CN109694874A - The clone of wheat cdna TaCPSF30 coded sequence and application - Google Patents
The clone of wheat cdna TaCPSF30 coded sequence and application Download PDFInfo
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Abstract
The clone of wheat cdna TaCPSF30 coded sequence of the present invention and application, belong to molecular biology of plants and gene clone technology field, the clone of wheat cdna TaCPSF30 is obtained first, the 35S promoter that the coding region sequence (CDS) and Gateway of the TaCPSF30 gene obtained using clone are compatible drives the pMDC83 carrier with GFP label, it constructs TaCPSF30 and is overexpressed recombinant vector pMDC83-TaCPSF30, then with mediated by agriculture bacillus flower-dipping method arabidopsis thaliana transformation cpsf30 mutant, and screen homozygous transgenic plant, it is built into the heterologous complement strain of TaCPSF30 arabidopsis thaliana transformation.
Description
Technical field
The invention belongs to molecular biology of plants and gene clone technology field, and in particular to a kind of wheat cdna
The clone of TaCPSF30 coded sequence and application.
Background technique
Wheat is the important cereal crops in China, improves wheat yield and improves its quality and is badly in need of as China's Wheat Production
The strategic issues of solution.Wheat is happiness nitrate nitrogen (NO3 -) crop, since crop utilization rate of nitrogen fertilizer is low, especially different cultivars
Wheat for nitrogen utilization efficiency difference, so that yield be caused larger difference occur, and then influence peasant income.Study wheat pair
NO3 -The metabolic processes such as absorbing, transport, assimilate and utilize to improve wheat nitrogen utilization rate is the key that solve the above problems.
Largely studies have shown that arabidopsis Polyadenylation factor CPSF30 is at target gene mRNA precursor (pre-mRNA)
It plays an important role in process, the processes such as wide participation gene expression regulation, oxidative stress, growth and development.We send out recently
Existing, nearest result of study discovery, arabidopsis CPSF30-L is an important regulation NO3 -The gene of metabolic process.The gene
Plant be can change to NO3 -Absorption efficiency；Meanwhile the gene has an effect on NO3 -Transhipment in root and overground part.In addition,
AtCPSF30 regulates and controls NO3 -The expression of assimilation genes NIA, NiR are to influence plant NO3 -Assimilation and accumulation in vivo.It is above-mentioned
As a result illustrate that AtCPSF30 gene is plant control NO3 -The key factor of metabolic pathway.
Wheat breed China spring is complicated hexaploid genome, clones in wheat and identifies that participation improves Nitrogen utility
The gene of rate is extremely difficult.In the work of early period, the present inventor has found the homologous base of arabidopsis CPSF30 in China spring
Because of TaCPSF30, it is located in A, B and D genome.Secondary protein structure analysis the result shows that, TaCPSF30 with
AtCPSF30 height is consistent.The above results show that TaCPSF30 very likely regulates and controls wheat to NO3 -The generations such as absorption, transhipment, assimilation
It apologizes for having done sth. wrong journey, is worth being furtherd investigate.
Summary of the invention
A kind of clone of wheat cdna TaCPSF30 coded sequence provided by the invention and its application, obtain wheat cdna
The clone of TaCPSF30 coded sequence, the research plant that is cloned in for providing wheat cdna TaCPSF30 coded sequence improve nitrogen
Application in utilization rate mechanism and genetic improvement.
To achieve the goals above, the technical solution adopted by the present invention are as follows:
The clone of wheat cdna TaCPSF30 coded sequence of the present invention, gram of the wheat cdna TaCPSF30 coded sequence
It is grand to obtain in accordance with the following methods:
(1) wheat total serum IgE is extracted, reverse transcription synthesizes cDNA；
(2) using cDNA as template, special primer is designed, PCR amplification gene TaCPSF30 coding region sequence is passed through, wherein
PCR amplification the primer sequence is as follows:
TaCPSF30 forward primer (F):
5'-CGGGGTACCATGGACGACGGCGACGGC-3'；
TaCPSF30 reverse primer (R):
5'-CCGCTCGAG TCGCTTCCTTGACCGCCT-3'；
(3) it recycles and purifies target fragment in the amplified production of (2) and be sequenced, sequencing is correctly wheat cdna
The clone of TaCPSF30 coded sequence.
The application of the clone of wheat cdna TaCPSF30 coded sequence of the present invention, wheat cdna TaCPSF30 coding
The application of sequence being cloned in research plant raising nitrogen utilization efficiency mechanism and genetic improvement.
