CN101597611B - Eukaryotic recombinant plasmid for enhancing pre-harvesting sprouting resistance and application thereof - Google Patents

Abstract

The invention relates to a gene for enhancing pre-harvesting sprouting resistance, with a nucleotide sequence disclosed as SEQ ID No: 1 in a sequence table. The nucleotide sequence of the SEQ ID No: 1 is inserted into a eukaryotic carrier to obtain a eukaryotic recombinant plasmid of the invention. The eukaryotic recombinant plasmid is converted into paddy rice and lycopersicon esculentum to obtain a transgenic plant. An experiment indicates that the SEQ ID No: 1 gene can suppress the germinating potential of seeds, such as the paddy rice and the like, reduces the germination speed but has noinfluence on the final germination rate of the seeds; and compared with wild type seeds, the suppression to the seed germination of the transgenic seed is more obvious along with the raising of the concentration of abscisic acid (ABA). Thereby, the gene can be applied to the improvement of the pre-harvesting sprouting resistance.

Description

A kind of eukaryon recombinant plasmid and application thereof that improves pre-harvesting sprouting resistance

Technical field

The invention belongs to plant genetic engineering field, particularly a kind of gene that improves pre-harvesting sprouting resistance, recombinant plasmid and in the application aspect the pre-harvest sprouting resistance when running into overcast and rainy wet environment of improvement plant.

Background technology

The seed vivipary is called as in grass that fringe germinates or fringe is sprouted or premature eruption, is the phenomenon that seed just germinates on the maternal plant of field when running into overcast and rainy wet environment before results.This phenomenon is very general in crops such as paddy rice, wheat, corn, barley, Chinese sorghum, rape.Fringe is sprouted and can be caused a series of biochemical reaction of the inner generation of crop seed, as improve the activity of carbohydrate degrading enzyme and proteolytic ferment etc. in the seed body, reserve substance in degraded embryo and the endosperm, thus cause the seed fertility to die down, and seed production and processing quality descend.Cause very big influence and financial loss to farm crop production.

In order to control the harm of seed premature eruption, people have taked many measures, comprise before reinforcement is to crop harvesting and the management after the results, and utilize the method for breeding to cultivate the pre-harvest sprouting resistance new variety.For paddy rice, the method that the most extensively adopts in production application is to use fringe to sprout inhibitor at present, wait as paclobutrazol, dormin and toxilic acid hydrazine and to suppress fringe and sprout, though this measure can make fringe germinate and be inhibited on certain degree, but require the working concentration of inhibitor higher, and inhibitor residual time in seed is longer.Therefore, no matter from the economic benefit angle still from the non-toxic and safe angle, these methods all are unworthy promoting.Simultaneously, utilizing traditional breeding way to cultivate new fringe, to sprout kind cycle of resistance plant long, generally needs 5-8, and takes effect slowly.

Summary of the invention

The purpose of this invention is to provide a kind of gene, eukaryon recombinant plasmid that improves pre-harvesting sprouting resistance, the overexpression of described gene can obviously strengthen the susceptibility of transgenic plant to plant hormone dormin (ABA), thereby promote seed maturity and induce seed dormancy, improve plant seed fringe in the overcast and rainy wet environment before results and sprout resistance, improve the particularly yield and quality of farm crop of plant with this.

Technical scheme of the present invention is as follows:

The gene of raising pre-harvesting sprouting resistance of the present invention, called after PHSR (pre-harvesting sproutingresistance), its nucleotide sequence is as described in the SEQ ID NO:1 in the sequence table, and expressed amino acid sequence of polypeptide is as described in the SEQ ID NO:2 in the sequence table.The clone of this gene: according to tomato cDNA sequence (accession number: AY911399) among the GenBank, utilize primer-design software Primer Premier 5.0 to design 2 pairs of nest-type PRC special primers, from the tomato mature seed, extract total RNA, utilize reverse transcription PCR (RT-PCR) technology from tomato, to be separated to the encoding sequence of PHSR, shown in SEQ ID NO:1 in the sequence table.

Eukaryon recombinant plasmid of the present invention is the described nucleotide sequence of SEQ ID NO:1 to be inserted in the carrier for expression of eukaryon obtain, and described carrier for expression of eukaryon is a kind of among pHB, pMON1772, pBE12, pBC7, the pBI121.

With above-mentioned eukaryon recombinant plasmid rice transformation and tomato, obtain transfer-gen plant.Experiment shows: respectively get the mature seed of the T2 that comes from independent transfer-gen plant of some paddy rice and tomato for seed and wild-type plant in the experiment, be positioned in the culture dish that has double-deck filter paper, be immersed in the distilled water, with the naked eye add up the quantity of the rice paddy seed that germinates and do not germinate every 24h.Discovery is in the early stage of sprouting, and the germination speed of transgenic paddy rice and transgenic Fructus Lycopersici esculenti will be well below the germination speed of the tomato of the paddy rice of wild-type and wild-type.But find that to the later stage of sprouting rising significantly appears early stage in the germinating energy of transgenic seed, the transfer-gen plant seed is the same with wild type seeds to the end, all germinates.This shows that the PHSR gene can suppress germinating energy, the reduction speed of germination of rice paddy seed and tomato seeds.Therefore, gene of the present invention can be used in the improvement of pre-harvesting sprouting resistance.

The present invention has following beneficial effect:

1, the present invention sprouts the plant of resistance a kind of new recombinant plasmid is provided for cultivating fringe, helps the food crop increasing both production and income.

2, gene of the present invention is the own gene of plant itself, so transgenic plant is safe.

3, the present invention cultivates the cycle shortening 4-6 that fringe is sprouted the new variety of plant of resistance than conventional breeding, saves the man power and material greatly, and the fringe that can solve farm crop is simultaneously again sprouted harm.

4, the clone of gene of the present invention and plant transgene are ordinary method, and material requested is easy to obtain.

Description of drawings

Fig. 1 is used for eukaryon recombinant plasmid (pHB-PHSR) synoptic diagram of expressing in paddy rice.

Fig. 2 is used for eukaryon recombinant plasmid (pBI121-PHSR) synoptic diagram of expressing tomato.

Fig. 3 is transgenosis resistance screening mark hygromycin gene (HPT) pcr amplification figure as a result in the transgenic rice plant, and among the figure, the 1-15 swimming lane: pcr template is candidate's transgenic rice plant DNA; 16 swimming lanes: positive control, pcr template are to be used for the eukaryon recombinant plasmid pHB-PHSR that expresses in paddy rice.

Fig. 4 is PHSR gene PCR amplification figure in the transgenic rice plant, and among the figure, the 1-9 swimming lane: pcr template is transgenic paddy rice positive plant DNA; 10 swimming lanes: negative control, pcr template are the wild-type paddy DNA; 11 swimming lanes: positive control, pcr template are eukaryon recombinant plasmid pHB-PHSR; M swimming lane: molecular weight marker DL1500 (available from Dalian Bao Bio-Engineering Company).

Fig. 5 is the pcr amplification figure as a result of transgenosis resistance screening mark kalamycin resistance gene (NPTII) in the transgenic Fructus Lycopersici esculenti plant, and among the figure, swimming lane 1: negative control, pcr template are wild-type tomatoes plant DNA; Swimming lane 2: positive control, pcr template are to be used for the eukaryon recombinant plasmid pBI121-PHSR that expresses tomato; Swimming lane 3-16:PCR template is candidate's transgenic Fructus Lycopersici esculenti plant DNA.

Fig. 6 is transgenic paddy rice positive-selecting plant RT-PCR qualification result figure, among the figure, and wild-type: the rice plant of wild-type; 1 swimming lane: No. 1 plant of pHB-PHSR transgenic paddy rice; 2 swimming lanes: No. 2 plant of pHB-PHSR transgenic paddy rice; 3 swimming lanes: No. 3 plant of pHB-PHSR transgenic paddy rice.

