CN108949780A - The Ravenna grass genoid EfNAC44 that Ravenna grass wild species are expressed by low temperature stress - Google Patents
The Ravenna grass genoid EfNAC44 that Ravenna grass wild species are expressed by low temperature stress Download PDFInfo
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Abstract
The invention discloses the Ravenna grass genoid EfNAC44 that Ravenna grass wild species are expressed by low temperature stress, and expand its primer special and detect the gene in Ravenna grass wild species by the differential expression method and its special primer of low temperature stress.The gene is persistently risen in Ravenna grass wild species by expression quantity under low temperature stress, in cultivation of sugar cane kind (being) without obvious expression, shows that the gene directly takes part in low temperature stress response.The present invention is to recognize the degeneration-resistant mechanism of the gene, and provide technical support using the valuable source that Ravenna grass wild species are developed as sugarcane new cold-resistant species.
Description
Technical field
The invention belongs to technical field of molecular biology, and in particular to the gene of Ravenna grass wild species under low temperature stress
The primer special of EfNAC44 and the gene, by RT-qPCR to the gene in strong and weak Ravenna grass wild species, cultivation species diversity table
The method reached.
Background technique
Ravenna grass (Erianthus fulvus Ness.) is the open country in saccharum derived from the torrid zone, subtropical zone and temperate regions
One of non-hibernating eggs, it is wide in distributed pole within Chinese territory, possess stronger drought resisting, cold-resistant and resistance to lean ability.Height of the Ravenna grass in very cold
Altitude Regions also can normal growth, it is also seen that there is the growth of Ravenna grass under other rugged environments.Ravenna grass also possesses its emblic
Characteristic not available for sugarcane Wild related germplasm, mature early, seed maturing rate is high, excellent pollen development, somatic chromosome number
Mesh is minimum, is not merely suitble to conventional breeding, also takes advantage in terms of molecular breeding, and sugar content is also higher in Ravenna grass stalk,
There is great value in cane breeding.It is rich in adversity gene abundant in Ravenna grass, can be sugar cane breed improvement and resistance to adverse circumstance
Breeding provides excellent GENE SOURCES.(the difference of the sugarcane sibling species Ravenna grass photosynthetic gas commutativity such as Xu Chaohua, Li Chunjia, Lu Xin
Different analysis [J] Scientia Agricultura Sinica, 2016,49 (15): 2909-2920. Li Fusheng, quality of stand husband, what develops and uses sugarcane along long
Thinking [J] living resources of thatch Wild ornamental resources, 2004,20 (4): the such as 266-270. Tian Chunyan, Wang Xianhong, Li Fusheng are sweet
Morphological Diversity [J] China's agronomy of sugarcane wild species Ravenna grass is notified to, 2015,31 (15): 97-102. Lu Xin, Su Huosheng,
The sugarcane wild species such as Lin Xiuqin Yunnan Ravenna grass germplasm innovation research on utilization [J] Agricultural University Of Hunan journal, 2012,38 (2): 121-
124.) it therefore, resists cold in breeding in sugarcane, excavates cold tolerance gene new in Ravenna grass wild species and be of great significance.
NAC transcription factor is the new transcription factor of discovered in recent years, and member is numerous, has set up huge transcription factor man
Race.NAC is divided into typical and atypia, and typical case is that N-terminal contains 151-159 amino acid residue and highly conserved, C-terminal turn
The die body of record control region conservative in subgroup is higher, and atypia NAC transcription factor is only one or two concatenated knots
Structure domain.It, equally can be in low temperature stress reaction research shows that NAC gene is in addition to other than drought resisting, salt tolerant Stress responses work
Play regulating and controlling effect: NAC gene by adjusting the expression of cold-resistant related gene, by regulate and control the expression quantity of cold-resistant related gene come
Further regulate and control plant cold-resistant.(identification of the tea tree NAC transcription factor such as Han Yapeng, Cheng Lin, Yang Lingxiao family and biology letter
It ceases credit and analyses the Journal of Henan University [J], 2017,47 (3): the .NAC transcription factor such as 301-309. Zhou Honghui, Huang Hong, Xu Binlei
In plant to function [J] plant physiology journal 2017,53 (8) in biology and abiotic stress response: 1372-1382. sections pretty
Branch, application progress [J] the journal of crops of the .NAC such as Li Ying, Zhao Mingzhong transcription factor in plant abiotic stress gene engineering,
2017,(2):14-22.)