The recombinant plant expression vector of Gateway system compatible is first constructed, there is 35S promoter driving on the expression vector
TaCPSF30 coded sequence clone and GFP gene, the recombinant expression carrier is then transformed into arabidopsis cpsf30 and is dashed forward
Variant, screening obtain the homozygous heterologous complement strain of transgenosis, study wheat cdna TaCPSF30 in complement strain and are improving nitrogen
Plain utilization rate mechanism and the application in genetic improvement.
The application of the clone of the wheat cdna TaCPSF30 coded sequence, the specific steps are as follows: S1, pENTR3C-
The building of TaCPSF30 recombinant plasmid: digestion connects clone and the cloning vector of the wheat cdna TaCPSF30 coded sequence
PENTR3C: to the wheat cdna TaCPSF30 coded sequence of purifying be cloned in respectively at 5 ' ends and 3 ' ends plus such as KpnI and
XhoI restriction enzyme site obtains the target gene fragment with cohesive terminus,cohesive termini after double digestion；Then it is carried out with cloning vector pENTR3C
Digestion connection, obtains recombinant plasmid pENTR3C-TaCPSF30；S2 constructs recombinant plant expression vector using Gateway technology
PMDC83-TaCPSF30: the plant table of correct recombinant plasmid pENTR3C-TaCPSF30 Yu Gateway system compatible will be sequenced
LR reaction is carried out up to carrier pMDC83, obtains recombinant plant expression vector pMDC83-TaCPSF30.S3, Agrobacterium infect quasi- south
Mustard: plantation arabidopsis cpsf30 mutant；Prepare Agrobacterium competent cell；Then recombinant expression carrier pMDC83- is used
TaCPSF30 converts Agrobacterium competent cell；The conversion Agrobacterium of picking positive colony infects arabidopsis cpsf30 mutant；
Obtain effectively cloning the transgenic plants of wheat cdna TaCPSF30 coded sequence.
The Agrobacterium is Agrobacterium GV3101.
The plant is arabidopsis cpsf30 mutant or wheat breed China spring.,
It after the conversion Agrobacterium of picking positive colony infects arabidopsis cpsf30 mutant in S3, is selfed by 3 generations, screening
Heterologous complement strain out containing wheat cdna TaCPSF30 coded sequence, for studying wheat cdna TaCPSF30 coded sequence
The application in nitrogen utilization efficiency mechanism and genetic improvement is improved in research plant.
The invention has the benefit that present invention obtains the clone of wheat cdna TaCPSF30 coded sequence, using gram
The plant table with GFP label of the 35S of grand obtained wheat cdna TaCPSF30 coded sequence and Gateway system compatible driving
Up to carrier pMDC83, constructs TaCPSF30 and be overexpressed recombinant vector pMDC83-TaCPSF30, then pass through by medium of Agrobacterium
Dip-flower sends out arabidopsis thaliana transformation cpsf30 mutant, and screening obtains the homozygous heterologous complement strain of transgenosis, studies in complement strain
Wheat cdna TaCPSF30 is improving nitrogen utilization efficiency mechanism and the application in genetic improvement.
Wheat breed China spring is complicated hexaploid genome, clones in wheat and identifies that participation improves Nitrogen utility
The gene of rate is extremely difficult, and Arabidopsis plant genome is small, is easy to convert.So the present invention passes through
Bioinformatics software predicts TaCPSF30 gene, recycles plant expression vector that wheat TaCPSF30 gene is made to exist
It is expressed in arabidopsis cpsf30 mutant, identifies wheat TaCPSF30 base by studying heterologous complement strain Physiology and biochemistry phenotype
The function of cause, while tissue positioning of the TaCPSF30 in transgenic arabidopsis is observed, it is the clone of wheat cdna TaCPSF30
Nitrogen utilization efficiency research is improved in crop and genetic improvement application lays the foundation.