Fig. 7 is transgenic Fructus Lycopersici esculenti positive-selecting plant RT-PCR qualification result figure, among the figure, and wild-type: the tomato plant of wild-type; 1 swimming lane: No. 1 plant of pBI121-PHSR transgenic Fructus Lycopersici esculenti; 2 swimming lanes: No. 2 plant of pBI121-PHSR transgenic Fructus Lycopersici esculenti; 3 swimming lanes: No. 3 plant of pBI121-PHSR transgenic Fructus Lycopersici esculenti; 4 swimming lanes: No. 4 plant of pBI121-PHSR transgenic Fructus Lycopersici esculenti.

Fig. 8 is No. 1 plant T2 of pHB-PHSR transgenic paddy rice for sprouting result and the growth of seedlings after 10 days figure as a result behind in the solution of the ABA of 0 μ M 4 days of seed and paddy rice wild type seeds, among the figure, (a): the wild-type rice paddy seed is sprouted the plant after 4 days, (b): No. 1 plant T2 of pHB-PHSR transgenic paddy rice is for the plant of seed germination after 4 days, (c): the paddy rice wild type seeds is sprouted the plant after 10 days, and (d): No. 1 plant T2 of pHB-PHSR transgenic paddy rice is for the plant of seed germination after 10 days.

Fig. 9 be pHB-PHSR transgenic paddy rice T2 for seed and wild-type rice paddy seed the germination speed figure in the solution of the ABA of 0 μ M, among the figure, pHB-PHSR-1 is that the T2 of No. 1 plant of pHB-PHSR transgenic paddy rice is for seed; PHB-PHSR-2 is that the T2 of No. 2 plant of pHB-PHSR transgenic paddy rice is for seed; PHB-PHSR-3 is that the T2 of No. 3 plant of pHB-PHSR transgenic paddy rice is for seed.

Figure 10 be pHB-PHSR transgenic paddy rice T2 for seed and wild-type rice paddy seed the germination speed figure in the solution of the ABA of 1.5 μ M, among the figure, pHB-PHSR-1 is that the T2 of No. 1 plant of pHB-PHSR transgenic paddy rice is for seed; PHB-PHSR-2 is that the T2 of No. 2 plant of pHB-PHSR transgenic paddy rice is for seed; PHB-PHSR-3 is that the T2 of No. 3 plant of pHB-PHSR transgenic paddy rice is for seed.

Figure 11 be pHB-PHSR transgenic paddy rice T2 for seed and wild-type rice paddy seed the germination speed figure in the solution of the ABA of 5 μ M, among the figure, pHB-PHSR-1 is that the T2 of No. 1 plant of pHB-PHSR transgenic paddy rice is for seed; PHB-PHSR-2 is that the T2 of No. 2 plant of pHB-PHSR transgenic paddy rice is for seed; PHB-PHSR-3 is that the T2 of No. 3 plant of pHB-PHSR transgenic paddy rice is for seed.

Figure 12 is the tomato wild type seeds, pBI121-PHSR transgenic Fructus Lycopersici esculenti plant T2 is for seed figure as a result of the sprouting after three days in the 1/2MS substratum, among the figure, (a): the wild-type tomatoes seed, (b): No. 1 plant T2 of pBI121-PHSR-1 transgenic Fructus Lycopersici esculenti is for seed, (c): No. 2 plant T2 of pBI121-PHSR-2 transgenic Fructus Lycopersici esculenti are for seed, (d): No. 3 plant T2 of PBI121-PHSR-3 transgenic Fructus Lycopersici esculenti are for seed.

To be No. 1 plant T2 of pBI121-PHSR transgenic Fructus Lycopersici esculenti place the sprouting of 1/2MS substratum after the last 8 day figure as a result that contains different concns ABA for seed and tomato wild type seeds to Figure 13, the left side 4 strains of every width of cloth figure are tomato wild-type plant, and the right 4 strains are that No. 1 T2 of pBI121-PHSR transgenic Fructus Lycopersici esculenti is for plant; Among the figure, (a): 0 μ MABA., (b): 0.5 μ M ABA, (c): 2.5 μ M ABA, (d): 5 μ M ABA, (e): 10 μ M ABA, (f): 50 μ MABA.

Embodiment

Below in conjunction with embodiment, the invention will be further described.Among the following embodiment, all unreceipted concrete experiment conditions, be according to normal condition well known to those skilled in the art, Sambrook for example, the molecular cloning of Russell: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

Embodiment 1: the clone who improves the gene of pre-harvesting sprouting resistance

1, reagent

Restriction enzyme, Taq archaeal dna polymerase, T4DNA ligase enzyme, PrimeStar warm start high-fidelity DNA polymerase, pMD18-T cloning vector etc. are available from Dalian Bao Bio-Engineering Company; Trizol reagent is Time Technology company limited available from sky, Beijing; The reverse transcription test kit is available from Japanese ToYoBo company; Plasmid extracts and DNA reclaims test kit available from company of the excellent brilliant biotechnology in Anhui company limited; The PCR primer is synthetic by the handsome biotech firm in Shanghai; All the other reagent are import packing or homemade analytical pure product.

2, coli strain and vegetable material

The escherichia coli cloning bacterial strain is E.coli JM109, available from Clontech company.The paddy rice wild type seeds is that Japan is fine, and the tomato wild type seeds is AC ⁺, can buy by market.

3, substratum and solution

LB substratum: Tryptones 10g/L, yeast powder 5g/L, NaCl 10g/L.Transfer pH to 7.0, autoclaving with NaOH.

SOB substratum: Tryptones 20g/L, yeast powder 5g/L, NaCl 0.58g/L, KCl 0.19g/L, 100 * Mg ²⁺10mL.Transfer pH to 7.0, autoclaving with NaOH.

SOC substratum: SOB+20mM glucose.

TB buffer (face and use preceding configuration): 1M KCl 4mL, 0.45M MnCl ₂2.4mL, 0.50M CaCl ₂0.6mL, 0.50M K-MES 0.5mL, ddH ₂O 12.5mL (cumulative volume 20mL).

100 * Mg ²⁺Solution: 20.33g MgCl ₂.6H ₂O and 24.65g MgSO ₄.7H ₂The O constant volume is in 100mL H ₂O, autoclaving.

20% glucose solution: 20g glucose constant volume is in 100mL H ₂O, filtration sterilization.

1M KCl solution: 7.45g KCl constant volume is in 100mL H ₂O, autoclaving.

0.45M MnCl ₂Solution: 8.9g MnCl ₂.4H ₂0 constant volume is in 100mL H ₂O, autoclaving.

0.50M CaCl ₂Solution: 7.35g CaCl ₂.2H ₂0 constant volume is in 100mL H ₂O, autoclaving.

0.50M K-MES solution: 9.76g MES constant volume is in 100mL H ₂O transfers pH to 6.3 with KOH, and filtration sterilization is distributed into the every pipe of 0.5mL ,-20 ℃ of storages.

DMSO: the fresh DMSO of packing 200 μ l ,-20 ℃ of storages.

4, experimental technique

4.1 the plasmid trace extracts

1) the E.coli JM109 that will have a cloning vector pMD18-T plasmid (available from Dalian Bao Bio-Engineering Company) is inoculated in and is equipped with in the 5ml LB nutrient solution test tube of (containing an amount of microbiotic), and 37 ℃ of shaking tables are cultivated 12～16hr, with the amplification plasmid.

2) get the bacterium liquid of 1.5～5ml, under room temperature 10, the centrifugal 1min of 000xg.The evacuation substratum adds the mixed liquid of ZL-I/RnaseA (available from the excellent brilliant biotechnology in Anhui company limited) of 250 μ l in the precipitation, and vortex oscillation suspends cell fully again.

3) add 250 μ l ZL-II (available from the excellent brilliant biotechnology in Anhui company limited) in the resuspended mixed liquid, 4～6 mixed solution of test tube of overturning gently are to obtain a clarifying lysate.

