CN112898393B

CN112898393B - TaAOS gene and application of protein coded by same

Info

Publication number: CN112898393B
Application number: CN202110160686.0A
Authority: CN
Inventors: 康国章; 李鸽子; 肖向红; 王鹏飞; 柳海涛; 刘金
Original assignee: Henan Agricultural University
Current assignee: Henan Agricultural University
Priority date: 2021-02-05
Filing date: 2021-02-05
Publication date: 2022-03-29
Anticipated expiration: 2041-02-05
Also published as: CN112898393A

Abstract

The invention provides a TaAOS gene, and a protein coded by the same and application thereof, belonging to the technical field of plant breeding, wherein the nucleotide sequence of the TaAOS gene is shown as SEQ ID No. 1. The TaAOS gene provided by the invention improves the potassium stress tolerance of plants.

Description

TaAOS gene and application of protein coded by same

Technical Field

The invention relates to the technical field of plant breeding, in particular to a TaAOS gene and a protein coded by the same and application of the TaAOS gene.

Background

Potassium (K) is one of the essential nutrient elements for plant growth and development. It is involved in a series of physiological and biochemical processes in plants (Armenglad et al, 2004). In addition, K is one of the constituent elements of the matrix minerals of the soil, although the content of K in most soils is high, the content of effective K available for plants in the soil of the cultivated land is low at present due to the fixation of secondary minerals in the soil and the influence of plants on the depletion of effective potassium in the rhizosphere. Plants have developed more complete stress response reactions in vivo to adapt to stress tolerance resulting from the constantly changing content of K ions in different soils, such as enhancing potassium uptake, potassium redistribution, and adjusting root morphology (Nieves-cores et al, 2007; 2008). Under the condition of K stress, plant root system infection can start an in-vivo stress related tolerance adaptation mechanism and activate a high-affinity potassium absorption system (Wang and Wu,2013) in a short period, and plant hormones such as jasmonic acid, ethylene, abscisic acid, auxin and the like can mediate a K signal path, so that different plant physiological reactions such as plant potassium absorption capacity, leaf stomata closing, reduction of the number of metarrhizae and the like can be adjusted; in addition, these signaling molecules can also regulate differential changes in transcription levels of certain hormone genes (Shankar et al, 2013). Therefore, there is a need to find new genes involved in potassium stress tolerance in plants.

Disclosure of Invention

In view of the above, the present invention aims to provide a TaAOS gene, a protein encoded by the TaAOS gene, and applications of the TaAOS gene.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a TaAOS gene, the nucleotide sequence of which is shown in SEQ ID No. 1.

The invention also provides a protein coded by the TaAOS gene in the technical scheme, and the amino acid sequence of the protein is shown as SEQ ID No. 2.

The invention also provides application of the TaAOS gene in the technical scheme in improving the potassium stress tolerance of plants.

Preferably, the plant comprises rice.

Preferably, the application comprises the following steps:

1) connecting the TaAOS gene into pUN1301 to obtain a pCAMBIA1300-TaAOS vector;

2) transforming the pCAMBIA1300-TaAOS vector obtained in the step 1) into agrobacterium to obtain engineering bacteria;

3) introducing the engineering bacteria obtained in the step 2) into the callus of the plant, and culturing to obtain the potassium stress tolerant plant.

Preferably, pUN1301 obtained in the step 1) is subjected to enzyme digestion and then is connected with TaAOS gene; the enzyme digestion system comprises: pUN 130110. mu.L, 10 Xdigestion buffer 5. mu.L, BamHI 1. mu.L at a concentration of 10U/. mu.L, KpnI 0.8. mu.L at a concentration of 10U/. mu.L, ddH₂O is complemented to 50 mu L; the enzyme digestion conditions comprise: the enzyme was cleaved at 37 ℃ for 3 h.

Preferably, the content of pUN1301 in the enzyme-digested system is 1 mg.

