CN108048469B - The Ravenna grass class Calmodulin Gene ErCML30 expressed in Ravenna grass wild species by low temperature stress - Google Patents
The Ravenna grass class Calmodulin Gene ErCML30 expressed in Ravenna grass wild species by low temperature stress Download PDFInfo
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Abstract
The invention discloses the Ravenna grass class Calmodulin Gene ErCML30 expressed in Ravenna grass wild species by low temperature stress, and expand its primer special and detect differential expression method and its special primer of the gene in Ravenna grass wild species by low temperature stress.The gene is persistently risen in Ravenna grass wild species by expression quantity under low temperature stress, directly takes part in low temperature stress response.The present invention is to recognize the degeneration-resistant mechanism of the gene, and the important germ plasm resource and utilization that Ravenna grass wild species are developed as sugarcane new cold-resistant species provide technical support.
Description
Technical field
The invention belongs to biomolecule clone technology fields, and in particular to the Ravenna grass wild species under low temperature stress encode class calcium
The gene of plain (Calmodulin-like, CML), and the primers based on the gene are adjusted, relative RT-PCR is passed through
The method for detecting calbindin D28K differential expression in Ravenna grass wild species.
Background technique
Ravenna grass (E.fulvus) is that one in the Plumegrass (Erianthus) of the nearly edge of saccharum (Saccharum) is wild
Kind, it is distributed mainly on Yunnan Province of China, Sichuan and Tibet, growth height above sea level has cold-resistant, drought resisting, resistance to lean, perennial root in 500~2700m
The excellent characteristics such as strong, the anti-embroidery disease of property, and easily bloom, pollen amount it is extremely more.Sugarcane research institute of Yunnan Prov Agriculture University is from Ravenna grass wild species
Main Distribution Area collect more than 150 part of clonal material of different ecological type, and concentrate child care this school sugarcane provide
In the garden of source (height above sea level 1930m).Original sugarcane produces torrid areas, is suitble to plantation in tropical and subtropical zone weather, is whole world cultivated area
Maximum sugar crop, China has basically formed Guangxi, the big sugarcane district in Yunnan and Guangdong three now at present.Winter is extreme in recent years
Weather takes place frequently, and sugarcane is seriously affected in growth and development process in seedling stage by low temperature stress, current sugar cane breed low temperature in seedling stage
Under usually there is growing point necrosis, root structure damage is serious, seriously constrains growth, sugar and the biological yield of sugarcane.Cause
How this, improve sugar cane breed to the resistance of low temperature stress, the new varieties of cultivation low-temperature resistance, have become Sugarcane Breeding and grind
Study carefully important topic (such as Li Fusheng develop and use Ravenna grass Wild ornamental resources thinking [J] living resources, 2004,20 (4):
266-270；The auspicious Sugarcane Seedlings Root morphology structure of Sun Bo, Liu Guangling, Yang Litao, Li Yang and protection system are to low temperature stress
Respond [J] China Agricultural University journal, 2014,19 (06): 71-80.；The sugarcane wild species such as Lu Xin Yunnan Ravenna grass germplasm innovation benefit
With research [J] plant genetic resources journal, 2012,13 (02): 321-324.).Therefore, by excavating in Ravenna grass wild species
Degeneration-resistant new gene, and therefrom clone and separate anti contravariance related gene, are of great significance to sugarcane breeding for stress tolerance.
