CN107937590A - Clone primer pair and its application of peanut embryonic development late period Abundant protein gene - Google Patents
Clone primer pair and its application of peanut embryonic development late period Abundant protein gene Download PDFInfo
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Abstract
The invention discloses a kind of primer pair for cloning peanut embryonic development late period Abundant protein gene and its application, belong to molecular biology and genetic engineering field.The primer pair of present invention clone's peanut embryonic development late period Abundant protein gene, sequence are as follows：5 ' CCCTCAAATAATCACAATAC, 3 ' and 5 ' AGAACAGAGTGGCAGAGT 3 '.The primer pair designed using the present invention, clone has obtained peanut embryonic development late period Abundant protein gene from peanut；The gene is obvious to low temperature, high salt and ABA stress responses in peanut leaf and root, to responses of drought stress unobvious in blade, in root, drought stress expression quantity is first risen and is declined afterwards；It can be used for cultivating and screening low temperature resistant peanut, resistance to high salt, drought-resistant and ABA new varieties.
Description
Technical field
The invention belongs to molecular biology and genetic engineering field, and in particular to clone's peanut embryonic development late period enriches egg
The primer pair of white gene and its application.
Background technology
Plant will often face various unfavorable conditions (such as arid, salt stress, low temperature) stress in natural environment and cause
Lack of moisture, in the development process of plant, also face sometimes because dehydration and caused by injure, in long-term evolution process
Middle plant forms my a series of protection mechanism.Under stress conditions plant produce protective substance such as low molecular weight protein,
Polysaccharide, proline etc. are exactly one of these protection mechanisms.
LEA (Late EmbryogenesisAbundant proteins) albumen be found in earliest in the early 1980s,
Because it is named as embryonic development late period Abundant protein in vegetable seeds development late period largely accumulation, many researchs are found in plant
LEA protein high level accumulation during the lack of moisture caused by reason such as response arid, cold, therefore LEA protein is also to plant
One kind in thing Protective substances.Research to LEA genes contributes to analysis to probe into Coping style and life of the plant to stress conditions
Change procedure is managed, the cultivation of breeding and new varieties to plant is of great significance.
The content of the invention
For problems of the prior art, the present invention provides a pair to clone peanut embryonic development late period Abundant protein
The primer pair of gene, and provide the method using primer pair clone's peanut embryonic development late period Abundant protein gene, this hair
Bright technical solution is as follows：
The primer pair of peanut embryonic development late period Abundant protein gene is cloned, sequence is as follows：
5’-CCCTCAAATAATCACAATAC-3’；
5’-AGAACAGAGTGGCAGAGT-3’。
Application of the above-mentioned primer pair in peanut embryonic development late period Abundant protein gene cloning.
The method for cloning peanut embryonic development late period Abundant protein gene, including the processing of vegetable material, the extraction of RNA with
The synthesis of cDNA, PCR clone gene total lengths；
Primer sequence wherein in PCR clone genes total length is：
5’-CCCTCAAATAATCACAATAC-3’；
5’-AGAACAGAGTGGCAGAGT-3’。
On the basis of such scheme, the vegetable material educates No. 33 peanuts for flower.
On the basis of such scheme, the vegetable material processing is：
Flower is educated into No. 33 peanut seed sowings in Nutrition Soil and vermiculite (mass ratio 2:1) in composite soil, in light after sprouting
According to being grown in the illumination box of 16h/8h dark (28/22 DEG C), handled after growth of seedling to tri-leaf period；At low temperature
Reason：Peanut seedling is placed in illumination box, temperature is set as 4 DEG C；NaCl, PEG6000 and ABA processing：By peanut seedling
Extracted from soil, 200mmol L are directly dipped into after carefully rinsing root soil well-1NaCl, 20%PEG6000 or 100 μ
molL-1In ABA solution；Take blade and root respectively after 0,1,3,6,12,24,48 and 72h is handled, liquid nitrogen frozen preserves, and makees
For follow-up test material.
On the basis of such scheme, the extraction of the RNA and synthesizing for cDNA：Use the RNeasy Mini of Tiangeng
The total serum IgE of material after Kit extraction processs, is handled with DNaseI and removes DNA pollution, DNA pollution will be removed with M-MLV reverse transcriptase
RNA reverse transcriptions into cDNA.
