CN109295075A

CN109295075A - NfOCP1 anti-drought gene, the amino acid sequence of its coding and its purposes in raising plant drought resistance

Info

Publication number: CN109295075A
Application number: CN201811285409.7A
Authority: CN
Inventors: 叶水烽; 高宁宁; 吴金红; 罗利军
Original assignee: SHANGHAI MUNICIPAL AGRICULTURAL BIOLOGICAL GENE CENTER
Current assignee: SHANGHAI MUNICIPAL AGRICULTURAL BIOLOGICAL GENE CENTER
Priority date: 2018-10-31
Filing date: 2018-10-31
Publication date: 2019-02-01
Anticipated expiration: 2038-10-31
Also published as: CN109295075B

Abstract

The invention belongs to genetic engineering fields, in particular to NfOCP1 anti-drought gene, the amino acid sequence of its coding and its purposes in raising plant drought resistance, NfOCP1 anti-drought gene derives from hair-like nostoc, its nucleotide sequence is as shown in sequence table SEQ ID NO.1, the amino acid sequence that it is encoded is as shown in sequence table SEQ ID NO.2, for improving the drought resistance of plant especially rice.

Description

NfOCP1 anti-drought gene, its coding amino acid sequence and its improve plant drought Purposes in property

Technical field

The invention belongs to genetic engineering field, in particular to a kind of NfOCP1 anti-drought gene from hair-like nostoc and Its amino acid sequence encoded, for improving the drought resistance of plant especially rice.

Background technique

Hair-like nostoc (Nostoc flagelliforme) is mainly distributed on the arid or semiarid desert in the Northern Hemisphere Area is often subject to the side of body of the abiotic factors such as saline and alkaline Extreme drought, the larger temperature difference, high concentration, nutritional deficiency, uv b radiation Compel.Stress factors may generate a series of damage, including the damage of nucleic acid damaging, Protein Damage, film rouge etc. to cell, To upset normal cell metabolism.By the adaptation of long-term environmental selection and self structure function, hair-like nostoc is The mechanism of a series of physiological ecological and molecule is evolved to resist various adverse circumstances.Therefore, hair-like nostoc has become research Adverse circumstance adaptation mechanism and one of the optimal material for excavating adversity gene.

Under strong light, cyanobacteria is subject to photooxidation stress.Singlet oxygen is the chief active oxygen form of photooxidation stress, With very high response, attack and oxidized protein, lipid and accounting can especially excite D1 albumen in photosynthetical system PSII Degradation generates photo oxidative damage and photoinhibition (Kirilovsky, 2015).In plant, carotenoid can be direct As the substrate of single line oxygen, to reduce the Dan Xianyang concentration of photosynthetic reaction center, alleviate photooxidation stress.It is main in cyanobacteria (Kirilovsky, 2015) is completed by orange carotenoids fibroin (Orange Carotenoid Protein, OCP). Under field conditions (factors), fine day noon plant upper layer blade is it occur frequently that Xanthophyll cycle, when strong light and other environmental stress factors, such as Arid, high temperature etc. exist simultaneously, and Xanthophyll cycle aggravation can occur under low light intensity.

China is the country that a natural calamity takes place frequently, and water resource is relatively poor, and distributed pole is uneven on geographical and space-time, The agricultural in China is set to frequently suffer from arid and cause grain drop in production.Rice is one of most important cereal crops in China, production The freshwater resources of the nearly half in China are consumed, the saving water, resisting drought performance of the crops such as rice is improved, are to ensure China's grain security And ecological safety, ensure the great demand (Luo, 2010) of agricultural sustainable development.Currently, transgenic technology is answered extensively In the cultivation for using drought resisting rice, used strategy is exactly to express drought-induced or gene related to drought tolerance in rice.Cause This, clone and excavation to hair-like nostoc anti-drought gene, and apply on biological gene engineering with extremely important meaning.

Summary of the invention

In consideration of it, the object of the present invention is to provide a kind of NfOCP1 anti-drought gene from hair-like nostoc, based on hair Shape nostoc drought stress transcribes to obtain, and expression quantity obviously rises (Fig. 1) under drought stress.

Another object of the present invention is to provide above-mentioned NfOCP1 anti-drought gene and is improving the purposes in plant drought resistance, especially It is purposes in rice.

