WO2009117853A1

WO2009117853A1 - Method for cultivating plants having increased ability of nitrogen uptake

Info

Publication number: WO2009117853A1
Application number: PCT/CN2008/000610
Authority: WO
Inventors: 王志兴; 刘昱辉; 贾士荣; 伍祥贵; 包骏
Original assignee: 北京优利康生物农业技术有限公司
Priority date: 2008-03-27
Filing date: 2008-03-27
Publication date: 2009-10-01

Abstract

Provided is a method for cultivating plants having increased ability of nitrogen uptake by transforming recombinant expression vectors containing root-specific promoter and NAT3 gene into plant tissues or cells to obtain transgenic plants growing normally at the low nitrogen condition. The method has wide application foreground in plant nitrogen fertilizer absorption and breeding plant variety with nitrogen deficiency tolerance.

Description

Method for cultivating plants with improved nitrogen absorption capacity

The present invention relates to a method of cultivating plants having improved nitrogen absorption capacity.

Background technique

Tobacco is an important economic crop in China. The national roasting tobacco planting area is 16.74 million mu, and the national tobacco leaf purchase volume is maintained at 2.1 million tons. It is an important source of tax revenue in China. Nitrogen fertilizer is a fertilizer that is necessary for various crops, especially tobacco growth. The application rate of nitrogen fertilizer directly affects the yield and quality of tobacco leaves.

In recent years, the application rate of nitrogen fertilizers of various crops has been increasing, and the application rate has exceeded the standard seriously. However, the utilization rate of nitrogen fertilizer in crops is very low, causing pollution to the environment and serious eutrophication of rivers.

(Diatom Nitrate Transporter) 3⁄4@ , cloned from the diatoms (O^^roi ^cs /ks / u's) grown in the sea by Mark Hildebrand, which has no introns. The 7\3⁄4Γ gene-encoded nitrate transporter has a very high binding capacity to nitrate, which allows diatoms to enrich nitrate from seawater. Studies have shown that Λ3⁄4 transgenic plants have the ability to grow normally in low-content nitrate soils. NAT can promote the growth of transgenic plants in low-nitrogen soils, and can reduce or even avoid the application of nitrogen fertilizers and avoid environmental pollution without affecting crop yield.

At present, a number of nitrogen transport-related genes have been cloned from plants, such as from gold algae.

The nitrate transporter (IgNRT) gene, the ammonia transporter (IgAMT) gene and the glutamate synthase (IgGSII) gene cloned in {Isochrysis galbana) have cDNA lengths of 540 bp, 658 bp and 852 bp, respectively. The results of real-time PCR showed that the number of IgNRT and IgAMT transcripts was 27.6 and 28.5 in the presence of nitrate, and the number of transcripts in nitrogen deficiency increased by 390 % and 178%, respectively. 4%和六五五。 The transcription of the IgNRT and the IgAMT gene was severely inhibited, the number of transcripts decreased by 2.4% and 6.5%. The highest expression level of IgGSII was in cells grown in nitrate, followed by cells grown in the absence of nitrogen (50%), and again in cells grown in ammonium (25%). Comparing the expression of nitrate transport protein genes in different diatoms and green algae, the expression patterns of IgNRT, IgAMT and IgGSII genes of different diatoms and green algae were similar, and were regulated by additional nitrogen concentration and nitrogen species. The results of homology analysis showed that the homology of IgNRT and diatom NAT was 47%, and the homology with diatom ammonia transporter (CylAMT) was 48%. The homology of IgGSII to glutamate synthase (SGSA) derived from Skeletonema costatura was 61%.

Higher plant gene expression is time-specific and spatially specific, and spatial specificity refers to a specific gene having a property of expression in a specific part of a living body, that is, tissue specificity. In the process of genetic improvement of crops by molecular biology, it is often expected that inserted foreign genes can be restricted in expression in specific tissues, thereby enabling plants to obtain useful traits, which requires efficient tissue-specific promoter pairs. The expression of the target gene is regulated.

