CN107603988B - Nostoc flagelliforme NfcrtO drought-resistant gene and amino acid sequence and application thereof - Google Patents

Abstract

The invention belongs to the field of genetic engineering, and particularly relates to a nostoc flagelliforme NfcrtO drought-resistant gene and an amino acid sequence thereof, and application of the nostoc flagelliforme NfcrtO drought-resistant gene in improving plant drought resistance. Nostoc flagelliforme NfcrtO drought-resistant gene is characterized in that the nucleotide sequence of the gene is shown in a sequence table SEQ ID NO. 1. The amino acid sequence of the gene is shown in a sequence table SEQ ID NO. 2. The Nostoc flagelliforme NfcrtO drought-resistant gene is applied to improving the drought resistance of plants, in particular to application to rice. And (3) simulating drought stress, stressing until the contrast is completely dead, then recovering nutrient solution culture, observing survival conditions, wherein experimental results show that after 10 days of rehydration culture, the wild contrast has no survived plant, and transgenic lines OE-4 and OE-5 respectively have 43% and 26% survival rates, which shows that the overexpression of the Nfcro gene in the rice indeed improves the recovery drought resistance of the transgenic plants.

Description

Nostoc flagelliforme NfcrtO drought-resistant gene and amino acid sequence and application thereof

Technical Field

The invention belongs to the field of genetic engineering, and particularly relates to a nostoc flagelliforme NfcrtO drought-resistant gene and an amino acid sequence thereof, and application of the nostoc flagelliforme NfcrtO drought-resistant gene in improving plant drought resistance.

Background

Nostoc flagelliforme (Nostoc flagelliforme) is mainly distributed in arid or semiarid desert regions of the northern hemisphere and is often stressed by abiotic factors such as extreme drought, large temperature difference, high-concentration saline and alkaline, nutrient deficiency, UV-B radiation and the like. Stress factors may cause a range of damage to cells including nucleic acid damage, protein damage, membrane lipid damage, etc., thereby disrupting normal cellular metabolism. After long-term environmental selection and adaptation of self structural functions, nostoc flagelliforme has evolved a series of physiological, ecological and molecular mechanisms to resist various adversities. Therefore, nostoc flagelliforme has become one of the best materials for researching stress adaptation mechanism and discovering stress resistance genes.

The gene of the invention is known to have the closest homology relation with the gene Npun _ F3745 of the Nostoc punctifolia for coding beta-carotene ketolase (CrtO) through evolutionary tree analysis, and is named as NfcrtO gene. In 1997, catalytic characteristics of slr0088 encoded products in synechocystis PCC 6803 were studied, and heterologous expression experiments, in vitro biochemical experiments and in vivo insertion inactivation experiments prove that the slr0088 encoded products asymmetrically introduce a ketone group into the ionone ring of beta-carotene (beta-carotene) to catalyze the beta-carotene to generate echinenone (echinenone) (Schoenmakers et al, 1997). The carotenoid composition in all 3744-knocked-out algal strains of anabaena PCC7120 is analyzed by a High Performance Liquid Chromatography (HPLC), and the result shows that compared with the carotenoid composition of wild type PCC7120, the proportion of beta-carotene in all 3744-knocked-out algal strains is remarkably increased (41% of wild type and 85% of all 3744-knocked-out algal strains), the echinenone content is remarkably reduced (37% of wild type and 3% of all 3744-knocked-out algal strains), the canthaxanthin content is also remarkably reduced (4% of wild type and no detection of all 3744-knocked-out algal strains), and therefore all3744 in anabaena PCC7120 catalyzes beta-carotene to generate echinenone and canthaxanthin, and the oxidation resistance is enhanced (Mochimaru et al, 2005).

China is a country with frequent natural disasters, water resources are relatively poor, and the distribution is extremely uneven in geography and space-time, so that the agriculture of China is often subjected to drought to cause grain yield reduction. Rice is one of the most important grain crops in China, nearly half of fresh water resources in China are consumed in production, the water-saving and drought-resisting performance of crops such as rice and the like is improved, and the rice is a great demand for guaranteeing grain safety and ecological safety in China and sustainable development of agriculture (Luo, 2010). At present, the transgenic technology is widely applied to the cultivation of drought-resistant rice, and the adopted strategy is to express drought-induced or drought-resistant related genes in the rice.

Therefore, the method has very important significance for cloning and excavating the nostoc flagelliforme drought-resistant gene and applying the nostoc flagelliforme drought-resistant gene to biological gene engineering. The Nfcro gene is obtained based on a nostoc flagelliforme drought stress transcriptome experiment, and the expression quantity of the Nfcro gene is obviously increased under drought stress. The drought resistance function and the function of the drought resistance function in improving the drought resistance of the rice are identified, and the method has very important significance for cultivating new varieties of drought-resistant rice.

Disclosure of Invention

The invention aims to provide a nostoc flagelliforme NfcrtO drought-resistant gene, the nucleotide sequence of which is shown in a sequence table SEQ ID NO. 1.

The sequence length is 1695bp, and specifically:

ATGCAAGAGTATGATGTTGTGCTGATCGGTGCCGGACACAATGGGTTAGTTTGTGCAGCTTATTTGCTGAAAGCTGGTTATAGCGTCCTGTTACTAGAAAAGCGTTCTGTTCCAGGCGGTGCAGCAACAACTGAAGAATGTTTACCACAAGAAGCTCCTGGATTTAAATTTAACTTGTGTGCTATTGACCATGAATTTATTCACTTGGGGCCGGTAGTTGAAGAATTAGAACTAGAAAAATACGGCTTGCATTATTTGGAGTGTGATCCAGTTGTTTTCTGTCCTCATCCTGATGGTAAGTATTTCTTAGCACATAAATCGCTGGAAAAAACTTGTGCAGAAATCGCTCGTTATAGTGAACGGGATGCCAAAAAATACGCAGAATTTGTAGATTATTGGCAACGAGCGATCGGTGCAATGGTTCCTATGTTTAATGCACCGCCAAAGTCAATTATAGATATCGTTGGCAACTACGATATCAAAAAATTAAAAGATTTATTTTCGATGATTGGTTCCCCAAACAAAACGCTGGACTTTATTCGCACCATGTTAACCAGCGCAGAGGATTTACTTAACGAGTGGTTTGATGAGGAATTTCTAAAAGCGCCACTAGCCAGGCTCGCAGCAGAACTTGGTGCGCCGCCATCGCAAAAAACCCTTGCCATTGGTGCAATTATGATGGCGATGCGTCACAACCCTGGAATGGCCAGGCCACGCGGCGGAACTGGCGCACTTGTGAAAGCTTTAGTGGATTTAGTCACAAGTAAAGGTGGCGTTATTCTGACAGATCAGCATGTTGAAAAAGTTTTAATTGATGATGGAAAAGCTGTTGGTGTACGAGTGGCTGGTAACACCGAATATCGGGCTAAATACGGCGTAATTTCTAATATTGATGCCAAGCGGTTATTTTTGCAAATGACTGATAAAAGCGATATTGATGAAGCCGATCCTGACTTATGGGAAAGATTAGAACGCCGCATCGTTAATAATAACGAAACTATCCTCAAGATAGACTTAGCTTTAGACGAACCACTGCGCTTTCCATACCACGCCCATAAAGACGAATATCTCGTTGGTTCTATCTTAATTGCCGATTCCGTGGCTCATGTAGAACAGGCTCATAGTAAATGTAGCTTAGGAGAAATTCCTGATGCTGACCCATCAATGTATGTGGTCATGCCTAGTTATTTAGATCCCACATTAGCACCACCAGGTAAGCACACTGTATGGATTGAGTATTTTGCCCCTTATCAAATTGCTGGTGCAGAAGGCACTGGTTTAAGAGGTACTGGTTGGACAGATGAATTGAAAAACAAAGTTGCAGATAAAGTGGTTGATAAGTTGGCAGACTATGCACCAAATGTCAAGAACGCAACTATCGCCCGTCGTGTAGAAAGTCCAGCAGAATTAGGAGAAAGATTAGGTGCGTATAAAGGGAATTATTACCATATTGACATGACTTTAGATCAGATGGTATTTTTCCGTCCCTTGCCAGAAATAGCAAACTACAAAACGCCAATTGAGAATCTATTTTTGACTGGTGCAGGTACTCATCCAGGTGGTTCGATTTCGGGAATGCCAGGACGCAATTGTGCGCGTGTATTTTTGCAGGCAAAACATCCCATTAGTCAAACTCTGAAGGATGCACGCGATTCGATTAAGTCAACTGTAGAGTCGGTGTTTGGAATTAATTAA

another purpose of the invention is to provide the amino acid sequence of the Nostoc flagelliforme NfcrtO drought-resistant gene, which is shown as a sequence table SEQ ID NO.2,

the amino acid sequences are 564, and specifically are as follows:

MQEYDVVLIGAGHNGLVCAAYLLKAGYSVLLLEKRSVPGGAATTEECLPQEAPGFKFNLCAIDHEFIHLGPVVEELELEKYGLHYLECDPVVFCPHPDGKYFLAHKSLEKTCAEIARYSERDAKKYAEFVDYWQRAIGAMVPMFNAPPKSIIDIVGNYDIKKLKDLFSMIGSPNKTLDFIRTMLTSAEDLLNEWFDEEFLKAPLARLAAELGAPPSQKTLAIGAIMMAMRHNPGMARPRGGTGALVKALVDLVTSKGGVILTDQHVEKVLIDDGKAVGVRVAGNTEYRAKYGVISNIDAKRLFLQMTDKSDIDEADPDLWERLERRIVNNNETILKIDLALDEPLRFPYHAHKDEYLVGSILIADSVAHVEQAHSKCSLGEIPDADPSMYVVMPSYLDPTLAPPGKHTVWIEYFAPYQIAGAEGTGLRGTGWTDELKNKVADKVVDKLADYAPNVKNATIARRVESPAELGERLGAYKGNYYHIDMTLDQMVFFRPLPEIANYKTPIENLFLTGAGTHPGGSISGMPGRNCARVFLQAKHPISQTLKDARDSIKSTVESVFGIN

the invention also provides application of the nostoc flagelliforme NfcrtO drought-resistant gene in improving plant drought resistance, in particular application in rice.

The invention separates and applies a DNA fragment containing Nfcro gene, which endows escherichia coli and rice with enhanced drought resistance under drought conditions. The Nfcro gene of the invention and any DNA of interest or homologous DNA thereof can be obtained by directly amplifying the gene sequence of the Nfcro gene of the invention from genome, mRNA and cDNA by using PCR (polymerase chain reaction) technology. The expression vector carrying the NfcrtO gene can be introduced into prokaryotic bacterial cells and plant cells by using Ti plasmids and plant virus vectors and by using conventional biotechnology methods such as direct DNA transformation, microinjection, electroporation and the like.

And (3) simulating drought stress, stressing until the contrast is completely dead, then recovering nutrient solution culture, observing survival conditions, wherein experimental results show that after 10 days of rehydration culture, the wild contrast has no survived plant, and transgenic lines OE-4 and OE-5 respectively have 43% and 26% survival rates, which shows that the overexpression of the Nfcro gene in the rice indeed improves the recovery drought resistance of the transgenic plants.

Drawings

FIG. 1 is a graph comparing the expression of the NfcrtO gene in the water-loss transcriptome of Nostoc flagelliforme in example 1.

FIG. 2 is a graph showing the growth of the Nfcro gene strain of example 3 and a control strain under normal conditions (A) and mannitol stress (B).