Detailed description of the invention
Fig. 1 is the clone of purpose wheat cdna TaCPSF30 coded sequence, and wherein M swimming lane is DL2000Marker；1-4 swimming
Road is the coding region sequence clone of wheat cdna TaCPSF30；
Fig. 2 constructs the recombinant plant expression vector process that TaCPSF30 is overexpressed；
Fig. 3 is that recombinant cloning vector pENTR3C-TaCPSF30 converts the positive bacteria after bacillus coli DH 5 alpha competent cell
Fall qualification result；Wherein M swimming lane is DL2000Marker, and 1-4 swimming lane is positive bacterium colony, and No. 5 swimming lanes are negative control；
Fig. 4 is that recombinant expression carrier pMDC83-TaCPSF30 converts the positive bacteria after bacillus coli DH 5 alpha competent cell
Fall qualification result；Wherein M swimming lane is DL2000Marker, and 1-4 swimming lane is positive bacterium colony, and No. 5 swimming lanes are negative control；
Fig. 5 is that recombinant expression carrier pMDC8383-TaCPSF30 converts the positive after Agrobacterium GV3101 competent cell
Colony identification result；Wherein M swimming lane is DL2000Marker, and 1-4 swimming lane is positive bacterium colony, and No. 5 swimming lanes are negative control；
Fig. 6 is TaCPSF30 expression quantity testing result in the heterologous complementary plant of transgenosis；
Growing state of Fig. 7 transgenosis heterologous complementary plant shoots phase on 1/2MS solid medium, wherein A is quasi- south
Mustard wild type；B is arabidopsis cpsf30 mutant；C is that TaCPSF30 turns the heterologous complement strain 1 of arabidopsis cpsf30 mutant；D
Turn the heterologous complement strain 2 of arabidopsis cpsf30 mutant for TaCPSF30；
Fig. 8 TaCPSF30 turns nitrate nitrogen assimilation genes and transporter gene in the heterologous complementary plant of arabidopsis cpsf30 mutant
Expression quantity testing result.
Specific embodiment
The present invention is described in detail combined with specific embodiments below, but should not be construed as limitation of the invention.It is following
The test method of actual conditions is not specified in embodiment, operates usually according to normal condition, due to not being related to inventive point, thus it is not right
Its step is described in detail.
A kind of clone of common wheat gene TaCPSF30 coded sequence provided by the invention and its application, including following reality
Apply example.
Material therefor and reagent are as follows in following embodiments:
Material: the cloning vector of arabidopsis Col-0 wild type, arabidopsis CPSF30 mutant cpsf30, Gateway system
PENTR3C and plant expression vector pMDC83 (invitrogen), bacillus coli DH 5 alpha (invitrogen), Agrobacterium GV3101
(invitrogen)。
Reagent: Phusion high fidelity enzyme kit, Taq DNA polymerase kit (CWBIO), DNA-Marker
(CWBIO), KpnI restriction enzyme XhoI restriction enzyme, DNA purification and recovery kit (Tiangeng), small amount plasmid preparation
Kit (CWBIO), T4DNA ligase (invitrogen), LR clone enzyme (invitrogen), RNA purification kit
(CWBIO), First Strand cDNA Synthesis Kit (Thermo Fisher), agarose (Promega), remaining examination
Agent such as Trizol, chloroform, isopropanol, dehydrated alcohol, sodium hypochlorite, MES, sucrose, agar etc. are that domestic analysis is pure.
Embodiment 1
A kind of clone of wheat cdna TaCPSF30 coded sequence, the common wheat gene TaCPSF30 coded sequence
Clone obtains in accordance with the following methods:
1, the extraction (RNA isolation kit) of arabidopsis total serum IgE
The total serum IgE of corresponding Arabidopsis plant is extracted using RNA isolation kit (pillar total RNA extraction reagent box), specific method is such as
Under:
(1) about 100mg Chinese spring blade is weighed in 1.5mL Eppendorf pipe, is fully ground in liquid nitrogen；
(2) 1mL Buffer RLT (10% of material volume less than or equal to Buffer RLT volume) is added, fullys shake
It mixes, is stored at room temperature 5min；
It (3) is sufficiently removal cell wall residue, albumen, fat, polysaccharide etc., 4 DEG C, 12000rpm is centrifuged 10min, by supernatant
It is transferred in new centrifuge tube；
(4) split-phase:
1. plus 0.2mL chloroform, acutely shake 15s, be stored at room temperature 2min；
2. being centrifuged (4 DEG C, 12000rpm, not more than 12000rpm) 10min；
(5) it precipitates, and Polysaccharide removing:
1. taking colourless aqueous phase (about originally the 50% of Buffer RLT volume, about 500 μ L) to a new Eppendorf pipe
In, it is sure not to be drawn onto middle layer；
2. plus 70% ethyl alcohol of 0.25mL, be mixed by inversion；
(6) upper step acquired solution is added in the collecting pipe for having been charged into adsorption column (if liquid volume is more than 700 μ L,
Shift in two times), 4 DEG C, 12000rpm is centrifuged 20s, outwells the waste liquid in collecting pipe, adsorption column is placed back in collecting pipe；
(7) 700 μ L Buffer RW1 are added into adsorption column, 4 DEG C, 12000rpm is centrifuged 20s, outwells in collecting pipe
Waste liquid places back in adsorption column in collecting pipe；
(8) 500 μ L Buffer RW2 are added into adsorption column, 4 DEG C, 12000rpm is centrifuged 20s, outwells in collecting pipe
Waste liquid places back in adsorption column in collecting pipe；
(9) step (8) are repeated
(10) 4 DEG C, 12000rpm sky is from 2min；
(11) empty adsorption column is placed in several minutes of room temperature, thoroughly to dry；
(12) by adsorption column be placed in one it is new without in RNase centrifuge tube, 30 μ L RNase-free water, room is added
Temperature places 1min, and 4 DEG C, 12000rpm is centrifuged 1min, collects RNA solution, is placed in -70 DEG C of preservations.