Forefathers are more deep for sugarcane research, but are mainly limited to the research on Physiology and biochemistry, for sugarcane genome
Research it is less, the hereditary information data of sugarcane are also extremely limited in ncbi database, have especially with respect to Ravenna grass wild species are cold-resistant
The NAC gene of pass is even more to have not been reported.
Summary of the invention
Object of the present invention is to be to solve the problems, such as that sugarcane is improved sweet by serious technicals such as freeze injuries in recent years from molecular breeding angle
The cold resistance of sugarcane cultivar provides a Ravenna grass genoid EfNAC44 that can quickly express after by low temperature induction, and provides
The amplimer of the gene, and the gene is detected in the method for the differential expression of Ravenna grass wild species, further resist cold for sugarcane
Breeding lays the foundation and provides candidate gene.
This research obtains a NAC gene using electronic cloning technology from the Ravenna grass wild species under the conditions of low temperature stress, leads to
It crosses ncbi database and compares analysis, find the gene order and the known sequence likelihood ratio up to 96%, thus it is speculated that gene coding
Albumen is NAC transcription factor, therefore is Ravenna grass genoid EfNAC44 by the unnamed gene.
Technical scheme is as follows:
1. the Ravenna grass genoid EfNAC44 that Ravenna grass wild species are expressed by low temperature stress, full length nucleotide sequence such as SEQ
Shown in ID NO:1.
2. the Ravenna grass genoid EfNAC44's that Ravenna grass wild species described in amplification technique scheme 1 are expressed by low temperature stress is special
With primer, the primer special is 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 the base sequence of the downstream primer GP-R is as shown in SEQ ID NO:3.
3. Ravenna grass genoid EfNAC44 described in a kind of detection technique scheme 1 is in Ravenna grass wild species by low temperature stress
Differential expression method, including low temperature stress processing, the extraction of total serum IgE, the synthesis of the first chain of cDNA, relative RT-PCR technology
Ravenna grass genoid EfNAC44 is detected in Ravenna grass wild species by the differential expression situation of low temperature stress, it is characterised in that:
In relative RT-PCR technology detection Ravenna grass genoid EfNAC44 by the difference of low temperature stress in Ravenna grass wild species
In different expression, Ravenna grass genoid EfNAC44 difference in control sample and processing sample is detected respectively using special primer
Expression, the special primer are made of upstream primer QF and downstream primer QR, the base sequence such as SEQ of the upstream primer QF
Shown in ID NO:4, the base sequence of the downstream primer QR is as shown in SEQ ID NO:5.
4. differential expression method according to technical solution 3, which is characterized in that in the relative RT-PCR skill
Art detects Ravenna grass genoid EfNAC44 in Ravenna grass wild species by the differential expression situation of low temperature stress, RT-qPCR reactant
System: 25 μ l of total volume, wherein 2 × SuperReal PreMix Plus 10 μ l, 10 μM of upstream primer QF 0.6 μ l, under 10 μM
Swim 0.6 μ l, cDNA template of primer QR, 1 μ l, 50 × ROX Reference Dye, 0.6 μ l, RNase-free ddH2O 12.2μ
l；RT-qPCR response procedures: 95 DEG C of initial denaturation 15min, 95 DEG C of denaturation 10s, 60 DEG C of annealing 32s, 40 recycle.
Compared with prior art, the beneficial effects of the present invention are:
Present invention firstly provides the Ravenna grass genoid EfNAC44 that Ravenna grass wild species are expressed by low temperature stress, and amplification
Its primer special detects special primer of the gene in different materials by the differential expression under low temperature stress, detection method.