FIG. 3 is a graph showing the growth of the Nfcro gene-transferred algal strain and the control algal strain in example 3 on a mannitol-added plate (A) and the growth of the rehydrated algal strain after the drought treatment (B).

FIG. 4 is a diagram of the plant overexpression vector ub-06 selected in the present invention.

FIG. 5 is a graph showing the expression level of the rice line transformed with NfcrtO gene in example 3.

FIG. 6 is a graph showing the comparison of the survival of rice lines OX-4, OX-5 transformed with Nfcro gene and wild-type CK in example 4 after 20% PEG6000 treatment and rehydration.

Detailed Description

The invention will now be further illustrated by reference to the following examples:

experimental procedures without specific conditions noted in the examples below, generally followed by conventional conditions, such as molecular cloning in Sambrook et al: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations.

Example 1

Cloning of Nostoc flagelliforme NfcrtO gene

(1) Culture of Nostoc flagelliforme

Nostoc flagelliforme cultured in liquid (natural wild sample was sampled from Sunit left flag of inner Mongolia autonomous region). Homogenizing strain with glass homogenizer to disperse filament before inoculation, inoculating into 300ml BG11 culture medium, and performing aseptic culture at culture temperatureLight intensity of 20 +/-2 mu mol photons m at 25 DEG C^-2s^-1(24h for continuous illumination), shaking the algae for three times every day in the morning, at noon and at night to enable the algae cells to be suspended and uniformly receive illumination;

(2) DNA extraction

Grinding the nostoc flagelliforme sample cultured in the step (1) in liquid nitrogen, subpackaging the ground nostoc flagelliforme sample into 1.5mL centrifuge tubes, adding 1mL of extracting solution (100mmol/L Tris-HCl, 500mmol/L NaCl, 50mmol/L EDTA, PH8.0), 100 microliter 20% SDS and 10 microliter 10mg/mL proteinase K, and fully and uniformly mixing; cracking at 37 deg.C for 30min, and mixing once every 5 min; centrifuging at 12000rpm for 10min, collecting supernatant, adding equal volume of Tris saturated phenol, mixing, and standing at room temperature for 2 min; centrifuging at 12000rpm for 10min, collecting supernatant, adding equal volume of chloroform/isoamyl alcohol, mixing, and standing at room temperature for 2 min; centrifuging at 12000rpm for 10min, collecting supernatant, adding equal volume of chloroform, mixing, and standing at room temperature for 2 min; centrifuging at 12000rpm for 10min, collecting supernatant, adding equal volume of isopropanol, adding 1/10 volume of 3M NaAc, and mixing to obtain white filament; picking up the white filiform precipitate with a10 microliter suction head, and washing with 70% ethanol for 2 times; air dried and dissolved by adding 30. mu.l of Buffer EB.

(3) Amplification of full-Length Nfcro sequence

In nostoc flagelliforme water loss transcriptome analysis, the expression of the NfcrtO gene is obviously improved under the conditions of water loss of 10%, 30%, 50%, 70% and 90%. The full-length sequence of the NfcrtO gene is obtained by transcriptome sequencing, and upstream and downstream primers (NfcrOF 1:5-ATGCAAGAGTATGATGTTGTGCTG-3, NfcrOR 1: 5-ACTTTGTCCTGTGCAAGGCTTA-3) are designed according to sequence information and are shown in a sequence table SEQ ID NO. 3-4.

The NfcrtO gene is obtained by cloning from the genome DNA of the nostoc flagelliforme, and the amplified product is the nucleotide sequence (1-1695bp) shown in SEQ ID NO.1 by sequencing verification.

Example 2

Construction of Nfcro gene-transferred anabaena strain

(1) Construction of shuttle expression vectors

The PmtE promoter fragment was amplified using pRL25N-PpetE-LGFP plasmid as a template, and dATP was added to perform a reaction, and the PmtE fragment of 500bp size was recovered by cutting the gel. And connecting the recovered PmtE fragment with a pMD-18T vector, transforming E.coliDH5 alpha, growing a transformant, and then carrying out colony PCR identification. The correct transformants were picked and grown in LB liquid medium for amplification and sent to the company for sequencing. The Pmepete-pMD-18T transformant with correct sequencing is subjected to amplification culture, and the Pmepete-pMD-18T plasmid is extracted. The PmppetE-pMD-18T plasmid is synchronously double-digested by BamH I and Xho I, and the PmppetE promoter fragment of about 500bp is recovered by gel cutting. Meanwhile, pRL25N-PpetE-LGFP plasmid is synchronously subjected to double enzyme digestion by using BamH I and Xho I, and a large fragment of about 7900bp is recovered by cutting gel. Connecting PmtE-pMD-18T (cut and recovered by BamH I and XhoI) and pRL25N-PpetE-LGFP (cut and recovered by BamH I and XhoI), transforming E.coliDH5 alpha, growing a transformant, performing colony PCR identification, identifying a correct transformant, performing amplification culture, and upgrading the plasmid pRL 25N-PpetE-LGFP. SmaI singly enzyme-cuts pRL 25N-PmtE-LGFP plasmid, the single enzyme-cut product is recovered by column chromatography, dTTP reaction is carried out on the single enzyme-cut product, and pRL 25N-PmtE-LGFP is recovered by column chromatography (after Sma I enzyme-cut, T is added for recovery).

Connecting the target gene with a vector: the target gene of example 1, together with the dATP recovery product, was digested with ligation vector to recover pRL 25N-PmtE-LGFP (after Sma I digestion, T recovery was added), E.coliDH5. alpha. was transformed, after transformants were grown, colony PCR was identified, and the correct transformants were sequenced.

Coli HB 101: and (5) carrying out amplification culture on the transformant with the correct sequencing, and upgrading the plasmid. Coli HB101 (containing helper plasmid pRL443 and binding plasmid pRL623), and colony PCR identifies the correct transformant strain for liquid scale-up culture for transformation of anabaena PCC 7120.