Studies have shown that the absorption of plant water and various nutrients is mainly through the root system, and the transporter gene that enhances the utilization of nitrogen is driven by the root-specific promoter, which can make the target gene function normally. It can maximize the metabolic cost of plants. Yamamoto et al. isolated from tobacco. TobRB7 gene (GenBank accession number: S45406), and proved to have root tissue specificity (Yamamoto YT, Taylor CG, Acedo GN, et al. Characterization of cis - acting sequences regulating root-specific gene expression in tobacco. Plant Cell, 1991 3: 37Γ82) Nan et al. further demonstrated that the promoter of TobRB7 gene is a bidirectional promoter with root tissue-specific expression regulation in both the positive and negative directions. Myrosinase is a class of isoenzymes that catalyze the hydrolysis of glucosinolates, and its coding gene is a large gene family. The degradation products of myrosinase are involved in plant defense against pests and diseases, sulfur and nitrogen metabolism, and plant growth and apoptosis. The myrosinase is produced in the Brassica plant. ΚΥΙΟ is a myrosinase isolated from Arabidopsis thaliana and specifically expressed in roots and hypocotyls of Arabidopsis (INKENITZ, HEIKE BEI KEFELD, P10TR S PUZ10, et al. jD, a seeding and root specific gene And promoter from Arabidopsis thaliana [J] . Plant Science, 2001, 161: 337-346).

Invention disclosure

It is an object of the present invention to provide a method of cultivating plants having improved nitrogen absorption capacity.

The method for cultivating a plant having improved nitrogen absorption capacity provided by the present invention comprises introducing a recombinant expression vector containing a gene encoding a nitrate transporter into a plant tissue or a cell to obtain a plant having improved nitrogen absorption capacity;

The gene encoding the nitrate transporter is a DNA molecule as follows 1) or 2) or 3):

1) the nucleotide sequence thereof is the DNA molecule shown in SEQ ID NO: 2 in the sequence listing;

2) a DNA molecule that hybridizes under stringent conditions to 1) a defined DNA sequence and encodes the same functional protein;

3) a DNA molecule having more than 90% homology with the DNA sequence defined by the sequence 2 in the sequence listing, and encoding the same functional protein;

The promoter of the gene encoding the gene encoding the nitrate transporter in the M recombinant expression vector may be a constitutive promoter or a root-specific promoter.

The promoter may specifically be a root-specific promoter.

The starting vector for constructing the recombinant expression vector may be ρΝΑΠ21 _;

The PNAT121 can be obtained by inserting the gene encoding the nitrate transporter gene at the BamH I site of pCV121;

The PCV121 is obtained by substituting a small fragment between the ffi/7d III and c^? I sites of pCAMBIA 2301 into a target fragment; the target fragment is a small fragment obtained by double digestion of pMV121 with 3⁄47d III and ^I;

The pMV121 is obtained by substituting a small fragment between the Ηίηά III and BamHI sites of pUC121 into a 35S promoter fragment; the 35S promoter fragment is a small fragment obtained by double digestion of _P BI121 with HindYil and BamHl;

The pUC121 is 3⁄4c 1 and ₀ 7 of pUC19? A small fragment between the I sites was substituted with a NOS terminator fragment; the NOS terminator fragment was a small fragment obtained by double digestion of PBI121 with _C I and ^o/PI.

The recombinant expression vector can replace a small fragment between the Hind III and Xba I sites of PNAT121 Obtained for the root-specific promoter.

The root-specific promoter may specifically be a DNA molecule as follows 1) or 2) or 3):

1) the nucleotide sequence thereof is a DNA molecule represented by deoxyribonucleotide at position 5704 of the 5' end of sequence 3 in the sequence listing;

2) a DNA molecule that hybridizes to a defined DNA sequence under stringent conditions and has a promoter function;

3) A DNA molecule having 90% or more homology with a defined DNA sequence and having a promoter function.

1) the nucleotide sequence thereof is the DNA molecule of the sequence 4 of the sequence 4 from the 5' end of the 10-1442 deoxyribonucleotide;

The plant can be a dicot.

The plant may specifically be tobacco.

In order to facilitate the identification and screening of transgenic plant cells or plants, the recombinant expression vector used can be processed, such as a gene encoding a color-changing enzyme or a luminescent compound (GUS gene, luciferase) which can be expressed in plants. Genes, etc., resistant antibiotic markers (gentamicin markers, kanamycin markers, etc.) or anti-chemical marker genes (such as anti-tuberculosis genes). From the safety of transgenic plants, transgenic plants can be directly screened for adversely without any selectable marker genes.

The recombinant expression vector of the present invention can transform plant cells or tissues by using conventional biological methods such as sputum plasmid, Ri plasmid, plant viral vector, direct DNA transformation, microinjection, conductance, Agrobacterium mediated, and transformed plant tissue. Cultivate into plants. The transformed plant host can be either a monocot or a dicot, such as: tobacco, soybean, Arabidopsis, rice, wheat, corn, cucumber, squash, poplar, turfgrass, scorpion, and the like.

DRAWINGS

Figure 1 shows the results of electrophoresis detection of PCR-amplified diatom nitrate transporter gene.