The gene a small amount of expression in Ravenna grass in the case where no low temperature stress in RT-qPCR detection, in cultivation of sugar cane kind
(being) hardly expresses, the expression quantity substantial increase after low-temperature treatment in Ravenna grass wild species, illustrates the gene in Ravenna grass open country
Non-hibernating eggs is risen by expression quantity after low temperature stress, and the strong Ravenna grass wild species expression quantity of cold resistance is above cold resistance before and after the processing
Weaker Ravenna grass wild species, and show the Ravenna grass genoid before and after the processing without obvious expression in cultivation of sugar cane kind (being)
EfNAC44 belong to by low temperature stress express inducible expression of gene, the Ravenna grass genoid EfNAC44 Ravenna grass wild species by
Low temperature stress response process can be rapidly participated in after low temperature stress, by the up-regulation of expression quantity to regulate and control the resistance to of Ravenna grass wild species
Cold mechanism carries out molecular breeding using Ravenna grass wild species and provides technology branch for the degeneration-resistant mechanism for recognizing Ravenna grass class NAC gene
Support.
The present invention detects Ravenna grass genoid EfNAC44 in Ravenna grass wild species by the method for the differential expression of low temperature stress, institute
The special primer of design is generated in RT-qPCR high specificity, no miscellaneous band, and melting curve shows that primer free dimer generates.Inside
In the selection for joining gene, 5 kinds of candidate reference genes, respectively 25SrRNA, GAPDH, β-actin, β-tubulin, PPO, this examination
The internal reference 25SrRNA for selecting most suitable sugarcane is tested, stability is higher.This RT-qPCR uses 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 genoid EfNAC44 shown in SEQ ID NO:1 in sequence table.
The upstream primer GP- of the primer special of Ravenna grass genoid EfNAC44 is expanded in sequence table shown in SEQ ID NO:2
The base sequence of F.
The downstream primer GP- of the primer special of Ravenna grass genoid EfNAC44 is expanded in sequence table shown in SEQ ID NO:3
The base sequence of R.
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 genoid EfNAC44.In Fig. 1, M:maker；Swimming lane 1, swimming lane 2, swimming lane 3 are respectively
Amplified production at 55 DEG C, 57 DEG C, 60 DEG C of annealing temperature.
Fig. 2: low temperature stress handles the RT-qPCR difference of Ravenna grass genoid EfNAC44 in lower different materials in same time
Expression figure.In Fig. 2, ordinate indicates that the relative expression quantity of gene, abscissa indicate the name of different materials under treatment of different temperature
Claim.99-3-CK indicates that the Ravenna grass 99-3 (cold resistance is strong) under room temperature processing, 99-3-LT indicate the Ravenna grass under low temperature stress processing
99-3, I91-8-CK indicate that the Ravenna grass I91-8 (cold resistance is weaker) under room temperature processing, I91-8-LT are indicated under low temperature stress processing
Ravenna grass I91-8,04-14-CK indicates the lower Yunnan sugarcane 04-14 (filial generation of Ravenna grass 99-3) of room temperature processing, and 04-14-LT expression is low
Yunnan sugarcane 04-14 under warm Stress treatment, Xin-CK indicate the new platform sugar 10 (female parent of Yunnan sugarcane 04-14) under room temperature processing, Xin-
LT indicates the new platform sugar 10 under low temperature stress processing.
Fig. 3: it is upstream primer QF and downstream that RT-PCR technology, which detects special primer used in Ravenna grass genoid EfNAC44,
Testing result of the primer QR under different annealing temperature.M:maker；Swimming lane 1, swimming lane 2, swimming lane 3 are with 3 DEG C of low-temperature treatments respectively
The cDNA of the Ravenna grass 99-3 of 72h is template, and annealing temperature is followed successively by the amplified production at 55 DEG C, 57 DEG C, 60 DEG C.
Specific embodiment
Following embodiment without specified otherwise is conventional method in each embodiment for the invention will be further described,
Following experiment (including control) sample repeats three times.