Above-mentioned purpose of the invention is achieved through the following technical solutions:

In a first aspect, NfOCP1 anti-drought gene, derives from hair-like nostoc, nucleotide sequence such as SEQ ID NO.1 institute Show, sequence length 441bp, specifically:

ATGACTTACACAGCTGATGAAGCAACAAAACCAGCTTTAGAAACTTTTCAAGGCTTTGA TGTAGATA CTCAGCTAGCCCTACTTTGGTTCGGTTATCTTGATCTTAAAGAGCAGCTAAA CCCAGCACCTCCTGAAAGCGTAG AAGCTATAGCTAAAGCAGTGTTTGACCAAATCCAAG AGTTGTCTCAAGAAGAGCAACTGCAAGCACAACGGGACA TTATTAATGGTGCAAGCCA AACCTATAATAGTCTAAGTCCCAATGCTAAATTAGACGTTTGGCTACTGCTGGCAC AAGG AATGGACAATGGAAGCGTCATCCAAGTACCGTCCGACTATCAACTACCTGGTGAAACGG ATGAATTTGTC GCATTGATCAAAAAGCTAGAGTTTGAGCAGCGCGTTAATTTTATGTTGA GTGTGGTGCAAGGGTTCGGTAGTTAA； Its amino acid sequence encoded is 146, as shown in SEQ ID NO.2, specifically:

MTYTADEATKPALETFQGFDVDTQLALLWFGYLDLKEQLNPAPPESVEAIAKAVFDQIQELS QEEQL QAQRDIINGASQTYNSLSPNAKLDVWLLLAQGMDNGSVIQVPSDYQLPGETDEFVA LIKKLEFEQRVNFMLSVVQGFGS。

Second aspect, above-mentioned NfOCP1 anti-drought gene are rice improving the purposes on plant drought resistance, the plant.

According to the NfOCP1 anti-drought gene sequence, PCR (Polymerase Chain Reaction) skill is directlyed adopt Art, from genome, mRNA and cDNA amplification obtain the NfOCP1 anti-drought gene and any interested section of DNA or The section of DNA homologous with it, the expression vector for carrying above-mentioned NfOCP1 anti-drought gene can be by using Ti-plasmids, plant virus Carrier, directly delivered DNA, microinjection, the standard biologics technical method such as electroporation imports prokaryotic bacterial cell, in plant cell. DNA fragmentation by separating and using in hair-like nostoc including NfOCP1 anti-drought gene, can make Escherichia coli and rice exist Drought-resistant ability enhancing under drought condition.

The beneficial effects of the present invention are: by wild type seeds and turns NfOCP1 trans-genetic hybrid rice T2 seed and carry out germination examination It tests, by behind the culture of 1/2MS culture medium 10 days of addition 120mM mannitol, wild type is averaged plant height as the small of 6.15cm Bud, and transgenic line growing way is substantially better than wild type control, 4 family mean plant heights of transgenosis respectively reach 11.68cm, 10.44cm, 12.41cm and 14.4cm.Illustrate that overexpression NfOCP1 gene improves transgenosis really in rice The drought resistance of plant.

Detailed description of the invention

Fig. 1 be in embodiment 1 NfOCP1 gene in the expression comparison diagram of dehydration transcript profile of delivering vegetables.

Fig. 2 is expression analysis of the NfOCP1 gene under Water deficit in embodiment 1.

Fig. 3 is the prokaryotic expression SDS-PAGE electrophoresis result figure of NfOCP1 gene in embodiment 2, wherein ck is empty carrier Negative control, 1,2,3,4,5,6 is 6 recombinant plasmid pGEX-NfOCP1 clones.

Fig. 4 is that prokaryotic expression coerces result in embodiment 2.PGEX is empty carrier, and NfOCP1 is recombinant plasmid.Wherein scheme 4A is experimental result picture under normal culture collating condition, and Fig. 4 B is experimental result picture under the conditions of addition sorbierite, and Fig. 4 C is simultaneously Add the experimental result picture of IPTG and sorbierite.

Fig. 5 is the plant overexpression carrier ub-06 carrier figure that the present invention selects.

Fig. 6 is the expression quantity detection figure of 3 transfer NfOCP1 trans-genetic hybrid rice strain of embodiment.

Fig. 7 is that NfOCP1 trans-genetic hybrid rice strain OE-1, OE-3, OE-7, the OE-8 and wild type WT that turns in embodiment 4 exists Add the plant height statistics (7A) after growing 10 days in the plate of 120mM mannitol；OE-1, OE-3, OE-7, OE-8 and wild type Growth after WT is grown 10 days in the plate of addition 120mM mannitol shows (7B).