2, reverse transcription
1 μ g total serum IgE is taken, adding 0.5 μ L of Oligd (T), supplement RNase-free water to total volume is 6 μ L.
5min is placed in 65 DEG C of water-baths, configures following inverse transcription reaction liquid in pipe after being slightly centrifuged.
After 42 DEG C of heat preservation 1h, 70 DEG C of processing 15min, cooled on ice.
3, PCR amplification is carried out using Phusion high-fidelity DNA polymerase
(1) 0.2mL PCR dedicated pipe is taken, following component is sequentially added:
Forward primer (F): 5 '-CGGGGTACCATGGACGACGGCGACGGC-3 '；
Reverse primer (R): 5 '-CCGCTCGAG TCGCTTCCTTGACCGCCT-3 '；
(2) PCR pipe is closed, is put into PCR instrument.Reaction condition are as follows: 98 DEG C of initial denaturations 2min, 98 DEG C of denaturation 20s, according to drawing
Annealing temperature annealing 20s, the 72 DEG C of extension 30s/Kb of object, extend 10min after recycling 30 times, 72 DEG C.
(3) after completion of the reaction, the electrophoresis in 1% Ago-Gel checks expanding effect.PCR product Ago-Gel electricity
Testing result of swimming at 1953bp as shown in Figure 1, be observed that clearly purpose band.
The present invention also provides the research plants that is cloned in of the wheat cdna TaCPSF30 coded sequence described in one kind to improve
Application in nitrogen utilization efficiency mechanism and genetic improvement.Recombinant plant expression vector is constructed first with Gateway technology, recombination is planted
The clone for being connected with wheat cdna TaCPSF30 coded sequence for thering is 35S promoter to drive on object expression vector and GFP gene (figure
2), then the recombinant expression carrier is transformed into arabidopsis cpsf30 mutant, obtains heterologous complementary transgenic plant, benefit
With the application of transgenic plant research wheat cdna TaCPSF30 improved in nitrogen utilization efficiency mechanism and genetic improvement.Specifically
Steps are as follows:
The building of cloning vector pENTR 3C-TaCPSF30
1.PCR amplification TaCPSF30 gene C DS is simultaneously connect with pENTR 3C
Forward primer (F): 5 '-CGGGGTACCATGGACGACGGCGACGGC-3 '；
Reverse primer (R): 5 '-CCGCTCGAG TCGCTTCCTTGACCGCCT-3 '；
Using the cDNA of extraction as template, PCR amplification is carried out by specific primer F and R, and in target gene upstream under
Trip introduces Kpn I and Xho I restriction enzyme site respectively.With Tiangeng (Beijing) plastic recovery kit recycle PCR product, PCR product with
Gateway compatible pENTR 3C carries out digestion connection, and operating procedure according to the form below carries out.
| Ingredient | Dosage |
| Target fragment recovery product | 0.5-10ng |
| pENT3C | 0.5μL |
| Salt solution | 0.5μL |
| H2O | Up to 3μL |
After mixing, 30 minutes are reacted under room temperature (25 DEG C), then ice bath 1 minute, for converting bacillus coli DH 5 alpha, surface
LB culture plate containing kanamycin is applied, 37 DEG C are cultivated 12 hours.Then, what the single white colonies coating of picking was new contains card
The LB culture plate of that mycin, 37 DEG C are cultivated 12 hours, carry out colony PCR amplification, as shown in Figure 3.Meanwhile it is correct by bacterium is sieved
Bacterial strain send Shanghai Sangon Biotech Company to carry out DNA sequencing.DNAMAN software is by the gene coding region TaCPSF30 of sequencing sequence and prediction
Sequence is compared, and illustrates that cloning vector pENTR 3C-TaCPSF30 is constructed successfully after comparing correctly, it is standby to extract positive plasmid
With.The extraction of plasmid uses Beijing Kang Wei company kit, and by specification step carries out.
