CN108728454A - A kind of potato StDWF1 genes and preparation method thereof and the gene is overexpressed to promote the method for potato Rapid Rooting - Google Patents

Abstract

The invention provides a potato StDWF1 gene, a preparation method thereof, and a method for overexpressing the gene to promote rapid potato rooting, which relate to the field of potato molecular biology and are realized through the steps of StDWF1 gene cloning, overexpression vector construction, and Agrobacterium dipping and transfer. The purpose of promoting rapid rooting of potatoes by overexpressing the potato StDWF1 gene is to root at least 10 days in advance, which is conducive to enhancing the growth and vigor of potato plants.

Description

A kind of potato StDWF1 gene and its preparation method and overexpressing the gene to promote The method of fast rooting in potatoes

technical field

The invention relates to the field of potato molecular biology, in particular to a potato StDWF1 gene, a preparation method thereof and a method for overexpressing the gene to promote rapid potato rooting.

Background technique

Potato is the fourth staple food in my country. It has the characteristics of high yield, rich nutrition, edible processing and so on, which is of great significance to food security. However, the losses caused by improper storage of potatoes every year are huge, which has a huge impact on the potato industry.

Potatoes are edible for men, women and children, and the market demand is large. Therefore, it is necessary to increase the yield of potatoes. However, in the process of potato growth, only when the roots grow to a certain length can fruit be produced. At present, the growth cycle of potatoes is 80 -120 days, the growth cycle cannot meet the market demand. To shorten the potato growth cycle, increase the rooting speed, and reduce the rooting time are effective means.

Contents of the invention

The invention provides a potato StDWF1 gene, which changes the dormancy time and rooting time of the potato through the gene, so as to balance the contradiction between potato storage and growth.

In order to solve the problems of the technologies described above, the technical scheme adopted in the present invention is as follows:

A potato StDWF1 gene, the base sequence of which is shown in SEQ ID NO:1.

A method for preparing a potato StDWF1 gene, comprising the steps of:

(1) extracting total RNA from the test-tube plantlet of potato variety Feureta;

(2) cDNA was synthesized by reverse transcription from the extracted total RNA;

(3) PCR amplification, using cDNA as a template, introducing XbaI and SmaI restriction sites before the CDS region of cDNA, designing amplification primers StDWF1PF and StDWF1PR, and adding primers StDWF1PF, primers StDWF1PR, 10× amplification buffer, Sterile water, dNTPs, and Taq DNA polymerase were mixed and then PCR amplified, wherein the volumes of cDNA, primer StDWF1PF, primer StDWF1PR, 10× amplification buffer, sterile water, dNTPs, and Taq DNA polymerase were: 1 μL, 1 μL, 1 μL, 2 μL, 15.3 μL, 0.5 μL, 0.2 μL, and the concentrations of cDNA, primer StDWF1PF, primer StDWF1PR and dNTPs are 10-100 ng/L, 2 μM/μl, 2 μM/μl, 10 mmol/L, respectively;

The base sequences of the amplification primers StDWF1 PF and StDWF1 PR are respectively shown in SEQ ID NO: 2 and SEQ ID NO: 3;

(4) Recover and purify the PCR amplification product, and obtain the potato StDWF1 gene through sequencing analysis.

Preferably, in step (1), the time to extract the total RNA of the Fevorita test-tube plantlet is the leaf emergence stage, and the extraction site is the young leaves of the Feureta test-tube plantlet.

As preferably, the extraction method of total RNA in step (1) adopts Trizol method, and concrete steps are as follows:

(1a) Grinding: Put 4 scoops of liquid nitrogen and the sample part of the potato variety Favorita into the mortar. After the liquid nitrogen evaporates, quickly grind it into a fine powder, quickly put it into the EP tube, add 1mL Trizol, mix vigorously, and put it at room temperature. 5-10min;

(1b) Centrifuge at 12000rpm at 4°C for 5min, pipette the supernatant into a new tube, add 200ml of chloroform, shake slightly up and down, and let stand at room temperature for 5min;

(1c) Centrifuge at 12000rpm at 4°C for 10min, pipette the supernatant into a new tube, add an equal volume of phenol/chloroform/isoamyl alcohol, and invert to mix;

(1d) Centrifuge at 12000rpm at 4°C for 20min, draw the supernatant into a new tube, gradually add absolute ethanol to make the final concentration 12%, mix upside down and then centrifuge quickly;

(1e) Centrifuge at 12000rpm at 4°C for 10min, pipette 400mL supernatant into a new tube, add 0.7 times the volume of isopropanol to mix up and down, and add 0.2 times the volume of 1mol/L NaAc-20°C to precipitate for 1h;

(1f) Centrifuge at 12000rpm at 4°C for 15min, discard the supernatant, add 1mL 75% ethanol to wash the precipitate, centrifuge at 8500rpm at 4°C for 3min, then wash twice with 1mL 75% ethanol;

(1g) Pour off the supernatant, absorb excess liquid as much as possible, blow air on the ultra-clean bench for 10 minutes to remove residual ethanol, add 80mL DEPC treated water, immediately put in 55°C water bath for 5min, -80°C for 60min, repeat twice;

(1h) Centrifuge at 12000rpm at 4°C for 20min, carefully pipette 70μL of the supernatant, pay attention not to touch the bottom, and obtain total RNA, and store it at -80°C for later use;

Wherein, steps (1c)-(1h) are all operated on ice.

Preferably, in step (2), the total RNA is reverse-transcribed to synthesize cDNA, and reverse-transcribed according to the instructions of the cDNA synthesis kit of TakaRa Company. The total reaction system volume is 20 μl, and an RNase free centrifuge tube is used. The reaction system is as follows:

Denature at 65°C for 5 minutes, rapidly cool on ice for at least 1 minute, centrifuge briefly, then add:

Mix well, react at 55°C for 60 minutes, inactivate the enzyme at 70°C for 15 minutes, and store at -20°C.

As preferably, the reaction condition of PCR amplification in step (3) is:

(3a) Perform a pre-denaturation reaction at 95°C for 3 minutes;

(3b) Perform denaturation reaction at 95°C for 30s;

(3c) Annealing at 54-58°C for 30s;

(3d) Extending at 72°C for 1-1.5min;

(3e) loop steps (3b)～(3d) thirty-five times;

(3f) Extending at 72°C for 10 minutes;

(3g) keep warm at 4°C until processing.

As preferably, the method for recovering the purified PCR amplification product in step (4) specifically includes the following steps:

(4a) Column equilibration step: add 600 μl equilibration solution to the adsorption column, centrifuge at 10,000 rpm for 1 min, discard the equilibration waste solution in the collection tube and put it back into the collection tube;

(4b) Put the PCR amplification product in 0.01g/ml agarose gel, transfer the agarose gel into a centrifuge tube, add 100μl potassium acetate solution to the centrifuge tube, bathe in 50°C water for 10min, and put it up and down Invert the centrifuge tube to fully dissolve the gel;

(4c) Introduce the dissolved gel into the adsorption column, centrifuge at 12000rpm for 1min, discard the waste liquid from the collection tube and put the adsorption column into the collection tube again;

(4d) Add 600 μl of solution DNA wash buffer to the adsorption column, centrifuge at 12000 rpm for 1 min, discard the waste liquid from the collection tube and put the adsorption column into the collection tube again;

(4e) Put the adsorption column into the collection tube, centrifuge at 12000rpm for 2min, and let stand for 10min;

(4f) Put the adsorption column into a clean centrifuge tube, add an appropriate amount of elution buffer dropwise to the middle of the adsorption membrane, place at room temperature for 2 minutes, and centrifuge at 12,000 rpm for 2 minutes to collect the DNA solution;

(4g) Pour the solution obtained by centrifugation into the adsorption column again, put it at room temperature for 1 min, and centrifuge at 12000 rpm for 2 min to increase the recovery rate, and obtain the potato StDWF1 gene product, and the obtained potato StDWF1 gene product is stored at -20°C for later use.

