CN106636132B - A kind of ultraviolet stress-related genes of highland barley and its application - Google Patents

A kind of ultraviolet stress-related genes of highland barley and its application Download PDF

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CN106636132B
CN106636132B CN201611147881.5A CN201611147881A CN106636132B CN 106636132 B CN106636132 B CN 106636132B CN 201611147881 A CN201611147881 A CN 201611147881A CN 106636132 B CN106636132 B CN 106636132B
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highland barley
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gene
ultraviolet
reagent
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CN106636132A (en
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曾兴权
原红军
徐齐君
韦泽秀
王玉林
扎桑
白丽军
尼玛扎西
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Chengdu Life Baseline Technology Co ltd
Institute Of Agriculture Tibet Autonomous Region Academy Of Agriculture And Animal Husbandry
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Institute Of Agriculture Tibet Autonomous Region Academy Of Agriculture And Animal Husbandry
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    • C12Q2600/158—Expression markers

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Abstract

The invention discloses a kind of gene relevant to the ultraviolet stress of highland barley and its cloning process, whether which can be used for detecting highland barley by ultraviolet stress.The present invention provides the ultraviolet stress-related genes of highland barley, and nucleotide sequence is as shown in SEQ ID NO.1.Whether the expression of gene of the present invention is related by livid purple external radiation stress to highland barley, expression by detecting gene of the present invention may determine that whether highland barley has received ultraviolet radioactive stress, the control for growing subsequent growth condition for highland barley provides theories integration, has good market application prospect.

Description

A kind of ultraviolet stress-related genes of highland barley and its application
Technical field
The present invention relates to a kind of ultraviolet stress-related genes of highland barley and its applications.
Background technique
Highland barley English name: hullessbarley is a kind of cereal crop of grass family Hordeum, because of glume inside and outside it Separation, seed is exposed, therefore also known as naked barley, wheat, barley.Mainly produced in Tibet, China, Qinghai, Sichuan, Yunnan and other places, it is The main food of Tibetan people.Highland barley is planted on Qinghai-Tibet Platean there are about history in 3500, extends among material and culture Field of spirit culture forms highland barley culture with rich connotation and ethnic characteristics on Qinghai-Tibet Platean.Have extensive medicinal And nutritive value, the highland barley products such as highland barley fine dried noodles, highland barley steamed bun, barley nutritional powder have been introduced.
Influence of the ultraviolet radioactive enhancing on ground to plant is reached caused by ozone layer is thinning has caused the whole society Extensive concern is the major subjects of current sphere of learning research.Ultraviolet stress can seriously affect the growth and development of highland barley, to final Yield cause large effect.
However, but having no effective means in terms of whether detection highland barley is by ultraviolet radioactive at present.
Summary of the invention
The present invention provides a kind of genes relevant to the ultraviolet stress of highland barley and its cloning process, the gene can be used for examining Highland barley is surveyed whether by ultraviolet stress.
The present invention provides the ultraviolet stress-related genes of highland barley, and nucleotide sequence is as shown in SEQ ID NO.1.
The present invention provides a kind of ultraviolet Stress Related Proteins of highland barley, and amino acid sequence is as shown in SEQ ID NO.2.
The present invention provides a kind of methods of ultraviolet stress-related genes for cloning aforementioned highland barley, and steps are as follows:
1) highland barley is taken, RNA is extracted；
2) reverse transcription, purifying are carried out by primer of primer pair shown in SEQ ID NO.3~SEQ ID NO.4.
In step 2), the system and condition of reverse transcription are as follows:
Reverse transcription reaction system:
Reverse transcription reaction condition:
In step 2), the method for purifying is: the method for purifying is that the side of target DNA fragments is recycled in agarose gel electrophoresis Method.
The present invention provides a kind of kit for coerce by ultraviolet radioactive of detection highland barley, including it is optional be used to detect it is aforementioned The reagent of gene expression dose or foregoing proteins content.