4) add 350ul ZL-III (available from the excellent brilliant biotechnology in Anhui company limited) in the above-mentioned mixed liquid, and leniently turn upside down centrifuge tube for several times, until forming white flocks.Under room temperature 10, the centrifugal 10min of 000xg.

5) getting a clean Mu-Pu (available from the excellent brilliant biotechnology in Anhui company limited) plasmid trace separator column is contained in a 2ml and collects on the test tube (being equipped with).Careful sucking-off supernatant, and it is gone in pillar, guaranteeing to go in the supernatant in the post does not have the cell contamination precipitation.Under room temperature 10, the centrifugal 1min of 000xg makes lysate flow through pillar fully.

6) discard the centrifugal liquid that throws away, the ZL damping fluid (available from the excellent brilliant biotechnology in Anhui company limited) that adds 500 μ l is to pillar, and under the room temperature 10, the centrifugal 1min washing of 000xg pillar guarantees to remove remaining protein to obtain the required high quality DNA of back operation.

7) discard collection liquid, add the DNA lavation buffer solution washing pillar of 720 μ l with the dehydrated alcohol dilution, room temperature 10, the centrifugal 10min of 000xg discards washings.

8) the optional step of doing: repeating step 7, the DNA lavation buffer solution with 720 μ l washs pillar once again.

9) under the room temperature 10, the centrifugal void column 1min of 000xg is to dry pillar matrix.

10) pillar is placed on the clean little centrifuge tube of 1.5ml, directly add 50～100 μ l sterilization deionized water or TE damping fluid liquid on pillar matrix (added amount depends on expection end product concentration), 10,000xg is centrifugal, and 1min goes out DNA with wash-out.

4.2DNA recovery scheme

1) handles sepharose-EB electrophoresis miscellany with difference DNA isolation fragment.The agarose of any kind or grade can use.

2) when the spacing of band between line reached enough amount, the dna fragmentation required careful on ultraviolet lamp scaled off.So can guarantee gel that handle contains DNA taking out as much as possible

3) by the gel thin slice being contained in the method that claims its weight in the little centrifuge tube of 1.5ml, determine its volume approx.For example its density is 1g/ml, so the volume of gel just can obtain by the following method: the weight of gel thin slice be 0.2g then its volume be 0.2ml.Adding volume is the NJ damping fluid of 3～4 times of gel volumes, and miscellany is placed 55～65 ℃ of water-bath temperature bath 7min, or melts fully to gel.

4) the DNA-agarose solution of 750 μ l is added to a Mu-Pu DNA and reclaims on the purification column, and pillar is contained in the 2ml collection tube of a dried Net, in 10, the centrifugal 1min of 000xg discards liquid under the room temperature.

5) step is done in choosing: wash pillar with 300 μ l NJ damping fluids, and 10, centrifugal 1min under the 000x g.

6) the DNA lavation buffer solution with the dilution of 750 μ l dehydrated alcohols washs pillar.Under the room temperature 10, the centrifugal 1min of 000xg.

7) discard effluent liquid, repeated for the 6th step once.

8) discard liquid, void column 10, the centrifugal 1min of 000xg is to dry the liquid of base for post matter remnants.

9) pillar is contained on the 1.5ml centrifuge tube of a dried Net, add 30～50 μ l 10, the sterilization deionized water of 000xg centrifugal (the end product concentration that specifically depends on expection) (the perhaps TE damping fluid of PH8.0) on base for post matter, 10, the centrifugal 1min of 000xg is with eluted dna.

4.3 tomato seeds RNA extracts

1) liquid nitrogen grinds the sophisticated seed of tomato rapidly, adds Trizol by 50-100mg tissue/ml Trizol (is Time Technology company limited available from sky, Beijing), concuss, and room temperature is placed 5min.

2) 12, the centrifugal 5min of 000rpm.

3) get supernatant, press 200ul chloroform/ml Trizol and add chloroform, concuss 15s, room temperature is placed 3min.

4) 4 ℃ 12, the centrifugal 15min of 000g.

5) get supernatant, press 0.5ml Virahol/ml Trizol and add the Virahol mixing, room temperature is placed 10min.

6) 4 ℃ 12, the centrifugal 10min of 000g abandons supernatant, and RNA is sunken to the pipe end.

7) add 75% ethanol by 1ml 75% ethanol/ml Trizol, gentle vibration centrifuge tube, precipitation suspends.

8) 4 ℃ 8, the centrifugal 5min of 000g abandons supernatant as far as possible.

9) (the RNA sample is too not dry, otherwise is difficult to dissolving for drying at room temperature 5-10min.)。

10) use 50ul DEPC-H ₂O dissolving RNA sample, 55-60 ℃, 5-10min.

4.4RT-PCR

1)RT

In one 200 μ l EP pipe, add following component on ice:

5×RT?Buffer 4μl

DNTP Mixture (each 10mM) 2 μ l

RNase?Inhibitor(10U/μl)?1μl

Oligo(dT) ₂₀(10pmol/μl) 1μl

Total?RNA Xμl

RNase-Free?H ₂O (11-X)μl

ReverTra?Ace 1μl

Carry out reverse transcription by following program: 4 ℃ of 5min of 99 ℃ of 5min of 42 ℃ of 20min (reaction) (enzyme denaturation) (preservation).

2)PCR

1)) the clone of commentaries on classics paddy rice PHSR gene

In one 200 μ l EP pipes, add following component on ice:

5×PrimeStar?Buffer 10μl

DNTP Mixture (each 2.5mM) 4 μ l

RT product 1 μ l

PHSR-F1 1μl

PHSR-R1 1μl

ddH ₂O 32.5μl

PrimeStar 0.5μl

Increase by following program: 98 ℃ of 3min (pre-sex change); 98 ℃ of 10s (sex change), 58 ℃ of 15s (renaturation), 72 ℃ of 2min (extension), described denaturation renaturation-30 circulations of extension; 72 ℃ of 5min (extending eventually).

With above-mentioned PCR product is template, carries out second with nested primer PHSR-F2 and PHSR-R2 and takes turns high-fidelity PCR, extends time 2min, and other condition is the same.

Primer sequence is as follows:

PHSR-F1：5’-CAAGTCAAAATCAAGAATGAAAAGG-3’

PHSR-R1：5’-ATTGATTTCTCTTTTCCTGGACTAT-3’

PHSR-F2：5’-AGAATGAAAAGGGAGTTAAATGATGT-3’

PHSR-R2：5’-ACTATCCAACAGCTTGTGCAAATG-3’

By aforesaid operations, obtained to be used to make up the PHSR gene of pHB-PHSR paddy rice recombinant expression.

2)) the clone of commentaries on classics tomato PHSR gene

In one 200 μ l EP pipes, add following component on ice:

5×PrimeStar?Buffer 10μl

DNTP Mixture (each 2.5mM) 4 μ l

RT product 1 μ l

PHSR-F1 1μl

PHSR-R1 1μl

ddH ₂O 32.5μl

PrimeStar 0.5μl

Increase by following program: 98 ℃ of 10s (sex change) of 98 ℃ of 3min (pre-sex change), 58 ℃ of 15s (renaturation), 72 ℃ of 2min (extension), described denaturation renaturation-30 circulations of extension; 72 ℃ of 5min (extending eventually).

With above-mentioned PCR product is template, carries out second with nested primer LEPBI-F1 and LEPBI-R1 and takes turns high-fidelity PCR, extends time 2min, and other condition is the same.

Primer sequence is as follows: LEPBI-F1:5 '- TCTAGAATGAAAAGGGAGTTAAATGA-3 '

LEPBI-R1：5’- GAGCTCTATCCAACAGCTTGTGCAAAT-3’

By aforesaid operations, obtained to be used to make up the PHSR gene of pBI121-PHSR tomato recombinant expression.