A method for overexpressing potato StDWF1 gene to promote quick rooting of potato, comprising the steps:

(1) Construction of overexpression vector

a. Ligation of the cloning vector: connect the potato StDWF1 gene with the pMD19-T cloning vector, first heat the potato StDWF1 gene at 65°C for 5 minutes, and then add the following reaction system:

After mixing and centrifuging, connect at 4°C for 18h, and then store at -20°C;

Wherein, solution I is an equal-volume mixed solution of 50mM glucose, 25mM Tris-HCL, and 10mM EDTA, with a pH value of 8.0;

b. Recombinant plasmid DNA transformation: Mix 5 μl cloning vector ligation product with 100 μl Escherichia coli DH5α competent cells on the ultra-clean workbench, ice-bath for 30 minutes; heat shock in 42°C water bath for 90 seconds, then ice-bath for 5 minutes; add 800 μl liquid medium , 37°C, 220rpm shaking culture for 40min; on the LB solid medium that has been added with Amp antibiotics, the content of Amp antibiotics is 100μg/ml, evenly smear 40μl of X-gal with a concentration of 20mg/ml and 4μl of X-gal with a concentration of 200mg/ml IPTG mixture; after the culture of the bacteria resumes, centrifuge the bacteria solution at room temperature at 3000 rpm for 2 minutes, take out 800 μl of the supernatant with a pipette, and resuspend the remaining 100 μl; spread the bacteria solution evenly on the plate on the ultra-clean workbench , put it for 30min-60min and wait for complete absorption, then culture it upside down at 37°C for 16-18h, and pick a single colony for identification after growth; add the single colony to be identified to 20μl ddH2O, mix well, and use it as a PCR identification reaction system for bacteria liquid The template and PCR products were subjected to agarose gel electrophoresis; the colonies that were positive by gel electrophoresis were picked into the liquid LB solid medium containing Amp antibiotics, cultured with shaking at 37°C for 8 hours, and the plasmid was extracted. After the plasmid was detected by PCR, the detection The obtained positive clones were identified by plasmid PCR, and the positive plasmids identified by PCR were sequenced and analyzed to obtain the pMD19-T vector containing the target fragment;

c. Use restriction endonucleases BamH1 and SmaI to double digest the pMD19-T vector containing the target fragment and the overexpression vector pBI121 respectively, recover the pBI121 vector and the target fragment obtained by digestion and connect them with T4-DNA ligase, digest The reaction was carried out at 37°C, and the ligation reaction was carried out at 4°C overnight; the ligation product pBI121-StDWF1 was transformed into Escherichia coli competent cells, a single colony was picked and multiplied, the plasmid was extracted, and transformed into root cancer after identification by PCR, enzyme digestion and sequencing Competent cells of Agrobacterium;

(2) Transform the plasmid containing the gene of interest into Agrobacterium

a. Pick a single colony of Agrobacterium tumefaciens GV3101, inoculate it in 5 ml of YEB liquid medium containing 50 μg/ml rifampicin and 15 μg/ml gentamicin, and cultivate overnight at 28° C. at 200 rpm;

b. Take 2ml of the overnight culture and add it to 50ml of YEB liquid medium containing the same antibiotic, culture it with shaking at 28°C until the OD600 is 0.5, and bathe the bacteria in ice for 30 minutes;

c. Centrifuge at 2500g for 5 minutes at 4°C to collect bacteria;

d. Add 10ml 0.15M NaCl to suspend Agrobacterium cells, and centrifuge at 2500g for 5 minutes;

e. Add 1ml of pre-cooled 20mmol/L CaCl ₂ suspension cells. This step is operated on ice, and the obtained Agrobacterium competent cells are used within 24 hours;

f. Take 100ml of Agrobacterium competent cells and melt on ice, add 1mg of pMD19-T carrier containing the target fragment, mix well and let stand on ice for 30 minutes, quick freeze in liquid nitrogen for 1min, and bathe in 37°C water for 5min, then Add 1ml of YEB medium without antibiotics, culture with slow shaking at 28°C for 4 hours; centrifuge at 5000rpm for 30 seconds to collect the bacteria, discard the supernatant, add 0.1ml of YEB medium without antibiotics to resuspend the cells, and spread on the medium containing 50μg/ ml rifampicin, 15μg/ml gentamycin and 50mg/ml Kana antibiotics on the YEB plate, cultivated at 28°C for about 48 hours; pick the single colonies grown on the plate, inoculate them with 50mg/ml Kana antibiotics and corresponding In the YEB liquid culture medium of antibiotics of Agrobacterium strains, shake and culture overnight at 28°C; extract part of the plasmid DNA, and use the plasmid DNA as a template for PCR amplification identification. If the identification is positive, the plasmid Agrobacterium containing the target gene will be transferred;

(3) Agrobacterium-infected test-tube seedling potato stem section

a. Pre-cultivate the stems of test-tube plantlets. On the ultra-clean workbench, cut the stems of the expanded test-tube plantlets, and place the cut stems flat on the solid medium for pre-cultivation, and cultivate them in the dark at about 20°C. -3d;

b. Agrobacterium activation, take the transformed plasmid Agrobacterium containing the target gene and inoculate it in a solid YEB medium containing 50mg/L Kan and 50mg/L Rif, culture at 27°C for 48h, seal the dish and store at 4°C; Add 4mL of YEB medium into a 10mL centrifuge tube, pick a single colony, shake the bacteria at 27°C for 16 hours at 220rpm, and store at 4°C for later use; inoculate at a ratio of 1:100 into fresh liquid YEB medium in a 10mL centrifuge tube, 220rpm, Shake the bacteria at 27°C for 12 hours, store at 4°C for later use;

c. Agrobacterium dipping and co-cultivation and screening, put the stem section into a sterile bottle on the ultra-clean workbench, take the shaken bacterial solution, centrifuge at 6000rpm for 5min, remove the supernatant, add MS liquid medium to resuspend; Transfer the cultured stem section from the solid medium to a small beaker; pour the resuspended bacteria into the small beaker, and infect the solution for 2-3 minutes; pour off the bacteria solution, pour sterile water into the stem section, and repeat twice Afterwards, transfer to sterile filter paper to absorb excess water, then transfer the stem segment to a symbiotic medium with two layers of sterile filter paper, and culture in the dark at 24°C for 36 hours; put the stem segment into a sterile solution containing 100mg/L Cef Wash in water for 3 to 4 times, absorb excess water and transfer to screening medium, 16h light/8h dark, light intensity 60μmol/m2.s1, 22±1℃;

d. Callus culture, change the medium every 10-14 days, remove the deadly, polluted and completely albino plants due to the overgrowth of Agrobacterium, and obtain the potato plants overexpressing the potato StDWF1 gene.

As preferably, in step (1) in the process of constructing the overexpression vector, the step of extracting the plasmid is:

I. Draw 3ml of bacterial liquid into a 5ml centrifuge tube, centrifuge at 5000rpm for 30 seconds, and collect the bacteria;

II. Add 200ml of solution I, and vibrate the suspended bacteria with an oscillator, wherein solution I is an equal-volume mixed solution of 50mM glucose, 25mM Tris-HCL, and 10mM EDTA, with a pH value of 8.0;

III. Add 300ml of prepared solution II, and mix it upside down, wherein solution II is an equal-volume mixed solution of 0.2nM NaOH and 1% mass fraction of SDS;

IV. Add 300ml pre-cooled solution III immediately after the solution is clarified, and ice bath for 5 to 10 minutes after mixing, wherein solution III is an equal volume mixed solution of alcohol of 3mM potassium acetate, 2mM acetic acid, and 75% mass fraction (;

V. Centrifuge at 5000 rpm for 15 minutes at 4°C;

VI. Take the supernatant, add an equal volume of chloroform/isoamyl alcohol, mix it upside down, and centrifuge at 5000rpm for 5 minutes to precipitate the protein;

VII. Aspirate the supernatant, add 0.7 times the volume of isopropanol, and mix well; centrifuge at 5000rpm for 10 minutes, discard the supernatant, then wash the precipitate with 70% ethanol, discard the supernatant at 8000rpm for 5 minutes; dry at room temperature for 10 minutes and dissolve in an appropriate amount ddH2O2, that is, the extracted target plasmid, and stored at 4°C for future use.

As preferably, in the process of constructing the overexpression vector in step (1), the plasmid PCR detection is first amplified and then detected, and the steps are as follows:

Add 20 μl of the plasmid to be tested into the reaction system, and add the following components in turn in the ice bath environment:

Mix well, centrifuge slightly to sink to the bottom, and carry out cyclic amplification reaction. The specific conditions of cyclic amplification reaction are as follows:

(3a) Perform a pre-denaturation reaction at 95°C for 3 minutes;

(3b) Perform denaturation reaction at 95°C for 30s;

(3c) Annealing at 54-58°C for 30s;

(3d) Extending at 72°C for 1-1.5min;

(3e) loop steps (3b)～(3d) thirty-five times;

(3f) Extending at 72°C for 10 minutes;

(3g) keep warm at 4°C;

The PCR amplification product was analyzed by 1.0% agarose gel electrophoresis, and the analysis result was obtained.