Wherein, the reagent is PCR method reagent.
Wherein, the reagent includes primer pair shown in SEQ ID NO.5~SEQ ID NO.6.
Wherein, the reagent further includes following reagent: 5 × RT Reaction Mix, Oligo (dT), TUREscript H-RTase/RI Mix and RNase Free dH2O, their volume ratio are 4:1.4:1:8.6.
Present invention finds highland barley ultraviolet radioactives to coerce relevant gene (nucleotide sequence is as shown in SEQ ID NO.1), Whether the expression of gene of the present invention is related by livid purple external radiation stress to highland barley, after ultraviolet radioactive stress, highland barley The expression of gene of the present invention can be obviously improved, can be with by the content of the expression or albumen that detect gene of the present invention Judge whether highland barley has received ultraviolet radioactive stress, the control for growing subsequent growth condition for highland barley provides theories integration, has good Good market application prospect.
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.
The specific embodiment of form by the following examples remakes further specifically above content of the invention It is bright.But the range that this should not be interpreted as to the above-mentioned theme of the present invention is only limitted to example below.It is all to be based on above content of the present invention The technology realized all belongs to the scope of the present invention.
Detailed description of the invention
The digestion verification figure of Fig. 1 pET28a- gene of the present invention
Fig. 2 expression result chart
Fig. 3 GAPDH detects amplification curve (green is control group, and red is ultraviolet processing group)
Fig. 4 GAPDH detects solubility curve (green is control group, and red is ultraviolet processing group)
Fig. 5 genetic test amplification curve of the present invention (green is control group, and red is ultraviolet processing group)
Fig. 6 genetic test solubility curve of the present invention (green is control group, and red is ultraviolet processing group)
The 2^- △ △ Ct result of Fig. 7 control group and experimental group of the present invention
Specific embodiment
The cloning and expression of the gene of the present invention of embodiment 1
One: gene cloning
(1) RNA extracting and reverse transcription
1. with highland barley 320 and happiness 8 germinations growth seedling.
2. taking fresh blade plant extract kit extract RNA and detecting.
3. extraction step is as follows:
3.1 instruments and reagent
Key instrument: SCILOGEX D3024R centrifuge, analytikjena-Easycycler；Scientz-48 high pass Measure tissue grinder instrument；
Main agents: reverse transcription reagent box (TUREscript 1st Stand cDNA SYNTHESIS Kit) (Ai De Come, article No. PC1802)；2 × premixed liquid (German DBI pcrMix).
3.2 experimental methods and step
3.2.1 in tissue samples RNA extraction
RNA extracts kit uses jena innuPREP RNA Mini Kit.
3.2.2 the synthesis (reverse transcription) of cDNA
Using Aidlab company reverse transcription reagent box (TUREscript 1st Stand cDNA SYNTHESIS Kit) into Row reverse transcription operation, using 20uL reaction system (each reaction 500ul system is used for subsequent PCR):
Reverse transcription reaction condition is as follows:
After reaction, cDNA, -20 DEG C of preservations are obtained.
Gene primer information of the present invention is as follows:
(2) PCR product electrophoresis and editor
1 instrument and reagent
Key instrument: power supply, electrophoresis apparatus, imaging system.
Main agents: agarose, 500ml 1XTAE, marker, Gold view.
2 experimental methods and step:
2.1 1% Ago-Gels of preparation: it weighs 0.5g agarose and is placed in conical flask, 50ml1 × TAE. electromagnetic oven is added Heating boil 3 times to agarose all melt, shake up, 1.0% Ago-Gel liquid
The preparation of 2.2 offset plates: organic glass inside groove (plastic tank) wash clean in electrophoresis tank is taken, dries, is put into glue glass Inside groove is placed in horizontal position by plate, and has been put comb in fixed bit and mixed the Ago-Gel liquid for being cooled to 65 DEG C or so It carefully pours on inside groove glass plate, glue is unfolded slowly, until to form uniform glue-line quiet at room temperature for entire glass pane surface It sets until gel solidify (30min) completely, vertically gently pulls out comb, remove adhesive tape, gel and inside groove are put into electrophoresis tank addition 1 × TAE electrophoretic buffer is not until crossing offset plate.