Figure 2 shows the results of restriction enzyme digestion of the positive clone plasmid pNAT.

Figure 3 shows the results of enzyme digestion identification of the intermediate vector _P UC121

Figure 4 shows the results of restriction enzyme digestion of the intermediate vector pMV121

Figure 5 shows the results of restriction enzyme digestion of the intermediate vector pCV121

Figure 6 shows the results of enzyme digestion of plant expression vector ρΝΑΤ121

Figure 7 is a flow chart of the construction of the intermediate carrier pCV121

Figure 8 is a flow chart showing the construction of the plant expression vector ρΝΑΤ121

Figure 9 is a flow chart showing the construction of the recombinant expression vector pTRNAT

Figure 10 is a flow chart showing the construction of the recombinant expression vector pKYNAT Figure 11 shows the results of restriction enzyme digestion of recombinant expression vector pTRNAT

Figure 12 shows the results of restriction enzyme digestion of recombinant expression vector pKYNAT

The best way to implement the invention

The following experimental methods are conventional methods unless otherwise specified.

Example 1. Construction of 表达3⁄47^ plant expression vector pNAT121

I. Cloning of the nitrate transporter-encoding gene

1. Total RNA extracted from diatoms

The total RNA of diatoms was extracted using Invitrogen's TRIZ0L ^R Reagent kit (Cat. No. 15596-026) and with reference to the kit instructions. The specific method includes the following steps:

1) Take 50-100 mg of diatoms, grind into powder in liquid nitrogen, and transfer to a 1.5 mL centrifuge tube.

2) Add 1 mL of TRIZ0L Reagent and mix thoroughly for 5 min at room temperature.

3) Add 200 μl of chloroform, shake for 15 s, and place at room temperature for 2_3 min, 4. C, centrifuge at 12000g for 10min.

4) The supernatant was taken, 500 μl of isopropanol was added, and the mixture was allowed to stand at room temperature for 10 min, centrifuged at 1 ° C for 5 min at 4 ° C, and a white flake RNA precipitate was observed at the bottom of the centrifuge tube.

5) Discard the supernatant, carefully add ImL 70% ethanol, do not destroy the RNA pellet, and use a sampler to aspirate all the liquid after 5 seconds.

6) Place 5- lOmin to evaporate the ethanol (do not let it dry completely, otherwise it will affect the solubility). Add 20μ1 DEPC water to dissolve the precipitate to obtain total diatom RNA.

2. Reverse transcription synthesis cDNA

Using the total diatom RNA obtained in step 1 as a template, the cDNA was synthesized by reverse transcription using Invitrogen's GeneRacer kit and the kit instructions. The specific method includes the following steps:

1) Take diatom total RNA 10μ1, add Ιμΐ Gene Racer Oligo dT Primer, Ιμΐ dNTPs, Ιμΐ sterile distilled water.

2) Warm the bath at 65 °C for 5 min to remove the RNA secondary structure, ice bath for 5 min, and briefly centrifuge.

3) Add 4μ1 5Χ first strand buffer, Ιμΐ 0.1M DTT, Ιμΐ RNaseOutTM, Ιμΐ

Mix SuperscriptTM III RT buffer to a total volume of 20μ1 and mix with a pipette.

4) After a brief centrifugation, 50 Ό warm bath for 50 min.

5) Warm bath at 70 °C for 15 min, place on ice for 2 min to stop the reaction, and centrifuge at maximum speed for a short time.

6) Add Ιμΐ RnaseH, warm bath at 37 °C for 20 min.

7) Centrifuge briefly to obtain cDNA, which can be used immediately for PCR amplification or storage at -20 °C.

3. PCR amplification of the target gene

Using the cDNA obtained in step 2 as a template, PCR amplification was carried out under the guidance of a pair of primers (P1 and P2). P1 (upstream primer): 5, -ATGAGTGGAACTGATGTTGCA-3 ' ,

P2 (downstream primer): 5, - TCTTCTCGGTATCAGGTTGGG-3, .

The PCR reaction conditions were: 95 ° C, 5 min → 94 ° C, 30 s, 67 ° C, lmin, 72 ° C, 2 min, after 30 cycles - 72 ° C extension for 10 min.

After the reaction, the PCR product was detected by 1% agarose gel electrophoresis, and the detection results are shown in Fig. 1. (lane 1 is Marker: λ D / Eco/? 1 + /find III; Lane 2 is a PCR amplification product). There is a distinct amplified band at 1449 bp.