Compared with the prior art, the beneficial effects of the present invention are:

The present invention discovers the potato StDWF1 gene that affects the dormancy time of potatoes and the later growth and rooting of potatoes, clones the sequence of the coding region of the potato StDWF1 gene, constructs the gene overexpression plant, and lays the foundation for the study of the StDWF1 gene function; through the overexpression of the DWF1 gene plant, the StDWF1 in potato is analyzed The mechanism of tuber germination lays the foundation for the study of the molecular mechanism of potato tuber storage and germination, and rationally regulates tuber storage and germination; a breeding method for new potato varieties is constructed.

Description of drawings

Fig. 1 is the agarose gel electrophoresis detection figure that potato total RNA extracts;

Fig. 2 is the agarose gel electrophoresis detection figure of PCR amplification result;

Fig. 3 is the target gene plasmid amplification agarose gel electrophoresis detection picture;

Fig. 4 is the detection diagram of pBI121-StDWF1 recombinant plasmid Bam H1/Sam double digestion agarose gel electrophoresis;

Fig. 5 is the state figure after the stem section of potato tube seedling is dipped in by Agrobacterium and co-cultivated;

Fig. 6 is the state figure after 2 days of soaking and co-cultivating the stem section of potato tube seedlings through Agrobacterium;

Fig. 7 is the state figure after 45 days of potato test-tube seedling stem section being dipped in by Agrobacterium and co-cultured;

Fig. 8 is the growth figure that the stem section of potato tube seedlings is transferred to the rooting medium and carried out rooting screening after Agrobacterium dipping and co-cultivation;

Fig. 9 is the growth figure of the propagation of potato test-tube plantlet strain;

Figure 10 is a diagram of the PCR detection results of the pBI121-StDWF1 transgenic line nptII;

Fig. 11 is a comparison chart of the root length of wild-type plants and StDWF1 overexpression transgenic plants at 40 days;

Figure 12 is a comparison of leaf shape and size between wild-type plants and StDWF1 overexpression transgenic plants;

Figure 13 is a comparison of the rooting situation of wild-type plants and StDWF1 overexpression transgenic plants at 20-30 days;

Figure 14 is a comparison of plant heights between wild-type plants and StDWF1 overexpression transgenic plants;

Fig. 15 is the agarose gel electrophoresis detection figure that leaf total RNA extracts;

Figure 16 is a graph showing the expression analysis of StDWF1 gene in different tissue parts;

Fig. 17 is a graph showing the expression analysis of StDWF1 gene in leaves at different stages.

Detailed ways

All the features disclosed in this specification, except mutually exclusive features and/or steps, can be combined in any way.

The present invention will be described in detail below in conjunction with the accompanying drawings.

Example 1

The base sequence of the potato StDWF1 gene is shown in SEQ ID NO:1.

StDWF1 gene cloning and the method for overexpressing the gene are as follows:

Test materials: tube seedlings of potato variety Fewruita (FW), provided by the Potato Research and Development Center of Sichuan Agricultural University.

Plasmid: pMDTM19-T Simple Vector was purchased from TaKaRa Company; pBI121 overexpression vector

Strains: Escherichia coli DH5α; Agrobacterium GV3101

Reagents: DEPC (diethyl pyrocarbonate) and Trizol were purchased from Invitrogen Company, and water-saturated phenol, H2O2, chloroform, isoamyl alcohol, isopropanol, ethanol, sodium acetate, etc. were purchased from Chengdu Kelong Chemical Reagent Factory. Reverse transcription kit RevertAid FirstStrand cDNA Synthesis was purchased from Thermo; DNA gel purification recovery kit, 50×TAE buffer was purchased from Tiangen Company; high-fidelity Pfu enzyme, restriction endonuclease, DNA Ligation Kit 2.0 were purchased from TaKaRa company.

Common reagent preparation:

① Reagents for Total RNA Extraction

0.1% DEPC: add 0.5mL DEPC to 500mL ddH2O2, 37°C, 150rpm, after 6h, it can be stored at room temperature for later use.

DEPC water: Dissolve 0.1ml of DEPC in 100ml of water, shake overnight, and sterilize at 120°C for 20min.

75% ethanol (V/V): add 25ml of ethanol to 75ml of DEPC water (ready to use)

② Agarose gel electrophoresis reagents

1×TAE buffer solution: Take 20ml of 50×TAE in a 1L graduated cylinder, and add ddH2O to make up to 1L.

③ Bacterial culture medium

Escherichia coli LB medium (1L): 10g trypsin, 5.0g yeast powder, 10.0g NaCl, add 1L water, adjust the pH to 7.0 with Tris, add 15g agar powder if it is solid, and sterilize at 121°C for 20min.

Agrobacterium YEB medium (1L): 5.0g trypsin, 5.0g sucrose, MgSO4.7H2O, 10g yeast powder, 5.0g beef extract, add 1L water, adjust the pH to 7.0 with Tris, if it is solid, add 15g agar powder, and extinguish at 121°C Bacteria 20min.

Main equipment:

PCR instrument (Bio-Rad), micropipette (Eppendorf), temperature-controlled shaker (Thermo), electric thermostat incubator, electrophoresis apparatus and horizontal electrophoresis tank (Bio-Rad), high-speed refrigerated centrifuge (Thermo), condensate Gel Imaging System (Bio-Rad).

1. StDWF1 gene cloning

⑴.Total RNA extraction

The mortar, EP tubes, pipette tips, ddH2O water, and 1mol/L NaAc used for RNA extraction were all treated with 0.1% DEPC for 24 hours, and DEPC was degraded at 121°C for 20 minutes, and the mortar, EP tubes, and pipette tips were dried at 80°C. DEPC treated water was used to prepare 75% ethanol.

RNA was extracted using the Trizol method, and the specific steps were as follows:

① Grinding: Put 4 scoops of liquid nitrogen and the sampled potatoes into the mortar. After the liquid nitrogen volatilizes, quickly grind it into a fine powder, quickly put it into an EP tube, add 1mL Trizol, mix vigorously, and let it stand at room temperature for 5-10 minutes.

②Centrifuge at 12000rpm at 4°C for 5min, pipette the supernatant into a new tube, add 200ml of chloroform, shake slightly up and down, and let stand at room temperature for 5min.

Operate on ice as follows:

③Centrifuge at 12000rpm at 4°C for 10min, pipette the supernatant into a new tube, add an equal volume of phenol/chloroform/isoamyl alcohol, and invert to mix.

④ Centrifuge at 12000rpm at 4°C for 20min, draw the supernatant into a new tube, gradually add absolute ethanol to make the final concentration 12%, mix upside down and then centrifuge quickly.

⑤ Centrifuge at 12,000 rpm at 4°C for 10 minutes, pipette 450 mL into a new tube, add 0.7 times the volume of isopropanol to mix by inverting up and down, and add 0.2 times the volume of 1mol/L NaAc to precipitate at -20°C for 1 hour.

⑥Centrifuge at 12000rpm at 4°C for 15min, discard the supernatant, add 1mL of 75% ethanol to wash the precipitate, centrifuge at 8500rpm at 4°C for 3min, then wash twice with 1mL of 75% ethanol.

⑦ Pour off the supernatant, absorb excess liquid as much as possible, blow air on the ultra-clean table for 10 minutes to remove residual ethanol, add 80mL of DEPC-treated water, immediately put in 55°C water bath for 5 minutes, -80°C for 60 minutes, repeat twice.

⑧Centrifuge at 12,000 rpm at 4°C for 20 minutes, carefully pipette 70 μL of the supernatant, taking care not to touch the bottom, and freeze at -80°C for later use.

RNA purity and concentration detection

RNA integrity was detected by agarose gel electrophoresis: 2 μL sample + 6 μL 1.5×Loading Buffer, 1% agarose, Gel Red dye, 80V, 40min, 1×TAE buffer.

Take 5 μL sample + 2mL water and dilute 400 times, measure OD230, OD260, OD280, calculate OD260/OD280, OD260/OD230, compare the purity of RNA, RNA concentration (ng/μL) = OD260×400×40.