Early period by the measurement of physical signs, filters out the Ravenna grass wild species Ravenna grass 99-3 for resisting cold strong, and cold resistance is weaker
Ravenna grass I91-8, the cultivar that Ravenna grass wild seed generation relatively resists cold (are) the new platform of female parent of Yunnan sugarcane 04-14 and Yunnan sugarcane 04-14
Sugared 10 materials (the .7 Ravenna grass wild species such as [1] Cao Zhequn, Xiao Furong, Li Fusheng and its identification of progeny material cold tolerance at the seedling stage
[J] journal of crops, 2017 (5): in the raw difference sugar cane breed blade of 43-48. [2] Shi Jingyu, He Lilian, Wang Xianhong, Li Fu
The optimizing research of general flavone content and extraction process, journal of crops, 2016 (5): 19-24).These materials can by acquisition or
Purchase is obtained, the applicant Yunnan Prov Agriculture University address: the Yunnan Province Kunming Panlong District source Feng road 452 by the applicant
Number, postcode 650201.
Experimental material: test material has different clones Ravenna grass wild species: Ravenna grass 99-3, Ravenna grass I91-8, cultivation of sugar cane product
Kind (being): Yunnan sugarcane 04-14, new platform sugar 10, totally 4 parts of materials, 4 parts of materials are planted in the sugarcane of sugarcane research institute of Yunnan Prov Agriculture University
In resource garden (Kunming, Yunnan, height above sea level 1950m).
The acquisition for the Ravenna grass genoid EfNAC44 that 1 Ravenna grass wild species of embodiment are expressed by low temperature stress:
One, the Ravenna grass class NAC gene EfNAC44 full length sequence that design amplification Ravenna grass wild species are expressed by low temperature stress is special
Use primer
The high-throughput transcript profile sequencing data of the Sugarcane smuts such as the Ravenna grass wild species obtained according to pre-stage test carries out biology
Bioinformatics analysis screening, dedicated the drawing of amplification Ravenna grass genoid EfNAC44 is designed using biosoftware Primer5.0, Oligo 7
Object, the primer special are made of upstream primer GP-F and downstream primer GP-R, the base sequence such as SEQ of the upstream primer GP-F
Shown in ID NO:2, for the base sequence of the downstream primer GP-R as shown in SEQ ID NO:3, which serves Hai Shenggong
Company's synthesis.
Two, the Ravenna grass wild species Total RNAs extraction of low temperature stress processing.
(1) low temperature stress is handled: the almost the same Ravenna grass 99-3 of the seedling growth state of potting, Ravenna grass I91-8 are all provided with
Control group and processing group, control group carry out low temperature stress processing, the low temperature in 25 DEG C of routinely management growths of room temperature, processing group
Stress treatment is then while respectively to acquire the blade of control group and processing group material in 3 DEG C of low-temperature epitaxy 72h, press respectively
Following methods carry out:
(2) blade of acquisition is placed in liquid feeding nitrogen in mortar quickly to grind, every 100mg sample is added 1ml TRIzol and extracts
Liquid, vortex oscillation 15s, each sample is in 15~30 DEG C of placement 5min；Chloroform is added, the dosage of chloroform uses 1ml to be every
200 μ l chloroforms are added in TRIzol, cover pipe lid, 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) it is added and uses RNase-Free ddH2The ethanol solution that the volume fraction that O is prepared is 75% washs precipitating, dosage
1ml TRIzol 1ml RNase-Free ddH is used to be every2The ethanol solution that the volume fraction that O is prepared is 75%.