Preferably, the enzyme used for ligation in step 1) comprises T4 ligase; the system of said connection comprises: 2. mu.L of pUN1301, 1. mu.L of TaAOS gene 6. mu. L, T4 ligase and 1. mu.L of 10 Xligase buffer; the conditions of the connection include: ligation was carried out at 16 ℃ for 10 h.

Preferably, the agrobacterium of step 2) comprises agrobacterium EHA 105.

Preferably, the rice variety includes nipponica.

The invention provides a TaAOS gene, and a protein coded by the same and application thereof, wherein the nucleotide sequence of the TaAOS gene is shown in SEQ ID No. 1. The TaAOS gene provided by the invention participates in the potassium stress tolerance of plants.

Drawings

FIG. 1 is a sequence amplified into the coding region of the TaAOS gene;

FIG. 2 is a physical map of overexpression vector pCUN 1301-TaAOS;

FIG. 3 shows the genetic transformation process of TaAOS-transgenic rice;

FIG. 4 shows the identification of TaAOS-transgenic rice, wherein A is the identification of transgenic plants using hygromycin gene (Hpt II); b, identifying the transgenic plant by using the vector and the target gene; c, identifying the protein expression level of the transgenic plant by using a tag antibody HA;

FIG. 5 is a phenotype of potassium stress tolerance in TaAOS-transgenic rice;

FIG. 6 is a bioassay, Root refers to roots, Leaf refers to leaves;

FIG. 7 shows the measurement of K content in the plant, Root refers to Root, Leaf refers to Leaf;

FIG. 8 shows the Jasmonic Acid (JA) content measurement in plants, Root for roots and Leaf for leaves.

Detailed Description

The invention provides a TaAOS gene, the nucleotide sequence of which is shown as SEQ ID No.1 and specifically comprises the following components:

ATGGCGGGCGGCGACGAGGGCTCCCTGGTGCCGAGGCAGGTGCCGGGCAGCTACGGCATGCCGTTCGTCTCGGCCATCCGCGACCGCCTCGACTTCTACTACTTCCAGGGCCAGGACAAGTACTTCGAGTCCCGTGTCGAGAAGTACGGCTCCACCGTCGTCCGCATCAACGTCCCGCCGGGCCCCTTCATGGCGCGCGACCCGCGGGTGGTCGCCGTGCTCGACGCCAAGAGCTTCCCCGTGCTCTTCGACGTCGACAAGGTCGAGAAGAAGAACCTCTTCACCGGCACCTACATGCCCTCCACCTCCCTCACCGGAGGCTTCCGCGTCTGCTCCTACCTCGACCCCTCCGAGCCCATCCACACCAAGGTCAAGCAGCTGCTCTTCTCCCTCCTTGCCTCCCGCAAGGACGCCTTCATCCCGGCCTTCCGTTCCCACTTCTCCTCGCTCCTCGCCACCGTGGAGTCGCAGATCGTGCTCGGCGGCAAGTCCAACTTCAACACGCTCAACGACGCCACCTCCTTCGAGTTCATCGGCGACGCCTACTTCGGCGTGCTCCCTTCTGCGTCAGACCTAGGTACCACCGGCCCGACCAAGGCCGCAAAGTGGCTCATATTCCAGCTCCACCCGCTCGTCACGCTCGGCCTCCCCATGATCCTCGAGGAGCCGCTCCTCCACACGGTGCACCTCCCTCCCATCCTCGTCAGCGGCGACTACAAGGCGCTCTACAAGTACTTCTTCGCCGCTGCGACCAAGGCGCTCGACACCGCCGAGGGCCTCGGACTGAAGCGGGACGAGGCATGCCACAACCTGTTGTTCGCCACCGTGTTCAACAGCTACGGTGGCCTCAAGGTGCTTCTCCCGGGGATCCTCGCGCGCATCGCGGGGGCCGGAGAGAAGTTCCACCAGAAGCTCGTCGCGGAGATACGCGCCGCCGTGGCGGACGCCGGCGGCAAGGTGACGATAGAGGCGCTGGAGAAGATGGAGCTGACCAAGTCGGCGGTGTGGGAGGCGCTGCGGCTGGACCCGCCCGTCAAGTTCCAGTACGGCCGCGCCAAGGCGGACATGAACATCGAGAGCCACGACGCGGTGTTCGCCGTGAAGAAGGGGGAGATGCTGTTCGGGTACCAGCCGTGCGCCACCAGGGACCCCCGCGTGTTCGGCTCCACGGCGAGGGAGTTCGTCGGCGACCGGTTCGTCGGGGAGGAGGGAAGGAAGCTGCTGCAGTACGTGTACTGGTCCAACGGGCGGGAGACCGAGAGCCCCAGCGTGGACAACAAGCAGTGCCCAGGCAAGAACCTGGTCGTGCTCGTGGGCAGGCTCCTGGTGGTGGAGCTGTTCCTCCGGTACGACACCTTCACCGCCGACGTCGGGGTCGACCTGCTCGGCACCAAGGTTGAGTTCACCGGCGTCACCAAGGCCACGTCCGGTCCTGAGAGCGCTGTTTAA。