On the basis of such scheme, the PCR clone genes total length is：Using the cDNA after reverse transcription as template, with 5 '-
CCCTCAAATAATCACAATAC-3 ' and 5 '-AGAACAGAGTGGCAGAGT-3 ' carries out RCR reactions, PCR reaction bars for primer
Part is as follows：94℃2min；94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 1min, 35 circulations；72℃10min.
Peanut is low temperature resistant, resistance to height cultivating and screen for the peanut embryonic development late period Abundant protein gene of above method clone
Application in salt, drought-resistant and ABA new varieties.
Beneficial effects of the present invention
The primer pair designed using the present invention, clone has obtained peanut embryonic development late period Abundant protein base from peanut
Cause, by sequence alignment and evolutionary analysis, is named as AhLEAL；The gene is studied low using fluorescent quantitative PCR technique
Expression pattern under temperature, arid, high salt and ABA stress, it turns out that：AhLEAL genes are in blade and root to low temperature, high salt
Obvious with ABA stress responses, AhLEAL genes, to responses of drought stress unobvious, in root, are expressed in blade in face of drought stress
Amount first rises to be declined afterwards；It can be seen from the above that the gene can be used for cultivating and screen low temperature resistant peanut, resistance to high salt, drought-resistant and ABA
New varieties；The cultivation of breeding and new varieties to peanut is of great significance
Brief description of the drawings
The nucleotide and amino acid sequence of Fig. 1 AhLEAL genes；
Fig. 2 AhLEAL and other species embryonics development late period Abundant protein amino acid alignment (Ah：Arachis
Hypogaea peanuts；Pj：Prosopisjuliflora algarrobas；Ck：Caragana korshinskii Caragana korshinskiis；Am：
Ammopiptanthusmongolicus Ammopiptanthus mongolicus；Protein sequence number：PjLEA：ABG66530；CkLEA1：AGN90999；
AmLEAL：AAW31666；AhLEA：ACF74336)；
Expression pattern analysis of Fig. 3 AhLEAL genes in peanut leaf and root under low temperature stress；
Expression pattern analysis of Fig. 4 AhLEAL genes in peanut leaf and root under drought stress；
Expression pattern analysis of Fig. 5 AhLEAL genes in peanut leaf and root under high-salt stress；
Expression pattern analysis of Fig. 6 AhLEAL genes in peanut leaf and root under ABA stress.
Embodiment
Used term in the present invention, it is unless otherwise specified, generally usual with those of ordinary skill in the art
The implication of understanding.
With reference to specific embodiment, and with reference to the data further detailed description present invention.Following embodiments simply be
Illustrate the present invention, rather than limit the scope of the invention in any way.
Embodiment 1
1.1 test methods and process
1.1.1 vegetable material and processing
Material therefor of the present invention educates No. 33 for peanut (Arachis hypogaea L.) kind flower, which is by this item
The degeneration-resistant new varieties of high yield that selection and breeding are arrived after the parents of mesh group initiative.By seed sowing Nutrition Soil and vermiculite (mass ratio 2:
1) in composite soil, grown after sprouting in the illumination box of illumination 16h/8h dark (28/22 DEG C), treat growth of seedling to three
Leaf is handled after the phase.
Low-temperature treatment：Peanut seedling is placed in illumination box, temperature is set as 4 DEG C；
NaCl, PEG6000 (polyethylene glycol, polyethylene glycol) and ABA processing：By peanut seedling from soil
Extract, 200mmolL is directly dipped into after carefully rinsing root soil well-1NaCl, 20%PEG6000 or 100 μm of olL- 1In ABA solution.
Take each group blade and root respectively after 0,1,3,6,12,24,48 and 72h is handled, liquid nitrogen frozen preserves, as rear
Continuous test material.
1.1.2 test reagent
Total RNA extraction reagent box, TOP10 competence are purchased from Tiangeng biochemical technology Co., Ltd.