The test results are shown in Figure 1. Total RNA was extracted from the cultivated potato variety Fevorita, and detected by agarose gel electrophoresis. There were two obvious bands (28S and 18S), indicating that the RNA quality was good.

⑵.RT-PCR

① cDNA synthesis by reverse transcription

Perform reverse transcription according to the instructions of the cDNA synthesis kit from TakaRa Company. The total reaction system is 20 μl, using RNase free centrifuge tubes. The reaction system is as follows:

Denature at 65°C for 5 minutes, rapidly cool on ice for at least 1 minute, centrifuge briefly, then add:

Mix slightly evenly, react at 55°C for 60 minutes, inactivate the enzyme at 70°C for 15 minutes, and store at -20°C.

②PCR amplification

In front of the CDS region of the target gene and introducing XbaI and SmaI restriction sites (to facilitate the connection and connection of the vector), the primers used for amplification are as follows:

The primers for StDWF1 CDS amplification are:

StDWF1PF:5'GGATCC-ATGGCAGATGTTCAGGC 3'

StDWF1 PR: 5'CCCGGG-TCAATCTTCAGGCTCAT 3'

The reaction system is 20 μl, and the following components are added in sequence:

Mix well, centrifuge slightly to sink to the bottom, and carry out the amplification reaction according to the following cycle, reaction conditions:

(a) Perform a pre-denaturation reaction at 95°C for 3 minutes;

(b) Perform denaturation reaction at 95°C for 30s;

(c) Annealing at 54-58°C for 30s;

(d) Extending at 72°C for 1-1.5 min;

(e) Cycle steps (b) to (d) 35 times;

(f) Extending at 72°C for 10 minutes;

(g) insulation at 4°C;

1.0% (w/v) agarose gel electrophoresis analysis PCR amplification results are shown in Figure 2, the results show that a 1704bp fragment was obtained.

⑶. Target fragment recovery and purification

Refer to the method of Tiangen DNA Gel Recovery Kit, as follows:

a. Column equilibration step: add 600 μl equilibration solution to the adsorption column, centrifuge at 10000 rpm for 1 min, discard the equilibration waste solution in the collection tube and put it back into the collection tube.

b. Cut out the agarose gel containing the target gene.

c. Add 100 μl of potassium acetate solution to the centrifuge tube containing the target gene gel, bathe in 50°C water for 10 minutes, and invert the centrifuge tube up and down to fully dissolve the gel.

d. Introduce the dissolved gel into the adsorption column, centrifuge at 12000rpm for 1min, discard the waste liquid in the collection tube and put the adsorption column into the collection tube again.

e. Add 600 μl of solution PW to the adsorption column, centrifuge at 12000 rpm for 1 min, discard the waste liquid from the collection tube and put the adsorption column into the collection tube again.

f. Repeat step 5.

g. Put the adsorption column into the collection tube, centrifuge at 12000rpm for 2min, and let stand for 10min.

h. Put the adsorption column into a clean centrifuge tube, add an appropriate amount of elution buffer dropwise to the middle of the adsorption membrane, and place it at room temperature for 2 minutes. Centrifuge at 12000rpm for 2min to collect the DNA solution.

i. Pour the solution obtained by centrifugation into the adsorption column again, put it at room temperature for 1 min, and centrifuge at 12000 rpm for 2 min to increase the recovery rate. The obtained DNA (StDWF1 gene) product was stored at -20°C for future use.

2. Construction of overexpression vector

(1) The target fragment is connected to the cloning vector and transformed

① connection

Connect the recovered and purified PCR product to the pMD19-T cloning vector, and the reaction system is as follows:

The target fragment was heated at 65°C for 5 minutes, and operated on ice:

After mixing and centrifuging, connect at 4°C for 18h and store at -20°C.

②Recombinant plasmid DNA transformation

a. Mix 5 μl of the ligation product with 100 μl of Escherichia coli DH5α competent cells on an ultra-clean workbench, and bathe in ice for 30 minutes.

b. Heat shock in a water bath at 42°C for 90 seconds and ice bath for 5 minutes.

c. Add 800 μl of liquid medium, shake at 220 rpm for 40 minutes at 37°C.

d. On the LB solid medium (100 μg/ml) to which Amp has been added, evenly smear a mixture of 40 μl of X-gal (20 mg/ml) and 4 μl of IPTG (200 mg/ml).

e. After the culture of the bacteria resumes, centrifuge the bacteria solution at room temperature at 3000 rpm for 2 minutes, take out 800 μl of the supernatant with a pipette, and resuspend the remaining 100 μl.

f. On the ultra-clean workbench, spread the bacterial solution evenly on the plate, place it for 30min-60min until it is completely absorbed, then incubate it upside down at 37°C for 16-18h, and pick a single colony for identification after growth.

(2) Identification of recombinant plasmids

Pick a single white colony from the blue-white screening medium, add it to 20 μl of ddH2O, mix well, and use it as a template for the PCR identification reaction system of the bacterial solution, and the PCR product is subjected to agarose gel electrophoresis. Pick the positive colonies by gel electrophoresis into the liquid LB medium containing Amp antibiotics, culture them with shaking at 37°C for 8 hours, extract the plasmids, and carry out plasmid PCR identification, then send the positive plasmids for sequencing.

Among them, plasmid extraction follows the steps of the OMEGA plasmid extraction kit:

(a) Pipette about 3ml of bacterial solution into a 5ml centrifuge tube, centrifuge at 5000rpm for 30 seconds to collect bacteria.

(b) Add 200ml of solution I, shake the suspended cells vigorously with a shaker.

(c) Add 300ml of prepared solution II, and mix evenly by inverting.

(d) Immediately add 300ml of pre-cooled solution III after the solution is clear, mix well and ice-bath for 5-10 minutes.

(e) Centrifuge at 5000 rpm for 15 minutes at 4°C.

(f) Take the supernatant, add an equal volume of chloroform/isoamyl alcohol, mix evenly by inversion, and centrifuge at 5000 rpm for 5 minutes to precipitate the protein.

(g) Aspirate the supernatant, add 0.7 times the volume of isopropanol, and mix well. Centrifuge at 5000rpm for 10 minutes, discard the supernatant, then wash the precipitate with 70% ethanol, discard the supernatant at 8000rpm for 5min. After drying at room temperature for 10 minutes, it was dissolved in an appropriate amount of ddH2O2 and stored at 4°C for future use.

Plasmid PCR detection, first amplification reaction, electrophoresis analysis, and then biological sequencing, the specific operations are as follows:

The reaction system is 20 μl, and the following components are added on ice:

Mix well, centrifuge slightly to sink to the bottom, and carry out cycle amplification reaction according to the following conditions:

(a) Perform a pre-denaturation reaction at 95°C for 3 minutes;

(b) Perform denaturation reaction at 95°C for 30s;

(c) Annealing at 54-58°C for 30s;

(d) Extending at 72°C for 1-1.5 min;

(e) Cycle steps (b) to (d) 35 times;

(f) Extending at 72°C for 10 minutes;

(g) insulation at 4°C;

After the PCR amplification products were analyzed by 0.8% agarose gel electrophoresis, the positive clones were sent to Shanghai Sangon for sequencing. The results are shown in Figure 3. The test results showed that the target fragment was successfully connected to the cloning vector, and the StDWF1 gene coding sequence fragment was obtained.

(3) Digestion of cloning vector and construction of overexpression vector

The pMD19-T vector containing the target fragment and the overexpression vector pBI121 (empty) were double-digested with restriction endonucleases BamH1 and SmaI respectively, and the obtained pBI121 vector and target fragment were recovered and ligated with T4 ligase. The digestion reaction was carried out at 37°C, and the ligation reaction was carried out at 4°C overnight.

Among them, the enzyme digestion reaction system and the ligation reaction system are as follows:

The ligation product pBI121-StDWF1 was transformed into Escherichia coli competent cells, and the medium resistance screening, PCR, enzyme digestion and sequencing identification were carried out.

(4) Transform the plasmid containing the gene of interest into Agrobacterium

Ⅰ Preparation of Agrobacterium Competent Cells

(a) Pick a single colony of Agrobacterium tumefaciens GV3101 from the YEB solid plate, inoculate it in 5ml of YEB liquid medium containing 50 μg/ml rifampicin and 15 μg/ml gentamicin, at 28°C, 200 rpm Minute overnight shaking culture;

(b) Take 2ml of the overnight culture and add it to 50ml of YEB liquid medium (containing the same antibiotic), shake it at 28°C until the OD600 is 0.5, and put the bacteria in ice bath for 30 minutes;

(c) Centrifuge at 2,500g for 5 minutes at 4°C to collect bacteria;

(d) Add 10ml 0.15M NaCl to suspend Agrobacterium cells, centrifuge at 2,500g for 5 minutes;

(e) Add 1ml of pre-cooled 20mmol/L CaCl2 to suspend the cells, and operate on ice in this step.