4. Freeze-thaw recovery PCR amplification products

The PCR-amplified DNA fragment of 1449 bp in length is recovered by freeze-thaw method, and the specific method comprises the following steps:

1) Cut the target fragment approximately 1449 bp in length from the agarose gel and place in a new centrifuge tube.

2) Add 200 μl of TE buffer, shake on a shaker for 5 min, and freeze in liquid nitrogen for 5 min.

3) Remove and bathe at 65 ° C for 5 min.

4) Repeat the freezing and thawing twice as in steps 2) -3).

5) The phenol, phenol/chloroform (mixing ratio 1: 1), chloroform was sequentially extracted, and the DNA was precipitated with absolute ethanol, and then 4 ul of sterile water was added to dissolve the precipitate, and the precipitate was the target fragment for recovery.

5. Cloning and sequencing of the target fragment

The target fragment was cloned by the vector pMD18_T (TaKaRa, Cat. No. D504A) by taking 4ul of the recovered PCR amplification product, adding lul pMD18-T vector, 5ul Ligase Solution I, and then 16°C. Connect 4h. The ligated product was transformed into E. coli DH5 ct competent cells, and the positive recombinant clone was screened. The plasmid was digested and identified. The results are shown in Figure 2 (lane 1 is Marker: λ DNA/EcoR I + Hind III; lane 2 is The digested product was obtained, and a 1449 bp fragment was obtained, which was consistent with the expected result, and the recombinant plasmid was named pNAT. Then sequencing, the sequencing results showed that the insert has the nucleotide sequence of sequence 2 in the sequence listing, and the sequence 2 in the sequence table is composed of 1449 deoxyribonucleotides, and the open reading frame (0RF) is in the sequence listing. Deoxyribonucleotides from positions 1 to 1449 of the sequence 2 from the 5' end, and the amino acid sequence encoding the sequence 1 in the sequence listing. The gene sequence and its encoded amino acid residue sequence were aligned and analyzed, and the nucleotide sequence homology of the gene with the diatom Λ 3⁄477 gene (GenBank No.: gi: 5733416) published in GenBank was 99.31%. The amino acid sequence homology of the encoded protein was 99.76%, indicating that the nitrate transporter gene of diatom was obtained, named Μ4Γ3, and its encoded protein was named NAT3.

Second, Λ3⁄4Γ·? plant expression vector pNAT121 acquisition

1. Construction of intermediate carrier PCV121

Referring to Figure 7, the intermediate carrier pCV121 is constructed. The specific process includes the following steps:

1) Construction of intermediate carrier PUC121

The vector pBI121 (Beijing Bayer Biotech Co., Ltd.) was digested with restriction endonucleases c 1 and ^3⁄40 I to recover a 260 bp T-N0S terminator fragment, which was digested with the same enzyme. The vector PUC19 (purchased from Dalian Bao Biotech Co., Ltd.) was ligated to obtain an intermediate vector pUC121.

The restriction endonuclease 3⁄4c I and I were used for double enzyme digestion. The results of restriction enzyme digestion were shown in Figure 3 (lane 1 is Marker: λ /EcoR I + Ηϊηά III; lanes 2 and 3 are digested products). A DNA fragment of approximately 260 bp in size was obtained by digestion, which was consistent with the expected results.

2) Construction of intermediate vector pMV121 The vector pΒΙ121 was digested with restriction endonucleases τ3⁄4'/7ί/ III and ^ I to recover the 800 bp CaMV 35S promoter fragment, which was ligated with the vector pUC121 constructed in step 1 of the same enzyme double digestion. The intermediate vector pMV121 was obtained.

Using restriction endonuclease 73⁄43⁄4^ III and double enzyme digestion, the results of restriction enzyme digestion are shown in Figure 4 (lane 1 is the enzyme digestion product; lane 2 is Marker: λ /EcoR I + Ηϊηά III), which is digested by enzyme. A DNA fragment of approximately 800 bp in size was obtained, which was consistent with the expected results.

3) Acquisition of the intermediate carrier pCV121

The vector pMV121 constructed in step 2 was digested with restriction endonucleases ζ' ί/ III and ^oR I to recover the 1.1 kb target fragment, which was digested with the same enzyme, pCAMBIA 2301 (Beijing). Bayer Biotech Co., Ltd.) was connected to obtain the intermediate vector pCV121.

The restriction endonuclease III and I were used for restriction enzyme digestion. The results of restriction enzyme digestion are shown in Figure 5 (lane 1 is a digested product; lane 2 is a Marker: λ ΌΝΑ/EcoR I + Hind III). The fragment size is approximately 1. lkb, which matches the expected result.