LATaq archaeal dna polymerases, pMD18-T carriers, quantitative fluorescent PCR are purchased from SYBR Premix Ex Taq polymerases
The precious biology in Dalian.
M-MLV reverse transcriptase is purchased from Promega.
Gel reclaims kit, plasmid extraction kit are purchased from Omega companies.
1.1.3RNA extraction is synthesized with cDNA
The RNeasy Mini Kit of the extraction Tiangeng of sample total serum IgE, method detailed refer to its operation instruction.
The RNA of extraction is handled to remove DNA pollution with DNase I before synthesis cDNA.
The synthesis of cDNA (complementary DNA, complementary DNA), every 25 μ L reactants are carried out with M-MLV reverse transcriptase
2 μ g RNA templates are added in system；Reverse transcription reaction system 42 DEG C carry out 1h, be placed on cooled on ice 5min.
1.1.4 the amplification of full length gene
According to the full length sequence of known peanut LEA protein in cDNA library, the primer of design amplification gene total length, primer
Sequence is as follows：5 '-CCCTCAAATAATCACAATAC-3 ' and 5 '-AGAACAGAGTGGCAGAGT-3 '.Using reverse transcription cDNA as
Template, reacts amplification gene by PCR.PCR reaction conditions are as follows：94℃2min；94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 1min, 35
A circulation；72℃10min.
1.1.5 sequence analysis
By measured sequence using NCBI websites (http://www.ncbi.nlm.nih.gov) on BLAST instruments
The similitude and homology search of gene order are carried out, and the comparison of genetic homology, cDNA total lengths are carried out using these sequences
Www.ncbi.nlm.nih.gov websites ORF is used in open reading frame (open reading frame, the ORF) analysis of sequence
Finder on-line analyses.Its amino acid sequence is derived according to the cDNA sequence of measured gene, and utilizes sequence analysis tools pair
It is analyzed：Using Protparam (http://web.expasy.org/protparam/) prediction albumen basic physics
Chemical property；CDSS (the Conserved Domain Search Service) database of the Structure and function domain of albumen in NCBI
On analyzed；The secondary structure prediction of albumen uses SOPMA secondary Structure Prediction Method
(https://npsa-prabi.ibcp.fr/cgi-bin/secpred_sopma.pl)；The domain analysis of protein transmembrane structure
(http://www.ch.embnet.org/software/TMPRED_form.html)；Using LOCtree3 (https:// rostlab.org/services/loctree3/) analysing protein Subcellular Localization situation.
1.1.6 Phylogenetic Analysis
The LEA protein of different plant species in GenBank is chosen, is carried out using the multiple alignment of DNAman softwares
Multiple Sequence Alignment is analyzed, and creates the comparison result of the LEA multisequencing of a separate sources；System occur and evolve analysis than
Completed on the basis of with MEGA4.0 softwares, genealogical tree is built using Neighbour-Joining methods.
1.1.7 quantitative fluorescent PCR carries out gene expression analysis
When carrying out quantitative fluorescent PCR, cDNA samples are first diluted to 8ng μ L-1, often in reaction system plus after 2 μ L dilutions
CDNA.Fluorescence quantitative PCR instrument uses the LightCycler 2.0 of Roche.Reaction condition：95℃10s；95 DEG C, 5s, 60 DEG C,
30s, 72 DEG C, 10s, 40 circulations；Solubility curve is drawn, temperature raises 0.5 DEG C per 10s.This experiment reference gene used is
Actin11, each sample are repeated 3 times, are averaged, and analyze data using delta-delta Cp methods, error line is 3 weights
Multiple standard deviation.
Fluorescent quantitation the primer is as follows：
Actin11 primers are：
5 '-TTGGAATGGGTCAGAAGGATGC-3 ' and 5 '-AGTGGTGCCTCAGTAAGAAGC-3 '；
AhLEAL primers are：
5 '-TAACTAACAAACTCGTCCTT-3 ' and 5 '-AACTATGGCATTGAATTGAA-3 '.