Use within 24 hours of Agrobacterium competence.

Ⅱ Transformation of Agrobacterium (freeze-thaw method)

Take 100ml of competent cells and thaw on ice, add 1mg of recombinant plasmid, mix well, let stand on ice for 30 minutes, freeze in liquid nitrogen for 1min, bathe in water at 37°C for 5min, then add 1ml of YEB medium (without any antibiotics) ), cultured with slow shaking at 28°C for 4 hours; centrifuged at 5000rpm for 30 seconds to collect the bacteria, discarded the supernatant, added 0.1ml of YEB medium to resuspend the cells, and spread the cells containing 50μg/ml rifampicin and 15μg/ml gentamicin (Gen) and 50mg/ml Kna on the YEB plate, cultured at 28°C for about 48 hours. Pick a single colony grown on the plate, inoculate it in YEB liquid culture solution (containing 50 mg/ml Kan and antibiotics corresponding to the Agrobacterium strain), and cultivate overnight at 28° C. with shaking. A small amount of plasmid DNA was extracted, and the plasmid DNA was used as a template for PCR amplification and identification to obtain Agrobacterium GV3101 containing pBI121-StDWF1.

Three pMD-StDWF1 recombinants were selected and sent for sequencing. The sequencing results of the three recombinants were the same, but there were differences with the published sequences: there were 10 differences in total, and only the mutation sites 22 and 133 caused changes in the amino acid sequence. Changed from A to C at 22bp, causing the 8th isoleucine in the amino acid sequence to change to leucine; at 133 bp, C changed to G, causing the 45th leucine to change to valine in the amino acid sequence acid. Due to the degeneracy of the codon, the difference of the other 8 bases did not cause changes in the amino acid sequence. Another three recombinants were selected for sequencing, and the results were still the same as before, indicating that differences in potato varieties may have caused the gene sequences to be different. So far, the clone of StDWF1 gene which is basically consistent with the published sequence has been obtained. In this experiment, the target gene was cloned from the leaves of Chuanyu 117 test-tube plantlets. The sequencing results differed from the published sequence by 11 bases. There were 2 amino acid changes in the same position, but the changed amino acids were different. So far, the CDS region of StDWF1 gene completely consistent with the published sequence was cloned.

The plant expression vector pBI121 and the recombinant cloning vector were digested separately, and the purified and recovered target fragment was ligated with the digested overexpression vector pBI121 by T4 ligase to construct the DWF1 gene overexpression vector driven by CaMV35S and transform it into competent Escherichia coli middle. The recombinant plasmid was digested with BamhI and SmaI to obtain a 1704bp fragment (Figure 4). A single colony was obtained in the YEB solid selection medium containing antibiotics, indicating that the Agrobacterium strains containing the pBI121-StDWF1 plasmid were obtained respectively.

In this study, the full-length StDWF1 coding sequence was cloned from the leaves of Feureta, and compared with the whole genome sequencing data, it was found that there were 10 base differences, of which 2 difference bases led to amino acid mutations, but the amino acid mutation position The point is not in the FAD binding domain, and such differences will not cause changes in protein functions. This sequence difference is mainly due to differences in plant growth environments and species differences, and will not change the function of the original protein. Three-dimensional structural analysis showed that the mutated amino acids did not cause differences in the protein's 3D structure.

3. Cultivation of potato plants overexpressing the StDWF1 gene

Material:

Test-tube seedlings of potato variety Chuanyu No. 10 (C10); laboratory-constructed Agrobacterium GV3101 containing pBI121-StDWF1.

Reagents: Kanamycin (Kan) and antibiotic cephalosporin (cef) were purchased from Vanke Reagent Company.

Used plant plant growth regulators include: 6-benzyl adenine (6-BA), gibberellin (GA3) thiadizuron (TDZ), indole acetic acid (IAA) etc. are all Sigma company products, 2,4- Dichlorophenoxyacetic acid (2, 4-D) was purchased from Vanke Reagent Co., Ltd., and after being made into a mother liquor, it was sterilized by filtration through a 0.22 μm filter membrane and stored at -20°C for future use. Each nutrient component of MS medium is analytically pure, and its formula is as follows: (unit.mg L-1)

Major elements: potassium nitrate (KNO3) 1900, ammonium nitrate (NH4NO3) 1650, potassium dihydrogen phosphate (KH2PO4) 170, magnesium sulfate (MgSO4 7H2O) 370, calcium chloride (CaCl2 2H2O) 440

Trace elements: potassium iodide (KI) 0.83, boric acid (H3BO3) 6.2, manganese sulfate (MnSO4 4H2O) 22.3, zinc sulfate (ZnSO4 7H2O) 8.6, sodium molybdate (Na2MoO4 2H2O) 0.25, copper sulfate (CuSO4 5H2O) 0.025, cobalt chloride (CoCl2 6H2O) 0.025

Iron salt: disodium ethylenediaminetetraacetic acid (Na2.EDTA) 37.25, ferrous sulfate (FeSO4 7H2O) 27.85

Organic Ingredients: Inositol 100, Glycine 2, Thiamine Hydrochloride (VB1) 0.1, Pyridoxine Hydrochloride (VB6) 0.5, Niacin (VB5) 0.5

Solid medium needs to add agar 6g/L, sucrose 30g/L. Except for agar, after adding the rest, adjust the pH to 5.8 with 1mol/LTris (6.057g to 50mL), add agar and boil for 3-4 times.

Agrobacterium-infected test tube tuber stem segment method to transfer the target gene:

① Pre-cultivation of test-tube seedling stems

On the ultra-clean workbench, cut the stems of the multiplied test-tube plantlets, and place the cut stems flat on the solid medium for pre-cultivation, and cultivate them in the dark at about 20°C for 2-3 days.

②Agrobacterium activation

Streak inoculate the original bacterial solution in solid YEB medium (containing 50mg/L Kan and 50mg/L Rif), culture at 27°C for about 48h, seal the dish and store at 4°C. Add 4mL YEB to a 10mL centrifuge tube, pick a single colony, shake the bacteria at 220rpm, 27°C for 16h, and store at 4°C for later use. Inoculate the bacteria in fresh liquid YEB in a 10mL centrifuge tube at a ratio of 1:100, shake the bacteria at 27°C for 12 hours at 220rpm, and store at 4°C for later use.

③ Culture medium preparation

Suspension liquid: liquid MS+3% sucrose, pH5.9-6.0.

Stem segment pre-medium and co-medium: MS+1.0 mg/L 6-BA+0.2 mg/L TDZ+0.05 mg/L 2,4-D+0.1 mg/LGA3+3% sucrose+0.6% agar.

Bud selection medium: MS+1.0mg/L 6-BA+0.2mg/L TDZ+0.05mg/L 2,4-D+0.1mg/L GA3+3% sucrose+0.6% agar+50mg/L Kan+ 100mg/L Cef+250mg/L Car.

Cut the differentiated shoots and insert them into rooting selection medium: MS+3% sucrose+0.6% agar+50mg/L Kan+100mg/L Cef+250mg/L Car.

④ Agrobacterium dipping co-cultivation and screening

Put the stem section into a sterile bottle on the ultra-clean workbench, take the shaken bacterial solution, centrifuge at 6000rpm for 5min, remove the supernatant, add MS liquid medium to resuspend.

Transfer the pre-cultured stem segments from the solid medium to a small beaker.

Pour the resuspended bacteria into a small beaker, and infect the solution for 2-3 minutes.

Pour off the bacterial solution, pour sterile water to wash the stems, repeat twice, transfer to sterile filter paper to absorb excess water, then transfer the stems to a symbiosis medium with two layers of sterile filter paper, at 24°C Incubate in the dark for 36h.

Wash the stem segments in sterile water containing 100mg/L Cef for 3 to 4 times, absorb excess water and transfer to screening medium, 16h light/8h dark, light intensity 60μmol m-2s-1, 22±1℃ .

⑤Callus culture

Change the medium every 10-14 days, the more frequent the change, the faster the differentiation. It takes 35-90 days to remove deadly, polluted and completely albino plants due to overgrowth of Agrobacterium.