Class calmodulin (Calmodulin-like, CML) belongs to one kind in calbindin, and calcium signal is plant
The major regulator of growth and development and stress response, Ca2+- CML signal system takes part in transduction of the plant to more adverse circumstance signals,
When plant is by environment stress, calcium ion concentration increases, and calcium ion leads to these calbindins in conjunction with calbindin
Hydrophobic region is exposed and in conjunction with biomembrane or hydrophobic biological macromolecular, thus adjust biomembrane or these target proteins function and
Enzymatic activity, i.e. calbindin be excited and experience and transmit Ca2+ oscillations (Liu Wei tomato calmodulin and class calmodulin
Disease-resistant adjusting function analysis [D] Zhejiang University, 2015.；The clone of the Japan silk floss mealybug Calmodulin gene such as Luo Mei and biology
Bioinformatics analysis [J] Hua Zhong Agriculture University journal, 2012,31 (03): 320-324.；The such as Liang Qiufen calmodulin is sent out in plant
Function [J] life science in educating, 2005, (S2): 1-5.).Class calmodulin research at present is less, in abiotic stress
Aspect, the rice CML gene OsMSR2 and CML4 cloned from rice by salt and drought-induced, effect in plant
The signal that mediates of hormone ABA transmit it is related；The CML10 found in arabidopsis participates in the formation of ascorbic acid, maintains plant
Intracorporal redox equilibrium；In terms of biotic, CML adjusts plant to the defense response of pathogen, arabidopsis CML9's
Expression is generated NO and hypersensitivity by pathogen and hormone Induced by Salicylic Acid, and the homologous gene APR134 in tomato is also positive to be adjusted
Save plant to the defense response of pathogen (progress [J] Chinese science of the plant calmodulin physiological function such as Zeng Houqing:
Life science, 2016,46 (06): 705-715.).But it yet there are no class Calmodulin Gene under plant low temperature stress
Mechanism of action report, class Calmodulin Gene is also not yet cloned into saccharum.
Summary of the invention
The invention aims to improve the resistance of existing cultivation sugar cane breed, make sugarcane can high yield in Chilling stress
The case where stable yields is to adapt to climatic deterioration in recent years provides the class calcium induced in a new Ravenna grass wild species by low temperature stress
Plain gene is adjusted, also provides a kind of primer for expanding the gene and a kind of detection gene in Ravenna grass wild species by low temperature stress
Differential expression method, to further lay the foundation and provide candidate gene for research of cold resistance in sugarcane breeding.
This research obtains a class calmodulin using electronic cloning technology clone from the Ravenna grass wild species by low temperature stress
Gene is compared through BLASTn in NCBI and is analyzed, and the homology with known CML gene is 95%, thus it is speculated that gene coding
Albumen is calmodulin like-protein, therefore is Ravenna grass class Calmodulin Gene ErCML30 by the unnamed gene.
Technical scheme is as follows:
1. the Ravenna grass class Calmodulin Gene ErCML30 expressed in Ravenna grass wild species by low temperature stress, full length nucleotide sequence
Column are as shown in SEQ ID NO:1.
2. the Ravenna grass class Calmodulin Gene expressed in Ravenna grass wild species described in amplification technique scheme 1 by low temperature stress
The primer special of ErCML30, it is described it is dedicated be made of upstream primer GP-F and downstream primer GP-R, the upstream primer GP-F's
Base sequence is as shown in SEQ ID NO:2, and the base sequence of the downstream primer GP-R is as shown in SEQ ID NO:3.
3. a kind of detection Ravenna grass class Calmodulin Gene ErCML30 is in Ravenna grass wild species by the differential expression side of low temperature stress
Method, the synthesis of extraction, the first chain of cDNA including total serum IgE, relative RT-PCR technology detect Ravenna grass class Calmodulin Gene
ErCML30 is in Ravenna grass wild species by the differential expression situation of low temperature stress, in which:
The Ravenna grass wild species in seedling stage are set as control group and experimental group in the extraction of total serum IgE, control group is at 25 DEG C of room temperature
Under the conditions of do not do other processing normal growths, experimental group is divided into processing I and processing II, and processing 1 is grown under the conditions of 4 DEG C of low temperature
For 24 hours, processing II grows 72h under the conditions of 4 DEG C of low temperature, respectively acquisition control, processing I and the blade for handling II, respectively liquid feeding nitrogen
It quickly grinds and obtains three samples, three samples extract total serum IgE respectively；
In relative RT-PCR technology detection Ravenna grass class Calmodulin Gene ErCML30 in Ravenna grass wild species by low temperature stress
Differential expression situation in, use designed special primer as RT-PCR primer carry out different disposal and control between gene
Differential expression detection, the special primer are made of upstream primer QF and downstream primer QR, the upstream primer QF base sequence
As shown in SEQ ID NO:4, the base sequence of the downstream primer QR is as shown in SEQ ID NO:5；The Ravenna grass class calmodulin
The full length nucleotide sequence of gene ErCML30 is as shown in SEQ ID NO:1.
