CN111778226A - Plasma membrane H related to alkali stress resistance of rice+-ATPase proteins and uses thereof - Google Patents

Abstract

The invention provides a rice alkali stress-resistant related plasma membrane H⁺-ATPase protein and application thereof, belonging to the technical field of plant stress resistance. The rice alkali stress resistant plasma membrane H provided by the invention⁺-ATPase protein encoded by the OsAHA3 gene and having the amino acid sequence shown in SEQ ID No. 1. The invention also provides a plant expression vector containing the rice alkali-resistant stress gene OsAHA 3. Transforming plants with plant expression vectors by Agrobacterium-mediated methodAfter alkali stress treatment, the phenotype of the transgenic plant is observed and the survival rate is counted, and the result shows that: plasma membrane H encoded by gene OsAHA3⁺The ATPase protein can effectively improve the tolerance of plants to alkali stress and simultaneously realize the overexpression of the gene OsAHA3 in transgenic plants.

Description

Plasma membrane H related to alkali stress resistance of rice+-ATPase proteins and uses thereof

Technical Field

The invention belongs to the technical field of plant stress resistance, and particularly relates to a rice alkali stress-resistant related plasma membrane H⁺-ATPase proteins and uses thereof.

Background

Environmental stress (Environmental stress) refers to the general term for various adverse effects on crops in the environment, such as alkali, salt, drought, fungi, etc. Among them, the alkali stress is one of the most threatening adversities to crops, and it is characterized by long duration, wide range of influence, etc. According to incomplete statistics of the United nations' textbook organization (UNESCO) and the Food and Agriculture Organization (FAO), the saline and alkaline area is 9.54 hundred million hectares [2] all over the world. The Chinese saline-alkali soil has a large amount, the northeast area is one of the largest concentrated distributions of the soda saline-alkali soil, and the area is up to 765 million hectares. Wherein, the saline-alkali soil area of 12 cities and counties in the west of Jilin province is more than 160 million hectares. As a solid organism, in order to reduce the harm of alkali stress to the plant, a set of complete alkali stress resisting mechanism is formed in the long-term evolution process. When subjected to alkaline stress, corresponding regulatory mechanisms are initiated to accommodate this adverse environment. The genetic engineering of plants shows huge potential in the development time of over 30 years, and the cloning of alkali-resistant stress related genes and the development of alkali-resistant molecular breeding by utilizing the genetic engineering show wide prospects.

The response of plants to alkali stress is multi-level and complex. At present, the research of plant response to alkali stress mainly focuses on model plant Arabidopsis thaliana, and H is found in the alkali stress response process⁺A key functional protein AHA2 and regulatory elements for reducing rhizosphere pH are discharged. Rice is one of important grain crops, more than half of the population of the world is cultivated, and the production of the rice has important significance on the global grain safety. The planting of rice is one of effective ways for efficiently utilizing saline-alkali soil, and in order to achieve the expected effect of the measure, the saline-alkali tolerance potential of the rice needs to be excavated as far as possible on the basis of vigorously improving soil and eliminating saline-alkali physical and chemical barriers. However, the research of alkali-resistant stress genes in rice is slow, and the alkali-resistant stress gene engineering is relatively weak. Separating the gene related to the alkali stress resistance of rice and applying the gene to rice genetic engineering for assistanceThe breeding assistance and the alkali stress resistance improvement have extremely important significance for effectively controlling the damage of alkali stress to rice, increasing the rice yield and improving the rice quality.

Disclosure of Invention

In view of the above, the present invention aims to provide a novel alkali stress resistant protein, namely, the rice alkali stress resistant associated plasma membrane H⁺-ATPase proteins and uses thereof.

The invention provides a rice alkali stress-resistant related plasma membrane H⁺-ATPase protein, the amino acid sequence of which is shown in seq id No. 1.

The invention provides a gene OsAHA3 related to alkali stress resistance of rice, and the nucleotide sequence of the gene OsAHA3 is shown in SEQ ID NO. 2.

The invention provides a plant expression vector containing the gene OsAHA3 related to the alkali stress resistance of rice.

Preferably, the plant expression vector is pCsV1300 as a basic vector, and the gene OsAHA3 is inserted into a multiple cloning site of the basic vector, wherein the multiple cloning site is XbaI and BamHI.

The invention provides a primer pair for amplifying a rice alkali stress-resistant related gene OsAHA3, which comprises an upstream primer and a downstream primer, wherein the nucleotide sequence of the upstream primer is shown as SEQ ID NO. 3; the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 4.

The invention provides the OsAHA3 and the plasma membrane H⁺-the use of an ATPase protein, said plant expression vector or said primer pair for the resistance of plants to alkali stress.

The invention provides the OsAHA3 and the plasma membrane H⁺-the use of an ATPase protein, said plant expression vector or said primer pair in a transgenic plant resistant to alkali stress.

Preferably, the plant includes a crop, a vegetable, and a fruit tree.

Preferably, the pH value of the alkaline solution in the environment is 8-12 during the alkali stress resistance.