the invention also provides a protein coded by the TaAOS gene in the technical scheme, wherein the amino acid sequence of the protein is shown as SEQ ID No.2, and specifically comprises the following steps:

MAGGDEGSLVPRQVPGSYGMPFVSAIRDRLDFYYFQGQDKYFESRVEKYGSTVVRINVPPGPFMARDPRVVAVLDAKSFPVLFDVDKVEKKNLFTGTYMPSTSLTGGFRVCSYLDPSEPIHTKVKQLLFSLLASRKDAFIPAFRSHFSSLLATVESQIVLGGKSNFNTLNDATSFEFIGDAYFGVLPSASDLGTTGPTKAAKWLIFQLHPLVTLGLPMILEEPLLHTVHLPPILVSGDYKALYKYFFAAATKALDTAEGLGLKRDEACHNLLFATVFNSYGGLKVLLPGILARIAGAGEKFHQKLVAEIRAAVADAGGKVTIEALEKMELTKSAVWEALRLDPPVKFQYGRAKADMNIESHDAVFAVKKGEMLFGYQPCATRDPRVFGSTAREFVGDRFVGEEGRKLLQYVYWSNGRETESPSVDNKQCPGKNLVVLVGRLLVVELFLRYDTFTADVGVDLLGTKVEFTGVTKATSGPESAV。

In the present invention, the plant preferably includes rice, and the variety of rice preferably includes nipponica.

In the present invention, the application preferably comprises the steps of:

1) connecting the TaAOS gene into pUN1301 to obtain a pCAMBIA1300-TaAOS vector;

The invention connects the TaAOS gene into pUN1301 to obtain pCAMBIA1300-TaAOS vector.

In the invention, pUN1301 is preferably connected with TaAOS gene after enzyme digestion; the enzyme digestion system preferably comprises: pUN 130110. mu.L, 10 Xdigestion buffer 5. mu.L, BamHI 1. mu.L at 10U/. mu.L, KpnI 0.8. mu.L at 10U/. mu.L, ddH₂O is complemented to 50 mu L; the enzyme cutting conditions preferably comprise: the enzyme was cleaved at 37 ℃ for 3 h. In the present invention, the amount of pUN1301 contained in the enzyme-digested system is preferably 1 mg.

In the present invention, the enzyme used for the ligation preferably includes T4 ligase; the linked system preferably comprises: 2 μ L of pUN1301(50 ng/. mu.L), TaAOS gene (50 ng/. mu.L), 6 μ L, T4 ligase 1 μ L and 10 Xligase buffer 1 μ L; the conditions for the connection preferably include: ligation was carried out at 16 ℃ for 10 h.

The obtained pCAMBIA1300-TaAOS vector is transformed into agrobacterium to obtain engineering bacteria. In the present invention, the Agrobacterium comprises Agrobacterium EHA 105. The invention has no special limitation on the transformation method for transforming the pCAMBIA1300-TaAOS vector into the agrobacterium, and the method for transforming the agrobacterium by adopting the conventional vector is only needed.