4.5 high-fidelity PCR product tailing and be connected with cloning vector pMD18-T

Take turns second and to add 1 μ lTaq archaeal dna polymerase in the high-fidelity PCR product, 72 ℃ of reaction 15min get final product.If the PCR product is impure, then must reclaim the purification of target fragment earlier, add an amount of PCR Buffer, dNTPs and Taq archaeal dna polymerase again and carry out tailing.

PCR product behind the tailing is connected than 3/1 by the mole molecule number with cloning vector pMD18-T, and reaction system is as follows:

10×ligase?Buffer 1μl

pMD?18-T(50μg/μl) 1μl

The PCR product of tailing (～150 μ g/ μ l) 1 μ l

Ligase(350U/μl) 1μl

ddH ₂O 6μl

16 ℃ of connections are spent the night.

4.6 intestinal bacteria transform

1) preparation of competent cell

A) inoculate the single bacterium colony of intestinal bacteria in 2mL SOB nutrient solution, 37 ℃ of incubated overnight;

B) transfer the 0.5mL overnight culture to 50mL SOB nutrient solution, 18 ℃ of concuss 18～24h to A ₆₀₀≈ 0.55;

C) nutrient solution is changed in the 50mL centrifuge tube over to ice bath 10min, 4 ℃ of centrifugal 10min of 4000rpm;

D) remove supernatant, add the TB damping fluid suspension cell (noting: rotate gently) of 16mL precooling, ice bath 10min, 4 ℃ of centrifugal 10min of 4000rpm not with vibrator or pressure-vaccum mixing;

E) remove supernatant, add the TB damping fluid suspension cell of 4mL precooling, add the DMSO of 280 μ l, soft mixing, ice bath 10min;

F) be sub-packed in precooling and get in the 1.5mLEP pipe liquid nitrogen cryopreservation.

2) transform

A) taking out a pipe competent cell ice bath from liquid nitrogen thaws;

B) 10 μ l are connected product and competent cell mixing gently, ice bath 30min;

C) 42 ℃ of thermal shocking 90s, ice bath 1-2min immediately;

D) add the SOC of 0.8mL, mixing, 37 ℃ of gentle shaking table 1h.

E) room temperature 13, and the centrifugal 1min of 000rpm outwells a part of supernatant liquor, stays the supernatant liquor of about 200 μ l, with supernatant liquor and cell mixing, are coated with LB+amp (50 μ g/ml) flat board, 37 ℃ of overnight incubation with the rifle head.

4.7 the quick cracking process of cell is identified recombinant plasmid

1) the single transformant of picking is inoculated in 500 μ l and contains in the corresponding antibiotic LB nutrient solution, and 37 ℃ of shaking culture are to A ₆₀₀Be 0.6～0.8.

2) get 200 μ l bacterium liquid to 0.5ml EP pipe, 13, the centrifugal 1min of 000rpm removes supernatant, stays about 20 μ l supernatants.

3) add 20 μ l 2 * quick lysate [0.2mol/L NaOH 50mL+SDS 0.5g, sucrose 27.2g adds distilled water to 200ml], thermal agitation.

4) the centrifugal 15min of 13000rpm.

5) get the direct electrophoresis of 5 μ l supernatants.With contrast ratio, what electrophoresis band lagged behind promptly may be recombinant vectors.

4.8 bacterium colony PCR identifies recombinant plasmid

Be bacterium colony PCR again to determine that inserting fragment is target fragment through the recombinant plasmid that quick cracking process is identified, reaction system is as follows:

10×PCR?Buffer 2μl

Mg ²⁺(1.5mM) 1.2μl

DNTP Mixture (each 2.5mM) .6 μ l

Bacterium liquid 1 μ l

PHSR--F2 0.4μl

PHSR-R2 0.4μl

ddH ₂O 12.4μl

Taq archaeal dna polymerase 1 μ l

Reaction conditions: 94 ℃ of 5min (pre-sex change); 94 ℃ of 40s (sex change), 58 ℃ of 30s (renaturation), 72 ℃ of 2min (extension), described denaturation renaturation-30 circulations of extension; 72 ℃ of 5min (extending eventually).

The recombinant plasmid that bacterium colony PCR is determined checks order, and sequencing result is the nucleotide sequence shown in the SEQ ID NO:1 in the sequence table.

Embodiment 2: the expression of gene in paddy rice and tomato that improves pre-harvesting sprouting resistance

1, material

1.1 reagent and material

Conventional reagent is identical with embodiment 1.

Being used for genetically modified agrobacterium strains is EHA105 (available from Clontech company); Carrier for expression of eukaryon pHB and PBI121 (available from Dalian Bao Bio-Engineering Company).

1.2 substratum and solution

YEB substratum: yeast extract 1g/L, extractum carnis 5g/L, peptone 5g/L, sucrose 5g/L, MgSO ₄.7H ₂O 0.5g/L.Transfer pH to 7.0, autoclaving with NaOH.YEP substratum: yeast extract 10g/L, peptone 10g/L, NaCl 5g/L.Transfer pH to 7.0, autoclaving with NaOH.

The conventional tissue culture medium (TCM) of using:

2, method

2.1PHSR the structure of the eukaryon recombinant plasmid that gene is expressed in paddy rice and tomato

2.1.1 it is as follows to make up the primer be used for the eukaryon recombinant plasmid (pHB-PHSR) of expressing in paddy rice:

PHSR-F2：5’-AGAATGAAAAGGGAGTTAAATGATGT-3’

PHSR-R2：5’-ACTATCCAACAGCTTGTGCAAATG-3’

Increase by following program: 94 ℃ of 5min (pre-sex change); 94 ℃ of 40s (sex change), 58 ℃ of 30s (renaturation), 72 ℃ of 2min (extension), described denaturation renaturation-30 circulations of extension; 72 ℃ of 5min (extending eventually).

High-fidelity PCR product carries out general T ag enzyme tailing then after electrophoresis reclaims.Again the fragment of tailing is connected with the pMD18-T cloning vector, transformed into escherichia coli (JM109) then, chose mono-clonal in second day, after carrying the positive colony plasmid, cut PstI and XbaI double digestion with restriction enzyme then, at last be connected (connection site: PstI and XbaI), obtain to contain the overexpression eukaryon recombinant plasmid of PHSR gene, called after pHB-PHSR (Fig. 1) with the pHB carrier of cutting with same restrictions enzyme enzyme.

2.1.2 it is as follows to make up the primer be used for the eukaryon recombinant plasmid (PBI121-PHSR) of expressing tomato:

LEPBI-F1：5’- TCTAGAATGAAAAGGGAGTTAAATGA-3’(XbaI)

LEPBI-R1：5’- GAGCTCTATCCAACAGCTTGTGCAAAT-3(SacI)

Increase by following program: 94 ℃ of 5min (pre-sex change); 94 ℃ of 40s (sex change), 58 ℃ of 30s (renaturation), 72 ℃ of 2min (extension), described denaturation renaturation-30 circulations of extension; 72 ℃ of 5min (extending eventually).

High-fidelity PCR product carries out general T ag enzyme tailing then after electrophoresis reclaims.Again the fragment of tailing is connected with the pMD18-T cloning vector, transformed into escherichia coli (JM109) then, chose mono-clonal in second day, after carrying the positive colony plasmid, cut XbaI and SacI double digestion with restriction enzyme then, at last be connected (connection site: XbaI and SacI), obtain to contain the overexpression eukaryon recombinant plasmid of PHSR gene, called after pBI121-PHSR (Fig. 2) with the pBI121 carrier of cutting with same restrictions enzyme enzyme.