(1) bacterium solution for taking 1-5mL to be incubated overnight is added in centrifuge tube, and 12000rpm is centrifuged 1min, inhales abandon supernatant as far as possible.
(2) 250 μ LBuffer P1 are added in the centrifuge tube of Xiang Liuyou bacterial sediment, use pipettor or turbula shaker
Thorough suspended bacterial precipitating.Note: if fungus block does not mix thoroughly, it will influence lytic effect, keep extracted amount and purity relatively low.
(3) 250 μ L Buffer P2 are added into centrifuge tube, leniently turns upside down and mixes 4-6 times, split thallus sufficiently
Solution, bacterium solution should become limpid sticky at this time.5min is not to be exceeded in time used, in case plasmid is destroyed.
(4) 350 μ L Buffer N3 are added into centrifuge tube, leniently turns upside down mix 4-6 times immediately, will go out at this time
Existing white flock precipitate, 12000rpm are centrifuged 10min, form precipitating in centrifugation bottom of the tube at this time.
(5) column equilibration: 200 μ L Buffer PS being added into the collecting pipe for having been charged into adsorption column (Spin Column),
12000rpm is centrifuged 2min, outwells the waste liquid in collecting pipe, adsorption column is placed back in collecting pipe.
(6) gained supernatant in step 4 is added in the collecting pipe for having been charged into adsorption column, is careful not to that precipitating is sucked out,
12000rpm is centrifuged 1min, outwells the waste liquid in collecting pipe, adsorption column is put back in collecting pipe.
(7) 600 μ L Buffer PW, 12000rpm centrifugation 1min are added into collecting pipe, outwell the waste liquid in collecting pipe.
(8) step 7 is repeated.
(9) adsorption column is placed back in collecting pipe, 12000rpm is centrifuged 1min, outwells the waste liquid in collecting pipe.It will inhale
Attached column is placed in several minutes of room temperature, thoroughly to dry.
(10) adsorption column is placed in a new centrifuge tube, 50-100 μ L is vacantly added dropwise to the intermediate position of adsorbed film
Buffer EB is placed at room temperature for several minutes, and 12000rpm is centrifuged 1min, and plasmid solution is collected into centrifuge tube.- 20 DEG C of preservations
Plasmid.
2, the building of plant expression vector pMDC83-TaCPSF30
The plant of correct cloning vector pENTR 3C-TaCPSF30 and 35S promoter and Gateway system compatible will be sequenced
Object expression vector pMDC83 carries out LR and is reacted, and obtains the TaCPSF30 Gene Fusion GFP reporter gene of 35S promoter driving
Plant expression vector.LR reaction system is as follows: cloning vector 1 μ L (about 100ng) carrier, 1 μ L of pMDC83 carrier is (about
100ng), 0.5 μ L of LR clone enzyme.Above-mentioned substance mixing is placed on (25 DEG C) of room temperature to react 1-2 hours, converts Escherichia coli
DH5α.The bacterial strain surface of conversion applies LB culture plate containing kanamycin, and 37 DEG C are cultivated 12 hours.Then, picking is individually white
The new LB culture plate containing kanamycin of color clone's coating, 37 DEG C are cultivated 12 hours, colony PCR amplification are carried out, such as Fig. 4 institute
Show.Illustrate that expression vector pMDC 83-TaCPSF30 is constructed successfully, extraction positive plasmid is spare, and extracting method is as described above.
The building of the heterologous complement strain of wheat cdna TaCPSF30 complement Arabidopsis cpsf30 mutant
1, the preparation and conversion of Agrobacterium tumefaciems GV3101 competent cell
The preparation method of Agrobacterium competent cell is as follows:
(1) picking single colonie GV3101 is inoculated in 5mL YEP fluid nutrient medium, and 28 DEG C, 200rpm is incubated overnight.
(2) take 2mL culture into 50mL liquid YEP, continuing to cultivate to OD600 is 0.5 or so.
(3) by culture ice bath 30min, 4 DEG C, 5000rpm, it is centrifuged 5min.
(4) it discards supernatant, 10mL 0.1mol/L cold NaCl suspension thalline；4 DEG C, 5000rpm, it is centrifuged 5min.
(5) supernatant is abandoned, precipitating is suspended with 1mL 20mmol/L cold CaCl2, is distributed into 200 μ L/ pipe, is freezed in liquid nitrogen
- 70 DEG C of preservations afterwards.