1.2 result of the tests and analysis
1.2.1 the clone of AhLEAL genes
PCR amplification of the present invention obtains LEA genes.The full length gene is 555bp, and open reading frame ORF is 291bp, coding
97 amino acid (Fig. 1).Blast analyses are carried out to the albumen of the gene code on NCBI websites, find the albumen and husky winter
Blue or green (Ammopiptanthus mongolicus), peanut (Arachis hypogaea), algarroba
(Prosopisjuliflora) the embryonic development late period and in the plant such as Caragana korshinskii (Caragana korshinskii) is rich
Rich albumen homology reaches nearly 65% (Fig. 2).It is AhLEAL (Arachis hypogaea Late- by the unnamed gene
embryogenesis abundant protein-likeprotein)。
1.2.2 AhLEAL albumen physicochemical property and structural analysis
Using ProtParam, analysis shows that, the theoretical molecular of AhLEAL is 10.19kDa, and theoretical isoelectric point is 10.06,
Average hydrophilic coefficient (grand average ofhydropathicity, GRAVY) is -0.494, and it is hydrophily to illustrate the albumen
Albumen；Predicted by proteins subcellular location instrument, this albumen can be located on mitochondria.Secondary protein structure is pre-
Survey the results show that α is spiral in AhLEAL accounts for 50.52%, β-pleated sheet accounts for 12.37%, and extended chain accounts for 6.19%, and random coil accounts for
30.93%；Conserved domain prediction result shows that the albumen has LEA_3 conserved domains.
1.2.3 expression change of the AhLEAL genes under abiotic stress
1.2.3.1 expression change of the AhLEAL genes in Roots of Peanut and blade under low temperature stress
Expression (figures of the AhLEAL in Roots of Peanut and blade under low temperature stress by fluorescence quantitative PCR detection
3).From figure 3, it can be seen that no matter in blade or root, expression of the AhLEAL on transcriptional level responds low temperature very bright
It is aobvious.The expression of AhLEAL has just been raised after low-temperature treatment 1h, with the extension of low-temperature treatment time, the expression of AhLEAL by
Cumulative height, expression quantity reaches highest (Fig. 3) when extremely processing 72 is small.Compared with untreated material, tables of the AhLEAL in blade
More than 100 times (Fig. 3 a) is up to up to amount, is reached as high as in root more than 12 times (Fig. 3 b).
1.2.3.2 expression change of the AhLEAL genes in Roots of Peanut and blade under drought stress
AhLEAL is in peanut leaf and root to drought stress response unobvious (Fig. 4).The gene is done in peanut leaf
To drought stress without significantly response (Fig. 4 a) under drought processing.Expression is increased slightly after Osmotic treatment in Roots of Peanut, to place
The 6h expression quantity of reason reaches highest, has then declined (Fig. 4 b) again.
1.2.3.3 expression change of the AhLEAL genes in Roots of Peanut and blade under high-salt stress
AhLEAL responds obvious (Fig. 5) high-salt stress in peanut leaf and root.From fig. 5, it can be seen that no matter in flower
In leave piece or root, the expression of the gene both starts to raise in salt stress 1h, reaches highest to 6h expression quantity, has then dropped
It is low.Maximum up-regulation multiple (more than 50 times) in root is higher than in blade (more than 10 times).
1.2.3.4AhLEAL expression change of the gene in Roots of Peanut and blade under ABA stress
Research shows that stress response gene may be regulated and controled by two kinds of signal pathways：ABA-dependent and ABA-
independent.In order to study the AhLEAL expression of abiotic stress induction and the relation of ABA, using Exogenous ABA respectively to flower
Take root after being handled with blade by fluorescence quantitative PCR detection the expression of AhLEAL genes.The result shows that the table of AhLEAL
Up in peanut leaf and root (Fig. 6) is induced by the obvious of ABA.From fig. 6, it can be seen that no matter in peanut leaf or root,
Expression quantity both reaches highest after ABA coerces 1h for the expression of the gene, then decreases.Maximum up-regulation multiple in root
(more than 25 times) are higher than in blade (more than 6 times).
The above described is only a preferred embodiment of the present invention, being not the limitation for making other forms to the present invention, appoint
What those skilled in the art changed or be modified as possibly also with the technology contents of the disclosure above equivalent variations etc.