The obtained engineering bacteria are introduced into the callus of the plant and cultured to obtain the potassium stress tolerant plant.

The method for introducing the engineering bacteria into the plant callus is not particularly limited, and the skilled person can perform routine operations. The method for culturing is not particularly limited, and a conventional plant callus culture method is adopted.

In order to further illustrate the present invention, the following detailed description of the invention is given in conjunction with examples, which should not be construed to limit the scope of the invention.

Example 1

Extracting the root system total RNA of the Zhoumai 18 wheat seedlings, and amplifying to 1449bp full-length cDNA for coding TaAOS by adopting an RT-PCR method. The specific operation process is as follows:

1. extraction of total RNA and cDNA from wheat

1) Extraction of total RNA of plants: selecting 0.1g of wheat seedling root system as a material, grinding the wheat seedling root system in liquid nitrogen, transferring freeze-dried powder ground in the liquid nitrogen into a 1.5mL centrifuge tube containing 1mL of Trizol reagent (Invitrogen), and fully and uniformly mixing; standing at 25 deg.C for 5 min; adding 0.2mL of precooled chloroform, shaking for 15s, and standing for 2-3 min at 25 ℃; centrifugation (4 ℃, 12,000rpm, 15 min); the supernatant was transferred to a new 1.5mL centrifuge tube (ca. 0.5mL) after which 0.5mL of pre-cooled isopropanol was added and left at 25 ℃ for 10 min; centrifugation (4 ℃, 12,000rpm, 15 min); removing the supernatant, washing the precipitate with 75% ethanol (1mL) for 2 times, and blowing on a super clean bench for 3-5 min; add 50. mu.L DEPC-ddH₂And (4) carrying out resuspension and precipitation to obtain an extracted total RNA solution, and storing the solution at-70 ℃ for later use after detecting the mass. A template for reverse transcription is prepared.

2) RT-PCR: diluting the above-mentioned extracted RNA concentration with reference to RT-PCR kit (Taraka) instructions; according to the quantitative result of RNA, 0.5. mu.g Oligo dT primer and DEPC-ddH were added to 1. mu.g RNA₂Supplementing O to 7.5 μ L, mixing, denaturing at 65 deg.C for 5min, and standing on ice for 5 min; after transient centrifugation, 12.5. mu.L of Reverse transcription mix (including 5 × Reaction Buffer, 2.5. mu.L; dNTP mix (2.5mM), 3.0. mu.L, Reverse Transcriptase Enzymes, 0.5. mu.L; RNase Inhibitor, 0.5. mu.L; DEPC-ddH) was added₂O, 6.0. mu.L). After mixing uniformly, the mixture is subjected to PCR for 1h at 42 ℃; at 95 ℃ for 10 min; the reverse transcription of cDNA is completed, and the concentration of cDNA is determined for use.

2. Amplification of TaAOS

According to the sequence of TaAOS searched in IWGSC database, designing primer (forward primer F (SEQ ID No. 3): ATGGCGGGCGGCGACGAG; reverse primer R (SEQ ID No. 4): CCACGAGCACGACCAGG), adding corresponding cDNA template, and configuring the reaction system as follows: prime Star (10 μ L); forward and reverse primers (1. mu.L each); cDNA (1. mu.L); distilled water (8. mu.L). PCR amplification was then used, program: pre-denaturation (94 ℃, 1 min); after 32 cycles of PCR amplification (denaturation at 94 ℃ for 10 min; renaturation at 56 ℃ for 15 s; extension at 72 ℃ for 1min), extension is continued at 72 ℃ for 10 min. Then, the PCR products were separated by agarose gel electrophoresis, and as a result, a band of the PCR product of about 1449bp was separated by electrophoresis as shown in FIG. 1 (FIG. 1).