The present invention provides the Ravenna grass class Calmodulin Gene ErCML30 that Ravenna grass wild species are expressed by low temperature stress for the first time, with
And it expands its primer special, detect special primer of the gene in Ravenna grass wild species by differential expression in low temperature stress.In phase
The gene in quantitative RT-PCR detection is hardly expressed in the case where no low temperature stress, after 4 DEG C of low-temperature treatments for 24 hours
Expression quantity of the gene in Ravenna grass is 55 times of control, the expression quantity after 4 DEG C of low-temperature treatments of 72h in Ravenna grass wild species
It is 75 times of control, illustrates that gene expression quantity after Ravenna grass wild species are by low temperature stress rises, and with the increasing of stress time
Add and increases, therefore, the Ravenna grass class Calmodulin Gene ErCML30 belongs to the inducible expression of gene expressed by low temperature stress,
Show the participation low temperature side of body that Ravenna grass class Calmodulin Gene ErCML30 can be rapid and lasting after Ravenna grass wild species are by low temperature stress
Compel response, for the benefit for recognizing this wild resource of the degeneration-resistant mechanism and Ravenna grass wild species of Ravenna grass class Calmodulin Gene ErCML30
With providing technical support.
The present invention detects Ravenna grass class Calmodulin Gene ErCML30 in Ravenna grass wild species by the side of the differential expression of low temperature stress
Method, designed specific primer high specificity in relative RT-PCR, no miscellaneous band generate, and melting curve shows primer free
Dimer generates.In the selection of reference gene, GAPDH gene, β-actin gene but the sugarcane that is more suitable for of selection are not selected
25SrRNA gene as internal reference, expression stability is higher (see Fig. 3) relative to other reference genes.This relative quantification RT-
PCR using SYBR Green I chimeric fluorescent method carry out, have high specificity, high sensitivity, it is reproducible the features such as.
It is the full length nucleotide sequence of Ravenna grass class Calmodulin Gene ErCML30 shown in SEQ ID NO:1 in sequence table.
It is the upstream primer GP-F for expanding Ravenna grass class Calmodulin Gene ErCML30 shown in SEQ ID NO:2 in sequence table
Base sequence.
It is the downstream primer GP-R for expanding Ravenna grass class Calmodulin Gene ErCML30 shown in SEQ ID NO:3 in sequence table
Base sequence.
It is the base sequence of the upstream primer QF of special primer shown in SEQ ID NO:4 in sequence table.
It is the base sequence of the downstream primer QR of special primer shown in SEQ ID NO:5 in sequence table.
It is the base sequence of the upstream primer 25S-F of reference gene 25SrRNA shown in SEQ ID NO:6 in sequence table.
It is the base sequence of the downstream primer 25S-R of reference gene 25SrRNA shown in SEQ ID NO:7 in sequence table.
Detailed description of the invention
Fig. 1: the amplification of Ravenna grass class Calmodulin Gene ErCML30.In Fig. 1, M:maker；Swimming lane 1, swimming lane 2, swimming lane 3
Amplified production at respectively 52 DEG C, 55 DEG C, 57 DEG C of annealing temperature.
Fig. 2: the phase of Ravenna grass class Calmodulin Gene ErCML30 in the lower Ravenna grass wild species blade of Different hypothermia stress time processing
To fluorescent quantitation differential expression figure.In Fig. 2, ordinate indicates that the relative expression quantity of gene, abscissa indicate Different hypothermia processing
The sample of different time.Ravenna grass blade under ER-1: 25 DEG C of room temperature processing, as control；ER-2:4 DEG C of low-temperature treatment for 24 hours after
Ravenna grass blade；Ravenna grass blade after ER-3:4 DEG C of low-temperature treatment 72h.
Fig. 3: specificity fluorescent quantifies primer detection result.M:maker；Swimming lane 1, swimming lane 2, swimming lane 3 are respectively temperature of annealing
Amplified production at 55 DEG C, 57 DEG C, 60 DEG C of degree.
Specific embodiment
Following embodiment is not limited to protection scope of the present invention for the invention will be further described.Each reality
Applying in example without specified otherwise is conventional method.
Experimental material: test material is Ravenna grass wild species (Kunming Ravenna grass 99-1), and plantation is ground in Yunnan Prov Agriculture University sugarcane
Study carefully sugarcane resource garden in (Kunming, Yunnan, height above sea level 1930m).