2, y1⁄2473⁄4t expression vector pNAT121 acquisition

See Figure 8 for the construction of a plant expression vector of Λ3⁄47^, as follows:

The plasmid vector pΝΑΤ constructed in the first step was digested with restriction endonuclease I to recover 144^; the target fragment of 3⁄44^, which was constructed with the intermediate vector PCV121 of step 2 which was digested with the same enzyme. The resulting plant expression vector ρΝΑΤ121 was obtained by ligation.

The restriction endonuclease was used for restriction enzyme digestion. The results of restriction enzyme digestion were shown in Figure 6 (lane 1 is Marker: λ ΌΜ/Eco? I + Hind III; lane 2 is the enzyme digestion product), and the size of the fragment was 1449 bp, in line with the expected results.

Example 2. TR promoter-driven Λ3⁄4 ^ 艮 specific expression vector pTRNAT construction

See Figure 9 for construction of the recombinant expression vector pTRNAT.

1. Cloning of the tobacco TR promoter

According to the published sequence (Yuri T. Yamamoto, The Plant Cell, Vol. 3, 371-382,

1991) Design a pair of primers (P3 and P4) to clone the TobRB7 promoter from total tobacco DNA. The above primers introduced a Hindlll site at the 5' end and a Xbal site at the 3' end.

P3 (upstream primer): 5, -TTTAAGCTTTCCTACACAATGTGAATTTG-3',

P4 (downstream primer): 5, -CTCTCTAGAAAAATGCCCCAAAAGAAGCTC-3'.

The PCR reaction conditions were: 94 ° C, 3 min → 94 ° C, 30 s, 62 ° C, 45 s, 72 ° C, lmin, 35 cycles and a 72 ° C extension for 10 min.

After the reaction, the PCR product was subjected to 1% agarose gel electrophoresis to obtain an expected molecular weight amplification band.

2. Cloning and sequencing of the target fragment

The target fragment was cloned by the vector pMD18-T (TaKaRa, Cat. No. D504A) by: PCR amplification product 4ul, followed by lul _P MD18-T carrier, 5ul Ligase Solution I, then 16° C is connected for 4h. The ligation product was transferred into E. coli DH5 α competent cells and screened. The positive recombinant clone was named pTRP. The pTRP digestion revealed that the linkage was correct. Then sequencing, the sequencing results showed that the insert has the nucleotide sequence of sequence 3 in the sequence listing, and the sequence 3 in the sequence table consists of 712 deoxyribonucleotides, from the 5' end of the 10th - 704 deoxyribose The nucleotide is the tobacco TobRB7 promoter sequence.

3, TR promoter-driven /Μ7^艮 specific expression vector pTRNAT construction

Use PNAT121

III and J3⁄4s I were digested and the vector was recovered.

Since the TobRB7 promoter sequence contains an A'm/ΙΠ site, in order to obtain the complete TobRB7 promoter sequence, pTRP was first completely digested with restriction endonuclease 3⁄4a I, and then partially digested with III, agarose. The fragment of about 700 bp was recovered by electrophoresis and ligated into the pNAT121 vector digested with W/7t/III and 3⁄4a I to obtain the 艮-specific expression vector PTRNAT.

The ligation products were digested with restriction endonucleases III and 3⁄4s I. The results of restriction enzyme digestion are shown in Figure 11 (lane 1 is pTRNAT/ffi/7i/ΙΠ+ Xba I, and lane 2 is Marker: λ k/ EcoR I + Ηϊηά III ) , the fragment size of the digested product was about 700 bp, which was consistent with the expected results.

Example 3. Construction of 7\3⁄3 3 specific expression vectors driven by KY promoter pKYNAT

1. Arabidopsis cloning of the YK10 promoter

See Figure 10 to construct the recombinant expression vector pKYNAT.

A pair of primers (P5 and P6) were designed according to the published sequence (INKE NITZ, HEIKE BEI KEFELD, P10TR S PUZ10, et al. Plant Science, 2001, 161: 337-346), and YK10 was cloned from Arabidopsis total DNA. The promoter introduces a τ3⁄4'/7ί ΙΠ position at the 5' end of the above primer, and the 3' end introduces a site.

P5 (upstream primer): 5, -GACAAGCTTCTGCAACGAAGTGTACCAAC -3, ,

P6 (downstream primer): 5, -TTGGAATTCTGATTTTATTCAAGAAAAATG -3, .

The PCR reaction conditions were: 94. C, 3rain—94. C, 30s, 62 ° C, 45 s, 72 ° C, 2 min, 35 cycles, a 72 ° C extension for 10 min.