Further, the PCR product was recovered and sequenced. The sequencing results showed that the nucleotide sequence of the PCR product was identical to the database entry sequence. The sequence of the TaAOS gene obtained by amplification is shown as SEQ ID No.1, and the sequence of the coded protein is shown as SEQ ID No. 2.

Example 2

TaAOS protein and application of coding gene thereof

1. Construction of TaAOS overexpression vector

KpnI and BamHI restriction sites were added to the upstream and downstream primers of TaAOS, respectively, according to the cloning site of the vector (forward primer F (SEQ ID No. 5): CCGGTACCATGGCGGGCGGCGACGAG; reverse primer R (SEQ ID No. 6): CCACGAGCACGACCAGGCCATGGCCG). And PCR amplifying the coding sequence of the TaAOS gene again, and recovering the fragment. Meanwhile, the vector pCUN-1301 plasmid is subjected to double enzyme digestion by using restriction enzymes KpnI and BamHI, wherein the enzyme digestion system is as follows: 10. mu.L (1mg) of plasmid, 5. mu. L, BamHI 1. mu.L (10U/. mu.L) of 10 Xdigestion buffer, 0.8. mu.L (10U/. mu.L) of KpnI, and ddH₂O replenishes the reaction system to 50. mu.L, and the enzyme is cleaved at 37 ℃ for 3 h. The cleavage products were separated by agarose gel electrophoresis and the linearized pUN1301 large fragment was recovered.

The recovered PCR was ligated with the fragments of the vector at a mass ratio of vector to fragment (1:3) using T4 ligase, 2.0. mu.L of vector and 6.0. mu.L of fragment were diluted according to the concentration, 1.0. mu.L of T4DNA ligase and 1.0. mu.L of 10 Xligase buffer were added, and they were mixed, centrifuged, and ligated at 16 ℃ for 10 hours. And transforming the ligation product into escherichia coli DH5 alpha competent cells, screening and culturing for 16h by using a resistance plate containing kanamycin to obtain a monoclonal, detecting the masculine monoclonal by PCR, and sequencing to verify the detection result of the PCR. Indicating that the pCAMBIA1300-TaAOS vector has been successfully constructed. As shown in fig. 2: the expression vectors contain the Ubi (maize ubiquitin gene) promoter, TaAOS gene, and Nos-T (Agrobacterium nopaline synthase terminator), respectively.

2. Acquisition and identification of TaAOS transgenic rice

(1) Acquisition of TaAOS transgenic Rice

Transferring the constructed over-expression vector into an agrobacterium EHA105 strain by using an electric stimulator, screening positive clones on a kanamycin resistant plate, and detecting whether TaAOS is transferred. Selecting and detecting correct positive clones to prepare engineering bacteria, and introducing the engineering bacteria into callus of Nipponbare paddy rice (Oryza sativa L.); then, washing the cefuroxime sodium powder for 4-5 times by using sterile water containing 300mg/L of cefuroxime sodium, and sucking water by using sterile filter paper; transferring it to N containing hygromycin and cefamycin₆D₂Screening on a culture medium; the culture medium enzyme is changed once in 2 weeks, 3 generations of culture are continuously carried out, callus with good growth state is selected and transferred to a differentiation culture medium for culture, the photoperiod is set as day light (12h, 28 ℃), and dark (12h, 24 ℃) at night; replacing the culture medium once a week until seedlings are differentiated; and (3) selecting seedlings which grow vigorously, transferring the seedlings to a rooting culture medium for rooting culture, opening a container sealing film when the seedlings grow to about 10cm, hardening the seedlings for 2-3 days, and then transferring the seedlings to a phytotron for cultivation. The specific process is shown in fig. 3.