Specific embodiment

Below with reference to embodiment, the invention will be further described:

Test method without specific conditions in following embodiments, usually according to normal condition, such as Sambrook etc. Molecular cloning: described in laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) Condition, or according to the normal condition proposed by manufacturer.

Embodiment 1: the clone of hair-like nostoc NfOCP1 gene

(1) culture of hair-like nostoc

The hair-like nostoc of Liquid Culture (wild sample is in Inner Mongolia Autonomous Region Sunite Left Banner).Before inoculation Disperse algal filament with glass homogenizer homogenate algae, is then seeded into 300ml, sterile culture is carried out in BG11 culture medium, cultivate 25 DEG C of temperature, 20 ± 2 μm of ol photons m of light intensity^-2s^-1(continuous illumination for 24 hours) shakes algae three times daily in the morning, afternoon and evening, keeps algae thin Born of the same parents suspend, uniformly receive illumination.

(2) DNA is extracted

The hair-like nostoc sample cultivated in step (1) is ground in liquid nitrogen, is dispensed into 1.5ml centrifuge tube, is added 1mL extracting solution (EDTA of the NaCl of the Tris-HCl of 100mmol/L, 500mmol/L, 50mmol/L, PH8.0), 100 microlitres 20%SDS, 10 microlitres of 10mg/mL Proteinase Ks, mixes well；37 DEG C of cracking 30min, every 5min mix primary； 12000rpm It is centrifuged 10min, takes supernatant that isometric Tris saturated phenol is added, mixes well, be stored at room temperature 2min；12000rpm centrifugation 10min takes supernatant that isometric chloroform/isoamyl alcohol is added, mixes well, be stored at room temperature 2min；12000rpm is centrifuged 10min, takes Isometric chloroform is added in supernatant, mixes well, is stored at room temperature 2min；12000rpm is centrifuged 10min, takes supernatant to be added isometric Isopropanol is added the 3M NaAc of 1/10 volume, mixes well, it is seen that white filiform；With 10 microlitres of suction nozzle picking white silks Shape precipitating, is transferred to 70% ethyl alcohol and washes 2 times；It is dried in air, 30 microlitres of Buffer EB dissolutions is added.

(3) amplification of NfOCP1 full length sequence

In hair-like nostoc dehydration transcriptome analysis, in dehydration 10%, dehydration 30%, dehydration 50, dehydration 70%, dehydration Under conditions of 90%, the expression of NfOCP1 gene is all significantly risen.The full length sequence for obtaining NfOCP1 gene is sequenced by transcript profile, Upstream and downstream primer (NfOCP1F1:5-ATGACTTACACAGCTGATGA-3, NfOCP1R1:5- are designed according to sequence information TTAACTACCGAACCCTTG-3), as shown in sequence table SEQ ID NO.3-4.

Clone obtains NfOCP1 gene from hair-like nostoc genomic DNA, and by sequence verification, amplified production is this Invent nucleotide sequence (1-441bp) shown in SEQ ID NO.1.

(4) expression pattern analysis of the NfOCP1 gene under dehydration processing

Solid is delivered vegetables and is immersed in BG11 culture medium, reaches fully saturated after 2 days.And start dehydration processing, in dehydration 0%, dehydration 10%, dehydration 30%, dehydration 50%, 90%6 point in time sampling of dehydration 70% and dehydration set in liquid nitrogen and grind. It is extracted using the RNAprep Pure polyphenol polysaccharide vegetable total RNA extraction reagent box (catalog number (Cat.No.): DP441) of TIANGEN company The total serum IgE delivered vegetables, electroresis appraisal total serum IgE quality, then measures rna content on spectrophotometer.

Reverse transcription synthesizes the first chain cDNA

It needs to digest extracted RNA sample with DNaseI before reverse transcription, reaction system is as follows:

After 37 DEG C of reaction 15min, 0.25 μ L 0.1M EDTA (guaranteeing final concentration > 2mM) is added, 70 DEG C of incubation 10min are whole It only reacts, of short duration centrifugation is placed on spare on ice.

The synthesis of first chain cDNA is referring to Promega reverse transcription system A3500 operation manual, the specific steps are as follows:

The reaction system of following 20 μ L of each preparation of reagents is sequentially added in the sample that DNaseI digested:

By upper reaction system in 42 DEG C of incubation 15min；Then 95 DEG C of heating 5min, inactivate AMV reverse transcriptase and prevent It is in conjunction with DNA；4 DEG C or 5min is placed on ice.The cDNA prepared can immediately using or deposit in -20 DEG C it is spare.