2. Cloning and sequencing of the target fragment

The target fragment was cloned by the vector pMD18-T (TaKaRa, Cat. No. D504A) by: PCR amplification product 4ul, followed by lul pMD18-T vector, 5ul Ligase Solution I, then 16 °C Connect 4h. The ligation product was transferred into E. coli DH5 a competent cells, and the positive recombinant clone was screened and named pYK. The ρΥΚ digestion showed that the connection was correct. This was sequenced and the sequencing revealed that the insert had the nucleotide sequence of SEQ ID NO: 4 in the Sequence Listing. Sequence 4 in the sequence listing consists of 1451 deoxyribonucleotides, and the deoxyribonucleotide from position 10 to 1442 at the 5' end is the Arabidopsis thaliana YK10 promoter sequence.

3, ΙΧ / promoter driven 71⁄24Γ4艮 specific expression vector ρΥΚΝΑΤ construction

1) After ρΝΑΤ121 was digested with 3⁄4s I, the ends were filled in with Klenow, and then digested with ^ ^τ^ III to recover the vector. 2) pKY was first digested with ^σΤΡ I, blunt-ended with Kl enow, and then digested with #i 7i/III to recover the promoter fragment of 1451b _P.

3) The digested fragment obtained in 2) was ligated to the obtained PNAT 121 vector after digestion, and the K-specific expression vector pYKNAT driven by the YK10 promoter was obtained.

The ligation products were identified by restriction enzymes '/^ 111 and ^3⁄43 I. The results of enzyme digestion were shown in Figure 12 (lane 1 was pYKNAT/ffi/7i riI+J3⁄4sI and lane 2 was Marker: λ). ΌΜ/BcoR I + Hind III ) , the fragment size of the digested product was about 1451 bp, which was consistent with the expected results.

Example 4: Detection of nitrogen absorption capacity of transgenic tobacco

First, the acquisition of genetically modified tobacco

The recombinant expression vectors pTRNAT and pYKNAT constructed in Example 2 and Example 3 were transformed into Agrobacterium tumefaciens LBA4404 by freeze-thaw method, respectively, and Agrobacterium tumefaciens LBA4404 integrated with pTRNAT and pYKNAT were transformed into tobacco NC89 by leaf disc method. Two rounds of screening were performed with MS medium containing 100 mg/L kanamycin, and each round was screened for 10-15 days to obtain positive transgenic plants. The positive transgenic plants screened were further screened by PCR, and the pair of primers used for PCR were P7 and P8.

P7 (upstream primer): 5, -ATGAGTGGAACTGATGTTGCA-3 ' ,

P8 (downstream primer): 5 ' - TCTTCTCGGTATCAGGTTGGG-3, .

The pTRNAT and pYKNAT transgenic tobacco were identified by PCR. The positive transgenic plants were amplified by PCR to obtain 1449 bp bands. The results showed that 30 strains of pTRNAT and pYKNAT transgenic tobacco were obtained, that is, 30 strains of pTRNAT transgenic tobacco and 30 strains were obtained. pYKNAT transgenic tobacco.

At the same time, pNAT121 was introduced into tobacco NC89, the method was the same as above, and as a control, 30 strains were obtained.

PNAT121 transgenic tobacco.

Screen the obtained transgenic tobacco with T. Generation; use T. The seed produced by the self-crossing and the plant grown by it are represented by the 1\ generation, and the seed is first germinated on the MS medium containing 100 mg/L kanamycin, and the 1\ generation plant is obtained by screening; The resulting seeds and the plants grown therefrom were expressed in T ₂ generation, and the _2nd generation seeds were germinated on MS medium containing 100 mg/L kanamycin, and the homozygous lines were obtained by screening.

2. Detection of nitrogen absorption capacity of transgenic tobacco

1, the preparation of the medium

The following media were prepared separately - 1) MS. Medium.

2) 1/8 N medium: N content (including ammonia nitrogen and nitrate nitrogen) is MS. 1/8, other components are the same as MS. Medium.

3) 1/16 N medium: N content (including ammonia nitrogen and nitrate nitrogen) is MS. 1/16, the other components are the same as MS. Medium.

4) 1/32 N medium: N content (including ammonia nitrogen and nitrate nitrogen) is MS. 1/32, the other components are the same as MS. Medium.

2. Nitrogen reduction sensitivity test of transgenic tobacco

Take the seed of the L-generation pTRNAT transgenic tobacco obtained in step 1, and after the seeds are strictly sterilized, Seeded separately in MS. , 1/8N (N content is 1/8 of MS, including ammonia nitrogen and nitrate nitrogen), 1/pendulum, 1/32N medium. Thirty lines were sown in each medium, and each seed contained 100 seeds. The test was repeated 3 times.