(2) Identification of TaAOS transgenic rice

The DNA of the T0 generation transgenic seedlings was extracted, and hygromycin primers were used to detect whether the TaAOS transgenic rice plants contained hygromycin genes, the results are shown in A in FIG. 4, except for wild type, the detected plants all contained hygromycin genes. Indicating that these strains carry the selection marker gene of the overexpression vector. On this basis, primer LBP (forward primer F (SEQ ID No. 7): 5'-AAAAGGAAGGTGGCTCCTAC-3') was designed near the upstream of the insertion site of the vector gene and combined with the downstream fragment of the inserted gene, the amplification result is shown in FIG. 4B, and the combination of vector primer LBP and gene downstream primer (RP (SEQ ID No. 8): 5'-CCACGAGCACGACCAGG-3') amplified a fragment (LP and RP combined band) larger than the target gene, indicating that the TaAOS gene is overexpressed in rice. Further, these plants were cultured in additional generations, and total proteins of transgenic plants containing the target gene and the marker gene were extracted from a large number of T2-generation plants. Further, the expression level of these strains at the protein level was measured using the HA-tag antibody. The results are shown in fig. 4 as C: the transgenic lines TaAOS-OE4(#4) and TaAOS-OE5(#5) express higher amounts at the protein level than the other lines. Therefore, these 2 lines were used for subsequent studies.

3. Identification of role of TaAOS-transgenic rice in K stress tolerance

The seeds of the screened homozygous strains TaAOS-OE4 and TaAOS-OE5 germinate independently, seedlings with consistent growth and development are selected and divided into 3 parts when the seeds grow to 3 leaf stages, and normal (+ K, 6.0mM), low potassium (LK, 0.3mM) and potassium deficiency (DK, 0mM) treatment is carried out on different types of plants. The results are shown in FIG. 5, where TaAOS-transgenic rice plants developed significantly better than wild-type plants under LK and DK conditions after 14d potassium stress. The biomass measurements are shown in FIG. 6 and Table 1, where the dry weight of TaAOS-transformed rice plants is increased compared to the control plants under LK and DK conditions, respectively. The measurement of the K content in the plant body shows that the K content of the TaAOS-transformed rice plant has insignificant accumulation difference in the root system and the leaf under the LK condition as shown in figure 7 and table 2; however, under the DK condition, the K content of the TaAOS-transformed rice plant is obviously higher than that of the wild type (1.77 and 2.8 times) only in the overground part, and is obviously reduced (0.69 and 0.57 times) in the root system, which indicates that under the potassium deficiency condition, the TaAOS-transformed rice plant influences the transfer of K from the root system of the rice to the leaf; in addition, Jasmonic Acid (JA) content in plants was determined as shown in fig. 8 and table 3, and JA content in transgenic plants was significantly higher than that of wild type, indicating that TaAOS-transgenic rice might respond to K stress tolerance by stimulating JA signal in vivo.

TABLE 1 Rice biomass assay data

TABLE 2 measurement of K-biomass in transgenic rice plants

TABLE 3 measurement data of JA content in vivo under K stress condition of rice

The foregoing is merely a preferred embodiment of the invention and is not intended to limit the invention in any manner. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be construed as the protection scope of the present invention.