Identification of regenerated shoots from cultured calli:

① Screening and identification of rooting

The regenerated shoots were cut off and added to the rooting screening medium, the light intensity was 60μmol m-2s-1, 16h light/8h dark, and cultured at 20±1°C. The strains that could grow roots could be preliminarily judged that the target gene had been transferred.

②PCR detection

Small amount of DNA extraction from tuber tuber leaves of potato by CTAB method

A. 2% CTAB extraction buffer is preheated in a 65°C water bath.

B. Get 8 test-tube seedling leaves in a mortar and grind to powder with liquid nitrogen;

C. Add 700ul of 2% CTAB extraction buffer, stir gently, place in a water bath or incubator at 65°C, shake gently every 10min, and take it out after 30-60min;

D. After cooling for 2 minutes, add chloroform-isoamyl alcohol (24:1) to fill the tube, shake gently for 2-3 minutes to mix the two evenly;

E. Put it into a centrifuge and centrifuge at 10000rpm for 10min. At the same time, add 600ul of isopropanol to another new sterilized centrifuge tube;

F. After centrifuging at 10 000rpm for 1min, pipette gently absorb the supernatant, transfer it into a centrifuge tube containing isopropanol, and shake the centrifuge tube slowly up and down for 30s, so that the isopropanol and the water layer are fully mixed until you can see DNA flocs;

G. After centrifuging at 10000rpm for 1min, pour off the liquid immediately, be careful not to pour out the white DNA precipitate, and place the centrifuge tube upside down on the spread paper towel;

After H.60s, upright the centrifuge tube, add 720ul of 75% ethanol and 80ul of 5M sodium acetate, rotate gently, and flick the tip of the tube with your fingers to make the precipitate and the DNA block at the bottom of the tube float in the liquid;

I. Place it for 30min to dissolve the impurity of the DNA block;

J. After centrifuging at 10000rpm for 1min, pour off the liquid, then add 800ul of 75% ethanol, and wash the DNA for another 30min;

K. After centrifuging at 10000rpm for 30 sec, pour off the liquid immediately, and place the centrifuge tube upside down on a spread paper towel; after a few minutes, stand the centrifuge tube upright, and dry the DNA (air-dried naturally or with a blower);

L. Add 50ul of 0.5×TE buffer to dissolve the DNA, take 4μl for electrophoresis detection, and store the rest at -20°C for later use.

Synthetic resistance screening marker gene nptⅡ primers:

nptII-P1: GCTATGACTGGGCACAACAG;

nptII-P2; ATACCGTAAAGCACGAGGAA.

③ Identification of expression level of positive transgenic lines

RNA was extracted from the semi-quantitatively identified strains, three bottles were selected for each strain, and cDNA was synthesized by reverse transcription. The expression of StDWF1 in the transgenic lines relative to the non-transgenic lines was determined by using the qRT-PCR method of the cDNA of each line.

④ Phenotypic Analysis of Potato Transplants

The transgenic strain StDWF1 gene expression level was multiplied, 3 bottles for each strain, 3 buds were inoculated in each bottle, the light temperature was 24°C, and the morphological characteristics were compared and observed after 15 days, including the transgenic strain Plant height, root length, fresh weight, stem diameter and other indicators of the line and wild-type test-tube plantlets.

⑤Data Analysis

The software spss17.0 (SPSS Inc, Chicago, USA) was used for significant difference analysis, and the significance level was P<0.05.

After the stems of potato test-tube seedlings were dipped and co-cultivated by Agrobacterium (Fig. 5), they were transferred to the bud screening medium. After about 2 weeks of cultivation, the stems began to expand and callus appeared (Fig. 6), and they healed after 45 days. Green shoots (Fig. 7) were formed from the injured tissue, and after being cultivated to about 1 cm in size, the shoots were cut off and transferred to the rooting medium for rooting screening. The transgenic plants could grow normally in the rooting medium, while the non-transgenic plants could grow in the medium but could not take root (Fig. 8). After a period of time, the transgenic lines multiplied, and 61 complete transgenic plants with resistant plants were obtained ( FIG. 9 ).

The total RNA of the leaves of the transgenic lines was extracted, reverse-transcribed into cDNA, and electrophoresis analysis of the PCR amplified product showed that a 676bp nptII gene fragment could be amplified in the pBI121-StDWF1 transgene (Fig. 10).

After PCR detection, positive plants were selected, and the root length, leaf size, rooting induction speed and plant height of the StDWF1 overexpressed transgenic plants were analyzed. The roots of the 40-day overexpressed material were significantly longer than those of the wild-type plants. The promotion of rooting induction (Fig. 11) found that the overexpression began to take root in 20 days, while the wild type did not take root in 30 days (Fig. 13), and the overexpression leaf size and plant height were all higher than the wild type (Fig. 12 and 14). In summary, StDWF1 promotes plant growth. As can be seen from the table below, the plant height, fresh weight and root length of the transgenic plant No. 4 were significantly higher than those of the non-transgenic line, and the differences were significant. There were different degrees of non-significant differences in the wild type, indicating that the growth of the heel of the plants was promoted after the overexpression of the StDWF1 gene.

Example 2

Expression analysis of StDWF1 in different parts of potato at different stages:

Total RNA was extracted from different tissues (roots, stolons, old leaves, young leaves, stems and tubers) and leaves of different growth stages (sampling began 2 weeks after sowing, and the sampling period was 1 week) in Fevoria, and was expressed as Oligo(dT ) as primers, cDNA was synthesized according to the First StrandcDNA Synthesis Kit Revertra Ace-α-reverse transcription method.

Internal reference EF1αL primers were designed according to the potato genome database,

That is, EF1αL F: 5'-CTTGTACACCACGCTAAGGAG-3';

EF1αL R: 5′-GTCAATGCAAACCATTCCTTG-3′.

Design StDWF1 quantitative primers based on the full-length cDNA sequence:

DWF1-P1: 5'-AGTTGGTGGACTTTCTTCTTTC-3';

DWF1-P2: 5'-TTCTGCATTCCTCTGTTCAAG-3'.

The quantitative PCR reaction system was: 4.5 μL cDNA, 0.25 μL each of upstream and downstream primers (10 μmol/L), 5 μL of 2×SsofastEva Green, and 10 μL of ddH2O.

The reaction program is 95°C for 30s; 95°C for 5s, 55°C for 5s, 39 cycles; 95°C for 10s, 65-95°C for the melting curve, each temperature rises at 0.5°C and stops for 5s.

Three replicates were set up for each test sample, and the gene expression was analyzed using the 2-△△Ct method on Bio-Rad CFX ManagerV and Excel software to determine the expression of StDWF1 in different tissue parts and leaves of different growth stages of potato.

After adding low-concentration ethanol, NaAc, and freezing and thawing to remove sugar, good quality RNA was obtained. The electrophoresis detection of some samples is shown in Figure 15, and three clear bands of 28S, 18S, and 5S can be seen. OD260/230 and OD260/280 indicated that the extracted RNA was free from sugar, salt and protein contamination, and the quality met the requirements for library construction and sequencing, and could be used in the next test.

Real-time fluorescent quantitative analysis of StDWF1 gene expression in potato roots, stolons, tubers, stems, old leaves and young leaves (Figure 16). The results showed that it was expressed in 5 kinds of tissues. After significant analysis (p≤0.05), the expression level of StDWF1 gene in young leaves, old leaves and stolons was significantly higher than that in other tissue parts, followed by tubers, roots and stems. It indicated that StDWF1 was mainly expressed in the young leaves, old leaves and stolons of Fevorita, and there were significant differences in the expression of each tissue.

Quantitative analysis was carried out on each stage of the leaves, and it was found that the StDWF1 gene was expressed in all growth stages, and the whole growth period was divided into three stages by using the significance analysis (p≤0.05): the first stage (14d, emergence stage) StDWF1 gene The expression level was the highest, and the highly expressed StDWF1 promoted the synthesis of growth substances such as BR in leaves and regulated plant growth; in the second stage (21d-28d), the plant growth was stable, and the expression of StDWF1 in leaves was lower than that in the previous stage; in the third stage ( 35d-56d) is the later stage of potato plant growth, the synthesis activities of various substances are significantly weakened, and the expression level of StDWF1 drops to the lowest in the growth period (as shown in Figure 17).