The seeds of the T ₂ generation pYKNAT transgenic tobacco obtained in the step 1 were taken, and the seeds were strictly sterilized and sown in the MS. , 1/8N (N content is 1/8 of MS, including ammonia nitrogen and nitrate nitrogen), 1/disordered 1/32N medium. Thirty lines were sown in each medium, and each seed contained 100 seeds. The test was repeated 3 times.

Seeds of PNAT121 transgenic tobacco (non-specific promoter) were taken, and the seeds were sterilized and sterilized, and then seeded in MS. , 1/8N (N content is MS. 1/8, including ammonia nitrogen and nitrate nitrogen), 1/pendulum, 1/32N medium. Thirty strains were sown in each medium, and each seed contained 100 seeds. The test was repeated 3 times.

The seeds of NC89 non-transgenic tobacco (CK) were taken and the seeds were strictly sterilized and sown in MS. , 1/8N (N content is MS. 1/8, including ammonia nitrogen and nitrate nitrogen), 1/16N, 1/32N medium. 100 seeds were sown in each medium. The test was repeated 3 times.

The seeds of the above four treatments were cultured under the same conditions for 60 days, and the growth of pTRNAT, pYKNAT, ρΝΑΊΊ21 transgenic tobacco and non-transgenic tobacco NC89 in different media, leaf size, were observed on the 30th day after sowing. The growth of pTRNAT, pYKNAT, pNAT121 transgenic tobacco and non-3w tobacco and NC89 non-transgenic tobacco in different media is shown in Table 1. The leaf diameter in Table 1 is the average of 30 lines.

Table 1 Effects of different nitrogen content on the growth of transgenic tobacco

Transgenic plant leaf diameter (mm)

Leaf diameter pNAT121 transformation pTRNAT transformation pYKNAT transformation plant (mm) plant plant strain

MS„ 1 30 7-8 7-8 7-8 7-8

1/8N 1 30 4-5 6-7 7-8 6-7

1/16N 1 30 3-4 5-6 6-7 6-7

1/32N 1 30 1-2 4-5 5-6 4-5 On the MS. In the medium, there was no significant difference in the growth status of pTRNAT, pYKNAT, pNAT121 transgenic tobacco and non-transgenic tobacco. pTRNAT, pYKNAT, pNAT121 transgenic tobacco in brain, 1/16N medium and non-transgenic tobacco sown in the same medium. In contrast, the leaves were slightly larger, and pTRNAT, pYKNAT transgenic tobacco was superior to pNAT121 transgenic tobacco. The pTRNAT transgenic tobacco, pYKNAT transgenic tobacco, PNAT121 transgenic tobacco and non-transgenic tobacco in 1/32N medium were severely yellowed and slow-growing; however, compared with non-transgenic tobacco, the leaves of transgenic tobacco were larger and greener. . Overall, the growth status of pTRNAT transgenic tobacco, pYKNAT transgenic tobacco, pNAT121 transgenic tobacco and non-transgenic tobacco in nitrogen-deficient medium was significantly different. pYKNAT transgenic tobacco and PNAT121 transgenic tobacco were 2-3 times larger than those of non-transgenic tobacco. ; pTRNAT transgenic tobacco leaves than 3-5 times large non-transgenic tobacco and transgenic tobacco compared pYKNAT _{P NAT121} transgenic tobacco leaves large 1. 5-2 times. The above results indicated that: PNAT121 transgenic tobacco can significantly improve the nitrogen use efficiency of transgenic plants; 釆 use root-specific promoter to drive the specific expression of NAT gene in roots, which can be improved more than constitutive promoters. Nitrogen use efficiency. The TR promoter-driven pTRNAT) is more efficient than the YK promoter-driven ΓρΥΚΝΑΤ), which improves the plant's nitrogen use efficiency and allows plants to grow normally under low nitrogen conditions.

After sowing 60, the fresh weight of transgenic tobacco and NC89 non-transgenic tobacco grown in different media were weighed, and then the plants were baked in an 80-inch oven for 1 hour, and the dry weight of each plant was weighed. The statistical results are shown in Table 2. Show. The fresh weight and dry weight of the plants in Table 2 are the average of 30 lines.