Sequence listing

<110> Henan university of agriculture

<120> TaAOS gene and application of protein coded by same

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1449

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

atggcgggcg gcgacgaggg ctccctggtg ccgaggcagg tgccgggcag ctacggcatg 60

ccgttcgtct cggccatccg cgaccgcctc gacttctact acttccaggg ccaggacaag 120

tacttcgagt cccgtgtcga gaagtacggc tccaccgtcg tccgcatcaa cgtcccgccg 180

ggccccttca tggcgcgcga cccgcgggtg gtcgccgtgc tcgacgccaa gagcttcccc 240

gtgctcttcg acgtcgacaa ggtcgagaag aagaacctct tcaccggcac ctacatgccc 300

tccacctccc tcaccggagg cttccgcgtc tgctcctacc tcgacccctc cgagcccatc 360

cacaccaagg tcaagcagct gctcttctcc ctccttgcct cccgcaagga cgccttcatc 420

ccggccttcc gttcccactt ctcctcgctc ctcgccaccg tggagtcgca gatcgtgctc 480

ggcggcaagt ccaacttcaa cacgctcaac gacgccacct ccttcgagtt catcggcgac 540

gcctacttcg gcgtgctccc ttctgcgtca gacctaggta ccaccggccc gaccaaggcc 600

gcaaagtggc tcatattcca gctccacccg ctcgtcacgc tcggcctccc catgatcctc 660

gaggagccgc tcctccacac ggtgcacctc cctcccatcc tcgtcagcgg cgactacaag 720

gcgctctaca agtacttctt cgccgctgcg accaaggcgc tcgacaccgc cgagggcctc 780

ggactgaagc gggacgaggc atgccacaac ctgttgttcg ccaccgtgtt caacagctac 840

ggtggcctca aggtgcttct cccggggatc ctcgcgcgca tcgcgggggc cggagagaag 900

ttccaccaga agctcgtcgc ggagatacgc gccgccgtgg cggacgccgg cggcaaggtg 960

acgatagagg cgctggagaa gatggagctg accaagtcgg cggtgtggga ggcgctgcgg 1020

ctggacccgc ccgtcaagtt ccagtacggc cgcgccaagg cggacatgaa catcgagagc 1080

cacgacgcgg tgttcgccgt gaagaagggg gagatgctgt tcgggtacca gccgtgcgcc 1140

accagggacc cccgcgtgtt cggctccacg gcgagggagt tcgtcggcga ccggttcgtc 1200

ggggaggagg gaaggaagct gctgcagtac gtgtactggt ccaacgggcg ggagaccgag 1260

agccccagcg tggacaacaa gcagtgccca ggcaagaacc tggtcgtgct cgtgggcagg 1320

ctcctggtgg tggagctgtt cctccggtac gacaccttca ccgccgacgt cggggtcgac 1380

ctgctcggca ccaaggttga gttcaccggc gtcaccaagg ccacgtccgg tcctgagagc 1440

gctgtttaa 1449

<210> 2

<211> 482

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met Ala Gly Gly Asp Glu Gly Ser Leu Val Pro Arg Gln Val Pro Gly

