence as shown in the amino acid sequence of sequence table SEQ ID NO.15,
SEQ ID NO.15 translation is complete.
The nucleotide sequence of the cDNA sequence of the cDNA sequence of the BnaLCR78 gene of low oleic acid material is as such as sequence table SEQ
Shown in the nucleotide sequence of ID NO.12；The amino acid of the amino acid sequence such as sequence table SEQ ID NO.16 of its albumen translated
Shown in sequence, SEQ ID NO.16 translation is complete.
The nucleotide sequence of the cDNA sequence of the cDNA sequence of BnaLCR78 gene disclosed in Peng Qi paper is as such as sequence table
Shown in the nucleotide sequence of SEQ ID NO.13；The ammonia of the amino acid sequence such as sequence table SEQ ID NO.17 of its albumen translated
Shown in base acid sequence, SEQ ID NO.17 translation is complete.
Claims (3)
1. a kind of measuring method of the cabbage type rape containing high oleic acid, which is characterized in that the cabbage type rape is to keep system
The offspring of 100B, its BnaLCR78 gene order is as shown in SEQ ID No.5；Detection holding is the offspring of 100B
The reverse transcription product of BnalCR78 gene, if amplification piece identical with nucleotide sequence shown in SEQ ID No.5 can be obtained
Section, and the amplified fragments of nucleotide sequence shown in SEQ ID No.6 can be obtained, then holding detected is that the offspring of 100B contains
There is high oleic acid；If can only obtain the amplified fragments of nucleotide sequence shown in SEQ ID No.6, holding detected is 100B
Offspring be low oleic acid；The high oleic acid is that the oleic acid content of rape is greater than or equal to 60%, and the low oleic acid is the oil of rape
Acid content is less than 60%.
2. measuring method according to claim 1, which is characterized in that comprise the steps of:
(1) mRNA of the BnaLCR78 gene for the offspring that holding to be determined is 100B is extracted, reverse transcription obtains the mRNA's
CDNA, the nucleotide sequence of reverse transcription primer is as shown in the nucleotide sequence of sequence table SEQ ID NO.1；
(2) PCR amplification, the nucleotide sequence of PCR amplification primer such as sequence table SEQ ID are carried out to the cDNA that step (1) obtains
Shown in the nucleotide sequence of NO.2 and SEQ ID NO.3；
(3) the cDNA amplified production that step (2) is prepared is sequenced, obtains the cDNA nucleotide sequence；
If expansion of the cDNA nucleotide sequence that step (3) sequencing obtains both containing nucleotide sequence shown in SEQ ID No.5
Increase segment, and the amplified fragments containing nucleotide sequence shown in SEQ ID No.6, then holding detected is the offspring of 100B
Contain high oleic acid；If can only obtain the amplified fragments of nucleotide sequence shown in SEQ ID No.6, holding system detected
The offspring of 100B is low oleic acid；The high oleic acid is that the oleic acid content of rape is greater than or equal to 60%, and the low oleic acid is rape
Oleic acid content less than 60%.
3. measuring method according to claim 2, which is characterized in that the measuring method comprises the steps of:
(1) reverse transcription: extracting the mRNA that holding is the offspring of 100B, on ice, extraction is added in sterile centrifuge tube
Then 1 μ L of mRNA10pg-5 μ g, 1 μ L, 10mM dNTPs of primer is added and is handled by pyrocarbonic acid diethyl ester and gone out through high temperature and pressure
The pure water of bacterium filtering is to 13 μ L；The nucleotide sequence of the primer is as shown in the nucleotide sequence of sequence table SEQ ID NO.1；
(2) it is uniformly mixed, 65 DEG C are kept for 5 minutes, then rapidly as holding 1 minute on ice；
(3) on ice, RNase inhibitor 1 the μ L, 0.1mol of 5 times of first 4 μ L, 40u/ μ L of chain buffer are added into centrifuge tube
1 μ L, 200u/ μ L of dithiothreitol (DTT) SuperScriptTM1 μ L of III reverse transcriptase, until 20 μ L of total volume；
(4) it mixes, is placed in 50 DEG C, kept for 60 minutes；
(5) 70 DEG C are placed in be kept for 15 minutes, reaction is terminated, obtains reverse transcription product cDNA；
(6) reverse transcription product prepared by step (5) is placed in subzero 20 DEG C of preservations；
(7) the PCR instrument T100 of Bio-Rad, response procedures PCR amplification: are used are as follows: 94 DEG C are kept for 4 minutes, and 94 DEG C are kept for 40 seconds,
58 DEG C are kept for 40 seconds, and 72 DEG C are kept for 30 seconds, are carried out 35 circulations, are kept for 7 minutes in 72 DEG C；Reaction system are as follows: 2 μ of cDNA template
The ES of each 0.8 μ L, 5U/ μ L of 0.5 μ L, 10mM dNTPs of upstream and downstream primer shown in L, SEQ ID NO.2 and SEQ ID NO.3
Then Taq archaeal dna polymerase 0.8 μ L, 10 times of 3 μ L of polymerase chain reaction buffer complement to 30 μ L with sterile water volume；
(8) the cDNA product obtained to step (7) amplification is sequenced, and obtains the nucleotide sequence of sequencing result cDNA；
If expansion of the cDNA nucleotide sequence that step (8) sequencing obtains both containing nucleotide sequence shown in SEQ ID No.5
Increase segment, and the amplified fragments containing nucleotide sequence shown in SEQ ID No.6, then holding detected is the offspring of 100B
Contain high oleic acid；If can only obtain the amplified fragments of nucleotide sequence shown in SEQ ID No.6, holding system detected
The offspring of 100B is low oleic acid；The high oleic acid is that the oleic acid content of rape is greater than or equal to 60%, and the low oleic acid is rape