(2) Transformation of anabaena PCC7120

The anabaena is transformed by using a triparental hybridization method, and the method comprises the following steps: (A) collecting algae: 100ml of wild type algae strain in late logarithmic growth stage is subpackaged into 50ml of sterilized large centrifuge tube, the room temperature is 5000rpm, the centrifugation is carried out for 5min, the supernatant is discarded, and the precipitated algae cells are collected. (B) Washing and resuspending algae cells: adding sterilized BG11 culture medium to the precipitated algae cells to 45ml, centrifuging at room temperature 5000rpm for 5min, and discarding the supernatant. This step was repeated once. The pelleted algal cells were resuspended in 10ml BG11 medium. (C) Collecting bacteria: 50ml of donor was transferred to a 50ml sterilized large centrifuge tube, centrifuged at 4000rpm at room temperature for 5min, and the supernatant was discarded. (D) And (3) cleaning and resuspending bacteria: adding sterilized LB liquid culture medium into the precipitation bacteria cells to 45ml scale, centrifuging at room temperature 4000rpm for 5min, and discarding the supernatant. This step was repeated once. Adding 1ml LB culture medium into the precipitation bacterial cells, and blowing and resuspending. (E) Mixing algae and bacteria: transferring the bacteria in the step (D) to the algae cells in the step (B), adding BG11 to 35ml scales, shaking and mixing uniformly to ensure that the algae and the bacteria are fully contacted. Centrifuge at 5000rpm for 5min at room temperature, and discard the supernatant. Adding 300-500 mu lBG11 into the precipitated algae, and uniformly mixing by blowing. The mixture of bacteria and algae was transferred to a 1.5ml sterilized EP tube. (F) And (3) weak light adaptation: and (E) placing the mixture of the bacteria and the algae in a constant-temperature illumination incubator at 30 ℃ for 1 hour under weak light, and reversing and uniformly mixing the mixture for 1 time. (G) Film laying and plate coating: sterilized 0.45 μm microporous filter membrane was plated onto solid non-resistant BG11 plate. Sucking 400-. Blow-drying in a superclean bench, sealing the plate, placing in a 30 ℃ constant-temperature illumination incubator, and adaptively culturing for 24h under weak light. (H) And (3) resistance screening: filters containing the mixture of bacteria and algae were transferred to BG11(Neo50BG11) solid plates containing 50. mu.g/ml Neo in a clean bench. Culturing at 30 deg.C under weak light. Transformants appeared in about 2 weeks.

(3) Screening and identification of over-expression strain

Screening of over-expression strain: after single-clone transformants appeared on Neo50BG11 solid plates, single clones were picked and streaked on Neo50BG11 solid plates for expansion culture. Growing-capable monoclonals were streaked onto Neo50BG11 solid plates for 2-3 generations.

Identification of overexpression algal strains: extraction of anabaena PCC7120 gDNA: 5ml of log phase algae are taken, centrifuged at room temperature of 6000rpm for 5min, and the supernatant is discarded. Add 400. mu.l 1 XTE buffer (pH8.0) and mix by pipetting. A small amount of quartz sand, 200. mu.l Tris saturated phenol and 200. mu.l chloroform were added: isopentanol (V/V24: 1) was shaken vigorously on a shaker for 1-2min at 12000rpm at room temperature, centrifuged for 5min, 400. mu.l of the supernatant was pipetted into another fresh 1.5ml EP tube, 0.5. mu.l of RNase A was added, and digested in a 37 ℃ constant temperature water bath for 30-40 min. Add 400. mu.l chloroform: isoamyl alcohol (V/V ═ 24: 1), mixed by inversion. After centrifugation at 12000rpm for 5min at room temperature, the supernatant was aspirated (about 300. mu.l) and transferred to another clean 1.5ml EP tube, 1/5 volumes (60. mu.l) of 5M NaCl was added, and 2 volumes (720. mu.l) of absolute ethanol were added thereto, followed by mixing by inverting the mixture upside down. The mixture was placed in a refrigerator at-20 ℃ and allowed to settle overnight. Centrifuging at 12000rpm for 20min at 4 deg.C, discarding supernatant to obtain colorless transparent precipitate as gDNA, adding 1ml 70% ethanol into the precipitate, and mixing by inversion. 7000rpm, 4 ℃, 5min, discard the supernatant, air dry the precipitate at room temperature (about 5min), add 50 μ l deionized water, blow and mix well, dissolve gDNA.

PCR identification of over-expressed algal strains: mu.l of gDNA was used as a template for PCR identification. The PCR system and procedure were as follows:

PCR reaction system

PCR procedure

Remarking: the number of cycles was 28 cycles.

Example 3

Resistance identification of Nfcro gene transferred anabaena strain

(1) Growth curve determination under mannitol stress

The logarithmic phase strain with consistent OD750 value was taken and the initial photosystem II (PSII) maximum photochemical efficiency Fv/Fm was determined using Handy PEA plant efficiency analyzer (Hansatech Co.). A1 ml sample was taken before treatment and the initial Fv/Fm was determined. Mannitol stress treatment: 1ml of algae solution was added to a 24-well plate, and 1ml of 0.8M Man BG11 medium was added thereto and mixed well, so that the final concentration of Man treatment was 0.4M. Control and treatment groups were each 3 replicates in parallel. Mixing, sampling, measuring initial OD750, standing at 30 deg.C under light intensity of 20 + -2 μmol · m-2 · s-1, shaking algae once every morning, noon and evening, and changing positions once every morning and evening. OD750 was measured every two days and growth curves were plotted. The result shows that the control algal strain (42) and the No. 20 algal strain (20) grow basically in the same way under the condition of normal culture medium culture; after the mannitol is added into the culture medium, the growth speed of the control algae strain is obviously reduced, which shows that the mannitol has a stress effect on the growth of algae, while the growth speed of the No. 20 algae strain is obviously faster than that of the control algae strain, which shows that the No. 20 algae strain can tolerate the stress of the mannitol, and is shown in figure 2.