2.3 sample-addings: sample is added respectively with 10ul micropipettor in the sample sulculus of offset plate (2ul), often adds one A sample, mono- feed head of Ying Genghuan, to prevent pollution, when sample-adding, does not break the gel face around sample well.
2.4 electrophoresis: the gel slab after sample-adding is powered immediately carries out electrophoresis, voltage 130V, 13min, and sample is (black by cathode Color) it is mobile to positive (red) direction.
2.5 observation photograph: observing in the UV lamp, and DNA, which exists, then shows red fluorescence band, using gel imaging system System is taken pictures preservation.
Two: recycling PCR product sequencing subclone and inducing expression
The building of recombinant strains
1. identified for genes
The recycling of 1.1 target DNA fragments
Pcr amplification product is separated with 10g/L agarose gel electrophoresis, gel reclaims kit recycles target fragment.
(1) it is smashed to pieces after cutting the agarose containing target fragment, by weight/volume ratio 1:3 (agar sugar weight: NE combination liquid Volume) be added NE combination liquid.As 200mg agarose adds 600mL NE combination liquid；
(2) 50~60 DEG C of 3~5min of water-bath, glue melt completely, can turn upside down therebetween 2~3 times；
(3) in being transferred to solution, standing 5min makes DNA sufficiently with pellosil ining conjunction with, 13000r/min centrifugation 10~20 seconds, Outwell the waste liquid in collecting pipe；
(4) 80% ethyl alcohol of 500 μ l is added in centrifugal purification column, 13000r/min is centrifuged 10~20 seconds, outwells collection Waste liquid in pipe；
(5) step (4) are repeated；
(6) 13000r/min is centrifuged 1min again, as far as possible removing residual ethanol；
(7) centrifugal purification column is placed in new centrifuge tube, uncap 2~3min of placement, is evaporated completely ethyl alcohol；
(8) 50 DEG C of sterile TE solution are preheated, 40~50 μ l is added into centrifugal purification column, stood 3~5min, make to elute Pellosil is sufficiently impregnated with by liquid；
(9) 13000r/min is centrifuged 1min, and tube bottom solution is required DNA.
1.2 prepare competent cell BL21 (DE3)
(1) one single colonie (diameter 2-3mm) of picking in the plate of 16~20h is cultivated from 37 DEG C, is gone to one and is contained In 100mL LB culture medium, after 37 DEG C of violent shaken cultivation 2h, OD600 to 0.5 or so is surveyed every 30min；
(2) by bacterium be transferred to one it is sterile, in ice-cold 50mL PA tube, culture is made to be cooled to 0 DEG C；
(3) 10min is centrifuged with 4000r/min in 4 DEG C, to recycle cell；
(4) culture solution is poured out, pipe is inverted 1min, so that last trace culture solution is flow to end；
(5) every 50mL initial incubation liquid 20mL, 0.1mol/L CaCl2 and 10mL, 0.1mol/L MgCl2 are put on ice 30min is set, solution is resuspended every part of precipitating, sets 10min in mixture of ice and water；
(6) 10min is centrifuged with 4000r/min in 4 DEG C, to recycle cell；
(7) supernatant is poured out, pipe is inverted 1min, so that last trace supernatant is flow to end；
(8) every 50mL initial incubation liquid ice-cold 0.1mol/L CaCl of 2mL2Every part of cell precipitation is resuspended in solution. Cell 4 DEG C save 12~for 24 hours, to improve transformation efficiency.
The above operation aseptically carries out.