2.2 the preparation of Agrobacterium competent cell

1) the single bacterium colony of picking Agrobacterium is in the YEB of 2ml liquid nutrient medium (containing Rif 50 μ g/ml), and 28 ℃ of shaking culture are spent the night;

2) get incubated overnight liquid 500 μ l and transfer in 50ml YEB (containing Rif 50 μ g/ml) liquid nutrient medium, 28 ℃ of shaking culture are to OD ₆₀₀=0.5;

3) 4 ℃ of centrifugal 5min of 5000rpm collect thalline, add the NaCl solution suspension thalline of 10ml 0.15M, ice bath 10min;

4) 4 ℃ of centrifugal 5min of 5000rpm collect thalline, with the 20mM CaCl of 1ml precooling ₂Solution suspension thalline, ice bath 10min;

5) cell for preparing uses immediately, or is distributed into 200 μ l/ pipe, quick-frozen 1min in the liquid nitrogen, and it is standby to put-70 ℃ of preservations.

2.3 the conversion of Agrobacterium

1) gets 200 μ l competent cells, thaw on ice;

2) add the eukaryon recombinant plasmid that 1 μ g builds, flick mixing, ice bath 30min;

3) quick-frozen 1min in the liquid nitrogen, 37 ℃ of water-bath 5min add 1ml YEB substratum then, 28 ℃ of shaking culture 4h at a slow speed;

4) culture is coated on the YEB flat board that contains 50 μ g/ml Kan and 50 μ g/ml Rif, cultivated about 48h for 28 ℃.

2.4 the evaluation of Agrobacterium positive colony

The single bacterium colony that grows on the picking flat board is inoculated in the YEB liquid nutrient medium that contains 50 μ g/ml Kan and 50 μ g/ml Rif, and 28 ℃ of shaking culture are spent the night, and is that template is carried out the pcr amplification evaluation with bacterium liquid.

2.5 agrobacterium-mediated transformation rice transformation

2.5.1 inducing and subculture of embryo callus

Manual the shelling of the fine mature seed of paddy rice Japan, 75% ethanol disinfection 1 minute, the vibration sterilization is 25 minutes among 25% chlorine bleach liquor, and sterile purified water washes 3 times again, is inoculated into then on the inducing culture, and evoked callus forms under the 24-26 ℃ of dark culture condition.

2.5.2 the pre-cultivation of callus

Choose the particulate state callus that nature dispersion, color cadmium yellow, diameter are about 2-3mm, be transferred in the pre-culture medium, place 27 ℃ of dark cultivations 4 days.

2.5.3 cultivation of Agrobacterium and processing

The bacterium liquid that scraping from-80 ℃ of frozen pipes of low temperature takes a morsel contains pHB-PHSR plant overexpression carrier is in additional kantlex (Kanamycin, Kan) 50mg/L and Rif 50mg/L YEB solid medium line activates at 28 ℃ of dark 36-72h that cultivate then; Bacterium is drawn in single bacterium colony switching of getting on the activation flat board, 28 ℃ of cultivations two days later, with the AAM substratum that is added with 100 μ M/L Syringylethanones in right amount with the bacterium wash-out, be suspended in and be added with in the 100 μ M/L Syringylethanone 20ml AAM substratum, after acutely shaking 1min, adjust bacterial concentration to OD600nm=0.8-1.0, leave standstill 1h, allow Agrobacterium form suspension.

2.5.4 cultivate altogether

The pre-incubated callus of learning from else's experience adds the Agrobacterium bacterium liquid of above-mentioned processing in the culturing bottle of sterilization, leave standstill 15min after shaking slightly, is inoculated in common substratum after drying callus on the aseptic filter paper, 25 ℃ of dark 3d that cultivate.

2.5.5 eccysis Agrobacterium

Callus after picking is cultivated is altogether used aseptic water washing 3-5 time in the wide-mouth culturing bottle, shake for several times at every turn, loses thread thalline in water.The last sterilized water that contains 250mg/L carboxylic Bian penicillin of using leaves standstill 1h, places on the aseptic filter paper then and dries.

2.5.6 the screening of callus

Callus is transferred to the selection substratum screening kanamycin-resistant callus tissue that contains hygromycin resistance, per two week switchings 1 time.

2.5.7 the subculture of resistant calli and the regeneration of plant

Per two weeks are transferred to callus on the new selection substratum, and to need for three weeks approximately be visible warty aureus kanamycin-resistant callus tissue grows from the shrivelled callus of brownization.Treat that a part of selecting kanamycin-resistant callus tissue after callus is grown up is transferred on the division culture medium, about 2 weeks or longer time back callus begin to change green, can put out new shoots after 3 weeks or longer time, and root also grows subsequently.Seedling is moved on the root media 1 clone of every culturing bottle.Treat seedling take root grow up to after, shift out culturing bottle, clean the substratum on the root after, move to greenhouse pot culture.

2.6 agrobacterium-mediated transformation transforms tomato

The eukaryon recombinant plasmid pBI121-PHSR that builds changes in the Agrobacterium standby.

Soak tomato seeds 10min with 10% clorox, use sterile water wash again 4～5 times, on the 1/2MS substratum, cultivate after blotting seed-coat moisture with aseptic filter paper, 25 ℃, secretly cultivate about 4d, show money or valuables one carries unintentionally the back transposition under illumination, illumination 1500lx, 16h/d takes off cotyledon before its rough leaf grows, place and cultivate 2d on the pre-culture medium.Conversion method adopts Ye Panfa.28 ℃ of incubated overnight of Agrobacterium that will have goal gene, 5000r/min, 5min, centrifugal under the room temperature, remove supernatant liquor, Agrobacterium is diluted to OD=0.1, the cotyledon of cultivating 2d is in advance immersed wherein 10～15min with inducing culture, outwell bacterium liquid cotyledon is placed on the aseptic filter paper, blot to forward on the common substratum behind the unnecessary bacterium liquid and cultivate 3d.Change subculture screening and culturing on the regeneration culture medium then over to, per 3 weeks are changed a subculture, when treating indefinite bud length to the 3cm left and right sides, downcut to be transferred on the root media and take root.Deng root development good after, shift out greenhouse pot culture.

(1) pre-culture medium: MS+1mg/L 6BA+0.04mg/L IAA.(2) inducing culture (100mL): 5mLAB salt+2mL MES buffer reagent+2mL sodium phosphate salt buffer reagent+91mL 1% glucose.(3) be total to substratum: MS+0.2mg/L KH2PO4+0.1mg/L Kinetin+0.2mg/L 2,4-D+15mg/L Syringylethanone.(4) regeneration culture medium: MS+500mg/L carboxylic Bian benzylpenicillin sodium+50mg/L sulphuric acid kanamycin+2mg/L6-BA+0.2mg/L IAA.(5) root media: MS+500mg/L carboxylic Bian benzylpenicillin sodium+2mg/L IAA.More than the pH value of each substratum be 6.0.

2.7 the evaluation of transfer-gen plant

2.7.1 the extraction of genomic dna

(1) gets the fresh material of 100mg, under the annex solution nitrogen status, be ground into fine powder, be sub-packed in the 1.5mL eppendorf pipe, 2 * CTAB that every pipe adds 65 ℃ of preheatings of 500 μ L extracts damping fluid (100mmol/L Tris-Hcl pH 8.0,20mmol/L EDTA pH 8.0,1.4mol/L NaCl, 40mmol/L 2 mercapto ethanol, 2%CTAB) mixing.

(2) 65 ℃ of water bath heat preservation 60min are cooled to room temperature, add isopyknic chloroform.

(3) the centrifugal 10min of 5000g gets supernatant liquor, adds isopyknic Virahol, and room temperature is placed 15min, deposit D NA.

(4) the centrifugal 10min of 12000g abandons supernatant and stays precipitation, washes twice at least with 70% ethanol, dries up precipitation.

(5) precipitation is dissolved among the 200 μ L TE, adds RNase A (10mg/mL), 37 ℃ of insulation 30min add isopyknic phenol/chloroform, mixing.

(6) the centrifugal 10min of 12000g gets supernatant, adds the dehydrated alcohol of 1/10 volume 3mol/L NaAc and 2.5 times of volumes, and room temperature is placed 10min.