The acquisition of the Ravenna grass class Calmodulin Gene ErCML30 expressed in 1 Ravenna grass wild species of embodiment by low temperature stress
One, the Ravenna grass class Calmodulin Gene ErCML30 overall length sequence expressed in design amplification Ravenna grass wild species by low temperature stress
The primer special of column.
Ravenna grass wild species after different disposal are sent to the high-throughput transcript profile sequencing of progress by early period, and the transcript profile of return is surveyed
Sequence result carries out bioinformatic analysis screening, the Unigene sequence of genetic transcription group data splicing is obtained, according to splicing
The Unigene sequence arrived utilizes the design amplification of biosoftware Primer5.0, Oligo 7 Ravenna grass class Calmodulin Gene ErCML30's
Primer special, the primer special are made of upstream primer GP-F and downstream primer GP-R, the base sequence of the upstream primer GP-F
Column are as shown in SEQ ID NO:2, and as shown in SEQ ID NO:3, which send the base sequence of the downstream primer GP-R
Shanghai Sangon Biotech Company's synthesis.
Two, the Total RNAs extraction of the Ravenna grass wild species blade of low-temperature treatment.
(1) processing of sample: 6 almost the same basin Ravenna grass wild species of seedling taking phase growth conditions are carried out using illumination box
Test process, if control group and experimental group, 2 basin room temperature (25 DEG C) grow other and are not processed as control (ER-1), 2 basin low temperature
For 24 hours (Er-LT2), 2 basin low-temperature treatment 72h (Er-LT3), low-temperature treatment temperature is 4 DEG C for processing.
(2) blade that processing is completed is respectively placed in liquid feeding nitrogen in mortar quickly to grind, every 100mg sample is added
1mlTRIzol extracting solution, vortex oscillation 15s, each period are sampled as a processing sample, totally 6 processing samples.Each sample
Product are in 15~30 DEG C of placement 5min；Chloroform is added, the dosage of chloroform is every using 1ml TRIzol 200 μ l chloroforms of addition, is covered
Pipe band, vortex oscillation 15s, 15~30 DEG C of placement 3min.
(3) 4 DEG C, 12000rpm is centrifuged 15min, and solution is divided into three layers at this time: yellow is organic phase, middle layer and upper layer
Colourless is water phase, and RNA is primarily present in water phase, water phase is transferred to another new without in RNase centrifuge tube.
(4) isometric isopropanol is added into obtained water phase, mixes, 15~30 DEG C of placement 20-30min.
(5) 4 DEG C, 12000rpm is centrifuged 10min, abandons supernatant.
(6) volume fraction that DEPC water is prepared is added and washs precipitating for 75% ethanol solution, dosage is every use
The ethanol solution that 1mlTRIzol is 75% with the volume fraction that 1mlDEPC water is prepared.
(7) 4 DEG C, 5000rpm is centrifuged 3min, and supernatant liquid is carefully sucked out with pipette tips, retains precipitating.
(8) 15~30 DEG C are deposited in and dries 2-3min, distilled water of the 30-100 μ l without RNase is added, sufficiently dissolution RNA
Afterwards, 2 control sample RNA mixing are saved as a total control sample in -70 DEG C, and the processing sample in 2 periods is respectively at -70
DEG C save；
The synthesis of three the first chains of .cDNA
(1) template ribonucleic acid is thawed on ice；5×gDNA Buffer,FQ-RT Primer Mix,10×Fast RT
Buffer、RNase-Free ddH2O thaws at (15-25 DEG C) of room temperature, is immediately placed on ice after defrosting.It is molten by every kind before use
Liquid vortex oscillation mixes, and brief centrifugation (revolving speed 6000rpm, 30s) remains in the liquid of tube wall to collect.In order to guarantee reaction solution
The accuracy of preparation is first configured to Mix, is then dispensed into each reaction tube again when carrying out every reaction.
(2) the gDNA removal system in RNA is carried out according to following system and prepare mixed liquor, thoroughly mix.Brief centrifugation (turns
Fast 6000rpm, 30s), 42 DEG C are placed in, 3min is incubated for.It is subsequently placed in and places on ice.