The foregoing is an embodiment of the present invention. The present invention is not limited to the above embodiments, and anyone should know that any structural changes made under the inspiration of the present invention, and any technical solutions that are the same as or similar to the present invention, all fall within the scope of protection of the present invention.

Claims (10)

1. a kind of potato StDWF1 genes, which is characterized in that its base sequence such as SEQ ID NO:Shown in 1.

2. a kind of preparation method of potato StDWF1 genes, which is characterized in that include the following steps：

(1) total serum IgE is extracted from Potato Cultivars Favorita test tube seedling；

(2) cDNA is synthesized according to the total serum IgE reverse transcription of extraction；

(3) PCR amplification introduces XbaI and SmaI restriction enzyme sites using cDNA as template before the areas CDS of cDNA, and design amplification is drawn Object StDWF1PF and StDWF1PR, and add primer StDWF1PF, primer StDWF1PR, 10 × amplification buffer, sterile water, DNTPs, Taq archaeal dna polymerase mixing simultaneously carry out PCR amplification, wherein cDNA, primer StDWF1PF, primer StDWF1PR, 10 × Amplification buffer, sterile water, dNTPs, Taq archaeal dna polymerase volume be respectively：1μL,1μL,1μL,2μL,15.3μL,0.5μ L, 0.2 μ L, and cDNA, primer StDWF1PF, primer StDWF1PR and dNTPs concentration be respectively 10~100ng/L, 2 μ M/μl,2μM/μl,10mmol/L；

The base sequence of amplimer StDWF1PF, StDWF1PR are respectively such as SEQ ID NO:2,SEQ ID NO:Shown in 3；

(4) recovery purifying pcr amplification product obtains potato StDWF1 genes through sequencing analysis.

3. a kind of preparation method of potato StDWF1 genes as claimed in claim 2, which is characterized in that in step (1), carry It is the blade seeding stage to take the opportunity of Favorita test tube seedling total serum IgE, and extract part is the tender leaf of Favorita test tube seedling.

4. a kind of preparation method of potato StDWF1 genes as claimed in claim 2, which is characterized in that total in step (1) The extracting method of RNA uses Trizol methods, is as follows：

(1a) is ground：Mortar is put into 4 spoonfuls of liquid nitrogen and Potato Cultivars Favorita specimen locations, and liquid nitrogen is worn into rapidly after being evaporated completely Fine powder, is quickly charged with after EP pipes plus 1mL Trizol, violent mixing, room temperature put 5-10min；

(1b) 4 DEG C of 12000rpm centrifuge 5min, draw in supernatant to new pipe, and 200ml chloroforms are added, slight to rock up and down, room Temperature stands 5min；

(1c) 4 DEG C of 12000rpm centrifuge 10min, and isometric phenol/chloroform/isoamyl alcohol is added to new pipe in Aspirate supernatant, overturns mixed It is even；

(1d) 4 DEG C of 12000rpm centrifuge 20min, and for Aspirate supernatant to new pipe, being gradually added into absolute ethyl alcohol keeps its final concentration of 12%, Quick spin after the mixing that turns upside down；

(1e) 4 DEG C of 12000rpm centrifuge 10min, draw 400mL supernatants to new pipe, 0.7 times of volume isopropanol is added and runs up and down Mixing, and 0.2 times of volume 1mol/L NaAc-20 DEG C is added and precipitates 1h；

(1f) 4 DEG C of 12000rpm centrifuge 15min, abandon supernatant, and 75% ethyl alcohol of 1mL is added and washes precipitation, 4 DEG C, 8500rpm centrifugations 3min, then 75% ethyl alcohol of 1mL is added to wash 2 times；

(1g) outwells supernatant, sops up surplus liquid as possible, and super-clean bench up-draught 10min removes residual ethanol, is added at 80mLDEPC Water is managed, 55 DEG C of water-bath 5min are immediately placed in, -80 DEG C of 60min are repeated 2 times；

(1h) 4 DEG C of 12000rpm centrifuge 20min, careful to draw 70 μ L of supernatant, pay attention to not contacting bottom to get to total serum IgE, in- 80 DEG C freeze it is spare；

Wherein, step (1c)-(1h) is operated on ice.

5. a kind of preparation method of potato StDWF1 genes as claimed in claim 2, which is characterized in that total in step (2) RNA reverse transcriptions synthesize cDNA, and reverse transcription, overall reaction system body are carried out by the cDNA synthetic agent box operation instructions of TakaRa companies Product is 20 μ l, and using RNase free centrifuge tubes, reaction system is as follows：

65 DEG C are denaturalized 5 minutes, at least 1 minute cooling on ice rapidly, slightly centrifuge, are then added：

It is uniformly mixed, 55 DEG C of reactions 60min, 70 DEG C of 15min make enzyme inactivate, -20 DEG C of preservations.

6. a kind of preparation method of potato StDWF1 genes as claimed in claim 2, which is characterized in that PCR in step (3) The reaction condition of amplification is：

(3a) carries out pre-degeneration reaction under the conditions of 95 DEG C, when a length of 3min；

(3b) carries out reaction of degeneration (RD) under the conditions of 95 DEG C, when a length of 30s；

(3c) anneals 30s under the conditions of 54-58 DEG C；

(3d) extends 1-1.5min under the conditions of 72 DEG C；

(3e) circulation step (3b)~(3d) 35 times；

(3f) extends 10min under the conditions of 72 DEG C；

(3g) keeps the temperature pending under the conditions of 4 DEG C.

7. a kind of preparation method of potato StDWF1 genes as claimed in claim 2, which is characterized in that returned in step (4) The method for receiving purifying pcr amplification product specifically comprises the following steps：

(4a) column equilibration step：600 μ l equilibrium liquids are added into adsorption column, 10000rpm centrifuges 1min, outwells the flat of collecting pipe Weighing apparatus waste liquid is simultaneously returned to collecting pipe again；

Pcr amplification product is placed in the Ago-Gel of 0.01g/ml by (4b), and Ago-Gel is transferred in centrifuge tube, to 100 μ l liquor kalii aceticis, 50 DEG C of water-bath 10min, and the centrifuge tube that turns upside down are added in centrifuge tube, fully dissolves gel；

(4c) imports dissolved gel in adsorption column, and 12000rpm centrifuges 1min, outwells collecting pipe waste liquid and by adsorption column It is put into collecting pipe again；

(4d) 600 μ l solution D NA wash buffer, 12000rpm are added into adsorption column and centrifuge 1min, and it is useless to outwell collecting pipe Adsorption column is simultaneously put into collecting pipe by liquid again；

Adsorption column is put into collecting pipe by (4e), and 12000rpm centrifuges 2min, and stands 10min；

Adsorption column is put into a clean centrifuge tube by (4f), and suitable elution buffer is vacantly added dropwise to adsorbed film centre position Liquid is placed at room temperature for 2min, and 12000rpm centrifuges 2min, collects DNA solution；

The solution that centrifugation obtains is re-poured into adsorption column by (4g), and room temperature puts 1min, and 12000rpm centrifuges 2min to improve recycling Rate to get the potato StDWF1 gene outcomes to potato StDWF1 gene outcomes, obtained be stored in -20 DEG C it is spare.

8. a kind of overexpression potato StDWF1 genes are to promote the method for potato Rapid Rooting, which is characterized in that including such as Lower step：

(1) over-express vector is built

A. cloning vector is connected：Potato StDWF1 genes are connect with pMD19-T cloning vectors, first by potato StDWF1 bases Because heating 5min at 65 DEG C, following reaction system is then added：

After mixing centrifugation, 4 DEG C of connection 18h, then in -20 DEG C of preservations；

Wherein, solution I is isometric mixed solution of 50mM glucose, 25mM Tris-HCL, 10mM EDTA, pH value 8.0；