(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 RNA points of 2 low temperature stress processing samples
It is not 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 thaws at (15 DEG C -25 DEG C) of room temperature, is immediately placed on ice after defrosting.By every kind before use
Solution vortex oscillation mixes, and brief centrifugation (revolving speed 6000rpm, 30s) remains in the liquid of tube wall to collect.In order to guarantee to react
The accuracy that liquid is prepared first is 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 genoid EfNAC44 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, expands the Ravenna grass genoid EfNAC44 expressed in Ravenna grass wild species Ravenna grass 99-3 blade by low temperature stress
Overall length, 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 denaturations
2min；95 DEG C of denaturation 30s；60 DEG C of annealing 30s；72 DEG C of extension 2min, 35 circulations；72 DEG C, 6min, rear 4 DEG C of preservations.
The base sequence of upstream primer GP-F is as shown in SEQ ID NO:2, the base sequence of downstream primer GP-R such as SEQ
Shown in ID NO:3.
Five, the recycling (the multifunctional dna purification and recovery kit that hundred Tyke biotech firms provide) of target gene
PCR product carries out glue recycling through 1% agarose gel electrophoresis, by the purpose band in gel, comprising the following steps:
(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
The volatilization of middle ethyl alcohol 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 without miscellaneous band as shown in Figure 1, generate.After carrying out gel extraction to amplified fragments, the recycling piece is sequenced
Duan Xulie is consistent with the Unigen sequence that transcript profile obtains, i.e., the recovery product is the sugarcane that Ravenna grass wild species are expressed by low temperature stress
Thatch genoid EfNAC44, the sequencing result of the recovery product is as shown in SEQ ID NO:1 in sequence table.
In addition, being also provided with the experiment of two different annealing temperatures, this two experiments are except PCR reaction interval in above-mentioned steps four
Annealing temperature in sequence is respectively outside 55 DEG C, 57 DEG C, remaining step is identical as above steps.
A kind of detection Ravenna grass genoid EfNAC44 provided by the present invention of embodiment 2 is coerced in Ravenna grass wild species by low temperature
Urgent differential expression method, comprising the following steps:
One, low temperature stress is handled:
By the almost the same Ravenna grass 99-3 of the seedling growth state of potting, Ravenna grass I91-8, Yunnan sugarcane 04-14, new platform sugar 10
Every part of material, which is all provided with, sets control group and processing group, and control group routinely manages growth in room temperature (25 DEG C), and processing group carries out low temperature
Stress treatment, the low temperature stress processing are then while respectively to acquire control group and processing group material in 3 DEG C of low-temperature epitaxy 72h
The blade of material carries out as follows respectively:
Two, the extraction of total serum IgE
(1) blade of acquisition is placed in liquid feeding nitrogen in mortar quickly to grind, every 100mg sample is added 1ml TRIzol and extracts
Liquid, vortex oscillation 15s, each sample is in 15~30 DEG C of placement 5min；Chloroform is added, the dosage of chloroform uses 1ml to be every
200 μ l chloroforms are added in TRIzol, cover pipe band, vortex oscillation 15s, 15~30 DEG C of placement 3min；4 DEG C, 12000rpm centrifugation
15min, solution is divided into three layers at this time: yellow is organic phase, middle layer and upper layer it is colourless be water phase, RNA is primarily present water
Xiang Zhong is transferred to water phase another new without 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) it is added and uses RNase-Free ddH2The ethanol solution that the volume fraction that O is prepared is 75% washs precipitating, dosage
1ml TRIzol 1ml RNase-Free ddH is used to be every2The ethanol solution that the volume fraction that O is prepared is 75%；4 DEG C,
5000rpm is centrifuged 3min, and supernatant liquid is carefully sucked out with pipette tips, 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 low temperature stress 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 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 for subsequent experimental or cryo-conservation.
Four, RT-qPCR technology detects Ravenna grass genoid EfNAC44 in Ravenna grass wild species and cultivar (being) by low temperature
The differential expression situation of expression is coerced, experiment carries out in 7500 fluorescence volume PCR instrument of ABI company, before and after the processing to low temperature stress
Expression conditions analyzed (see Fig. 2).
(1) RT-qPCR reaction system:
The RT-qPCR reaction system of Ravenna grass genoid EfNAC44 configures:
25 μ l of total volume
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 ID
Shown in NO:5.
