A method of improving nitrogen assimilation efficiency in plants

0001Method for improving plant nitrogen assimilation efficiency

0002The present invention relates to a method for increasing the efficiency of plant nitrogen assimilation.

0003technical background

0004Nitrogen is the first major nutrient element required for plant growth. At present, only 30%-40% of the nitrogen fertilizer applied in agriculture is absorbed and utilized by plants, and most of them are wasted. Some of it is converted into nitrates that are lost to the soil, causing environmental pollution. The GDH-transformed crop can effectively increase the absorption of nitrogen in plants and increase the utilization rate of nitrogen fertilizer, thereby saving the application amount of nitrogen fertilizer, and the economic benefit is enormous. At the same time, GDH is widely found in animals, plants and microorganisms, so plants that turn GDH do not harm humans, animals and plants.

0005At present, there are mainly studies on transgenic plants of glutamate dehydrogenase in the United States, Australia and other countries (US Patent N0.5955651; 5985634; 5998700). Their glutamate dehydrogenase genes are mainly derived from chlorella and Escherichia coli, and the model plants are economic crops such as tobacco and corn. Studies have found that the nitrogen utilization rate of plants transgenic with glutamate dehydrogenase is increased, which is manifested in larger and more abundant plant leaves. The nitrogen content of different tissues of plants transgenic with E. coli GDH was determined to be about 16% higher than that of the control group, and the amino acid content of different tissues was changed. Significantly, the content of glutamic acid is significantly lower, while the content of alanine, lysine, aspartic acid and the like is increased. The starch volume of the GDH gene potato was significantly increased compared with the control group. However, the activity of glutamate dehydrogenase used so far is much lower.

0006SUMMARY OF THE INVENTION - It is an object of the present invention to provide a method for increasing the efficiency of plant nitrogen assimilation.

0007To achieve the above object, the present invention provides a method for improving the efficiency of plant nitrogen assimilation, comprising: (a) a fungal glutamate dehydrogenase (GDH) gene and a protein capable of directing expression of a foreign gene in a plant. The promoter is ligated to construct a chimeric gene: (b) The constructed chimeric gene is introduced into a plant cell, and the transformed plant is selected and cultured.

0008In the method of the present invention, the promoter capable of directing expression of a foreign gene in a plant may be any one known in the art to direct expression of a foreign gene in a plant. Preferably, the glutamate dehydrogenase gene is a prosthetic NADP glutamate dehydrogenase gene or a prosthetic NAD glutamate dehydrogenation gene. The fungal glutamate dehydrogenase gene may be derived from a filamentous fungus of the genus Neurospora, including Neurospora, Neurospora, and Neurospora crassa. The glutamate dehydrogenase gene may also be derived from a yeast fungus, such as Saccharomyces cerevisiae. The glutamate dehydrogenase gene may also be derived from Basidiomycetes, such as Agaricus gar. The plant may be tobacco, corn, cotton or rice.

0009In the method of the present invention, the glutamate dehydrogenase may be derived from a glutamate dehydrogenase of Neurospora intermedia (Ni) having the sequence of SEQ ID NO: 1. The gene encoding the above glutamate dehydrogenase may have the sequence shown in SEQ ID NO: 2.

0010In the method of the present invention, the glutamate dehydrogenase may be derived from Neurospora sitophila (Ns) having the amino acid sequence of SEQ ID NO: 3. The glutamate dehydrogenase gene encoding the above may have the sequence shown in SEQ ID NO: 4.