B. recombinant plasmid dna converts：By 5 μ l cloning vectors connection products and 100 μ l bacillus coli DH 5 alpha senses on superclean bench It is uniform by state mixing with cells, ice bath 30min；42 DEG C of water-bath heat shock 90s, then ice bath 5min；It is added 800 μ l fluid nutrient mediums, 37 DEG C, 220rpm shaken cultivations 40min；On the LB solid mediums of Amp antibiotic have been added, the content of Amp antibiotic is 100 μ g/ml uniformly apply the IPTG mixed liquors of the X-gal and 4 a concentration of 200mg/ml of μ l that spread 40 a concentration of 20mg/ml of μ l；Thalline After renewal cultivation, room temperature 3000rpm, 2min centrifuge bacterium solution, take out 800 μ l supernatants with pipettor, remaining 100 μ l are resuspended；? On superclean bench, bacterium solution is uniformly coated in tablet, 30min-60min is placed and is inverted training then at 37 DEG C after fully absorbing 16-18h is supported, single bacterium colony identification is chosen after growing；Single bacterium colony to be identified is added in the ddH2O of 20 μ l, is uniformly mixed, as The template of bacterium solution PCR identification reaction systems, PCR product is into row agarose gel electrophoresis；The bacterium colony that picking gel electrophoresis is positive To in the liquid LB solid mediums containing Amp antibiotic, 37 DEG C of shaken cultivation 8h extract plasmid, after plasmid PCR detection, and The positive colony that detection is obtained carries out plasmid PCR identification, and positive plasmid is accredited as through PCR, obtains containing mesh through sequencing analysis Segment pMD19-T carriers；

C. restriction enzyme BamH1 and SmaI is used to distinguish pMD19-T carrier and over-express vector of the double digestion containing target fragment The pBI121 carriers that digestion obtains are recycled with target fragment and are connected with T4-DNA ligases by pBI121, and endonuclease reaction is 37 DEG C carry out, connection react on 4 DEG C overnight；Connection product pBI121-StDWF1 is converted into competent escherichia coli cell, picking again Single bacterium colony expands numerous, extraction plasmid, is transformed into the competent cell of Agrobacterium tumefaciems after PCR, digestion and sequencing identification；

(2) plasmid containing target gene is transferred to Agrobacterium

A. the single bacterium colony of picking Agrobacterium tumefaciems GV3101 is inoculated in 5ml containing 50 μ g/ml rifampins, 15 μ g/ml gentamicins In YEB fluid nutrient mediums, in 28 DEG C, 200 revs/min of overnight shaking cultures；

B. absorption is incubated overnight bacterium solution 2ml and is added in YEB fluid nutrient mediums of the 50ml containing identical antibiotic, 28 DEG C of shaken cultivations It is 0.5 to OD600, bacterium solution ice bath 30 minutes；

C.4 DEG C, 2500g centrifuges 5 minutes collection bacterium；

D. plus 10ml 0.15M NaCl suspension agrobatcerium cells, 2500g are centrifuged 5 minutes；

E. the 20mmol/L CaCl of 1ml precoolings are added₂Suspension cell, this step operate on ice, obtained Agrobacterium competence Cell, and used in 24 hours；

F. it takes 100ml Agrobacteriums competent cell and melts on ice, the pMD19-T carriers containing target fragment of 1mg are added, mix 30 minutes are stood after even on ice, then the not antibiotic YEB trainings of 1ml are added in the quick-frozen 1min in liquid nitrogen, 37 DEG C of water-bath 5min Support base, 28 DEG C of shaken cultivation 4 hours at a slow speed；5000rpm centrifuges 30 seconds collection bacterium, abandons supernatant, it is not antibiotic that 0.1ml is added YEB culture mediums suspension cell again is coated on anti-containing 50 μ g/ml rifampins, 15 μ g/ml gentamicins and 50mg/ml Kana On the YEB tablets of raw element, 28 DEG C are cultivated about 48 hours；The single bacterium colony grown on picking tablet, is inoculated in containing 50mg/ml Kana antibiotic and corresponding in the YEB liquid mediums of the antibiotic of agrobacterium strains, 28 DEG C of shaken cultivations are stayed overnight；Extraction unit Divide Plasmid DNA, carries out PCR amplification identification by template of Plasmid DNA, identification, which is positive, obtaining being transferred to the plasmid containing target gene Agrobacterium；

(3) During Agrobacterium test tube seedling potato stem section

A. test tube seedling stem section preculture, on superclean bench, numerous test tube seedling stem section is expanded in shearing, and the stem section after shearing is put down It is placed on solid medium and carries out preculture, 20 DEG C or so dark culturing 2-3d；

B. Agrobacterium activate, take be transferred to the plasmid Agro-Bacterium bacterium solution streak inoculation containing target gene in solid Kan containing 50mg/L with In the YEB culture mediums of 50mg/L Rif, 27 DEG C of culture 48h, envelope ware is in 4 DEG C of preservations；4mL YEB trainings are added in 10mL centrifuge tubes Support base, choose single bacterium colony, 220rpm, 27 DEG C are shaken bacterium 16h, be stored in 4 DEG C it is spare；With 1：100 to connect bacterium new in being filled with 10mL centrifuge tubes In fresh liquid YEB culture mediums, 220rpm, 27 DEG C are shaken bacterium 12h, be stored in 4 DEG C it is spare；

C. During Agrobacterium co-cultures and screening takes the bacterium solution shaken by stem section income aseptic bottle on superclean bench, 6000rpm centrifuges 5min, removes supernatant, and MS fluid nutrient mediums are added and are resuspended；By the good stem section of preculture from solid medium transfer Move to small beaker；The bacterium after being resuspended is poured into small beaker, liquid infects 2-3min；Bacterium solution is outwelled, sterile water wash stem section is poured into, weight Multiple be transferred on aseptic filter paper afterwards twice sucks excessive moisture, and stem section is then transferred to the symbiosis training for being lined with two layers of aseptic filter paper It supports in base, 24 DEG C of dark culturing 36h；Stem section is put into the sterile water of the Cef containing 100mg/L and is cleaned 3~4 times, sops up superfluous water It is transferred to screening and culturing medium after point, 16h light/8h is dark, light intensity 60 μm of ol/m2.s1,22 ± 1 DEG C；

D. callus tissue culture changes 1 subculture every 10-14d, removes because of the lethal of Agrobacteriuna overgrowth, pollution and complete Full albefaction plant is to get to the potato plant for being overexpressed potato StDWF1 genes.

9. a kind of overexpression potato StDWF1 genes as claimed in claim 8 to be to promote the method for potato Rapid Rooting, It is characterized in that, step (1) structure over-express vector during, extract plasmid the step of be：

I. it draws in 3ml bacterium solutions to 5ml centrifuge tubes, 5000rpm is centrifuged 30 seconds, collects bacterium；

II. plus 200ml solution Is, suspension thalline is acutely vibrated with oscillator, wherein solution I is 50mM glucose, 25mM Tris- Isometric mixed solution of HCL, 10mM EDTA, pH value 8.0；

III. plus the solution IIs that have configured of 300ml, overturn mixing, wherein solution II be 0.2nM NaOH, 1% mass fraction Isometric mixed solution of SDS；

IV. the solution III of 300ml precoolings is added after solution clarification immediately, ice bath 5~10 minutes after mixing, wherein solution III is The isometric mixed solution of alcohol of 3mM potassium acetates, 2mM acetic acid, 75% mass fraction；

V.4 DEG C, 5000rpm is centrifuged 15 minutes；

VI. supernatant is taken, isometric chloroform/isoamyl alcohol is added, after overturning mixing, 5000rpm centrifuges 5 minutes protein precipitations；

VII. supernatant is drawn, 0.7 times of volume isopropanol, mixing are added；5000rpm is centrifuged 10 minutes, and supernatant is abandoned in suction, then with 70% second Alcohol washing precipitation, 8000rpm, 5min abandon supernatant；Drying at room temperature is dissolved in appropriate ddH2O2 to get to the target of extraction after ten minutes Plasmid, 4 DEG C save backup.

10. a kind of overexpression potato StDWF1 genes as claimed in claim 8 to be to promote the method for potato Rapid Rooting, It is characterized in that, during step (1) structure over-express vector, plasmid PCR detection first expands to be detected again, and steps are as follows：

20 μ l plasmids to be checked are added in reaction system, ice bath environment sequentially adds following components：

Mixing, slightly centrifugation are sunk to the bottom, and carry out cyclic amplification reaction, and the condition of cyclic amplification reaction is specially：

(3a) carries out pre-degeneration reaction under the conditions of 95 DEG C, when a length of 3min；

(3b) carries out reaction of degeneration (RD) under the conditions of 95 DEG C, when a length of 30s；

(3c) anneals 30s under the conditions of 54-58 DEG C；

(3d) extends 1-1.5min under the conditions of 72 DEG C；

(3e) circulation step (3b)~(3d) 35 times；

(3f) extends 10min under the conditions of 72 DEG C；

(3g) is kept the temperature under the conditions of 4 DEG C；

Pcr amplification product is analyzed through 1.0% agarose gel electrophoresis, obtains analysis result.