Table 2 Statistical Results of Dry and Fresh Weight of Transgenic Tobacco and Non-GMO Tobacco Medium Fresh Weight (mg) Dry Weight (mg)

Types of

NC89 PNAT121 pTRNAT pYKNAT NC89 PNAT121 pTRNAT pYKNAT

MSo 255 258 256 257 26. 3 26. 8 26. 4 27. 3

1/8N 149 159 173 166 15. 0 19. 7 24. 7 21. 1

1/16N 56. 0 62. 0 76. 0 66. 0 8. 0 11. 8 16. 2 13. 1

1/32N 31. 0 46. 0 54. 0 48. 0 5. 0 6. 5 9. 6 7. 2 The fresh weight and dry weight of transgenic tobacco grown on different media were significantly higher than those of non-transgenic plants; The root-specific promoter-driven NAT transgenic tobacco had higher fresh weight and dry weight than the constitutively expressed PNAT121 transgenic tobacco. Different root-specific promoters had different degrees of nitrogen use efficiency, and the TRP promoter was superior to the YK10 promoter. On 1/16N medium, the dry weight of pTRNAT transgenic tobacco was twice as high as that of tobacco. The results further demonstrate that the TR promoter-driven rot 3 (pTRNAT) is more effective than the YK promoter-driven solid (pYKNAT) in improving plant nitrogen use, allowing plants to grow normally under low nitrogen conditions.

Industrial application

The transgenic plants obtained by the method provided by the present invention have greatly improved nitrogen absorption ability and can grow normally under low nitrogen conditions. The growth status of pTRNAT transgenic tobacco, pYKNAT transgenic tobacco and non-transgenic tobacco in nitrogen-deficient medium was significantly different. pYKNAT transgenic tobacco was 2-3 times larger than that of non-transgenic tobacco; pTRNAT transgenic tobacco was larger than non-transgenic tobacco.倍倍。 - 6 times, larger than the leaves of pYKNAT transgenic tobacco 1. 5- 2 times. The fresh weight and dry weight of pTRNAT transgenic tobacco grown on different media were significantly higher than that of pYKNA transgenic tobacco and control plants. On 1/16N medium, the dry weight of pTRNAT transgenic tobacco was twice as high as that of control tobacco. The invention will play an important role in the field of plant nitrogen fertilizer absorption and breeding of low-nitrogen-resistant plant varieties, and has broad application prospects.

Claims

Claims: A method for cultivating a plant having improved nitrogen uptake ability, which comprises introducing a recombinant expression vector containing a gene encoding a nitrate transporter into a plant tissue or a cell to obtain a plant having improved nitrogen absorption capacity; encoding the nitrate transporter The gene is a DNA molecule as follows 1) or 2) or 3):

1) its nucleotide sequence is the DNA molecule shown in SEQ ID NO: 2 in the Sequence Listing;

The promoter that activates transcription of the gene encoding the nitrate transporter in the recombinant expression vector is a constitutive promoter or a root-specific promoter.

2. The method of claim 1 wherein: the promoter is a root specific promoter.

3. The method according to claim 2, wherein: the starting vector for constructing the recombinant expression vector is ρΝΑ 21;

The PNAT121 is obtained by inserting a coding gene of the nitrate transporter between the BamH I sites of pCV121;

The PCV121 is obtained by substituting a small fragment between the ^III and coR I sites of pCAMBIA 2301 into a target fragment; the target fragment is a small fragment obtained by double digestion of pMV121 with III and ^RR I;

The pMV121 is obtained by substituting a small fragment between the Ηίηά III and a/a¥ I sites of pUC121 into a 35S promoter fragment; the 35S promoter fragment is a small fragment obtained by double digestion of ρΒΠ21 with Hi III and BamHl. ;

The PUC121 is obtained by substituting a small fragment between the feie I and oT? I sites of pUC19 into a NOS terminator fragment; the NOS terminator fragment is a small fragment obtained by ceps11 and _C1 . Fragment.

4. The method of claim 3, wherein the recombinant expression vector is obtained by substituting a small fragment between the Hind III and Xba I sites of pNAT121 into a root-specific promoter.

5. The method according to claim 4, wherein the root-specific promoter is a DNA molecule of the following 1) or 2) or 3):

2) a DNA molecule which hybridizes under stringent conditions to a defined DNA sequence and has a promoter function;

6. The method according to claim 4, wherein: the root-specific promoter is a DNA molecule of the following 1) or 2) or 3):

1) Its nucleotide sequence is the 10th to 1442th deoxyribonucleoside from the 5' end of sequence 4 in the sequence listing. a DNA molecule represented by an acid;

2) a DNA molecule which hybridizes under stringent conditions to a defined DMA sequence and has a promoter function;

7. A method according to any one of claims 1 to 6 wherein the plant is a dicot.

8. The method of claim 7 wherein: the plant is tobacco.