GDNA removes reaction system:
5×gDNA Buffer 2μl
Total RNA 2μl
RNase-Free ddH2O is supplied to 10 μ l.
(3) mixed liquor is prepared according to the reverse transcription reaction system of following system:
Reverse transcription reaction system:
10×Fast RT Buffer 2μl
RT Enzyme Mix 1μl
FQ-RT Primer Mix 2μl
RNase-Free ddH2O is supplied to 10 μ l.
(4) it by the Mix in reverse transcription reaction, is added in the reaction solution of gDNA removal step, mixes well.It 42 DEG C, is incubated for
15min 95 DEG C, is incubated for 3min and is put on ice later, obtains cDNA and save for subsequent experimental or -20 DEG C of low temperature.
Four, the amplification of the Ravenna grass class Calmodulin Gene ErCML30 of Ravenna grass wild species
The template that the cDNA obtained using step 3 (4) reverse transcription is reacted as PCR, with the primer special designed in step 1
Primer is reacted as PCR, is expanded complete by the Ravenna grass class Calmodulin Gene ErCML30 of low temperature stress expression in Ravenna grass wild species blade
It is long, PCR reaction system: 1 μ l of Template；1 μ l of upstream primer GP-F；1 μ l of downstream primer GP-R；Buffer 2μl；dNTP
2μl；0.5 μ l of Pfu enzyme；ddH2O is supplied to 20 μ l；Total volume is 20 μ l.PCR response procedures are as follows: 95 DEG C of initial denaturation 2min；95℃
It is denaturalized 30s；57 DEG C of annealing 30s；72 DEG C of extension 2min, 35 circulations；72 DEG C, 6min, rear 4 DEG C of preservations.
Five, the recycling of target gene
PCR product carries out glue recycling through 1% agarose gel electrophoresis, by the purpose band in gel, and steps are as follows:
(1) purpose band blob of viscose is cut in the UV lamp, is put into after as far as possible removing the Blank gel without DNA fragmentation
In 1.5ml centrifuge tube.
(2) sol solutions are added into blob of viscose in the ratio that 500 μ l sol solutions are added in every 100 μ l blob of viscose, set 60 DEG C of colloidal sols about
During which 10min constantly shakes.
(3) sol solutions are transferred in adsorption column after blob of viscose is completely dissolved, 18~22 DEG C, 12000rpm is centrifuged 30s, removes
Waste liquid.
(4) 500 μ l rinsing liquids are added into adsorption column, 18~22 DEG C, 12000rpm is centrifuged 30s, outwells waste liquid.
(5) step (4) are repeated, the centrifuge tube 12000rpm centrifugation 2min after outwelling waste liquid removes waste liquid completely, retains and inhales
Attached column.
(6) adsorption column is moved in the centrifuge tube of clean 1.5ml, 18~22 DEG C of placement 2min make it remain in rinsing liquid
Middle ethyl alcohol volatilization is dry.
(7) to adsorption column film center be added 30 μ l-50 μ l elution buffer, 18~22 DEG C of placement 1-2min,
12000rpm is centrifuged 2min eluted dna, and the solution eluted on the slave adsorption column film collected in centrifuge tube is the pure of target fragment
Change product recovered liquid.
Amplification is as shown in Figure 1, it is when annealing temperature is 57 DEG C, and expanding effect is best, and no miscellaneous band generates.To amplification
After segment carries out gel extraction, the Unigen sequence through the recycling fragment sequence being sequenced with transcript profile obtains is consistent, i.e. the recycling
Product is the Ravenna grass class Calmodulin Gene ErCML30 expressed in Ravenna grass wild species blade by low temperature stress, the survey of the recovery product
Sequence result is as shown in SEQ ID NO:1 in sequence table.
A kind of detection Ravenna grass class Calmodulin Gene ErCML30 provided by the present invention of embodiment 2 is in Ravenna grass wild species by low
The differential expression method of temperature stress, comprising the following steps:
One, the processing of sample:
6 almost the same basin Ravenna grass wild species of seedling taking phase growth conditions carry out test process using illumination box, if right
According to group and experimental group, (25 DEG C) growths of 2 basin room temperature as control (ER-1), 2 basin low-temperature treatments for 24 hours (ER-2), 2 basin low-temperature treatments
72h (ER-3), low-temperature treatment temperature are 4 DEG C.