The quantitative analysis of gene expression uses Takara companyPremix Ex Taq^TM(Perfect Real Time) kit and American AB I7000 quantitative PCR apparatus carry out.Primer is quantified according to NfOCP1 sequence design (NfOCP1QF:5-GCTGATGAAGCAACAAAACC-3；NfOCP1QR:5-CAAGATAACCGAACCAAAGT-3, primer sequence Column are as shown in sequence table SEQ ID NO.5-6).With rice housekeeping gene actin (GenBank accession It No.AY212324 is) reference gene, according to its cDNA sequence design primer.

The preparation of 20 μ l reaction systems:

Reaction condition are as follows: 95 DEG C of 30s are recycled 40 times then in 95 DEG C of 5s, 60 DEG C of 31s, and add Dissociation Stage.Data are collected when setting 60 DEG C of 31s in each cycle, other concrete operations are carried out by instrument operation instructions.Meter The mean CT-number and △ CT value for calculating target gene and reference gene, utilize 2^-ΔΔCTMethod carries out interpretation of result, finally by data The relative expression quantity histogram that GraphPad Prism5.0 makes target gene is imported, sees Fig. 2.

Embodiment 2: the prokaryotic expression of hair-like nostoc NfOCP1 albumen and stress identification

Utilize the prokaryotic expression carrier of One Step Cloning Kit recombinant technique building NfOCP1.In embodiment 1 Having obtained NfOCP1 gene is template, with preceding primer NfOCP1F2:5-ttccaggggcccctgggatccATGACTTACAC AGCTGATGA-3, rear primer NfOCP1R2:5-gtcacgatgcggccgctcgagTTAACTACCGAACCCTTG-3, primer As shown in sequence table SEQ ID NO.7-8, PCR amplification is carried out, product is recovered after purification, passes through One Step Cloning Amplified production segment is cloned on vector pGEX -6P-14984 by Kit recombining reaction.Detailed process is as follows:

(1), PCR amplification

20 μ L reaction systems are as follows:

Amplification program: 94 DEG C of initial denaturations 3min, 94 DEG C of 1min, 56 DEG C of 45sec, 72 DEG C of 1min, 30 circulations, 72 DEG C 10min.5 μ L electrophoresis detections are taken after reaction.

(2) recycling of PCR product

Purification and recovery is carried out using plain agar sugar gel DNA QIAquick Gel Extraction Kit (Tiangeng biochemical technology Co., Ltd).

(3) recombining reaction

Recombinate using the One Step Cloning Kit kit of Nanjing Vazyme Biotechnology Co., Ltd. anti- It answers.It is specific as follows:

2 μ g ring-type pGEX-6P-14984 plasmids are added in 20 μ l endonuclease reaction systems, 37 DEG C of digestion 2hr.It is restricted Restriction endonuclease usage amount is each 1 μ l of BamHI and XhoI.After the completion of digestion, digestion products are placed in 65 DEG C of heating 20min, in inactivation Enzyme cutting.Reaction system is formulated as follows in ice-water bath.

After the completion of system is prepared, is gently blown and beaten up and down with pipettor and mix each component several times, be placed in 37 DEG C of reaction 30min. To after the reaction was completed, reaction tube is placed in ice-water bath cooling 5min immediately.Then the cooling reaction solution of 20 μ l is taken, is added to 200 μ l is expressed in competence BL21 cell, is flicked and is mixed under tube wall number, places 30min on ice.42 DEG C heat shock 45~90 seconds, ice Water-bath is incubated for 2min.900 μ l LB culture mediums are added, 37 DEG C of incubation 10min sufficiently recover.37 DEG C are shaken bacterium 45min.Take 100 μ l Bacterium solution is uniformly coated on the plate containing antibiotic Amp+.Plate is inverted, is incubated overnight in 37 DEG C, sequencing is contained The recombinant plasmid pGEX-NfOCP1 of NfOCP1.