The configuration of the RT-qPCR reaction system of reference gene 25SrRNA:
25 μ l of total volume
The base sequence of upstream primer 25S-F is as shown in SEQ ID NO:6, the base sequence of downstream primer 25S-R such as SEQ
Shown in ID NO:7.
(2) RT-qPCR response procedures: 95 DEG C of initial denaturation 15min, 95 DEG C of denaturation 10s, 60 DEG C of annealing 32s, 40 recycle.
Test data is analyzed on 7500 fluorescence quantitative PCR instrument of ABI company.
Separately set that only annealing temperature is respectively in RT-PCR response procedures with the cDNA template of the Ravenna grass 99-3 of processing group
55 DEG C, 57 DEG C, 60 DEG C, special primer are upstream primer QF and downstream primer QR, remaining operating procedure is corresponding with the present embodiment 1
The identical experiment of step finds that the special primer (upstream primer QF and downstream primer QR) has specific (see Fig. 3).
Interpretation of result: Ravenna grass genoid EfNAC44 is in the case where no low temperature stress in Ravenna grass in RT-qPCR detection
A small amount of expression, is hardly expressed in cultivar (being), after 3 DEG C of low temperature stress processing of 72h in Ravenna grass wild species
Expression quantity substantial increase illustrates that Ravenna grass genoid EfNAC44 expression quantity after Ravenna grass wild species are by low temperature stress rises, and
The strong Ravenna grass wild species expression quantity of cold resistance is above the weaker Ravenna grass wild species of cold resistance before and after the processing, and cultivar (being)
Without obvious expression, the Ravenna grass genoid EfNAC44 belongs to the inducible expression of gene expressed by low temperature stress, shows Ravenna grass
Genoid EfNAC44 can rapidly participate in low temperature stress response process after Ravenna grass wild species are by low temperature stress, the Ravenna grass class
Gene EfNAC44 regulates and controls the cold-resistant mechanism of Ravenna grass wild species by the up-regulation of expression quantity.
Sequence table
<110>Yunnan Prov Agriculture University
<120>the Ravenna grass genoid EfNAC44 that Ravenna grass wild species are expressed by low temperature stress
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 591
<212> DNA
<213>Ravenna grass (Erianthus fulvus Ness.)
<400> 1
atgtcggtgc cgccgctctc cgaccacgag ctgcaggacg agccctgcgg tggcttcgac 60
gataacccgt acgccgcgac gagcgccgcc atgctcctgc agggcgcgtc gttcccggcg 120
ctgcatgccg cgtccgccgg cgcgcagagg atgcccagga tcccgtcctt ctcggagctg 180
ttcaatgacc cctcgctgct ggcgcacttc ttcgaggagg gcggcatgca gcaggacatg 240
gcacggctcg gtaaccagca gcagcacgcc cctctcctcg gccgccccgt cacgagccaa 300
ctgctggtca acagcggcag cagcatgtcc ccggggcaga tccagcagat ggatccgcct 360
gcctcgacgt cggccgctgg agatggcgca gctggcaagc gcaagagatc atcagaggcg 420
agtactgcta gtgccagtgc gctgtcgagc cagcagcagg cgtctgcagc caagaaaccg 480
aacggctact gttttggtgc aacgacgacg ttccaaatag gcaacgggct gcaggggtcg 540
tcgctgggcc accagatgca gcttcattct tctaacatgg ggatgaactg a 591
<210> 2
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
catcccatct tctcttccta at 22
<210> 3
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
tcagtggaga acagcaatc 19
<210> 4
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
tgatgatgag atcccacact agc 23
<210> 5
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
tcagggtgca tcggtgattg 20
<210> 6
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
ataaccgcat caggtctcca ag 22
<210> 7
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
cctcagagcc aatccttttc c 21
Claims (4)
1. the Ravenna grass genoid EfNAC44 that Ravenna grass wild species are expressed by low temperature stress, full length nucleotide sequence such as SEQ ID
Shown in NO:1.