1 5 10 15

Ser Tyr Gly Met Pro Phe Val Ser Ala Ile Arg Asp Arg Leu Asp Phe

20 25 30

Tyr Tyr Phe Gln Gly Gln Asp Lys Tyr Phe Glu Ser Arg Val Glu Lys

35 40 45

Tyr Gly Ser Thr Val Val Arg Ile Asn Val Pro Pro Gly Pro Phe Met

50 55 60

Ala Arg Asp Pro Arg Val Val Ala Val Leu Asp Ala Lys Ser Phe Pro

65 70 75 80

Val Leu Phe Asp Val Asp Lys Val Glu Lys Lys Asn Leu Phe Thr Gly

85 90 95

Thr Tyr Met Pro Ser Thr Ser Leu Thr Gly Gly Phe Arg Val Cys Ser

100 105 110

Tyr Leu Asp Pro Ser Glu Pro Ile His Thr Lys Val Lys Gln Leu Leu

115 120 125

Phe Ser Leu Leu Ala Ser Arg Lys Asp Ala Phe Ile Pro Ala Phe Arg

130 135 140

Ser His Phe Ser Ser Leu Leu Ala Thr Val Glu Ser Gln Ile Val Leu

145 150 155 160

Gly Gly Lys Ser Asn Phe Asn Thr Leu Asn Asp Ala Thr Ser Phe Glu

165 170 175

Phe Ile Gly Asp Ala Tyr Phe Gly Val Leu Pro Ser Ala Ser Asp Leu

180 185 190

Gly Thr Thr Gly Pro Thr Lys Ala Ala Lys Trp Leu Ile Phe Gln Leu

195 200 205

His Pro Leu Val Thr Leu Gly Leu Pro Met Ile Leu Glu Glu Pro Leu

210 215 220

Leu His Thr Val His Leu Pro Pro Ile Leu Val Ser Gly Asp Tyr Lys

225 230 235 240

Ala Leu Tyr Lys Tyr Phe Phe Ala Ala Ala Thr Lys Ala Leu Asp Thr

245 250 255

Ala Glu Gly Leu Gly Leu Lys Arg Asp Glu Ala Cys His Asn Leu Leu

260 265 270

Phe Ala Thr Val Phe Asn Ser Tyr Gly Gly Leu Lys Val Leu Leu Pro

275 280 285

Gly Ile Leu Ala Arg Ile Ala Gly Ala Gly Glu Lys Phe His Gln Lys

290 295 300

Leu Val Ala Glu Ile Arg Ala Ala Val Ala Asp Ala Gly Gly Lys Val

305 310 315 320

Thr Ile Glu Ala Leu Glu Lys Met Glu Leu Thr Lys Ser Ala Val Trp

325 330 335

Glu Ala Leu Arg Leu Asp Pro Pro Val Lys Phe Gln Tyr Gly Arg Ala

340 345 350

Lys Ala Asp Met Asn Ile Glu Ser His Asp Ala Val Phe Ala Val Lys

355 360 365

Lys Gly Glu Met Leu Phe Gly Tyr Gln Pro Cys Ala Thr Arg Asp Pro

370 375 380

Arg Val Phe Gly Ser Thr Ala Arg Glu Phe Val Gly Asp Arg Phe Val

385 390 395 400

Gly Glu Glu Gly Arg Lys Leu Leu Gln Tyr Val Tyr Trp Ser Asn Gly

405 410 415

Arg Glu Thr Glu Ser Pro Ser Val Asp Asn Lys Gln Cys Pro Gly Lys

420 425 430

Asn Leu Val Val Leu Val Gly Arg Leu Leu Val Val Glu Leu Phe Leu

435 440 445

Arg Tyr Asp Thr Phe Thr Ala Asp Val Gly Val Asp Leu Leu Gly Thr

450 455 460

Lys Val Glu Phe Thr Gly Val Thr Lys Ala Thr Ser Gly Pro Glu Ser

465 470 475 480

Ala Val

<210> 3

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atggcgggcg gcgacgag 18

<210> 4

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

ccacgagcac gaccagg 17

<210> 5

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

ccggtaccat ggcgggcggc gacgag 26

<210> 6

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ccacgagcac gaccaggcca tggccg 26

<210> 7

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

aaaaggaagg tggctcctac 20

<210> 8

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

ccacgagcac gaccagg 17

Claims

1.TaAOSThe application of the gene in improving the potassium stress tolerance of rice;

the nucleotide sequence of the TaAOS gene is shown in SEQ ID No. 1.

2. The application according to claim 1, characterized in that it comprises the following steps:

1) will be described inTaAOSThe gene is connected into pUN1301 to obtain a pCAMBIA1300-TaAOS vector;

3) introducing the engineering bacteria obtained in the step 2) into the callus of the rice, and culturing to obtain the potassium stress tolerant rice.

3. The use of claim 2, wherein the pUN1301 of step 1) is subjected to enzyme digestion and then is subjected to enzyme digestion with pUN1301TaAOSGene connection; the enzyme digestion system is as follows: pUN 130110. mu.L, 10 Xdigestion buffer 5. mu.L, BamHI 1. mu.L at a concentration of 10U/. mu.L, KpnI 0.8. mu.L at a concentration of 10U/. mu.L, ddH₂O is complemented to 50 mu L; the enzyme digestion conditions are as follows: the enzyme was cleaved at 37 ℃ for 3 h.

4. The use according to claim 3, characterized in that the amount of pUN1301 in the digested system is 1 mg.

5. The use according to claim 2, wherein the enzyme used in the ligation of step 1) comprises T4 ligase; the connecting system is as follows: 2. mu.L of pUN1301, 1. mu.L of TaAOS gene 6. mu. L, T4 ligase and 1. mu.L of 10 Xligase buffer; the connection conditions are as follows: ligation was carried out at 16 ℃ for 10 h.

6. The use according to claim 2, wherein the agrobacterium of step 2) is agrobacterium EHA 105.

7. The use according to any one of claims 1 to 6, wherein the rice variety is Nipponbare.