(4) expression of NfOCP1 albumen

The bacterial strain monoclonal of the recombinant plasmid containing NfOCP1 is placed in 2ml LB (g/ml of μ containing Amp50) and is incubated overnight for 37 DEG C. Bacterium is stayed overnight by 1: 50 dilution proportion, 37 DEG C of shake cultures to OD600 ≌ 0.6 take partially liq as the control group not induced, Remaining addition IPTG inducer is to final concentration 0.4mM as experimental group, two groups of continuation, 37 DEG C of shake culture 3hr.Bacterium is taken respectively Body 1ml, centrifugation 12000g × 30s harvest precipitating, is resuspended with 100 μ L 1%SDS, is mixed, 70 DEG C of 10min.Centrifugation 12000g × 1min takes supernatant as sample, SDS-PAGE electrophoresis detection, and concrete outcome is as shown in Figure 3.Wherein, CK1 and CK2 is empty carrier Negative control, 1,2,3,4,5,6 is 6 recombinant plasmid pGEX-NfOCP1 clones.

(5) prokaryotic expression is coerced

Wait for that OD600 is between 0.4 to 0.6 by bacterium described in step (4), is shaken, pGEX empty carrier adjusts each pipe as control Initial OD 600 it is consistent, then take isodose to expand numerous bacterium solution and be added in the LB of benzyl containing ammonia of various processing, 3 kinds of processing are respectively Any additional reagent is not added, sorbierite (Sorbitol) is added, inducer (IPTG) and sorbierite reagent is added.Every one The section time (is measured) with spectrophotometric determination OD600 every 1h, and statistical result draws line chart, is not being added as the result is shown Under IPTG inductive condition, recombinant plasmid and empty vector control bacterium growing way do not have difference, and after inducing, recombinant plasmid pGEX- NfOCP1 growing way under sorbierite stress is more preferable, and concrete outcome is as shown in figure 4, wherein Fig. 4 A is under normal culture collating condition Experimental result picture, Fig. 4 B are experimental result picture under the conditions of addition sorbierite, and Fig. 4 C is while adding the experiment of IPTG and sorbierite Result figure.

Embodiment 3: hair-like nostoc NfOCP1 overexpression rice transformation

To have obtained NfOCP1 gene in embodiment 1 as template, with preceding primer NfOCP1F3:5-caggtcgactctag AggatccATGACTTACACAGCTGATGA-3, rear primer NfOCP1R3:5-gggaaattcgagctggtcaccTTAACTA CCGAACCCTTG-3, primer carry out PCR amplification as shown in sequence table SEQ ID NO.9-10, and product is recovered after purification, leads to One Step Cloning Kit recombining reaction is crossed, amplified production segment is cloned into plant overexpression carrier ub-06, ub- 06 carrier figure is as shown in Figure 5.Detailed process is as follows:

(1) PCR amplification, by described in case study on implementation 1.

(2) recycling of PCR product, by described in case study on implementation 2.

(3) recombining reaction

Recombinate using the One Step Cloning Kit kit of Nanjing Vazyme Biotechnology Co., Ltd. anti- It should be as follows:

2 μ g ring-type ub-06 plasmids are added in 20 μ l endonuclease reaction systems, 37 DEG C of digestion 2hr.Restriction enzyme makes Dosage is each 1 μ l of BamHI and XhoI.After the completion of digestion, digestion products are placed in 65 DEG C of heating 20min, inactivate restriction endonuclease.

Reaction system is formulated as follows in ice-water bath.

After the completion of system is prepared, is gently blown and beaten up and down with pipettor and mix each component several times, be placed in 37 DEG C of reaction 30min. To after the reaction was completed, reaction tube is placed in ice-water bath cooling 5min immediately.Then the cooling reaction solution of 20 μ l is taken, is added to 200 μ l is expressed in competence DH5 α cell, is flicked and is mixed under tube wall number, places 30min on ice.42 DEG C heat shock 45~90 seconds, ice Water-bath is incubated for 2min.900 μ l LB culture mediums are added, 37 DEG C of incubation 10min sufficiently recover.37 DEG C are shaken bacterium 45min.Take 100 μ l Bacterium solution is uniformly coated on the plate containing antibiotic Amp+.Plate is inverted, is incubated overnight in 37 DEG C, sequencing is contained The plasmid u6-NfOCP1 of NfOCP1.