The nucleotide sequence (SEQ ID NO.1) of the object of the invention genetic fragment is as follows:
ATGGTGCACGCTGCGACGGCCGAGTGCTTCAGCAGCGTGTTCGCCTCATTCGACCACGACGCCGACGGC AGGATCTCGGCGGCGAAGCTGCGGCTGTGCATGAAGGCGACGCTAGGCGAGGACGTGTCGGCGGAGGACGCCGAGGC GCTCTTGGCGTCGGCCAACGCCGACGGATACCAGCTGCTGGACGAGCAGGAGTTCCTCCGGCTGGTGGCGCGGCCGG AGACGGAGGAGGAGGAGGAGCGGTGCAGGGGGCTGAGGGAGGCATTCGCGATGTACGAGGTGAAGGGCGAAGGGTGC ATCACGCCGTCGAGCCTGATGCGGATGCTCGCCAGGCTGGGGTCCGAACAGGGCATCGAGGAGTGCCGCGCCATGAT CCGCATGTTTGATTTGAATGAAGACGGACTGGTTTGCTACGACGAGTTCAAGGTTATGATGGATGCGTAG
Extraction, digestion and the recycling of 1.3 gene PCR product purification and recoveries of the present invention and expression plasmid pET28a (+)
After will be containing the strain culturing of plasmid pet128- plasmid and pET28a (+), plasmid be extracted, and with BamHI and XhoI Double digestion, by genetic fragment PCR purification and recovery product of the present invention (genetic fragment of the present invention can be prepared according to preceding method, Can directly synthesize) BamHI and XhoI double digestion is used, digestion system see the table below, 37 DEG C of effect 2h, gel reclaims kit recycling Target DNA.
Plasmid enzyme restriction reaction system:
The connection of 1.4 digestion recovery product pET28a (+) and genetic fragment of the present invention
0.2mL is taken to sterilize Eppendorf pipe one, 16 DEG C of connections are stayed overnight after each ingredient is added by table 4-2.
Coupled reaction system:
The cleavage map of pET28a- gene of the present invention is as shown in Figure 1, obtained the recombination containing gene of the present invention PET28a plasmid.1.5 inducing expression
1.5.1 recombinant expression plasmid pET28a- genetic transformation expressive host bacterium BL21 (DE3) competent cell of the present invention. Positive control is made into the empty carrier pET28a of extraction (+) conversion expression host strain BL21 (DE3) simultaneously；BL21 (DE3) competence Cell does not convert group and makees negative control.
1.5.2 the optimization of the inducing expression of recombinant protein and expression condition
1.5.3 the culture and expression of recombinant strains
What picking LB solid medium was grown on (containing Kan100ug/mL) has been transformed into pET28a- gene of the present invention BL21 (DE3) colony inoculation is in 5mL LB liquid medium (containing Kan100ug/mL)；
When 37 DEG C of shaken cultivations are about 0.5 to OD600,1mL is taken to be inoculated in the LB Liquid Culture containing Kan in 1:100 ratio In base；
The 100mL culture solution of culture is divided into 2 parts, a copy of it adds IPTG inducing expression, and IPTG conduct is not added in another Control.IPTG to final concentration of 1mmol/L is added, continues shaken cultivation
Change IPTG concentration, inducing temperature and incubation time and obtains best inducing expression condition.
Bacterium solution 1mL after taking IPTG to induce is centrifuged, abandoning supernatant, and after precipitating brine, 100ul physiology salt is added Water and 5 × SDS sample-loading buffer 25ul, 100 DEG C are boiled 10min, and centrifugation takes supernatant to carry out SDS-PAGE.To not inducing bacterium solution Same processing is done, expression is observed.
Simultaneously using LB Liquid Culture is unconverted and the BL21 of empty carrier pET28a (+) conversion is as compareing.