Two, the extraction of total serum IgE
(1) blade that processing is completed is respectively placed in liquid feeding nitrogen in mortar quickly to grind, 1ml is added in every 100mg sample
TRIzol extracting solution, vortex oscillation 15s, each period are sampled as a processing sample, totally 6 processing samples.Each sample
In 15~30 DEG C of placement 5min；Chloroform is added, the dosage of chloroform is every using 1ml TRIzol 200 μ l chloroforms of addition, covers pipe
Band, vortex oscillation 15s, 15~30 DEG C of placement 3min；4 DEG C, 12000rpm is centrifuged 15min, and solution is divided into three layers at this time: yellow
Organic phase, middle layer and upper layer it is colourless be water phase, RNA is primarily present in water phase, and water phase is transferred to another new nothing
In RNase centrifuge tube；
(2) isometric isopropanol is added into obtained water phase, mixes, 15~30 DEG C of placement 20-30min；4 DEG C,
12000rpm is centrifuged 10min, abandons supernatant；
(3) ethanol solution that the volume fraction that addition DEPC water is prepared is 75% washs precipitating, and dosage uses 1ml to be every
The ethanol solution that TRIzol is 75% with the volume fraction that 1mlDEPC water is prepared；4 DEG C, 5000rpm is centrifuged 3min, small with pipette tips
Supernatant liquid is sucked out in the heart, retains precipitating；
(4) 15~30 DEG C are deposited in and dries 2-3min, distilled water of the 30-100 μ l without RNase is added, sufficiently dissolution RNA
Afterwards, the sample RNA of control and 2 processing is saved in -70 DEG C.
The synthesis of three the first chains of .cDNA
(1) template ribonucleic acid is thawed on ice；5×gDNA Buffer,FQ-RT Primer Mix,10×Fast RT
Buffer, RNase-Free ddH2O thaw at (15-25 DEG C) of room temperature, are immediately placed on ice after defrosting.It is molten by every kind before use
Liquid vortex oscillation mixes, and brief centrifugation (revolving speed 6000rpm, 30s) remains in the liquid of tube wall to collect.In order to guarantee reaction solution
The accuracy of preparation is first configured to Mix, is then dispensed into each reaction tube again when carrying out every reaction.
(2) the gDNA removal system in RNA is carried out according to following system and prepare mixed liquor, thoroughly mix.Brief centrifugation (turns
Fast 6000rpm, 30s), 42 DEG C are placed in, 3min is incubated for.It is subsequently placed in and places on ice.
GDNA removes reaction system:
5×gDNA Buffer 2μl
Total RNA 2μl
RNase-Free ddH2O is supplied to 10 μ l.
(3) mixed liquor is prepared according to the reverse transcription reaction system of following system:
Reverse transcription reaction system:
10×Fast RT Buffer 2μl
RT Enzyme Mix 1μl
FQ-RT Primer Mix 2μl
RNase-Free ddH2O is supplied to 10 μ l.
(4) it by the Mix in reverse transcription reaction, is added in the reaction solution of gDNA removal step, mixes well.It 42 DEG C, is incubated for
15min 95 DEG C, is incubated for 3min and is put on ice later, obtains cDNA for subsequent experimental or cryo-conservation.
Four, the Ravenna grass class calmodulin expressed in relative RT-PCR technology detection Ravenna grass wild species blade by low temperature stress
The differential expression situation of gene ErCML30, experiment carries out in 7500 fluorescence volume PCR instrument of ABI company, before and after low-temperature treatment
Expression conditions are analyzed (see Fig. 2).
(1) relative RT-PCR reaction system:
The configuration of class Calmodulin Gene ErCML30 relative RT-PCR reaction system:
The base sequence of upstream primer QF is as shown in SEQ ID NO:4, the base sequence of downstream primer QR such as SEQ IDNO:
Shown in 5.
The configuration of reference gene 25SrRNA relative RT-PCR reaction system:
The base sequence of upstream primer 25S-F is as shown in SEQ ID NO:6, and the base sequence of downstream primer 25S-R is such as
Shown in SEQID NO:7.