Expression vector converts Agrobacterium and uses freeze-thaw method, and conversion process is as follows:
(1) it from -80 DEG C of taking-up GV3101 competent cells (250 μ L), thaws on ice, 1-2 μ L Plasmid DNA is added immediately.
(2) 5min is stood on ice.
(3) 5min is freezed in liquid nitrogen.
(4) 37 DEG C of water-bath heat shock 5min.
(5) YEP the fluid nutrient medium 1mL, 28 DEG C of shaking table culture 2-4h of antibiotic-free is added.
(6) 4000rpm room temperature is centrifuged 5min, thallus is collected, with 80-100 μ L YEP solution again suspension thalline.
(7) bacterium solution is spread evenly across YEP solid selection medium, 28 DEG C stationary culture 2-3 days.
(8) after growing bacterium colony, picking single colonie is lined again on antibiotic YEP solid plate, and bacterium is carried out after 36h
PCR identification is fallen, as a result as shown in Figure 5.
2, the conversion of arabidopsis
The genetic transformation of arabidopsis uses inflorescence dip method, the method is as follows:
(1) cutting the main inflorescence of arabidopsis, to induce the generation of side inflorescence, and side inflorescence is bloomed simultaneously, convenient for turning
Change.Material is irrigated into nutrient solution before conversion, watering or nutrient solution are controlled after conversion, in order to which seed is mature in time.
(2) Agrobacterium required for conversion is got out on the day before mentioning, the bacterium solution for taking 2-5mL to be incubated overnight is added to 250mL
Culture medium in shaking table culture, can be used for arabidopsis thaliana transformation in order to second day.
(3) the Agrobacterium thallus being incubated overnight is collected, is resuspended in 50-100mL dip dyeing liquid for shell.
(4) by arabidopsis elongation kind pod and opened spend whole cut after, by titbit immersion dip dyeing liquid for shell in, up and down
Titbit is shaken, in favor of the entrance of dip dyeing liquid for shell.Immerged time generally continues 1h or so.Arabidopsis after dip dyeing is laid flat into big basin
In, film is covered with moisturizing, while outer cover black thin film is protected from light.Arabidopsis taking-up is placed under light after dark processing, to maturation
Sowing afterwards.
It is as follows that arabidopsis disseminates formula of liquid:
With 1M KOH tune pH value to 5.7.
3, the screening of transgenic line
The arabidopsis T0 after infecting is collected for seed, carry out disinfection processing, the method is as follows:
(1) it takes appropriate seed in 1.5mL Eppendorf pipe, indicates relevant information；
(2) 70% ethyl alcohol of configured in advance takes 1mL Yu Guanzhong with pipettor, and suction plays mixing, connects seed fully
Ethanol solution is touched, stewing process 5min (pays attention to rinsing the seed that tube wall and pipe cover to guarantee that sterilizing is thorough), it
(being careful not to for seed being sucked out) is sucked out in the ethyl alcohol in centrifuge tube with 1mL pipettor afterwards；
(3) it spits warm water washing one time；
(4) 2.6% liquor natrii hypochloritis Yu Guanzhong of 1mL, mixing of turning upside down, stewing process 10min are taken with pipettor；
(5) it spits warm water washing five times；
(6) 4 DEG C are placed in save for use.
The screening of transgenic plant
It is dedicated in plant by the T0 after disinfection for seed program request on the MS solid medium containing 50 μ g/mL hygromycin
7d is grown in culturing room, condition of culture is 22 DEG C, the long-day (16h illumination/8h is dark).Selecting in T1 generation can be in hygromycin
The plant for sprouting and growing on MS plate, is transferred on vermiculite respectively and cultivates, condition of culture is same as above.
Collect T1 for seed, sterilize according to the above method, program request on the MS solid medium containing 50 μ g/mL hygromycin,
7d is grown in the dedicated culturing room of plant, condition of culture is 22 DEG C, the long-day (16h illumination/8h is dark).Energy is selected in T2 generation
Enough plant for sprouting and growing on hygromycin MS plate, are transferred on vermiculite respectively and cultivate, condition of culture is same as above.
Collect T2 for seed, sterilize according to the above method, program request on the MS solid medium containing 50 μ g/mL hygromycin,
7d is grown in the dedicated culturing room of plant, condition of culture is 22 DEG C, the long-day (16h illumination/8h is dark).Energy is selected in T3 generation
It is enough that isolated plant is all sprouted and grow and do not occurred on hygromycin MS plate, obtain heterologous complement strain TaCPSF30/
Cpsf30-1 and TaCPSF30/cpsf30-2.