(2) Flat plate stress treatment

Mannitol plate stress treatment: logarithmic phase algae with consistent OD750 values were dropped at 50. mu.l/drop onto solid Neo50BG11 plates with different mannitol concentrations. Observing the growth condition of the algae under the conditions of 30 ℃ and weak light. Mannitol concentration gradients were 0.6M and 0.8M, respectively. On the 15 th day of mannitol stress, the 20 th algal strain (20) grows better than the control algal strain (42), namely the 20 th algal strain shows the tolerance to mannitol osmotic stress, and after 23 days of treatment, the 20 th algal strain has more obvious phenotype of mannitol osmotic stress tolerance, as shown in figure 3A;

drought stress treatment: the algae is dripped on a solid Neo50BG11 flat plate, the flat plate is only covered but not sealed, the flat plate is naturally evaporated, dehydrated and dried (namely, the algae strains suffer from drought stress while growing, and the drought stress is more serious along with the prolonging of time), when the moisture in the solid culture medium is almost completely evaporated to form a transparent film, 2ml BG11 is added for rehydration, and the recovery condition of the algae is observed. As can be seen from FIG. 3B, the growth vigor of the two algal strains was substantially consistent upon rehydration. However, the control strain (42) will die gradually with the time after rehydration, i.e. the growth cannot be recovered after drying, while the growth can be recovered by the No. 20 strain, which shows that the No. 20 strain can resist dehydration drying adversity and has the capacity of resisting drought stress. Remarking: in the figure, 0d, 35d and 52d respectively represent days 0, 35 and 52 after rehydration.

Example 4

Nostoc flagelliforme NfcrtO overexpression transformation rice

Using the NfcrtO gene obtained in example 1 as a template, the primer nfcrof 3 was used: 5-caggtcgactctagaggatcc ATGCAAGAGTATGATGTTGTGCTG-3, rear primer NfcritOR 3: 5-gggaaattcgagctggtcacc ACTTTGTCCTGTGCAAGGCTTA-3, performing PCR amplification, wherein the primer sequence is shown in SEQ ID NO.5-6 of the sequence table, recovering and purifying the product, and Cloning the amplified product fragment to a vector ub-06 through One Step Cloning Kit recombination reaction. The specific process is as follows:

(1) PCR amplification was performed as described in example 1.

(2) The PCR product was recovered as described in example 1.

(3) Recombination reactions

The recombination reaction was performed using One Step Cloning Kit from Nanjing Novowed Biotechnology Ltd. The method comprises the following specific steps:

mu.g of the circular ub-06 plasmid was added to 20. mu.l of the digestion reaction system and digested at 37 ℃ for 2 hr. The restriction enzymes were used in an amount of 1. mu.l each of BamHI and XhoI. After the enzyme digestion is finished, the enzyme digestion product is heated for 20min at 65 ℃ to inactivate the endonuclease.

The following reaction system was prepared in an ice-water bath.

After the system is prepared, the components are mixed evenly by gently blowing and beating the components up and down for several times by a pipette and are placed at 37 ℃ for reaction for 30 min. After the reaction was completed, the reaction tube was immediately placed in an ice-water bath to cool for 5 min. Then 20. mu.l of the cooled reaction mixture was added to 200. mu.l of expression competent DH 5. alpha. cells, gently flicked down the tube wall and mixed, and placed on ice for 30 min. And (3) thermally shocking for 45-90 seconds at 42 ℃, and incubating for 2min in an ice water bath. 900. mu.l of LB medium was added and incubated at 37 ℃ for 10min for sufficient recovery. Shake the bacteria for 45min at 37 ℃. 100 mul of the bacterial solution was evenly spread on a plate containing antibiotic Amp +. The plate was inverted, cultured overnight at 37 ℃ and sequenced to obtain plasmid u6-NfcrtO containing NfcrtO.

(4) Agrobacterium transformation

Adding 1 mu L u6-NfcrtO plasmid into 100 mu L agrobacterium EHA105 competent cells, gently mixing, carrying out ice water bath for 30min, and carrying out quick freezing and cold shock in liquid nitrogen for 2 min; adding 400-800 μ LYEP culture medium (Kan)⁺) Shaking and culturing at 28 deg.C and 200r/min for 3-5 h; centrifuging at room temperature (5000r/min, 5min), retaining 100 μ L supernatant, resuspending thallus, and spreading on LA solid medium (Kan)⁺) Inverted culture at 28 ℃ for 2 days until outgrowthAnd selecting a colony with a proper size, and carrying out PCR detection on the single colony to obtain a positive strain.

(5) Callus induction: rinsing the seeds with sterile water for 15-20min, sterilizing with 75% ethanol for 1min, and sterilizing with sodium hypochlorite (1.5% effective concentration) solution under shaking for 20 min. Finally, the mixture is washed with sterile water for 5 times. The washed seeds were blotted dry with absorbent paper and inoculated in an induction callus medium and cultured in the dark at 25 ℃ for 2 weeks.

Callus induction medium: the induction medium shown in Table 1 was added with proline 0.3g, casein hydrolysate 0.6g, sucrose 30g and 2.5ml of 2,4-D (concentration 1mg/ml) to prepare a 1L solution, the pH was adjusted to 5.9, agar powder 7g was added, and the solution was sterilized at high temperature and high pressure.

(6) Subculturing: the embryogenic callus was excised, inoculated into a subculture medium, and cultured in the dark at 25 ℃ for 2 weeks.

Subculture medium: adopting the subculture medium shown in Table 1, adding 0.5g of proline, 0.6g of hydrolyzed casein protease, 30g of sucrose and 2ml of 2,4-D (concentration 1mg/ml) to prepare 1L solution, adjusting pH to 5.9, adding 7g of agar powder, and sterilizing at high temperature and high pressure.