1.5.4 recombinant expression plasmid SDS-PAGE electrophoresis detection
(1) Vertial electrophorestic tank is illustratively installed, guarantees sealing of the sealing adhesive strip to notch.According to the size of destination protein and Electrophoresis tank specification determines separation gel and concentration and volume that glue is concentrated；
(2) separation gel of concentration needed for configuring, speed are injected into device of gel, and space needed for reserving perfusion concentration glue is used Suction pipe carefully covers one layer of water in separation glue surface, and gel is disposed vertically at room temperature；
(3) after separation gel polymerization completely, occur an apparent boundary line between gel and water, pour out coating liquid, Residual liquid is blotted with filter paper；
(4) the concentration glue of concentration needed for preparing, the perfusion concentration glue on the separation gel having polymerize, and comb is inserted into and is concentrated In glue, generation bubble should be avoided, gel is disposed vertically at room temperature, polymerization 45min or so；
(5) after the polymerization completely of concentration glue, comb is carefully taken out.Gel is fixed on electrophoresis tank, is perfused in upper and lower slot Tris- glycine running buffer；
(6) it is loaded in a predetermined order, opens power supply, constant pressure electrophoresis.First with 60V low-voltage electrophoresis, when dye front enters After separation gel, voltage is adjusted to 120V, continues electrophoresis until bromophenol blue reaches separation gel bottom；
(7) gel is taken out after electrophoresis, rinses the electrophoretic buffer on net glue with deionized water, gel is put into fixation 15min is fixed in liquid, with the dyeing of Coomassie brilliant blue dye liquor, in 65 DEG C of 15~20min of dyeing, is then gently shaken with destainer de- 3~5h of color observes protein band.
As shown in Fig. 2, the present invention expresses to have obtained target protein.
The amino acid sequence (SEQ ID NO.2) for the target protein that gene expression of the present invention obtains is as follows:
MVHAATAECFSSVFASFDHDADGRISAAKLRLCMKATLGEDVSAEDAEALLASANADGYQLLDEQEFLR LVARPETEEEEERCRGLREAFAMYEVKGEGCITPSSLMRMLARLGSEQGIEECRAMIRMFDLNEDGLVCYDEFKVMM DA
The correlation that the gene of the present invention of embodiment 2 is coerced with highland barley by ultraviolet radioactive
1. material
2 seedling of the highland barley having been provided, sample message are as follows:
2. instrument and reagent
2.1 key instrument
Analytikjena-qTOWER2.2 type fluorescence quantitative PCR instrument (Germany), SCILOGEX D3024R centrifuge (beauty State), regular-PCR instrument analytikjena-Easycycler (Germany)；Ultramicron nucleic acid-protein analyzer scandrop100 (moral State)；Pipettor (U.S. bio-rad), DNA electrophorogram scope (monarch's instrument), electrophoresis apparatus (monarch's instrument).
2.2 main agents consumptive materials
Reverse transcription reagent box (TUREscript 1st Stand cDNA SYNTHESIS Kit) (Ai Delai (being brand) Article No. PC1802)；Green premixed liquid (German DBI).10ul pipette tips (U.S. GCS), the 200ul pipette tips (U.S. GCS), 1ml pipette tips (U.S. GCS), 200ul is without RNA enzyme PCR reaction tube (AXGEN)；1.5Ml manages (GCS) without RNA enzyme EP.
3. experimental method and step
The extraction of RNA in 3.1 tissue samples
(1) chloroform of 200ul is added in the sample centrifuge tube that trizal reagent has been added, vibrates 30 seconds on the oscillator It mixes, is placed at room temperature for 3min.
It (2) 4 DEG C, 12000 × g, is centrifuged 15 minutes, honest and upright and thrifty 600uL in collection is transferred in the EP pipe of new 1.5mL.
(3) isometric isopropanol is added, is sufficiently mixed by inversion, stands 10min at room temperature.
(4) 4 DEG C, 12000 × g is centrifuged 10 minutes, abandons supernatant.
(5) ethyl alcohol of 1ml75% is added, is sufficiently mixed by inversion, 4 DEG C, 7500 × g is centrifuged 5 minutes.
(6) alcohol is outwelled, is dried, 50ul DEPC water is added and dissolves RNA, immediately detectable concentration.
(13) 4 DEG C, 12000 × g is centrifuged 2 minutes, and solution is RNA sample in centrifuge tube.