0011We cloned three strains of Ni-GDH, Ns-GDH and Nc-GDH into Escherichia coli BL21 (DE3) for expression, and purified Ni-GDH, Ns-GDH, Nc-GDH for enzyme activity determination. It was found that the enzyme activities of the three GDHs were higher than those of the other genera. It has strong affinity and stability to ammonia. These three GDH genes were subcloned into the plant expression vector pROKII, and transformed into tobacco, corn, cotton and other crops by Agrobacterium transformation, electroporation transformation and pollen tube channel method. P8 'sex transformants were screened by PCR, Southern, Northern and enzymatic activity staining. It was transferred to a medium with different nitrogen concentration, and it was found that the tobacco expressing GDH could grow normally at a low nitrogen concentration of 5 mM to a lower ammonia ion concentration, while the leaves of the untransformed control group were yellowed and hindered in development. Nitrogen deficiency symptoms. The total nitrogen content and the unused nitrogen residues of the plants under low nitrogen conditions were determined. It was found that the nitrogen content of the transgenic fungal GDH plants was 20% higher than that of the unconverted, and the nitrogen residuals were reduced by 20-30%. The total nitrogen content of the plants in the nitrogen-depleted soil was determined, and the result was about 40% higher than that of the untransformed plants. At the same time, we constructed economic crops such as corn, rice, and cotton that have been transferred to the GDH gene, and found that the nitrogen content in the soil is 20-30% higher than that of the final transformed crop, and the nitrogen residue in the soil is reduced by more than 20%. The test showed that GDH was highly expressed in the above plants, which accelerated the oxidative deamination of glutamic acid and the reductive amination of α-ketoglutarate, which initiated a new nitrogen in plants. The utilization route improves the utilization of ammonia. Although higher plants have GDH, their affinity for ammonia is only 1 / 10 to 1 / 100 of fungal GDH. Therefore, the role of assimilation of ammonia cannot be exerted. The fungal GDH oxidative deamination of glutamate is accompanied by the release of large amounts of ATP and the formation of ct-glutoglutarate, which provides plants with sufficient energy and a large amount of carbohydrates involved in the tricarboxylic acid cycle. Nitrogen is the first major nutrient element required for plant growth. At present, only 30%-40% of the nitrogen fertilizer applied in agriculture is absorbed and utilized by crops, and most of them are lost, causing environmental pollution. The crops that are transferred to GDH can effectively increase the nitrogen utilization rate, thereby saving nitrogen utilization and reducing environmental pollution, and the economic benefits are enormous. EXAMPLES Example 1. Culture of Neurospora, Saccharomyces Cerevisiae and Agaricus bisporus and induction of glutamate dehydrogenase

00121. Transfer the three fungi from the solid slope to the wort medium, centrifuge for 48 hours at 250 rpm, collect the mycelia, and transfer to ammonia induction medium (4% glucose, 0.02MNH4AC, 0.04M N05). After induction for 3 hours, the mycelium was collected by centrifugation.

00132. Total RA extraction and reverse transcription-polymerase chain reaction (RT-PCR method to amplify glutamate dehydrogenase gene: After induction of mycelium by liquid nitrogen grinding and crushing, using guanidinium isothiocyanate The total RNA was extracted by reverse transcription-polymerase chain reaction.

0014The primers used are as follows:

0015Primer 1: 5 ' GCTCAGAATGTCTAACCTTCCCTCTGAG 3 '

0016Bow 1 2: 5 ' GCGAGCTCTAGTCTTGGACCACCAGTCACC 3 '

0017The reverse transcription reaction conditions are:

001865 V for 1 minute,

001930 ° C for 5 minutes,

002030 ° C - 65 ° C for 30 minutes,

002198 ° C for 5 minutes,

00225. C 5 minutes.

0023Polymerase chain reaction conditions:

002494 °C for 3 minutes, - 94 °C for 1 minute

002555 Ό 1 minute 25 cycles

002672 ° C 2 minutes

002772 10 min 3. Sequence analysis of Neurospora glutamate dehydrogenase (GDH) gene:

0028The glutamate dehydrogenase (GDH) gene amplified by the RT-PCR method was recovered by agarose gel electrophoresis. Three microliters (ul) of recovered product was added, and lul of pGEM-T eas vector, 5 ul of 2xT4 ligase buffer, lul DNA ligase, and 4' C enzyme were added overnight. The next day, the enzyme product was transformed into E. coli 'DH50a for colony screening. The screening method was identified by colony PCR and restriction enzyme digestion, and the restriction sites were Xbal and Sacl. The restriction enzymes produced two bands of 3.0 kb and 1.4 kb as the positive clone pT-GDH. Positive clones were picked and sequenced. 4. Saccharomyces cerevisiae and Agaricus bisporus glutamate dehydrogenase (GDH) gene sequencing:

0029The glutamate dehydrogenase (GDH) gene amplified by the RT-PCR method was recovered by agarose gel electrophoresis. Three microliters (ul) of recovered product was added, and lul of pGEM-T easy vector, 5 ul of 2xT4 ligase buffer, lul DNA ligase, and enzyme overnight at 4 °C were added. The next day, the enzyme-linked product was transformed into E. coli DH50a for colony screening. The screening method was identified by colony PCR and restriction enzyme digestion. The restriction enzyme sites were Xbal and SacL, and the two bands of 3.0 kb and 1.4 kb were positive clone pT-GDH. Positive clones were picked and sequenced.

00305. Expression of Neurospora, Saccharomyces Cerevisiae and Agaricus bisporus glutamate dehydrogenase gene in Escherichia coli

0031The vector pT-GDH was digested with Xbal, Sad, and recovered by agarose electrophoresis to obtain a GDH fragment, which was ligated overnight with 4 pΌ of the pBluescript vector digested with the same enzyme. The next day, E. coli DH50a was transformed, and the positive clone pBlueGDH was identified by colony PCR and restriction enzyme digestion. pBlueGDH was digested with ECoRV and SacI, and the GDH fragment was recovered by agarose electrophoresis, and ligated with pET30a vector digested with the same EcoRV and Sad at 12 °C overnight. The next day, E. coli DH50a was transformed and identified by colony PCR and restriction enzyme digestion. The positive clone was pETGDH. The next day, positive clones were picked and transformed into E. coli BL21 (DE3). The cells were cultured to an OD of 0.4, induced by ImM IPTG for 4 hr, and the cells were harvested. After the cells were washed with deionized water, they were sonicated. Centrifugation, supernatant and pellet were separately subjected to 10% SDS-PAGE electrophoresis. The results demonstrate that the expression product exists as an inclusion body.

00326. Metal-integrated affinity chromatography for purification of glutamate dehydrogenase.

0033Formulated in a solution of 8M urea in MCAC-10 (20mm/L Tris · CI, pH 7.9, 0.5mol/L NaCl,

003410% (v / v) glycerol, ImmOl several PMSF). At the same time, MCAC-40, MCAC-60, MCAC-80, MCAC-100, MCAC-200, MCAC-500 were prepared, that is, O.4mol/L, 0.6mol/L, O.8mol/L were added to MCAC-0. , lmol / L, 2mol / K 5mol / L of imidazole.

0035The glutamate dehydrogenase inclusion body was dissolved in the MCAC-0 solution containing 8 M urea, and applied to the NTA column. The column was washed with 5 ml of 8M Urea-MCAC buffer at a flow rate of 20-30 ml / h, and discarded. Effluent. The column was washed sequentially with 5 ml of the following buffer, 8 MUrea-MCAC40, 8M Urea-MCAC60, 8M Urea-MCAC80, 8M Urea-MCACIOO, 8Μ Urea-MCAC200, 8Μ Urea-MCAC500, respectively, and the effluent was collected. Perform 10% SDS-PAGE electrophoresis and silver staining for purity identification. 8M Urea "MCAC200 after the sinking liquid was collected for dialysis renaturation. The dialysis buffer was divided into two types, 0.1 mol/L Tris.HCl, pH 8.5 and pH 7.4, and 1 mM EDTA. 7. Dehydrogenation of glutamic acid Enzyme activity assay

0036The renatured refolding protein was centrifuged overnight, and the supernatant was collected. The protein concentration was determined by UV detection at 280 nm. The concentration (mg / ml) = A280 x 0.825 ο The enzyme activity was measured in the system Α and system C, respectively.

0037System A measures the reductive amination of GDH.