In order to verify the feasibility of the method for the present invention, we are prepared for homozygous wheat cdna referring to the method for embodiment 1
The heterologous complement strain of TaCPSF30 complement Arabidopsis cpsf30 mutant, and have studied being cloned in for wheat cdna TaCPSF30
Crop improves nitrogen utilization efficiency research and genetic improvement application, as follows:
1, the expression quantity of TaCPSF30 measures in heterologous complement strain
(1) in 1/2MS culture medium heterologous complement strain culture
In order to detect the expression quantity of TaCPSF30 in heterologous complement strain, first by heterologous complement strain TaCPSF30/
The seed disinfection of cpsf30-1 and TaCPSF30/cpsf30-2 is then transferred on 1/2 fluid nutrient medium of solid in plant illumination
It is cultivated in incubator, cultivation temperature is 22 DEG C, long-day (16h illumination/8h is dark) culture 7d.
(2) the expression quantity measurement of TaCPSF30
Using real-time fluorescence quantitative PCR (qRT-PCR) detect heterologous complement strain TaCPSF30/cpsf30-1 and
The expression quantity of TaCPSF30 in TaCPSF30/cpsf30-2.
By what is obtained after the RNA extracted in TaCPSF30/cpsf30-1 and TaCPSF30/cpsf30-2 progress reverse transcription
CDNA sample is diluted to 0.5-2ng/ μ L, and reaction system is as shown in the table:
QRT-PCR forward primer: 5 '-GACTACGGCGAGTGCCGCGAG-3 '；
QRT-PCR reverse primer: 5 '-AAGCCTGAACCAGGTGG-3 '
PCR pipe is closed, (ABI7500FAST) is put into fluorescence quantitative PCR instrument.Reaction condition are as follows: first 50 DEG C of initial denaturations
20s, then 95 DEG C of initial denaturation 10s, then 95 DEG C of denaturation 15s, 60 DEG C of annealing 1min carry out plate read, since step 3 later
Circulation 40 times, solubility curve are 95 DEG C of 15s, 60 DEG C of 1min, 95 DEG C of 30s, 60 DEG C of 15s.
Reaction guarantees the accuracy of result using tublin2 as reference gene, and each processing has biology repetition three times,
Three systems repeat.The expression of TaCPSF30 in heterologous complement strain TaCPSF30/cpsf30-1 and TaCPSF30/cpsf30-2
Amount is as shown in Figure 6, the results showed that wheat cdna TaCPSF30 can in heterologous complement strain high efficient expression.
2, upgrowth situation of the heterologous complement strain under Seedling Stage 1/2MS condition of culture
Nitrogen is the most important nutrient for influencing plant growth and development, and nitrogen metabolism will lead to the blade middle period extremely
Green element decline and the long shortening of main root.We observe upgrowth situation of the heterologous complement strain under Seedling Stage 1/2MS condition of culture,
As a result as shown in Figure 7.After 1/2MS solid culture grows 7d, arabidopsis wild type growth is all right, leaf green；Main root
Growth is normal, and length is about 4cm (Fig. 7 A).However, arabidopsis cpsf30 mutant is poor in growing state, the obvious chlorosis of blade；
Main root length shortens, length about 3cm (Fig. 7 B).Heterologous complement strain TaCPSF30/cpsf30-1's and TaCPSF30/cpsf30-2
Growing state is similar with wild type, leaf green；Main root growth is normal, and length is about 4cm (Fig. 7 C and D).The above results show
Wheat cdna TaCPSF30 can restore arabidopsis cpsf30 mutant to wild type phenotype under 1/2MS condition of culture, can mention
The nitrogen utilization efficiency of high plant is to maintain higher chlorophyll content and main root to grow.
3, TaCPSF30 can regulate and control arabidopsis NO3 -The expression of responsive genes
Plant utilizes and metabolism nitrogen NO3 -, the first step is wanted in sensing external environment with the presence or absence of NO3 -.More and more
Studies have shown that being not only a kind of nutriment or a kind of signaling molecule.Root system of plant perceives the external world, and there are NO3 -When, it can cause
Internal numerous gene expressions change.In these genes, NO3 -Assimilation genes NIA and NiR, NRT2.1 couples of transporter gene
NO3 -Response it is most sensitive.Therefore, the expression for commonly using these three genes measures the reaction feelings of Nitrate in Plants signal
Condition.We have detected the influence (Fig. 8) that TaCPSF30 expresses arabidopsis NIA, NiR and NRT2.1 in heterologous complement strain.Knot
Fruit shows the expression quantity decline of NIA, NiR and NRT2.1 in arabidopsis after AtCPSF30 mutation, illustrates that AtCPSF30 affects plant
Object is to extraneous NO3 -Perception is to affect nitrogen utilization efficiency.TaCPSF30 can restore in cpsf30 mutant NIA, NiR and
The expression of NRT2.1 illustrates that TaCPSF30 can regulate and control these three important NO of NIA, NiR and NRT2.1 to wild-type levels3 -It rings
The expression of gene is answered, the gene its vital effect in terms of improving nitrogen utilization efficiency.