3.2 RNA extract result
3.2.1 UV spectrophotometer measuring result
The synthesis (reverse transcription) of 3.3cDNA
Using Aidlab company reverse transcription reagent box (TUREscript 1st Stand cDNA SYNTHESIS Kit) into Row reverse transcription operation, using 40uL reaction system:
Reverse transcription reaction condition is as follows:
After reaction, cDNA, -20 DEG C of preservations are obtained.
Primer information is as follows:
3.4 primer information
The optimization of 3.5 Real-time PCR reaction conditions
Reaction condition system:
Step1-95℃-3Min
Step2-95℃-10s
Step3-TM℃-30s+plate read
Step5-Go to step2,39 cycles
Step6-Melt curve analysis (60 DEG C~95 DEG C ,+1 DEG C/cycle, holding time 4s).
QPCR reaction solution ingredient:
4 experimental results
4.1 all gene primer Tm value amplification experiments
The specific primer of all genes is configured by above-mentioned qPCR reaction system, 4 DEG C on PCR plate centrifuge 6000rpm is centrifuged 30s.Quantitative PCR apparatus is placed in again to be expanded by above procedure.
The amplification curve of GADPH and gene of the present invention are as shown in Fig. 3.
The detection of 4.2 test specimens
According to the annealing temperature (Tm value) of each gene-specific primer, sample detection is carried out to each sample respectively, if Set 2 secondary orifices.The calculating of target gene relative expression quantity is by instrument software in each sample
QPCRsoft3.0 is executed automatically, and using Pfaffl method, formula is as follows:
4.3 test result
Gene internal reference is done with GADPH, calculates 2^- △ △ Ct result as shown in Fig. 7 and following table:
2^-△△Ct
Control group (1#) 1
Experimental group (2#) 34.7756
Test result explanation, the expression of gene of the present invention and highland barley are positively correlated by ultraviolet radioactive stress, pass through The expression for detecting gene of the present invention is coerced when can measure highland barley by ultraviolet radioactive.
To sum up, present invention finds highland barley ultraviolet radioactives to coerce relevant gene (nucleotide sequence such as SEQ ID NO.1 institute Show) expression it is whether related by livid purple external radiation stress to highland barley, the expression by detecting gene of the present invention can be with Judge whether highland barley has received ultraviolet radioactive stress, the control for growing subsequent growth condition for highland barley provides theories integration, has good Good application prospect.
SEQUENCE LISTING
<110>Tibet Autonomous Region's animal husbandry academy of sciences Agricultural Research Institute
Chengdu life baseline Science and Technology Ltd.
<120>a kind of ultraviolet stress-related genes of highland barley and its application
<130> GY462-16P1477
<160> 8
<170> PatentIn version 3.5
<210> 1
<211> 447
<212> DNA
<213>nucleotide sequence of gene of the present invention
<400> 1
atggtgcacg ctgcgacggc cgagtgcttc agcagcgtgt tcgcctcatt cgaccacgac 60
gccgacggca ggatctcggc ggcgaagctg cggctgtgca tgaaggcgac gctaggcgag 120
gacgtgtcgg cggaggacgc cgaggcgctc ttggcgtcgg ccaacgccga cggataccag 180
ctgctggacg agcaggagtt cctccggctg gtggcgcggc cggagacgga ggaggaggag 240
gagcggtgca gggggctgag ggaggcattc gcgatgtacg aggtgaaggg cgaagggtgc 300
atcacgccgt cgagcctgat gcggatgctc gccaggctgg ggtccgaaca gggcatcgag 360
gagtgccgcg ccatgatccg catgtttgat ttgaatgaag acggactggt ttgctacgac 420
gagttcaagg ttatgatgga tgcgtag 447