00382.55ml 0.1MTris.HCl, ImMEDTA, pH 7.4

00390.1ml 0.1MNH4C1

00400.15ml 0.2M a-ketoglutaric acid

00410.2ml 0.15% (W / V) NADPH

00422 ul renatured protein, incubated at 25 ° C for 10 minutes at 25. The change in the absorbance of the system A at 340 nm was measured at a constant temperature of C. System C, Determination of oxidative deamination of GDH:

00432.8 ml of 0.16 M glutamic acid monosodium salt was dissolved in 0.1 M Tris.HCl, 1 mM EDTA, pH 8.5 buffer, 0.2 ml 0.2% NADP, 2 ul of refolding protein, and incubated at 37 ° C for 10 minutes. 340mm change in light absorption value. The unit of activity is one micromolar NADP reduction per minute⁺One micromolar NADPH is one unit per unit or minute of oxidation. - The results were determined to be 109.92 UI mg for Ni-GDH activity in System A, 72.93 U / mg for Ni-GDH in System C, and 95.37 U/mg for System A, in System C. It is 63013 U/mg, Nc-GDH is 100.25 U/mg in System A, and 65.00 U/mg in System C. 8. The subfamily of the cytosine dehydrogenase gene is subcloned into the plant expression vector pROKII

0044The GDH gene fragment was digested with Xbal and Saci from the pT-GDH vector, and after recovery by agarose electrophoresis, the pROKII vector was digested with the same enzyme. C enzyme overnight. The next day, E. coli DH5a was transformed, positive clone pROKII-GDH was identified by colony PCR and restriction enzyme digestion, and positive clones were selected and transformed into Agrobacterium LBA4404. 9. Saccharomyces cerevisiae, Agaricus bisporus glutamate dehydrogenase gene subcloned into plant expression vector pROKII

0045The GDH gene fragment was digested with Xbal and Sad from the pT~GDH vector, and recovered by agarose electrophoresis, and then ligated with the same digested pROKII vector at 4 过夜 overnight. The next day, E. coli DH5a was transformed, and the positive clone pROKII-GDH was identified by colony PCR and restriction enzyme digestion, and the positive clone was selected and transformed into Agrobacterium LBA4404.

004610. Agrobacterium tumefaciens-mediated leaf disc transformation for transformation of tobacco

0047(1) Cultivation of tobacco sterile seedlings

0048The surface of the tobacco seeds was sterilized and cultured on a hormone-free MS medium (MS salt of 10 15 g/L sucrose, 10 g/L agar). 25-28Ό , 80uE(m²S) Illumination for 16 hours, with the growth of the seedlings (after 1 to 1.5 months), the shoot tips were cut and transferred to a new MS medium to produce plantlets.

0049(2) Co-cultivation of tobacco leaf discs

0050The Agrobacterium tumefaciens containing the recombinant plasmid was inoculated into 5 ml of a medium containing kanamycin and rifampicin, and cultured overnight at 28 ° C at 200 rpm on a shaker, and the cells were collected by centrifugation. The edge and middle vein of the sterile seedlings were cut with a scalpel, and the leaves were cut into 5-8 mm wide strips along the vertical direction of the midrib. The leaves were immediately immersed in the Agrobacterium liquid for 30-40 minutes after cutting. The co-cultured leaves were taken out with tweezers, placed on a sterile filter paper to remove too much bacterial liquid, and the leaf strips were transferred to a medium containing co-culture (MS inorganic salt ten 0.6 mg 1 12, 4 D D 30 g / 1 sucrose ten 8g 11 agar) in a plate. The plate is sealed with a membrane to reduce evaporation and contamination of the water. Incubate at 28 ° C for 48 hr.

005111. Screening of transformed plants - Transfer of co-cultured leaves to callus induction medium (MS inorganic salt 100.6 mg 1 12,4-D 10 300 mg 1 1 kanamycin 10 500 mg / 1 jicomycin 10 30 g 1 1 sucrose on 10 8g 1 1 agar), making the transformed leaves fully contact with the medium is beneficial to the absorption of nutrients and hormones. After two weeks of culture on the callus induction medium, the leaves are transferred to the bud medium (MS inorganic salt, Lmg / 1 IAA eleven mg / 16 - BA ten 300 mg I 1 kanamycin ten 500 mg 11 carbenicillin ten 30 g 1 1 sucrose ten 8 g I 1 agar) culture. The small shoots were cut with a scalpel and transferred to a rooting medium (MS inorganic salt ten 0.4 mg / 11 BA ten 100 mg I 1 kanamycin ten 30 g 1 1 sucrose ten 8 g / l agar).