(2) relative RT-PCR response procedures:
95 DEG C of initial denaturation 15min,
95 DEG C of denaturation 10s,
60 DEG C of annealing 32s, 40 circulations；Test data is analyzed on 7500 fluorescence quantitative PCR instrument of ABI company.
Interpretation of result: Ravenna grass class Calmodulin Gene ErCML30 is had detected by relative RT-PCR and is coerced in Different hypothermia
Compel the differential expression situation (see Fig. 2) in 2 periods in the Ravenna grass wild species blade under the time, the Ravenna grass after finding low-temperature treatment
Ravenna grass class Calmodulin Gene ErCML30 differential expression is significant in wild species blade, and Ravenna grass class Calmodulin Gene ErCML30 is not having
It is hardly expressed in the case where having low temperature stress, Ravenna grass class Calmodulin Gene ErCML30 is in Ravenna grass wild species after processing for 24 hours
Expression quantity is 50 times of control, and expression quantity of the Ravenna grass class Calmodulin Gene ErCML30 in Ravenna grass wild species is pair after handling 72h
According to 75 times, illustrate Ravenna grass class Calmodulin Gene ErCML30 after Ravenna grass wild species are by low temperature stress expression quantity rise, and with
The increase of stress time and increase.Therefore Ravenna grass class Calmodulin Gene ErCML30 belongs to the induction type expressed by low temperature stress
Expressing gene shows the participation low temperature that Ravenna grass class Calmodulin Gene ErCML30 can be rapid and lasting after by low temperature stress
Stress response.
Sequence table
<110>Yunnan Prov Agriculture University
<120>the Ravenna grass class Calmodulin Gene ErCML30 expressed in Ravenna grass wild species by low temperature stress
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 795
<212> DNA
<213>Ravenna grass wild species (E.fulvus)
<220>
<221> CDS
<222> (82)..(645)
<223>coded sequence
<220>
<221> 5’UTR
<222> (1)..(81)
<223>it is not translated into the 5' terminal region of the mature transcript of protein
<220>
<221> 3’UTR
<222> (646)..(795)
<223>it is not translated into the 3' terminal region of the mature transcript of protein
<220>
<221> protein_bind
<222> (163)..(636)
<223>conserved domain EF structural domain
<220>
<221> primer_bind
<222> (1)..(20)
<223>site of PCR primer element
<220>
<221> primer_bind
<222> (775)..(795)
<223>site of PCR primer element
<400> 1
agaaataacc agccctcgca tccgcacgcc cacaaaacca cgaagaaaag aagaggagga 60
acgccgcaag ttcacgcgac g atg gag aac gcc act ccc gcc atc gcc aag 111
Met Glu Asn Ala Thr Pro Ala Ile Ala Lys
1 5 10
ccg tcg ctg tcc aag aag ccc tcg ccg tcg ttc cgc ctc cgg aac ggc 159
Pro Ser Leu Ser Lys Lys Pro Ser Pro Ser Phe Arg Leu Arg Asn Gly
15 20 25
agc ctc aac gct ctg cgc ctg cgc cgc gtg ttc gac ctc ttc gac cgc 207
Ser Leu Asn Ala Leu Arg Leu Arg Arg Val Phe Asp Leu Phe Asp Arg
30 35 40
aac ggc gac ggc gag atc acc ctc gac gag atg gcc tcg gcg ctc gac 255
Asn Gly Asp Gly Glu Ile Thr Leu Asp Glu Met Ala Ser Ala Leu Asp
45 50 55
tcg ctc ggc ctc ggc gcc gac cgc tcc ggc ctg gag gcc gcg gtg ggc 303
Ser Leu Gly Leu Gly Ala Asp Arg Ser Gly Leu Glu Ala Ala Val Gly
60 65 70
ggg tac atc ccg gcc ggc gcc gcg ggg ctc cgc ttc gac gac ttc gag 351
Gly Tyr Ile Pro Ala Gly Ala Ala Gly Leu Arg Phe Asp Asp Phe Glu
75 80 85 90
tcc ctc cac cgc gcg ctc ggg gac gcg ctg ttc ggc ccc atc ccg gag 399
Ser Leu His Arg Ala Leu Gly Asp Ala Leu Phe Gly Pro Ile Pro Glu
95 100 105
gag gtg cct gag gac gac gac gag ggg gac atg aag gag gcg ttc cgg 447
Glu Val Pro Glu Asp Asp Asp Glu Gly Asp Met Lys Glu Ala Phe Arg
110 115 120
gtg ttc gac gag gac ggt gac ggc ttc atc tcg gcc gcc gag ctg cag 495