Oleic acid content less than 60%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610118586.0A CN105671161B (en) | 2016-03-02 | 2016-03-02 | A method of containing high oleic acid according to BnaLCR78 genetic testing cabbage type rape |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610118586.0A CN105671161B (en) | 2016-03-02 | 2016-03-02 | A method of containing high oleic acid according to BnaLCR78 genetic testing cabbage type rape |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105671161A CN105671161A (en) | 2016-06-15 |
| CN105671161B true CN105671161B (en) | 2019-06-14 |
Family
ID=56306448
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610118586.0A Expired - Fee Related CN105671161B (en) | 2016-03-02 | 2016-03-02 | A method of containing high oleic acid according to BnaLCR78 genetic testing cabbage type rape |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105671161B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106749573B (en) * | 2016-12-15 | 2021-01-01 | 湖南农业大学 | Low-molecular-weight cysteine-rich protein of brassica napus and coding gene thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007138444A2 (en) * | 2006-05-29 | 2007-12-06 | Institut National De La Recherche Agronomique (Inra) | Genetic markers for high oleic acid content in plants |
| CN101550414A (en) * | 2008-12-31 | 2009-10-07 | 刘春林 | Method for obtaining cole with high oleic acid and low erucic acid by dual-approach gene expression synchronous suppression |
| CN101824472A (en) * | 2009-12-28 | 2010-09-08 | 华中农业大学 | Cabbage type rape high oleic acid molecular marker, preparation method and application thereof |
-
2016
- 2016-03-02 CN CN201610118586.0A patent/CN105671161B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007138444A2 (en) * | 2006-05-29 | 2007-12-06 | Institut National De La Recherche Agronomique (Inra) | Genetic markers for high oleic acid content in plants |
| CN101550414A (en) * | 2008-12-31 | 2009-10-07 | 刘春林 | Method for obtaining cole with high oleic acid and low erucic acid by dual-approach gene expression synchronous suppression |
| CN101824472A (en) * | 2009-12-28 | 2010-09-08 | 华中农业大学 | Cabbage type rape high oleic acid molecular marker, preparation method and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 与甘蓝型油菜脂肪酸组分形成相关新基因的克隆;彭琦;《中国博士学位论文全文数据库 农业科技辑》;20111215(第12期);第D047-13页 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105671161A (en) | 2016-06-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| TR201808715T4 (en) | Potatoes with low cold induced sweetening. | |
| CN108018316A (en) | A kind of method of gene knockout selection and breeding rmnd5b Gene Deletion zebra fish | |
| CN105647969A (en) | Method for breeding stat1a (signal transducer and activator of transcription 1) gene-deleted zebra fish through gene knockout | |
| CN110951838B (en) | Primer, probe and kit for detecting meloidogyne incognita based on RPA-LFD technology and application | |
| Soler et al. | A potato skin SSH library yields new candidate genes for suberin biosynthesis and periderm formation | |
| An et al. | Regulation of HbPIP2; 3, a latex-abundant water transporter, is associated with latex dilution and yield in the rubber tree (Hevea brasiliensis Muell. Arg.) | |
| Mario et al. | The haplotype-resolved reference genome of lemon (Citrus limon L. Burm f.) | |
| Moon et al. | Construction of a CRISPR/Cas9-mediated genome editing system in Lentinula edodes | |
| CN111100945B (en) | Reference gene of toona sinensis, primer and application thereof | |
| Zhang et al. | Identification of differentially expressed genes associated with apple fruit ripening and softening by suppression subtractive hybridization | |
| Xu et al. | Selection of relatively exact reference genes for gene expression studies in flixweed (Descurainia sophia) by quantitative real-time polymerase chain reaction | |
| CN105671161B (en) | A method of containing high oleic acid according to BnaLCR78 genetic testing cabbage type rape | |
| CN112795631B (en) | Fluorescent quantitative reference gene under abiotic stress of cryptomeria fortunei, and special primer and application thereof | |
| Hu et al. | Ethylene-regulated immature fruit abscission is associated with higher expression of CoACO genes in Camellia oleifera | |
| CN113774065B (en) | Fluorescent quantitative internal reference gene for different adults of fall webworm, primer and application thereof | |
| Gialdi et al. | Identification and characterization of contrasting sunflower genotypes to early leaf senescence process combining molecular and physiological studies (Helianthus annuus L.) | |
| Bade et al. | Genome-wide identification and analysis of the SGR gene family in Cucumis melo L | |
| CN108950050B (en) | Detection primer and kit for cytoplasmic male sterility restoring gene Rf (DW)10 of Dongfu-type rice, application and detection method | |
| CN108570473B (en) | Phalaenopsis cyclophilin CYP gene and application thereof | |
| CN105567856B (en) | A kind of genotype detection method of the fad2 genes of cabbage type rape | |
| Marino et al. | Guardians of quality: advancing Castanea sativa traceability using DNA analysis from seed to processed food | |
| CN110982928B (en) | Reference gene of red Chinese toon leaf and stem tissues under insect pest stress, primer and application thereof | |
| CN108611334A (en) | Mulberry tree class glycogen synthase kinase gene and its detection and application | |
| CN108866231B (en) | Detection primer and kit for cytoplasmic male sterility restoring gene Rf (DW)8 of Dongfu-type rice, application and detection method | |
| CN112877466A (en) | Fluorescent quantitative internal reference gene of cryptomeria fortunei under hormone stress and application of primer thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20190614 Termination date: 20200302 |