(4) Agrobacterium-mediated Transformation

1 μ L u6-NfOCP1 plasmid is added in 100 μ L Agrobacterium EHA105 competent cells, is mixed gently, ice water After bathing 30min, the quick-frozen cold shock 2min in liquid nitrogen；400-800 μ LYEP culture solution (Kan is added⁺), 28 DEG C, 200r/min shake Swing culture 3-5h；Room temperature is centrifuged (5000r/min, 5min), retains 100 μ L supernatants and thallus is resuspended, be coated on LA solid medium (Kan⁺), 28 DEG C are inverted culture 2 days until growing the bacterium colony of suitable size, and picking monoclonal carries out PCR detection, obtains positive Bacterial strain.

(5) callus induces: seed rinsed with sterile water 15-20min, then uses 75% ethanol disinfection 1min, then with secondary Sodium chlorate (1.5% effective concentration) solution oscillation disinfection 20min.Finally use aseptic water washing 5 times again.Washed seed is used Blotting paper, which blots, to be seeded in callus induction culture medium, 25 DEG C dark culture 2 weeks.

Calli induction media: using the induced medium of table 1, be added 0.3g proline, 0.6g hydrolyzed casein, 30g sucrose and 2.5ml 2,4-D (concentration 1mg/ml) are made into 1L solution, adjust pH to 5.9, and 7g agar powder, high temperature and pressure is added Sterilizing.

(6) squamous subculture: cutting embryo callus, access subculture medium in, 25 DEG C dark culture 2 weeks.

Subculture medium: using the subculture medium of table 1,0.5g proline, 0.6g hydrolyzed casein, 30g sugarcane is added Sugar and 2ml 2,4-D (concentration 1mg/ml) are made into 1L solution, adjust pH to 5.9, and 7g agar powder, autoclave sterilization is added.

(7) During Agrobacterium and callus co-culture: culture Agrobacterium, and picking positive single colonie is trained in 1ml Agrobacterium In nutrient solution (containing antibiotic), 28 DEG C of overnight incubations；The above culture is taken, is added in 50ml Agrobacterium culture solution (containing antibiotic), 28 DEG C are cultivated to OD600=0.6-1.0.The Agrobacterium bacterium solution of acquisition is centrifuged, suspending nutrient solution is added in the thallus being collected into In, shake culture 30min to OD600=0.6-1.0.Then callus is put into the suspending nutrient solution containing Agrobacterium bacterium solution In, shaken cultivation 20min or so.Callus is dried in sterilizing filter paper, is transferred in co-culture medium, 25 DEG C of dark trainings Support 5d.

Suspending nutrient solution: using the suspending nutrient solution of table 1, being added 0.08g hydrolyzed casein, 2g sucrose and 0.2ml 2, 4-D (concentration 1mg/ml) is made into 100ml solution, adjusts pH to 5.4, is divided into two bottles (every bottle of 50ml), autoclave sterilization.Make With the glucose and 100 μ L AS (100mM) that 1ml 50% is added before.

Co-culture medium: using the co-culture medium of table 1, be added 0.8g hydrolyzed casein, 20g sucrose and 3.0ml 2,4-D (concentration 1mg/ml) are made into 1L solution, adjust pH to 5.6, and 7g agar powder, autoclave sterilization is added.It uses The glucose and 1ml AS (100mmol/L) of 20ml 50% are added before.

(8) screening and culturing: after co-culturing 3d, the callus chosen is transferred in screening and culturing medium, 25 DEG C of dark cultures 2 In week, screening is twice.Screening and culturing medium: using the screening and culturing medium of table 2, be added 0.6g hydrolyzed casein, 30g sucrose and 2.5ml 2,4-D (concentration 1mg/ml) are made into 1L solution, adjust pH to 6.0, and 7g agar powder, autoclave sterilization is added.It uses 1ml Hn and 1ml Cn (100ppm) is added before.

(9) differentiation culture: picking embryo callus accesses differential medium, and 24 DEG C, 16h/8h brightness culture induction divides Change bud (4-6 weeks).Differential medium: using the differential medium of table 2,2.0mg/L 6-BA, 2.0mg/L KT, 0.2 is added Mg/L NAA, 0.2mg/L IAA, 1.0g hydrolyzed casein and 30g sucrose are made into 1L solution, adjust pH to 6.0, and 7g fine jade is added Cosmetics, autoclave sterilization.

(10) culture of rootage: when bud length to 2cm or so, young shoot being cut, is inserted into root media, 25 DEG C or so, 16h/8h brightness culture, root induction.Root media: using the root media of table 2,30g sucrose is added, it is molten to be made into 1 L Liquid adjusts pH to 5.8, and 7g agar powder, autoclave sterilization is added.