Claims (7)
1. a kind of clone of wheat cdna TaCPSF30 coded sequence, which is characterized in that the wheat cdna TaCPSF30 coding
The clone of sequence obtains in accordance with the following methods:
(1) wheat total serum IgE is extracted, reverse transcription synthesizes cDNA；
(2) using cDNA as template, special primer is designed, PCR amplification gene TaCPSF30 coding region sequence is passed through, wherein PCR expands
It is as follows to increase the primer sequence:
TaCPSF30 forward primer (F):
5'-CGGGGTACCATGGACGACGGCGACGGC-3'；
TaCPSF30 reverse primer (R):
5'-CCGCTCGAG TCGCTTCCTTGACCGCCT-3'；
(3) it recycles and purifies target fragment in the amplified production of (2) and be sequenced, sequencing is correctly wheat cdna
The clone of TaCPSF30 coded sequence.
2. the application of the clone of wheat cdna TaCPSF30 coded sequence according to claim 1, which is characterized in that wheat
The application of gene TaCPSF30 coded sequence being cloned in research plant raising nitrogen utilization efficiency mechanism and genetic improvement.
3. the application of the clone of wheat cdna TaCPSF30 coded sequence according to claim 2, which is characterized in that first structure
The recombinant plant expression vector of Gateway system compatible is built out, the TaCPSF30 for having 35S promoter to drive on the expression vector is compiled
The clone of code sequence and GFP gene, are then transformed into arabidopsis cpsf30 mutant for the recombinant expression carrier, screening obtains
The homozygous heterologous complement strain of transgenosis, study in complement strain wheat cdna TaCPSF30 improve nitrogen utilization efficiency mechanism and
Application in genetic improvement.
4. the application of the clone of wheat cdna TaCPSF30 coded sequence according to claim 3, which is characterized in that specific
Steps are as follows:
The building of S1, pENTR3C-TaCPSF30 recombinant plasmid:
Digestion connects clone and the cloning vector pENTR3C of the wheat cdna TaCPSF30 coded sequence: to the wheat of purifying
Being cloned in for gene TaCPSF30 coded sequence adds such as KpnI and XhoI restriction enzyme site at 5 ' ends and 3 ' ends respectively, after double digestion
To the target gene fragment for having cohesive terminus,cohesive termini；Then digestion connection is carried out with cloning vector pENTR3C, obtains recombinant plasmid
pENTR3C-TaCPSF30；
S2 constructs recombinant plant expression vector pMDC83-TaCPSF30 using Gateway technology:
The plant expression vector pMDC83 of correct recombinant plasmid pENTR3C-TaCPSF30 Yu Gateway system compatible will be sequenced
LR reaction is carried out, recombinant plant expression vector pMDC83-TaCPSF30 is obtained；
S3, Agrobacterium infect arabidopsis:
Plant arabidopsis cpsf30 mutant；Prepare Agrobacterium competent cell；Then recombinant expression carrier pMDC83- is used
TaCPSF30 converts Agrobacterium competent cell；The conversion Agrobacterium of picking positive colony infects arabidopsis cpsf30 mutant；
Obtain effectively cloning the transgenic plants of wheat cdna TaCPSF30 coded sequence.
5. the application of the clone of wheat cdna TaCPSF30 coded sequence according to claim 4, which is characterized in that described
Agrobacterium is Agrobacterium GV3101.
6. the application of the clone of wheat cdna TaCPSF30 coded sequence according to claim 4, which is characterized in that described
Plant is arabidopsis cpsf30 mutant or wheat breed China spring.
7. the application of the clone of wheat cdna TaCPSF30 coded sequence according to claim 4, which is characterized in that in S3
After the conversion Agrobacterium of picking positive colony infects arabidopsis cpsf30 mutant, it is selfed, is filtered out containing wheat-based by 3 generations
Because of the heterologous complement strain of TaCPSF30 coded sequence, mentioned for studying wheat cdna TaCPSF30 coded sequence in research plant
Application in high nitrogen utilization efficiency mechanism and genetic improvement.
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