2. expanding dedicated the drawing for the Ravenna grass genoid EfNAC44 that Ravenna grass wild species described in claim 1 are expressed by low temperature stress
Object, the primer special are made of upstream primer GP-F and downstream primer GP-R, and the base sequence of the upstream primer GP-F is such as
Shown in SEQ ID NO:2, the base sequence of the downstream primer GP-R is as shown in SEQ ID NO:3.
3. a kind of detect Ravenna grass genoid EfNAC44 described in claim 1 in Ravenna grass wild species by the difference of low temperature stress
Expression, including low temperature stress processing, the extraction of total serum IgE, the synthesis of the first chain of cDNA, the detection of relative RT-PCR technology
Ravenna grass genoid EfNAC44 is in Ravenna grass wild species by the differential expression situation of low temperature stress, it is characterised in that:
In relative RT-PCR technology detection Ravenna grass genoid EfNAC44 by the difference table of low temperature stress in Ravenna grass wild species
Up in situation, Ravenna grass genoid EfNAC44 differential expression in control sample and processing sample is detected respectively using special primer,
The special primer is made of upstream primer QF and downstream primer QR, the base sequence of the upstream primer QF such as SEQ ID NO:
Shown in 4, the base sequence of the downstream primer QR is as shown in SEQ ID NO:5.
4. differential expression method according to claim 3, which is characterized in that examined in the relative RT-PCR technology
Ravenna grass genoid EfNAC44 is surveyed in Ravenna grass wild species by the differential expression situation of low temperature stress, RT-qPCR reaction system:
25 μ l of total volume, wherein 2 × SuperReal PreMix Plus, 10 μ l, 10 μM of 0.6 μ l of upstream primer QF, 10 μM of downstreams are drawn
0.6 μ l, cDNA template of object QR, 1 μ l, 50 × ROX Reference Dye, 0.6 μ l, RNase-free ddH2O 12.2μl；
RT-qPCR response procedures: 95 DEG C of initial denaturation 15min, 95 DEG C of denaturation 10s, 60 DEG C of annealing 32s, 40 recycle.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103392586A (en) * | 2013-06-21 | 2013-11-20 | 云南农业大学 | Breeding method of drought resistant and cold tolerant Saccharum varieties/lines containing Erianthus fulvus Ness consanguinity |
| CN108048469A (en) * | 2017-12-26 | 2018-05-18 | 云南农业大学 | The Ravenna grass class Calmodulin Gene ErCML30 expressed in Ravenna grass wild species by low temperature stress |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103392586A (en) * | 2013-06-21 | 2013-11-20 | 云南农业大学 | Breeding method of drought resistant and cold tolerant Saccharum varieties/lines containing Erianthus fulvus Ness consanguinity |
| CN108048469A (en) * | 2017-12-26 | 2018-05-18 | 云南农业大学 | The Ravenna grass class Calmodulin Gene ErCML30 expressed in Ravenna grass wild species by low temperature stress |
Non-Patent Citations (4)
| Title |
|---|
| FABIO T.S.NOGUEIRA ET AL.: ""SsNAC23, a member of the NAC domain protein family, is associated with cold, herbivory and water stress in sugarcane"", 《PLANT SCIENCE》 * |
| RENATA FAVA DITT ET AL.: ""Analysis of the stress-inducible transcription factor SsNAC23 in sugarcane plants"", 《SCI. AGRIC.(PIRACICABA, BRAZ.)》 * |
| 孟玉 等: ""蔗茅野生种响应低温胁迫的数字基因表达谱"", 《分子植物育种》 * |
| 曹哲群 等: ""7个蔗茅野生种及其后代材料苗期耐寒性鉴定"", 《作物杂志》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113403322A (en) * | 2021-05-14 | 2021-09-17 | 云南大学 | Tea tree drought response gene CsNAC168 and encoding protein and application thereof |
| CN113403322B (en) * | 2021-05-14 | 2022-09-16 | 云南大学 | Tea tree drought response gene CsNAC168 and encoding protein and application thereof |
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