(7) the centrifugal 10min of 12000g abandons supernatant, with drying up precipitation behind 70% alcohol flushing, is dissolved among the 20 μ L sterilization ddH2O.

2.7.2 the genomic dna PCR of paddy rice and tomato detects

In order to determine that the PHSR gene has been incorporated into the genome of positive-selecting paddy rice and tomato, we are template with paddy rice and tomato dna group DNA respectively, to PHSR full length gene encoding sequence, paddy rice hygromycin resistance marker gene (HPT) and tomato kalamycin resistance marker gene (NPTII) have been carried out pcr amplification respectively.The PHSR gene specific primer is lseq-F1 and lseq-R1, and the hygromycin gene special primer is HPT-f and HPT-r, and the kalamycin resistance gene special primer is NPTII F1 and NPTII R1.

lseq-F1：5’-CACGAGCTCCGAAACGATTGGGAAGTACTA-3’

lseq-R1：5’-GACCTGCAGTGATTCTGAGTCTGATGACGATA-3’

Increase by following program: 94 ℃ of 5min (pre-sex change); 94 ℃ of 40s (sex change), 58 ℃ of 40s (renaturation), 72 ℃ of 40s (extension), described denaturation renaturation-30 circulations of extension; 72 ℃ of 5min (extending eventually)

HPT-f：：5′-TCGTTATGTTTATCGGCACTTTG-3′

HPT-r：5′-GCGTCTGCTGCTCCATACAAG-3′

Increase by following program: 94 ℃ of 5min (pre-sex change); 94 ℃ of 40s (sex change), 58 ℃ of 40s (renaturation), 72 ℃ of 40s (extension), described denaturation renaturation-30 circulations of extension; 72 ℃ of 5min (extending eventually)

NPTII-F1：5’-TCTCATGCTGGAGTTCTTCGC-3’

NPTII-R1：5’-GTCACCGACTTGAGCCATTTG-3’

Increase by following program: 94 ℃ of 5min (pre-sex change); 94 ℃ of 40s (sex change), 60 ℃ of 40s (renaturation), 72 ℃ of 1min (extension), described denaturation renaturation-30 circulations of extension; 72 ℃ of 5min (extending eventually).

2.7.3 transgenic paddy rice positive-selecting plant RT-PCR identifies

Because the expression of PHSR gene in the wild-type paddy rice has tissue specificity, it is only expressed in sophisticated seed, do not express in other tissue, and PHSR is a constitutive expression in the pHB-PHSR transgenic paddy rice.So, can identify the expression of PHSR gene on transcriptional level by being the RT-PCR of wild-type rice seedling and pHB-PHSR transgenic paddy rice seedling.So we get wild-type paddy rice (Japan fine) and the strain of pHB-PHSR transgenic positive 3 independently the mature seed in T2 generation of plant send out seedling, after 20 days, extract the mRNA of seedling respectively, after the reverse transcription, be RT-PCR and analyze, simultaneously we with paddy rice internal control gene (Actin) as contrast.PHSR Gene RT-PCR amplimer is: RT-PHSRF1 and RT-PHSR1; Actin Gene RT-PCR amplimer is: RT-ActinF1 and RT-ActinR1.

RT-PHSRF1：5’-GTTCACCGATTCGTCCTCTG-3’

RT-PHSRR1：5’-GGATTGTTAGCTTGAGATATTGC-3’

Increase by following program: 94 ℃ of 5min (pre-sex change); 94 ℃ of 40s (sex change), 55 ℃ of 40s (renaturation), 72 ℃ of 30s (extension), described denaturation renaturation-26 circulations of extension; 72 ℃ of 5min (extending eventually)

RT-ActinF1：5′-AAGATCCTGACGGAGCGTGGTTAC-3′

RT-ActinR1：5′CTTCCTAATATCCACGTCGCACTTC-3′

Increase by following program: 94 ℃ of 5min (pre-sex change); 94 ℃ of 40s (sex change), 60 ℃ of 40s (renaturation), 72 ℃ of 30s (extension), described denaturation renaturation-26 circulations of extension; 72 ℃ of 5min (extending eventually)

2.7.4 transgenic Fructus Lycopersici esculenti positive-selecting plant RT-PCR identifies

Because the expression of PHSR gene in wild-type tomatoes has tissue specificity, it is only expressed in sophisticated seed, do not express in other tissue, and PHSR is a constitutive expression in the pBI121-PHSR transgenic Fructus Lycopersici esculenti.So, can identify the expression of PHSR gene on transcriptional level by being the RT-PCR of wild-type tomatoes seedling and pBI121-PHSR transgenic Fructus Lycopersici esculenti seedling.So we get wild-type tomatoes (AC+) and the strain of pBI121-PHSR transgenic positive 4 independently the mature seed in T2 generation of plant send out seedling, after 10 days, extract the mRNA of seedling respectively, after the reverse transcription, be RT-PCR and analyze, simultaneously we with tomato internal control gene (UBI3) as contrast.PHSR gene RT-PCR amplimer is: RT-PHSRF1 and RT-PHSR1; UBI3 Gene RT-PCR amplimer is: RT-UBI3F1 and RT-UBI3R1.

RT-PHSRF1：5’-GTTCACCGATTCGTCCTCTG-3’

RT-PHSRR1：5’-GGATTGTTAGCTTGAGATATTGC-3’

Increase by following program: 94 ℃ of 5min (pre-sex change); 94 ℃ of 40s (sex change), 55 ℃ of 40s (renaturation), 72 ℃ of 30s (extension), described denaturation renaturation-30 circulations of extension; 72 ℃ of 5min (extending eventually)

RT-UBI3F1：5’-AGAAGAAGACCTACACCAAGCC-3’

RT-UBI3R1：5’-TCCCAAGGGTTGTCACATACATC-3’

Increase by following program: 94 ℃ of 5min (pre-sex change); 94 ℃ of 40s (sex change), 60 ℃ of 40s (renaturation), 72 ℃ of 30s (extension), described denaturation renaturation-26 circulations of extension; 72 ℃ of 5min (extending eventually)

2.8 transgenic paddy rice seed and wild-type rice paddy seed are sprouted experiment

The selective maturation degree is good, three different plants (pHB-PHSR-1 of the wild-type rice paddy seed of full seed and transgenic paddy rice, pHB-PHSR-2 and pHB-PHSR-3) T2 for mature seed each 40, evenly be positioned in the culture dish that has double-deck filter paper, be immersed in and contain different ABA (0 μ M, 1.5 μ M and 5 μ M) in the distilled water handled of concentration, the quantity of with the naked eye adding up the rice paddy seed that germinates and do not germinate every 24h.Treat in the culture dish to take a picture when transgenic paddy rice and wild-type rice seedling have enough difference, it is long and root is long (seedling of each independent plant is surveyed 10 strains, and mean value is removed in the triplicate experiment at last) to measure seedling.

2.9 transgenic Fructus Lycopersici esculenti seed and the experiment of wild-type tomatoes seed germination

The selective maturation degree is good, the T2 of three different plants (pBI121-PHSR-1, pBI121-PHSR-2 and pBI121-PHSR-3) of the wild-type tomatoes seed of full seed and transgenic Fructus Lycopersici esculenti is for becoming each 50 in seed, with 70% alcohol rinsing 1min; Be soaked among the chlorine bleach liquor of active chlorine content 2～5% vibration 15～20min then; With sterilized water washing 4～5 times, blot redundant moisture with sterilization filter paper, be inoculated on the 1/2MS substratum of the processing that contains different ABA (0 μ M., 0.5 μ M, 2.5 μ M, 5 μ M, 10 μ M, 50 μ M) concentration behind the 8d sprouting situation of observation seed and take pictures.