005212. Gene gun method for transforming corn

0053Place the gene gun in a larger, clean bench to facilitate aseptic processing. A 6 ul DNA-coated metal particle anhydrous ethanol suspension (about 0.6 ug of plasmid and 0.37 ug of metal particles) was spotted at the center of the microparticle carrier, immediately dried in a desiccator, or blown dry on a bench. Place the target tissue to be transformed in a 1-2 layer filter paper moistened with liquid medium or a 9 cm culture dish containing a solid culture dish, and evacuate when the vacuum reaches the desired value (660-760 mmHg, lmmHg). At =133, 322Pa), bombardment, a shot in about 12 seconds. The bombarded explants are transferred to callus induction medium or bud differentiation medium, 28' C, cultured in dark or weak light, and no screening pressure is added to the medium, and the transition culture is generally 1 to 2 weeks. . The explants after the transient culture are selectively cultured on a medium containing appropriate canarerein, and transferred to a subculture medium for about 1 month.

005413. Pollen tube pathway mediates fungal GDH gene transformation in cotton

0055The pROKII-GDH vector containing GDH gene was dissolved in 1 X SSC solution, and the normal flower was selected for emasculation, bagged isolation, cotton filaments were cut one day before pollination, and a thin layer of pollen germination was added to the sterile culture dish. Base, collect the fresh germinated pollen into the medium, incubate for 3 minutes under 3CTC conditions, add 30mm per 10ml medium³Pollen, when about 1 / 10 of the pollen has sprouted, add 1 / 10 volume of DNA solution carefully and mix it. After mixing with the pollen, the final concentration of DNA is 5ug / ml. Apply a mixture of DNA and pollen to the stigma. About 10mm³The treated pollen is given an ear, and after pollination, the bag is re-packed to isolate the seed.

005614. Agrobacterium-mediated transformation of fungal GDH gene into rice

0057Agrobacterium tumefaciens containing recombinant GDH gene was inoculated into 5 ml of kanamycin-containing and rifampicin solution, and cultured at 28 ° C for 200 rpm overnight. The cells were collected by centrifugation, and the sterile seedlings were cut into Agrobacterium immediately after cutting. Soak in the liquid for 30 minutes, take out the leaves, and then remove too much bacterial liquid onto the sterile filter paper and transfer to the co-culture medium. Incubate at 28 ° C for 48 hr.

005815. Screening and identification of plant-positive transformants of transgenic fungi GDH gene

0059(1) Method for extracting DNA from plants

0060Take lg plant leaves and add liquid nitrogen to grind into powder. Add 700 to 2xCTAB extract (2% (WIV) CTAB (hexadecanoyltriethylammonium bromide), 100 mmol / l Tris 'CI, pH 8.0, 20 mmol / L EDTA, 1.4 mol / INaCI), shake gently Evenly, water bath at 65 ° C for 30 minutes, shaking from time to time. Add 700 ul of phenol/chloroform/isoamyl alcohol (25:24:1) and gently shake until the solution is emulsified, and centrifuge at 7000 rpm for 5 minutes. The supernatant was taken and further extracted with phenol/chloroform/isoamyl alcohol (25:24:1) 2-3 times, 2 volumes of absolute ethanol were added, and the mixture was precipitated at 20 ° C overnight. After 7000 rpm/separation of the heart for 10 minutes, the nucleic acid precipitate was collected and dissolved in deionized water. Store at -20 °C for later use. (2) Identification of positive transformants by nucleic acid hybridization

0061The extracted plant total DNA was digested overnight, and the restriction site was Xbal, Sad, and agarose gel electrophoresis was performed the next day. The DNA fragment was transferred from a gel electrophoresis to a nylon membrane, and the DNA fragment was fixed by ultraviolet irradiation for 3 minutes. The probe labeled with digoxin (DIG) was hybridized with DNA immobilized on a nylon membrane at a hybridization temperature of 68 V for 20 hours. After the hybridization was completed, the nylon membrane was washed and reacted with digoxin-resistant alkaline phosphatase (anti DIG-AP) for 30 minutes, and washed for NBT I BCIP color development.