(11) transformed plant culture: after well developed root system, test tube mouth is opened, after sterile water hardening 2-3d is added, by plant It takes out, the solid medium of attachment is cleaned with sterile water, move into soil, just start wind sheltering of shading, carried out after robust plant Conventional field or greenhouse management culture.

Table 1: minimal medium ingredient 1

Table 2: minimal medium ingredient 2

(12) positive detection of overexpression plant

Clip regeneration plant blade, using CTAB method extract DNA, using hpt special primer (hptF: ACACTACATGGCGTGATTTCAT；HptR:TCCACTATCGGCGAGTACTTCT, primer such as sequence table SEQ ID NO.11- Shown in 12) carry out PCR detection.

PCR system:

PCR response procedures:

(13) expression of target gene detects in overexpression positive plant

Take T₀For rice leaf, it is placed in liquid nitrogen grinding, extracts RNA.It is specific as follows:

The extraction of RNA: for samples taken with powdery is ground into after liquid nitrogen frozen in mortar, addition fills 1ml TRNzol-A⁺The 2mL EP of reagent (Tiangeng biochemical technology Co., Ltd) is managed, and sufficiently after oscillation, is placed at room temperature for 5min, later plus 0.2ml chlorine It is imitative, after acutely shaking 15s, it is placed at room temperature for 3min；Afterwards in 4 DEG C, after 12000rpm is centrifuged 10min, supernatant moves to new 2mL In EP pipe, add isometric isopropanol precipitating RNA, adds 100 μ l RNase-free ddH₂O dissolution.Electroresis appraisal total serum IgE quality, Then rna content is measured on spectrophotometer.

Reverse transcription synthesizes the first chain cDNA

The quantitative analysis of gene expression uses Takara companyPremix Ex Taq^TM(Perfect Real Time) kit and American AB I7000 quantitative PCR apparatus carry out.Primer is quantified according to NfOCP1 sequence design (NfOCP1QF:5-GCTGATGAAGCAACAAAACC-3；NfOCP1QR:5-CAAGATAACCGAACCAAAGT-3), primer is such as Shown in sequence table SEQ ID NO.5-6).With rice housekeeping gene actin (GenBank accession No.AY212324) For reference gene, according to its cDNA sequence design primer.

The preparation of 20 μ l reaction systems:

Reaction condition are as follows: 95 DEG C of 30s are recycled 40 times then in 95 DEG C of 5s, 60 DEG C of 31s, and add Dissociation Stage.Data are collected when setting 60 DEG C of 31s in each cycle, other concrete operations are carried out by instrument operation instructions.Meter The mean CT-number and △ CT value for calculating target gene and reference gene, utilize 2^-ΔΔCTMethod carries out interpretation of result, finally by data The relative expression quantity histogram that GraphPad Prism5.0 makes target gene is imported, as shown in Figure 6.

Embodiment 4: transgenic paddy rice T₂For the sprouting stage mannitol simulating drought processing of seed

By overexpression transgenic lines seed decladding disinfection (75% ethanol postincubation 1min, 1.5%NaClO processing 20min, sterile water wash 5 times), it germinates on the 1/2MS culture medium containing 50mg/L hygromycin, one day evening of wild type control It is sowed on the 1/2MS culture medium without hygromycin.The good and consistent seed of growing way that germinates, transfer are selected after germinateing 2~3 days It is cultivated into the 1/2MS culture medium containing 120mM, counts growing state after 10 days.Experimental result is shown by 10 days coercing cultivations Afterwards, the average plant height of wild type control is 6.15cm, and transgenic line growing way is substantially better than wild type control, transgenosis 4 Family mean plant height has respectively reached 11.68cm, 10.44cm, 12.41cm and 14.4cm.Specifically as shown in fig. 6, explanation is in water The drought resistance that NfOCP1 gene improves transgenic plant really is overexpressed in rice.

The above is presently preferred embodiments of the present invention, but the present invention should not be limited to disclosed in the embodiment Content.So all do not depart from the lower equivalent or modification completed of spirit disclosed in this invention, the model that the present invention protects is both fallen within It encloses.