Val Phe Asp Glu Asp Gly Asp Gly Phe Ile Ser Ala Ala Glu Leu Gln
125 130 135
gcc gtg ctc aag aag ctc ggt ctg ccg gag gct cgg aac ctg gcc act 543
Ala Val Leu Lys Lys Leu Gly Leu Pro Glu Ala Arg Asn Leu Ala Thr
140 145 150
gtg cag gag atg atc tgc aac gtc gac cgc aac tgc gac ggc cgc gtc 591
Val Gln Glu Met Ile Cys Asn Val Asp Arg Asn Cys Asp Gly Arg Val
155 160 165 170
gac ttc ggc gaa ttc aag aac atg atg cag ggg atc acc gtg tgg ggc 639
Asp Phe Gly Glu Phe Lys Asn Met Met Gln Gly Ile Thr Val Trp Gly
175 180 185
gct tag ctgcagagcg cctgcgttgt cagcccctct actgcgtggt ttcatgtatg 695
Ala
tatctaggca ccgtagaatg cgcttttcta cccggcgctt cttcctgttc ttgatcgcgc 755
agtggattct tgctgttctt ggctttgctc tctgactcga 795
<210> 2
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<221> misc_
<222> (1)..(20)
<223>upstream primer GP-F
<400> 2
agaaataacc agccctcgca 20
<210> 3
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<221> misc_
<222> (1)..(20)
<223>downstream primer GP-R
<400> 3
tcgagtcaga gagcaaagcc 20
<210> 4
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<221> misc_
<222> (1)..(20)
<223>upstream primer QF
<400> 4
ccctctactg cgtggtttca 20
<210> 5
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<221> misc_
<222> (1)..(20)
<223>downstream primer QR
<400> 5
tcgagtcaga gagcaaagcc 20
<210> 6
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<221> misc_
<222> (1)..(22)
<223>upstream primer 25S-F
<400> 6
ataaccgcat caggtctcca ag 22
<210> 7
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<221> misc_
<222> (1)..(21)
<223>downstream primer 25S-R
<400> 7
cctcagagcc aatccttttc c 21
Claims (1)
1. a kind of detection Ravenna grass class Calmodulin Gene ErCML30 is in Ravenna grass wild species by the differential expression method of low temperature stress, packet
Include the extraction of total serum IgE, the synthesis of the first chain of cDNA, relative RT-PCR technology detection Ravenna grass class Calmodulin Gene ErCML30
In Ravenna grass wild species by the differential expression situation of low temperature stress, it is characterised in that:
The Ravenna grass wild species in seedling stage are set as control group and experimental group in the extraction of total serum IgE, control group is in 25 DEG C of conditions of room temperature
Lower growth, experimental group are divided into processing I and processing II, and processing I is grown for 24 hours under the conditions of 4 DEG C of low temperature, and processing II is in 4 DEG C of items of low temperature
72h growing under part, respectively acquisition control, processing I and the blade for handling II, liquid feeding nitrogen, which is ground, respectively obtains three samples, and three
Sample extracts total serum IgE respectively；
In relative RT-PCR technology detection Ravenna grass class Calmodulin Gene ErCML30 in Ravenna grass wild species by the difference of low temperature stress
In different expression, designed special primer is used to carry out the gene difference between different disposal and control as RT-PCR primer
Detection of expression, the special primer are made of upstream primer QF and downstream primer QR, the upstream primer QF base sequence such as SEQ
Shown in ID NO:4, the base sequence of the downstream primer QR is as shown in SEQ ID NO:5；The Ravenna grass class Calmodulin Gene
The full length nucleotide sequence of ErCML30 is as shown in SEQ ID NO:1.
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| CN108841842B (en) * | 2018-07-27 | 2021-06-29 | 云南农业大学 | Festuca arundinacea gene EfWRKY62 expressed by wild species of the saccharum arundinacea under low temperature stress |
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