3, result

3.1 the evaluation of transfer-gen plant

3.1.1 transgenic paddy rice positive-selecting plant PCR qualification result

Obtain 40 strain reuse water rice plants through tissue culture and resistance screening, identify through PCR and successfully to have obtained 15 strain transgenic paddy rices, Fig. 3 carries out hygromycin resistance selection markers gene (HPT) pcr amplification result for the part transgenic rice plant, amplification shows that resistant plant all amplifies and positive control amplified production specific band of the same size, proves that the HPT gene has been incorporated in the genome of rice transformation; Positive plant carries out pcr amplification with the special primer of tomato PHSR gene to this Gene Partial sequence fragment again, the result shows that the positive transfer-gen plant special length that all increases is the specific band of 568bp, shows that foreign gene PHSR has been incorporated into (Fig. 4) in the rice genome

3.1.2 transgenic Fructus Lycopersici esculenti positive-selecting plant PCR qualification result

Obtain 30 strains regeneration tomato plant through tissue culture and resistance screening, identify through PCR and successfully to have obtained 12 strain transgenic paddy rices, Fig. 5 carries out kalamycin resistance selection markers gene (NPTII) pcr amplification result for part transgenic Fructus Lycopersici esculenti plant, amplification shows that resistant plant all amplifies and positive control amplified production specific band of the same size, proves that the NPTII gene has been incorporated in the genome of rice transformation.

3.1.3 transgenic paddy rice positive-selecting plant RT-PCR qualification result

After identifying, find 3 of the pHB-PHSR transgenic paddy rice independently the amount of the mRNA of plant (pHB-PHSR-1, pHB-PHSR-2 and pHB-PHSR-3) PHSR obviously raise, and detect mRNA (Fig. 6) in the wild-type paddy rice less than PHSR.

3.1.4 transgenic Fructus Lycopersici esculenti positive-selecting plant RT-PCR qualification result

After identifying, find 4 of the pBI121-PHSR transgenic Fructus Lycopersici esculenti independently plant (pBI121-PHSR-1, pBI121-PHSR-2, pBI121-PHSR-3 and pBI121-PHSR-4) amount of mRNA of PHSR obviously raises, and detect mRNA (Fig. 7) in the wild-type tomatoes less than PHSR.

3.2 transgenic paddy rice seed and wild-type rice paddy seed are sprouted experimental result

In the early stage of sprouting, the T2 of pHB-PHSR transgenic rice plant will be well below the germination speed (Fig. 8) of wild-type rice paddy seed for seed germination speed.After sprouting 2 days, the T2 of wild-type rice paddy seed and pHB-PHSR transgenic rice plant does not have to sprout for seed; After sprouting 3 days, the germination rate 30% of wild-type rice paddy seed, and the T2 of pHB--PHSR transgenic rice plant does not have to sprout for seed; After sprouting 4 days, the germination rate 100% of wild-type rice paddy seed, and the T2 of pHB-PHSR transgenic rice plant has only small part to sprout for seed, germination rate is less than 30%; After sprouting 5 days, the T2 of pHB-PHSR transgenic rice plant reaches about 50% for seed germination rate; After this in time, the T2 of pHB-PHSR transgenic rice plant occurs rising significantly for the germinating energy rate of seed early stage, sprouts after 8 days, and the T2 of pHB-PHSR transgenic rice plant is for basic all germinate (Fig. 9) of seed.And along with the T2 of the rising transgenic rice plant of ABA concentration for the germination speed of seed decrease (Figure 10, Figure 11).This shows, the PHSR gene can suppress rice paddy seed germinating energy, reduce speed of germination, but to the nothing influence of the final percentage of germination of transgenic seed.In sum, the PHSR gene is sprouted resistance at the fringe that the intravital overexpression of rice plants can improve paddy rice effectively.

3.3 transgenic Fructus Lycopersici esculenti seed and wild-type tomatoes seed germination experimental result

The early stage of sprouting, the T2 of pBI121-PHSR transgenic Fructus Lycopersici esculenti plant will be well below the germination speed (Figure 12) of wild-type tomatoes seed for the germination speed of seed, and the T2 of wild-type tomatoes seed and pBI121-PHSR transgenic Fructus Lycopersici esculenti plant is put into the situation of observing the sprouting of seed in the 1/2MS substratum that the ABA of different concns handles for seed.After 8 days that sprout, find: along with the rising of ABA concentration, the T2 of pBI121-PHSR transgenic Fructus Lycopersici esculenti plant compares with the wild-type tomatoes seed for seed, the suffered inhibition of seed germination is also serious more, under the condition of 0 μ M ABA, the T2 of pBI121-PHSR transgenic Fructus Lycopersici esculenti plant can both sprout normally for seed and wild-type tomatoes seed, at 0.5 μ M, 2.5 μ M, under the concentration of 5 μ M ABA, the inhibition that the T2 of pBI121-PHSR transgenic Fructus Lycopersici esculenti plant is subjected to for the sprouting of seed is serious than wild-type tomatoes seed obviously, at 10 μ M, under the ABA concentration of 50 μ M, the T2 of pBI121-PHSR transgenic Fructus Lycopersici esculenti plant can not sprout normally for seed, but the wild-type tomatoes seed can also normally sprout, but growth of seedlings and growth also have been subjected to obvious suppression (Figure 13).This shows that the PHSR gene can effectively suppress the sprouting of seed at the intravital overexpression of tomato plants, thereby the fringe that improves tomato is sprouted resistance.

Sequence table

＜110〉Sichuan University

＜120〉a kind of eukaryon recombinant plasmid and application thereof that improves pre-harvesting sprouting resistance

<160>2

<170>PatentIn?Version?3.2

<210>1

<211>1754

<212>DNA

＜213〉tomato (Lycopersicon esculentum)

<220>

<221>mRNA

<222>(1).....(1754)