(7) Agrobacteria dip dyeing and callus co-culture: culturing agrobacterium, selecting positive single colony, culturing in 1ml agrobacterium culture solution (containing antibiotic) at 28 deg.C overnight; the above culture was added to 50ml of Agrobacterium culture medium (containing antibiotics) and cultured at 28 ℃ until OD600 became 0.6-1.0. And centrifuging the obtained agrobacterium liquid, adding the collected thalli into a suspension culture solution, and performing shake culture for 30min until OD600 is 0.6-1.0. Then placing the callus into suspension culture solution containing agrobacterium liquid, and carrying out shake culture for about 20 min. Air drying the callus on sterilized filter paper, transferring into co-culture medium, and dark culturing at 25 deg.C for 5 d.

Suspension culture solution: using the suspension culture medium shown in Table 1, 0.08g of hydrolyzed casein, 2g of sucrose and 0.2ml of 2,4-D (concentration: 1mg/ml) were added to prepare 100ml of a solution, the pH was adjusted to 5.4, the solution was divided into two bottles (50 ml each), and the solution was sterilized by autoclaving at high temperature. 1ml of 50% glucose and 100. mu.L AS (100mM) were added before use.

Co-culture medium: the co-culture medium shown in Table 1 was used, and 0.8g of hydrolyzed casein protease, 20g of sucrose and 3.0ml of 2,4-D (concentration: 1mg/ml) were added to prepare 1L of a solution, the pH was adjusted to 5.6, 7g of agar powder was added, and high-temperature and high-pressure sterilization was carried out. 20ml of 50% glucose and 1ml of AS (100mM) were added before use.

(8) Screening and culturing: after co-culturing for 3 days, selecting the good callus, transferring the callus into a screening culture medium, carrying out dark culture at 25 ℃ for 2 weeks, and screening twice.

Screening a culture medium: the screening medium shown in Table 2 was used, and 0.6g of hydrolyzed casein protease, 30g of sucrose and 2.5ml of 2,4-D (concentration: 1mg/ml) were added to prepare 1L of a solution, the pH was adjusted to 6.0, 7g of agar powder was added, and high-temperature autoclaving was performed. 1ml Hn and 1ml Cn (100ppm) were added before use.

(9) Differentiation culture: selecting embryogenic callus, inoculating into differentiation culture medium, culturing at 24 deg.C for 16h/8h in light and dark to induce differentiation bud (4-6 weeks).

Differentiation medium: adopting the differentiation culture medium shown in Table 2, adding 2.0 mg/L6-BA, 2.0mg/L KT, 0.2mg/L NAA, 0.2mg/L IAA, 1.0g of hydrolytic casein and 30g of sucrose to prepare 1L solution, adjusting pH to 6.0, adding 7g of agar powder, and sterilizing at high temperature and high pressure.

(10) Rooting culture: when the bud grows to about 2cm, cutting off the bud, inserting the bud into a rooting culture medium, culturing at about 25 ℃ in 16h/8h in light and dark, and inducing to root.

Rooting culture medium: the rooting medium shown in Table 2 was added with 30g of sucrose to prepare 1L of solution, the pH was adjusted to 5.8, 7g of agar powder was added, and the solution was sterilized at high temperature and high pressure.

(11) Culturing transformed plants: opening the test tube mouth after the root system is developed, adding sterile water to harden the seedlings for 2-3d, taking out the plants, washing the attached solid culture medium with sterile water, transferring the solid culture medium into soil, shading and avoiding wind at the beginning, and performing conventional field or greenhouse management culture after the plants are robust.

TABLE 1 minimal Medium composition 1

TABLE 2 minimal Medium composition 2

(12) Positive detection of overexpressing plants

Cutting regenerated plant leaves, extracting DNA by using a CTAB method, and carrying out PCR detection by using hpt specific primers (hptF: ACACTACATGGCGTGATTTCAT; hptR: TCCACTATCGGCGAGTACTTCT), wherein the primer sequences are shown in a sequence table SEQ ID NO. 7-8.

And (3) PCR system:

PCR reaction procedure:

(13) detection of expression level of target gene in over-expression positive plant

Get T₀And (3) replacing rice leaves, grinding in liquid nitrogen, and extracting RNA. The method comprises the following specific steps:

extraction of RNA: freezing the sample with liquid nitrogen in mortar, grinding into powder, adding 1ml TRNzol-A⁺2mL of an EP tube (Tiangen Biochemical technology Co., Ltd.) containing the reagent was shaken sufficiently, and then left at room temperature for 5min, followed by adding 0.2mL of chloroform, shaking vigorously for 15s, and then left at room temperature for 3 min; after centrifugation at 12000rpm for 10min at 4 ℃ the supernatant was transferred to a new 2mL EP tube, an equal volume of isopropanol was added to precipitate RNA, and 100. mu.L of RNase-free ddH was added₂And dissolving the O. The total RNA quality is identified by electrophoresis, and then the RNA content is determined on a spectrophotometer.

Reverse transcription to synthesize first strand cDNA

The extracted RNA sample was digested with DNaseI before reverse transcription in the following reaction scheme:

after 15min at 37 ℃ the reaction was stopped by adding 0.25. mu.L of 0.1M EDTA (to ensure a final concentration >2mM), incubating at 70 ℃ for 10min, and briefly centrifuged and placed on ice for further use.

First strand cDNA was synthesized according to the Promega reverse transcription System A3500 handbook, with the following steps:

the following reagents were added to the DNaseI digested sample in order to prepare a 20. mu.L reaction system:

incubating the reaction system at 42 ℃ for 15 min; then heating at 95 deg.C for 5min to inactivate AMV reverse transcriptase and prevent it from binding to DNA; standing at 4 deg.C or on ice for 5 min. The prepared cDNA can be used immediately or stored at-20 ℃ for use.