<210> 2
<211> 148
<212> PRT
<213>amino acid sequence of the albumen of gene expression of the present invention
<400> 2
Met Val His Ala Ala Thr Ala Glu Cys Phe Ser Ser Val Phe Ala Ser
1 5 10 15
Phe Asp His Asp Ala Asp Gly Arg Ile Ser Ala Ala Lys Leu Arg Leu
20 25 30
Cys Met Lys Ala Thr Leu Gly Glu Asp Val Ser Ala Glu Asp Ala Glu
35 40 45
Ala Leu Leu Ala Ser Ala Asn Ala Asp Gly Tyr Gln Leu Leu Asp Glu
50 55 60
Gln Glu Phe Leu Arg Leu Val Ala Arg Pro Glu Thr Glu Glu Glu Glu
65 70 75 80
Glu Arg Cys Arg Gly Leu Arg Glu Ala Phe Ala Met Tyr Glu Val Lys
85 90 95
Gly Glu Gly Cys Ile Thr Pro Ser Ser Leu Met Arg Met Leu Ala Arg
100 105 110
Leu Gly Ser Glu Gln Gly Ile Glu Glu Cys Arg Ala Met Ile Arg Met
115 120 125
Phe Asp Leu Asn Glu Asp Gly Leu Val Cys Tyr Asp Glu Phe Lys Val
130 135 140
Met Met Asp Ala
145
<210> 3
<211> 29
<212> DNA
<213>primer-F of gene of the present invention
<400> 3
attgcgggat ccatggtgca cgctgcgac 29
<210> 4
<211> 31
<212> DNA
<213>primer-R of gene of the present invention
<400> 4
attgccctcg agctacgcat ccatcataac c 31
<210> 5
<211> 17
<212> DNA
<213>primer-S of gene of the present invention
<400> 5
ggtccgaaca gggcatc 17
<210> 6
<211> 20
<212> DNA
<213>primer-A of gene of the present invention
<400> 6
accttgaact cgtcgtagca 20
<210> 7
<211> 19
<212> DNA
<213> GAPDH-S
<400> 7
ccaagccagc cacctatga 19
<210> 8
<211> 20
<212> DNA
<213> GAPDH-A
<400> 8
tggaaacaag gtcctcatcg 20

Claims (7)

1. a kind of method for the ultraviolet stress-related genes for cloning highland barley, it is characterised in that: steps are as follows:
1) highland barley is taken, RNA is extracted；
2) reverse transcription, purifying are carried out by primer of primer pair shown in SEQ ID NO.3~SEQ ID NO.4；
The nucleotide sequence of the ultraviolet stress-related genes of the highland barley is as shown in SEQ ID NO.1.
2. according to the method described in claim 1, it is characterized by: the system and condition of reverse transcription are as follows in step 2):
Reverse transcription reaction system:
Reverse transcription reaction condition:
3. according to the method described in claim 1, it is characterized by: the method for purifying is agarose gel electrophoresis in step 2) The method of middle recycling target DNA fragments.
4. a kind of kit that detection highland barley is coerced by ultraviolet radioactive, it is characterised in that: including for detecting described in claim 1 The reagent of gene expression dose；
The gene expression dose refers to: the amount by the RNA of the genetic transcription or the protein that is gone out by the gene expression Amount.
5. kit according to claim 4, it is characterised in that: the reagent is that gene described in detection claim 1 turns The reagent of the amount of the RNA of record.
6. kit according to claim 4, it is characterised in that: the reagent is PCR method reagent.
7. kit according to claim 5, it is characterised in that: the reagent includes SEQ ID NO.3~SEQ ID Primer pair shown in primer pair shown in NO.4 and SEQ ID NO.5~SEQ ID NO.6.
CN201611147881.5A 2016-12-13 2016-12-13 A kind of ultraviolet stress-related genes of highland barley and its application Active CN106636132B (en)

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Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Ling,H.Q等.Putative calcium¬binding protein CML19.《GenBank》.2015,第1-3页. *
植物类钙调素生理功能的研究进展;曾后清等;《中国科学 生命科学》;20160531;第46卷(第6期);第705-715页 *

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