＜223〉gene of raising pre-harvesting sprouting resistance

<400>1

aagttgtcaa?gtcaaaatca?agaatgaaaa?gggagttaaa?tgatgtttat?tatggtggca?60

acaaagaaga?tgtgcatgga?tttgagaata?ttaatatggt?gcctactgat?tttgatccta?120

tggatgatac?agatatttgg?ttaaatggaa?atttctctaa?tgattttact?tctcttcaag?180

attttccatg?catgtcttct?tcatcatcaa?cttccaattc?ccttcctact?gaacaatctg?240

atccatcttc?tggttgggct?gttcaaaaat?ctgatgctga?tgagcaagat?ttcgacacaa?300

ttagtgatca?agaatgtcta?aatgtgatgg?atttaatcga?cggggatcat?gaatttcttg?360

accctatgat?atcttttttc?aatcaacaac?aaaaagaaca?agcaaatgag?gagcaagtgt?420

ctatttttca?aggggatagt?gaacttgcac?taatgttttt?ggattggtta?aagcaaaaca?480

aagataacat?ttctgctgaa?gatatgagga?gtattaagct?taaacgttcg?acaattgaga?540

gcgcgtcgaa?acgattggga?agtactaaag?aagggaaaaa?acagttgttg?agactcattc?600

ttgattgggt?tgaacaacac?aggttgcaaa?agaaacaaat?gagagaggaa?cagagtatcc?660

aaaactctgc?cccttttaac?tttaataatc?caaatgcttg?cttttataat?gcctcgttca?720

ccgattcgtc?ctctgtaatg?acagggccaa?tacaggggta?cttcgtgcct?ccgtataatc?780

aaacaatgag?tggaagttca?acttctcagt?cctggtctca?gtcgcaattt?ataatggcga?840

acgcttctca?atacaatcga?tttcctgaga?ataatattac?gaataatgtt?gctatacctg?900

atcagccttt?attcagtgct?caatatgatc?agtaccaaat?tttcgatggg?agcggtgaga?960

gattggcaag?gttgggcact?tgtgctacta?aagaagccag?gaagatcaga?atggctaggc?1020

agagacgagt?accattgcat?cattatcgtc?atcagactca?gaatcaaaga?caaattagta?1080

atgagaaaag?tgtaatgatg?ggtagaaaga?ttaataattg?tgcaatatct?caagctaaca?1140

atccaggaaa?ttgggtatat?tggccttgtg?ctgctgctgc?tccgccaatc?gccatggtac?1200

cattggctga?tactccgcaa?tcacttccta?tggagaggtc?acccgtgcaa?tcacagaaac?1260

atcagaaaaa?cggttcaaca?gacaaaagac?aggcttgcaa?aacagagaag?aatctcaaat?1320

ttctcatgca?aaaagtgctg?aagcaaagtg?atgttggcca?tctaggaaga?attgtgttac?1380

caaagaaaga?agcagaaagt?catctcccgc?aacttgaaac?aagagacgga?atctcaattg?1440

ccatggaaga?cattgggact?tgtcgtgttt?ggaacatgaa?atatagattt?tggccaaata?1520

acaaaagcag?gatgtacctt?ctcgagaaca?caggtgattt?tgtcgtagct?aatggacttc?1580

aagaaggtga?tttcatagta?atatacgccg?acataaagtg?tggcaaatat?ttgatacgag?1620

gggtaaaagt?gaggccgaat?ggagcaaaat?cagatggcat?gcagccagca?aagaaaattg?1680

ttcgtaaaat?agctgctgtt?gcgtcatctc?catttgcaca?agctgttgga?tagtccagga?1740

aaagagaaat?caat 1754

<210>2

<211>569

<212>PRT

＜213〉tomato (Lycopersicon esculentum)

＜223〉polypeptide of raising stress resistance of plant

<400>2

MET?Lys?Arg?Glu?Leu?Asn?Asp?Val?Tyr?Tyr?Gly?Gly?Asn?Lys?Glu

1 5 10 15

Asp?Val?His?Gly?Phe?Glu?Asn?Ile?Asn?MET?Val?Pro?Thr?Asp?Phe

20 25 30

Asp?Pro?MET?Asp?Asp?Thr?Asp?Ile?Trp?Leu?Asn?Gly?Asn?Phe?Ser

35 40 45

Asn?Asp?Phe?Thr?Ser?Leu?Gln?Asp?Phe?Pro?Cys?MET?Ser?Ser?Ser

50 55 60

Ser?Ser?Thr?Ser?Asn?Ser?Leu?Pro?Thr?Glu?Gln?Ser?Asp?Pro?Ser

65 70 75

Ser?Gly?Trp?Ala?Val?Gln?Lys?Ser?Asp?Ala?Asp?Glu?Gln?Asp?Phe

80 85 90

Asp?Thr?Ile?Ser?Asp?Gln?Glu?Cys?Leu?Asn?Val?MET?Asp?Leu?Ile

95 100 105

Asp?Gly?Asp?His?Glu?Phe?Leu?Asp?Pro?MET?Ile?Ser?Phe?Phe?Asn

110 115 120

Gln?Gln?Gln?Lys?Glu?Gln?Ala?Asn?Glu?Glu?Gln?Val?Ser?Ile?Phe

125 130 135

Gln?Gly?Asp?Ser?Glu?Leu?Ala?Leu?MET?Phe?Leu?Asp?Trp?Leu?Lys

140 145 150

Gln?Asn?Lys?Asp?Asn?Ile?Ser?Ala?Glu?Asp?MET?Arg?Ser?Ile?Lys

155 160 165

Leu?Lys?Arg?Ser?Thr?Ile?Glu?Ser?Ala?Ser?Lys?Arg?Leu?Gly?Ser

170 175 180

Thr?Lys?Glu?Gly?Lys?Lys?Gln?Leu?Leu?Arg?Leu?Ile?Leu?Asp?Trp

185 190 195

Val?Glu?Gln?His?Arg?Leu?Gln?Lys?Lys?Gln?MET?Arg?Glu?Glu?Gln

200 205 210

Ser?Ile?Gln?Asn?Ser?Ala?Pro?Phe?Asn?Phe?Asn?Asn?Pro?Asn?Ala

215 220 225

Cys?Phe?Tyr?Asn?Ala?Ser?Phe?Thr?Asp?Ser?Ser?Ser?Val?MET?Thr

230 235 240

Gly?Pro?Ile?Gln?Gly?Tyr?Phe?Val?Pro?Pro?Tyr?Asn?Gln?Thr?MET

245 250 255

Ser?Gly?Ser?Ser?Thr?Ser?Gln?Ser?Trp?Ser?Gln?Ser?Gln?Phe?Ile

260 265 270

MET?Ala?Asn?Ala?Ser?Gln?Tyr?Asn?Arg?Phe?Pro?Glu?Asn?Asn?Ile

275 280 285

Thr?Asn?Asn?Val?Ala?Ile?Pro?Asp?Gln?Pro?Leu?Phe?Ser?Ala?Gln

290 295 300

Tyr?Asp?Gln?Tyr?Gln?Ile?Phe?Asp?Gly?Ser?Gly?Glu?Arg?Leu?Ala

305 310 315

Arg?Leu?Gly?Thr?Cys?Ala?Thr?Lys?Glu?Ala?Arg?Lys?Ile?Arg?MET

320 325 330

Ala?Arg?Gln?Arg?Arg?Val?Pro?Leu?His?His?Tyr?Arg?His?Gln?Thr

335 340 345

Gln?Asn?Gln?Arg?Gln?Ile?Ser?Asn?Glu?Lys?Ser?Val?MET?MET?Gly

350 355 360

Arg?Lys?Ile?Asn?Asn?Cys?Ala?Ile?Ser?Gln?Ala?Asn?Asn?Pro?Gly

365 370 375

Asn?Trp?Val?Tyr?Trp?Pro?Cys?Ala?Ala?Ala?Ala?Pro?Pro?Ile?Ala

380 385 390

MET?Val?Pro?Leu?Ala?Asp?Thr?Pro?Gln?Ser?Leu?Pro?MET?Glu?Arg

395 400 405

Ser?Pro?Val?Gln?Ser?Gln?Lys?His?Gln?Lys?Asn?Gly?Ser?Thr?Asp

410 415 420

Lys?Arg?Gln?Ala?Cys?Lys?Thr?Glu?Lys?Asn?Leu?Lys?Phe?Leu?MET

425 430 435

Gln?Lys?Val?Leu?Lys?Gln?Ser?Asp?Val?Gly?His?Leu?Gly?Arg?Ile

440 445 450

Val?Leu?Pro?Lys?Lys?Glu?Ala?Glu?Ser?His?Leu?Pro?Gln?Leu?Glu

455 460 465

Thr?Arg?Asp?Gly?Ile?Ser?Ile?Ala?MET?Glu?Asp?Ile?Gly?Thr?Cys

470 475 480

Arg?Val?Trp?Asn?MET?Lys?Tyr?Arg?Phe?Trp?Pro?Asn?Asn?Lys?Ser

485 490 495

Arg?MET?Tyr?Leu?Leu?Glu?Asn?Thr?Gly?Asp?Phe?Val?Val?Ala?Asn

500 505 510

Gly?Leu?Gln?Glu?Gly?Asp?Phe?Ile?Val?Ile?Tyr?Ala?Asp?Ile?Lys

515 520 525

Cys?Gly?Lys?Tyr?Leu?Ile?Arg?Gly?Val?Lys?Val?Arg?Pro?Asn?Gly

530 535 540

Ala?Lys?Ser?Asp?Gly?MET?Gln?Pro?Ala?Lys?Lys?Ile?Val?Arg?Lys

545 550 555

Ile?Ala?Ala?Val?Ala?Ser?Ser?Pro?Phe?Ala?Gln?Ala?Val?Gly

560 565

Claims (1)

1. be inserted into the application of eukaryon recombinant plasmid in the pre-harvesting sprouting resistance improvement that obtains in the carrier for expression of eukaryon by the described nucleotide sequence of SEQ ID NO:1 in the sequence table, described plant is paddy rice and tomato, and described carrier for expression of eukaryon is a kind of among pHB, pMON1772, pBE12, pBC7, the pBI121.

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