Quantitative analysis of Gene expression Using Takara

Premix Ex Taq^TM(Perfect Real Time) kit, and American ABI

7000 quantitative PCR instrument. Quantitative primers (NtcrtOQF: 5-GGACTTTATTCGCACCAT-3; NtcrtOQR: 5-TAACGCCACCTTTACTTG-3, and the primer sequences are shown in a sequence table SEQ ID NO. 9-10) are designed according to the NfcrtO sequence. A primer was designed based on the cDNA sequence of a rice housekeeping gene actin (GenBank accession No. AY212324) as a reference gene.

Preparation of 20. mu.l reaction System:

the reaction conditions are as follows: the temperature is 95 ℃ for 30s, then the circulation is carried out for 40 times at 95 ℃ for 5s and 60 ℃ for 31s, and the Dissociation Stage is additionally arranged. Data was collected at 60 ℃ for 31s for each cycle and other detailed operations were performed according to the instrument instructions. Calculating the average CT value and the Delta CT value of the target gene and the reference gene, using 2^-ΔΔCTThe method is used for analyzing results, and finally, data are introduced into GraphPad prism5.0 to make a histogram of the relative expression amount of the target gene, which is shown in figure 5.

Example 4

Seedling-stage PEG simulated drought treatment of transgenic rice

Over-expressing transgenic pedigree seeds were dehulled and sterilized (75% alcohol treatment for 1min, 1.5% NaClO treatment for 20min, 5 washes in sterile water), germinated on 1/2MS medium containing 50mg/L hygromycin, and wild type controls were sown overnight on 1/2MS medium without hygromycin. And selecting seeds which have good germination and consistent growth vigor after 2-3 days of germination, and transferring the seeds to a 96-hole plate for water culture (by adopting a nutrient solution formula of International Rice institute). And (3) adding 20% of PEG6000 to simulate drought stress in the three-leaf one-heart stage, stressing until all controls die, then recovering nutrient solution for culture, and observing survival conditions. The experimental results show that after 10 days of rehydration culture, the wild type control has no survived plants, and the transgenic lines OE-4 and OE-5 have 43% and 26% survival rates respectively, and particularly as shown in FIG. 6, the fact that the overexpression of the Nfcro gene in rice indeed improves the restoration drought resistance of the transgenic plants is demonstrated.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention, and the present invention should not be limited by the disclosure of the preferred embodiments. Therefore, it is intended that all equivalents and modifications which do not depart from the spirit of the invention disclosed herein are deemed to be within the scope of the invention.

Sequence listing

<110> Shanghai city agricultural biological gene center

<120> Nostoc flagelliforme NfcrtO drought-resistant gene and amino acid sequence and application thereof