Imitate embodiment.But it is every without departing from technical solution of the present invention content, the technical spirit according to the present invention is to above example institute
Any simple modification, equivalent variations and the remodeling made, still fall within the protection domain of technical solution of the present invention.
Claims (8)
1. clone the primer pair of peanut embryonic development late period Abundant protein gene, it is characterised in that：Sequence is as follows：
5’-CCCTCAAATAATCACAATAC-3’；
5’-AGAACAGAGTGGCAGAGT-3’。
2. application of the primer pair described in claim 1 in peanut embryonic development late period Abundant protein gene cloning.
3. clone the method for peanut embryonic development late period Abundant protein gene, it is characterised in that：Processing including vegetable material,
The extraction of RNA and synthesis, the PCR clone gene total lengths of cDNA；
Primer sequence wherein in PCR clone genes total length is：
5’-CCCTCAAATAATCACAATAC-3’；
5’-AGAACAGAGTGGCAGAGT-3’。
4. the method for peanut embryonic development late period Abundant protein gene is cloned according to claim 3, it is characterised in that：It is described
Vegetable material educates No. 33 peanuts for flower.
5. according to the method for the clone's peanut embryonic development late period Abundant protein gene of claim 3 or 4, it is characterised in that：
The vegetable material is handled：
Flower is educated into No. 33 peanut seed sowings in Nutrition Soil and vermiculite (mass ratio 2:1) in composite soil, in illumination after sprouting
Grow in the illumination box of 16h/8h dark (28/22 DEG C), handled after growth of seedling to tri-leaf period；Low-temperature treatment：
Peanut seedling is placed in illumination box, temperature is set as 4 DEG C；NaCl, PEG6000 and ABA processing：By peanut seedling from soil
Middle extraction, 200mmol L are directly dipped into after carefully rinsing root soil well-1NaCl, 20%PEG6000 or 100 μm of ol L- 1In ABA solution；Take blade and root respectively after 0,1,3,6,12,24,48 and 72h is handled, liquid nitrogen frozen preserves, as rear
Continuous test material.
6. according to the method for the clone's peanut embryonic development late period Abundant protein gene of claim 3 or 4, it is characterised in that：
The extraction of the RNA and synthesizing for cDNA：Using the total serum IgE of material after the RNeasy Mini Kit extraction processs of Tiangeng, use
DNaseI processing removes DNA pollution, and the RNA reverse transcriptions of DNA pollution will be removed into cDNA with M-MLV reverse transcriptase.
7. according to the method for the clone's peanut embryonic development late period Abundant protein gene of claim 3 or 4, it is characterised in that：
The PCR clone genes total length is：Using the cDNA after reverse transcription as template, with 5 '-CCCTCAAATAATCACAATAC-3 ' and
5 '-AGAACAGAGTGGCAGAGT-3 ' carry out RCR reactions for primer, and PCR reaction conditions are as follows：94℃2min；94 DEG C of 30s, 55
DEG C 30s, 72 DEG C of 1min, 35 circulations；72℃10min.
8. the peanut embryonic development late period Abundant protein gene of any one of claim 3~7 the method clone is being cultivated and sieved
Select the application in low temperature resistant peanut, resistance to high salt, drought-resistant and ABA new varieties.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1687417A (en) * | 2005-04-27 | 2005-10-26 | 东北林业大学 | Gene sequence in enrichment protein of late stage embryo in third group of vinca rosea |
| CN101218346A (en) * | 2005-07-06 | 2008-07-09 | 康乃尔研究基金会 | Dehydrin genes and promoters from coffee |
-
2017
- 2017-12-12 CN CN201711318059.5A patent/CN107937590A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1687417A (en) * | 2005-04-27 | 2005-10-26 | 东北林业大学 | Gene sequence in enrichment protein of late stage embryo in third group of vinca rosea |
| CN101218346A (en) * | 2005-07-06 | 2008-07-09 | 康乃尔研究基金会 | Dehydrin genes and promoters from coffee |
Non-Patent Citations (1)
| Title |
|---|
| 尹红: "与花生发育以及抗耐胁迫相关基因的克隆与表达研究", 《中国优秀硕士学位论文全文数据库农业科技辑》 * |
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