Sequence table

<110>Shanghai City Agricultural biological Gene Center

<120>NfOCP1 anti-drought gene, the amino acid sequence of its coding and its purposes in raising plant drought resistance

<130> 2018-10-29

<141> 2018-10-31

<160> 12

<170> SIPOSequenceListing 1.0

<210> 1

<211> 441

<212> DNA

<213>unknown ()

<400> 1

atgacttaca cagctgatga agcaacaaaa ccagctttag aaacttttca aggctttgat 60

gtagatactc agctagccct actttggttc ggttatcttg atcttaaaga gcagctaaac 120

ccagcacctc ctgaaagcgt agaagctata gctaaagcag tgtttgacca aatccaagag 180

ttgtctcaag aagagcaact gcaagcacaa cgggacatta ttaatggtgc aagccaaacc 240

tataatagtc taagtcccaa tgctaaatta gacgtttggc tactgctggc acaaggaatg 300

gacaatggaa gcgtcatcca agtaccgtcc gactatcaac tacctggtga aacggatgaa 360

tttgtcgcat tgatcaaaaa gctagagttt gagcagcgcg ttaattttat gttgagtgtg 420

gtgcaagggt tcggtagtta a 441

<210> 2

<211> 146

<212> PRT

<213>unknown ()

<400> 2

Met Thr Tyr Thr Ala Asp Glu Ala Thr Lys Pro Ala Leu Glu Thr Phe

1 5 10 15

Gln Gly Phe Asp Val Asp Thr Gln Leu Ala Leu Leu Trp Phe Gly Tyr

20 25 30

Leu Asp Leu Lys Glu Gln Leu Asn Pro Ala Pro Pro Glu Ser Val Glu

35 40 45

Ala Ile Ala Lys Ala Val Phe Asp Gln Ile Gln Glu Leu Ser Gln Glu

50 55 60

Glu Gln Leu Gln Ala Gln Arg Asp Ile Ile Asn Gly Ala Ser Gln Thr

65 70 75 80

Tyr Asn Ser Leu Ser Pro Asn Ala Lys Leu Asp Val Trp Leu Leu Leu

85 90 95

Ala Gln Gly Met Asp Asn Gly Ser Val Ile Gln Val Pro Ser Asp Tyr

100 105 110

Gln Leu Pro Gly Glu Thr Asp Glu Phe Val Ala Leu Ile Lys Lys Leu

115 120 125

Glu Phe Glu Gln Arg Val Asn Phe Met Leu Ser Val Val Gln Gly Phe

130 135 140

Gly Ser

145

<210> 3

<211> 20

<212> DNA

<213>unknown ()

<400> 3

atgacttaca cagctgatga 20

<210> 4

<211> 18

<212> DNA

<213>unknown ()

<400> 4

ttaactaccg aacccttg 18

<210> 5

<211> 20

<212> DNA

<213>unknown ()

<400> 5

gctgatgaag caacaaaacc 20

<210> 6

<211> 20

<212> DNA

<213>unknown ()

<400> 6

caagataacc gaaccaaagt 20

<210> 7

<211> 41

<212> DNA

<213>unknown ()

<400> 7

ttccaggggc ccctgggatc catgacttac acagctgatg a 41

<210> 8

<211> 39

<212> DNA

<213>unknown ()

<400> 8

gtcacgatgc ggccgctcga gttaactacc gaacccttg 39

<210> 9

<211> 41

<212> DNA

<213>unknown ()

<400> 9

caggtcgact ctagaggatc catgacttac acagctgatg a 41

<210> 10

<211> 39

<212> DNA

<213>unknown ()

<400> 10

gggaaattcg agctggtcac cttaactacc gaacccttg 39

<210> 11

<211> 22

<212> DNA

<213>unknown ()

<400> 11

acactacatg gcgtgatttc at 22

<210> 12

<211> 22

<212> DNA

<213>unknown ()

<400> 12

tccactatcg gcgagtactt ct 22

Claims

1.NfOCP1 anti-drought gene, nucleotide sequence derive from hair-like nostoc as shown in SEQ ID NO.1.

2. NfOCP1 anti-drought gene according to claim 1, which is characterized in that the ammonia of the NfOCP1 anti-drought gene coding Base acid sequence is as shown in SEQ ID NO.2.

3. NfOCP1 anti-drought gene described in claim 1 is improving the purposes in plant drought resistance, which is characterized in that described For the nucleotide sequence of NfOCP1 anti-drought gene as shown in SEQ ID NO.1, the plant is rice.

4. purposes according to claim 3, which is characterized in that the NfOCP1 anti-drought gene derives from hair-like nostoc.

5. purposes according to claim 3, which is characterized in that the amino acid sequence of the NfOCP1 anti-drought gene coding is such as Shown in SEQ ID NO.2.