<141> 2017-10-23

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1695

<212> DNA

<213> Nostoc flagelliforme

<400> 1

atgcaagagt atgatgttgt gctgatcggt gccggacaca atgggttagt ttgtgcagct 60

tatttgctga aagctggtta tagcgtcctg ttactagaaa agcgttctgt tccaggcggt 120

gcagcaacaa ctgaagaatg tttaccacaa gaagctcctg gatttaaatt taacttgtgt 180

gctattgacc atgaatttat tcacttgggg ccggtagttg aagaattaga actagaaaaa 240

tacggcttgc attatttgga gtgtgatcca gttgttttct gtcctcatcc tgatggtaag 300

tatttcttag cacataaatc gctggaaaaa acttgtgcag aaatcgctcg ttatagtgaa 360

cgggatgcca aaaaatacgc agaatttgta gattattggc aacgagcgat cggtgcaatg 420

gttcctatgt ttaatgcacc gccaaagtca attatagata tcgttggcaa ctacgatatc 480

aaaaaattaa aagatttatt ttcgatgatt ggttccccaa acaaaacgct ggactttatt 540

cgcaccatgt taaccagcgc agaggattta cttaacgagt ggtttgatga ggaatttcta 600

aaagcgccac tagccaggct cgcagcagaa cttggtgcgc cgccatcgca aaaaaccctt 660

gccattggtg caattatgat ggcgatgcgt cacaaccctg gaatggccag gccacgcggc 720

ggaactggcg cacttgtgaa agctttagtg gatttagtca caagtaaagg tggcgttatt 780

ctgacagatc agcatgttga aaaagtttta attgatgatg gaaaagctgt tggtgtacga 840

gtggctggta acaccgaata tcgggctaaa tacggcgtaa tttctaatat tgatgccaag 900

cggttatttt tgcaaatgac tgataaaagc gatattgatg aagccgatcc tgacttatgg 960

gaaagattag aacgccgcat cgttaataat aacgaaacta tcctcaagat agacttagct 1020

ttagacgaac cactgcgctt tccataccac gcccataaag acgaatatct cgttggttct 1080

atcttaattg ccgattccgt ggctcatgta gaacaggctc atagtaaatg tagcttagga 1140

gaaattcctg atgctgaccc atcaatgtat gtggtcatgc ctagttattt agatcccaca 1200

ttagcaccac caggtaagca cactgtatgg attgagtatt ttgcccctta tcaaattgct 1260

ggtgcagaag gcactggttt aagaggtact ggttggacag atgaattgaa aaacaaagtt 1320

gcagataaag tggttgataa gttggcagac tatgcaccaa atgtcaagaa cgcaactatc 1380

gcccgtcgtg tagaaagtcc agcagaatta ggagaaagat taggtgcgta taaagggaat 1440

tattaccata ttgacatgac tttagatcag atggtatttt tccgtccctt gccagaaata 1500

gcaaactaca aaacgccaat tgagaatcta tttttgactg gtgcaggtac tcatccaggt 1560

ggttcgattt cgggaatgcc aggacgcaat tgtgcgcgtg tatttttgca ggcaaaacat 1620

cccattagtc aaactctgaa ggatgcacgc gattcgatta agtcaactgt agagtcggtg 1680

tttggaatta attaa 1695

<210> 2

<211> 564

<212> PRT

<213> Nostoc flagelliforme

<400> 2

Met Gln Glu Tyr Asp Val Val Leu Ile Gly Ala Gly His Asn Gly Leu

1 5 10 15

Val Cys Ala Ala Tyr Leu Leu Lys Ala Gly Tyr Ser Val Leu Leu Leu

20 25 30

Glu Lys Arg Ser Val Pro Gly Gly Ala Ala Thr Thr Glu Glu Cys Leu

35 40 45

Pro Gln Glu Ala Pro Gly Phe Lys Phe Asn Leu Cys Ala Ile Asp His

50 55 60

Glu Phe Ile His Leu Gly Pro Val Val Glu Glu Leu Glu Leu Glu Lys

65 70 75 80

Tyr Gly Leu His Tyr Leu Glu Cys Asp Pro Val Val Phe Cys Pro His

85 90 95

Pro Asp Gly Lys Tyr Phe Leu Ala His Lys Ser Leu Glu Lys Thr Cys

100 105 110

Ala Glu Ile Ala Arg Tyr Ser Glu Arg Asp Ala Lys Lys Tyr Ala Glu

115 120 125

Phe Val Asp Tyr Trp Gln Arg Ala Ile Gly Ala Met Val Pro Met Phe

130 135 140

Asn Ala Pro Pro Lys Ser Ile Ile Asp Ile Val Gly Asn Tyr Asp Ile

145 150 155 160

Lys Lys Leu Lys Asp Leu Phe Ser Met Ile Gly Ser Pro Asn Lys Thr

165 170 175

Leu Asp Phe Ile Arg Thr Met Leu Thr Ser Ala Glu Asp Leu Leu Asn

180 185 190

Glu Trp Phe Asp Glu Glu Phe Leu Lys Ala Pro Leu Ala Arg Leu Ala

195 200 205

Ala Glu Leu Gly Ala Pro Pro Ser Gln Lys Thr Leu Ala Ile Gly Ala

210 215 220

Ile Met Met Ala Met Arg His Asn Pro Gly Met Ala Arg Pro Arg Gly

225 230 235 240

Gly Thr Gly Ala Leu Val Lys Ala Leu Val Asp Leu Val Thr Ser Lys

245 250 255

Gly Gly Val Ile Leu Thr Asp Gln His Val Glu Lys Val Leu Ile Asp

260 265 270

Asp Gly Lys Ala Val Gly Val Arg Val Ala Gly Asn Thr Glu Tyr Arg

275 280 285

Ala Lys Tyr Gly Val Ile Ser Asn Ile Asp Ala Lys Arg Leu Phe Leu

290 295 300

Gln Met Thr Asp Lys Ser Asp Ile Asp Glu Ala Asp Pro Asp Leu Trp

305 310 315 320

Glu Arg Leu Glu Arg Arg Ile Val Asn Asn Asn Glu Thr Ile Leu Lys

325 330 335

Ile Asp Leu Ala Leu Asp Glu Pro Leu Arg Phe Pro Tyr His Ala His

340 345 350

Lys Asp Glu Tyr Leu Val Gly Ser Ile Leu Ile Ala Asp Ser Val Ala

355 360 365

His Val Glu Gln Ala His Ser Lys Cys Ser Leu Gly Glu Ile Pro Asp

370 375 380

Ala Asp Pro Ser Met Tyr Val Val Met Pro Ser Tyr Leu Asp Pro Thr

385 390 395 400

Leu Ala Pro Pro Gly Lys His Thr Val Trp Ile Glu Tyr Phe Ala Pro

405 410 415

Tyr Gln Ile Ala Gly Ala Glu Gly Thr Gly Leu Arg Gly Thr Gly Trp

420 425 430

Thr Asp Glu Leu Lys Asn Lys Val Ala Asp Lys Val Val Asp Lys Leu

435 440 445

Ala Asp Tyr Ala Pro Asn Val Lys Asn Ala Thr Ile Ala Arg Arg Val

450 455 460

Glu Ser Pro Ala Glu Leu Gly Glu Arg Leu Gly Ala Tyr Lys Gly Asn

465 470 475 480

Tyr Tyr His Ile Asp Met Thr Leu Asp Gln Met Val Phe Phe Arg Pro

485 490 495

Leu Pro Glu Ile Ala Asn Tyr Lys Thr Pro Ile Glu Asn Leu Phe Leu

500 505 510

Thr Gly Ala Gly Thr His Pro Gly Gly Ser Ile Ser Gly Met Pro Gly

515 520 525

Arg Asn Cys Ala Arg Val Phe Leu Gln Ala Lys His Pro Ile Ser Gln

530 535 540

Thr Leu Lys Asp Ala Arg Asp Ser Ile Lys Ser Thr Val Glu Ser Val

545 550 555 560

Phe Gly Ile Asn

<210> 3

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 3

atgcaagagt atgatgttgt gctg 24

<210> 4

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 4

actttgtcct gtgcaaggct ta 22

<210> 5

<211> 45

<212> DNA

<213> Artificial Sequence

<400> 5

caggtcgact ctagaggatc catgcaagag tatgatgttg tgctg 45

<210> 6

<211> 43

<212> DNA

<213> Artificial Sequence

<400> 6

gggaaattcg agctggtcac cactttgtcc tgtgcaaggc tta 43

<210> 7

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 7

acactacatg gcgtgatttc at 22

<210> 8

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 8

tccactatcg gcgagtactt ct 22

<210> 9

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 9

ggactttatt cgcaccat 18

<210> 10

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 10

taacgccacc tttacttg 18

Claims (2)

1. An application of Nostoc flagelliforme NfcrtO drought-resistant gene in improving the drought resistance of rice is characterized in that the nucleotide sequence of the gene is shown in a sequence table SEQ ID NO. 1.

2. The application of the nostoc flagelliforme NfcrtO drought-resistant gene in improving the drought resistance of rice according to claim 1, which is characterized in that: the amino acid sequence of the gene is shown in a sequence table SEQ ID NO. 2.

Images