0062As a result, a hybridization band appeared at a position of about 1400 bp, which was identical to the positive control, indicating that the GDH gene has been integrated into the plant genome.

006316. Transplantation and progeny analysis of transformed plants

0064Carefully remove the agar from the root of the plant, transfer the plant to a larger container containing the MS inorganic salt solution, open the lid, allow the gas to diffuse for a few hours, add some sterile water to replenish the evaporated liquid, and then transplant the plants. Into the moist soil. The resulting seeds were isolated by nucleic acid hybridization and GDH enzyme live staining assays until a homozygous transgenic line was obtained.

006517. Gel analysis and staining for glutamate dehydrogenase activity

0066Grinding in liquid ammonia, extracting plant leaf protein, performing 5% non-denaturing gel electrophoresis, i20V for 72 hours. The gel was soaked in the following staining solution (50 mM Tris pH 9.3, 8 mg / ml glutamic acid, 0.04 mg I ml NADP, 0.04 mg / ml MTT, 0.04 mg / ml sulfuric acid, cough, 0.08 mg / -mICaC12). After the gel was immersed in the staining solution, there was a band at the site containing GDH, indicating that the transformed GDH expressed active glutamate dehydrogenase in the plant.

006718. Transgenic Plant Growth and Nitrogen Use Efficiency Test - After the fungus GDH positive plants were harvested, the sterile seedlings were propagated (Same 8(1)). As the seedlings grow, the shoot tips are cut and transferred to MS medium with different nitrogen contents. The ammonia concentrations were 20 mM, 10 mM, 5 m, 2.5 mM, respectively, to test their growth. As a result, there was no significant difference between the positive transformants and the control progeny on the 20 mM, 10 mM MS medium. On 5 mM and 2.5 mM medium, the growth of positive transformants was significantly better than that of the control group, while the control showed symptoms of nitrogen deficiency such as leaf chlorosis. After the plants are grown for 1 to 1.5 months, the plants are cleaned, dried, and tested for dry weight. The results showed that the positive transformants increased the dry weight by about 20% compared with the control group. At the same time, using the Kjeldahl method, the nitrogen content of the transgenic plants was increased by 20-30% compared with the control group. At the same time, the nitrogen utilization rate of the plants grown in the MS medium was measured, and the nitrogen utilization rate of the transformed plants was increased by 20-30% compared with the untransformed plants.

0068Sequence table

0069<110> Institute of Microbiology, Chinese Academy of Sciences <120> A method for improving the efficiency of plant nitrogen assimilation

0070<160> 4

0071<210>SEQID O: 1

0072<211>454 amino acids

0073<212>protein

0074<213> Neurospora intermedia

0075<400>

0076<img file="WO0202776A1_D0001.tif" />

ELPSLVAGSNIAGFV VAQAMHDQGDWWSKN

0078<210>SEQIDNO: 2

0079<211> 1365 base pairs

0080<212> DNA

0081<213> Neurospora intermedia

0082<400>

AGGGTGACTGGTGGTCCAAGAACTAA

0084<210>SEQIDNO: 3

0085<211>454 amino acids

0086<212>protein

0087<213>Good food Neurospora sitophila

0088<400>

0089MSNLPSEPEFEQAY ELAYTLENSSLFQ HPEYRTALAVASIPERVIQFRWWEDDNGNVQ GELPSLVAGSNIAGFVKVPQAMHDQGDWWSKN

0090<210>SEQID O: 4

0091<211> 1365 base pairs

0092<212> DNA

0093<213>Neurospora sitophila <400>

0094Ει

0095WIDWOW. . Work 0. SD OVDiDOS

0096<img file="WO0202776A1_D0002.tif" />

0097^6J00/10N3/X3d 9 0 OAV