The invention provides a rice alkali stress-resistant related plasma membrane H⁺-ATPase protein having the amino acid sequence of seq id NO.1 is shown. The invention utilizes the full-length cDNA sequence of the primer pair amplification gene OsAHA3 shown in SEQ ID NO.3 and SEQ ID NO.4 to connect the amplification product with the vector to obtain a plant expression vector, constructs a rice genetic transformation system through agrobacterium mediation, and generates a large amount of plasma membrane H for OsAHA3 through expression⁺-ATPase protein, tested for alkali resistance, the results show that: compared with wild Kitaake, the tolerance of the OsAHA3 overexpression plant to alkali stress is enhanced, and the survival rates of the OsAHA3 overexpression plants OsAHA3OX-1, OsAHA3OX-2 and OsAHA3OX-3 are obviously higher than that of the wild Kitaake.

The invention provides a plant expression vector containing the gene OsAHA3 related to the alkali stress resistance of rice. The plant expression vector is mediated by agrobacterium to construct a rice genetic transformation system, the transformation system not only has higher alkali resistance, but also carries out RT-qPCR detection on the expression level of OsAHA3 in transgenic rice, and the result shows that: compared with wild type, the expression level of OsAHA3 in 3 independent OsAHA3 overexpression plants (OsAHA3OX-1, OsAHA3OX-2 and OsAHA3OX-3) is increased by more than 20 times. The plant expression vector provided by the invention can successfully transform the OsAHA3 overexpression vector into rice.

Drawings

FIG. 1 is a schematic structural diagram of a genetic transformation vector pCsV 1300;

FIG. 2 is a schematic diagram showing the results of detecting the expression level of a rice alkali stress-resistant related gene OsAHA3 in a transgenic rice plant and a wild type Kitaake;

FIG. 3 is a schematic representation of the phenotype of transgenic rice plants after treatment with wild type Kitaake alkali stress;

FIG. 4 is a statistical representation of the survival rate of transgenic rice plants after base stress treatment with wild type Kitaake.

Detailed Description

The invention provides a plasma membrane H encoded by the gene OsAHA3 sequence⁺-ATPase protein, consisting of 956 amino acids, the amino acid sequence of which is shown in SEQ ID No.1 (MAEKEGNLDAVLKEAVDLENIPLEEVFENLRCSREGLTTQQAQQRLEIFGPNKLEEKEESKFLKFLGFMWNPLSWVMEAAAIMAIALANGGGKPPDWQDFVGIITLLVINSTISFIEENNAGNAAAALMARLAPKAKVLRDGRWTEEEAAILVPG)DIVSIKLGDIIPADARLLEGDPLKIDQSALTGESLPVTKGPGDGVYSGSTVKQGEIEAIVIATGVHTFFGKAAHLVDSTNQVGHFQKVLTAIGNFCICSIAVGMFVEIIVMYPIQHRAYRPGIDNLLVLLIGGIPIAMPTVLSVTMAIGSHRLSQQGAITKRMTAIEEMAGMDVLCSDKTGTLTLNKLTVDKNLIDVFERGITQDQVILMAARASRTENQDAIDTAIVGMLADPKEARAGIQEVHFLPFNPTDKRTALTYIDGDGKMYRVSKGAPEQILHLAHNKPEIERRVHAVIDKFAERGLRSLAVAYQEVPEGTKESPGGPWHFVGLMPLFDPPRHDSAETIRRALNLGVNVKMITGDQLAIGKETGRRLGMGTNMYPSSALLGQNKDESIAALPVDDLIEKADGFAGVFPEHKYEIVKRLQARKHICGMTGDGVNDAPALKKADIGIAVADATDAARSASDIVLTEPGLSVIISAVLTSRAIFQRMKNYTIYAVSITIRIVLGFMLLALIWKFDFPPFMVLIIAILNDGTIMTISKDRVKPSPLPDSWKLAEIFTTGVVLGGYLAMMTVIFFWAAYKTDFFPRIFHVESLEKTAQDDFQKLASAVYLQVSTISQALIFVTRSRSWSFVERPGFLLVFAFLVAQLIATLIAVYADWAFTSIKGIGWGWAGIVWLYNLIFYFPLDIIKFLIRYALSGKAWDLVIEQRIAFTRKKDFGKEERELKWAHAQRTLHGLQPPDAKMFSEKAGYNELNQMAEEAKRRAEIARLRELHTLKGHVESVVKLKGLDIETIQQSYTV)。

The invention provides a rice alkali stress resistance related gene OsAHA3, the total length is 3856bp, comprising an Open Reading Frame (ORF) of 2871bp, a 5 'UTR of 240bp and a 3' UTR of 745bp, an initiation codon is ATG, a termination codon is TGA, and a nucleotide sequence is shown as SEQ ID No.2 (atggctgagaaggagggcaacctcgacgccgtcctcaaggaggccgtcgacttggagaatattcccctcgaggaagtgtttgagaacctgagatgcagccgcgagggtctcacaactcagcaggcgcagcagcgcctcgaaatctttggccccaacaagcttgaggagaaggaggagagcaagttcctcaagtttttggggttcatgtggaatccactctcatgggtcatggaagctgcagctatcatggccattgcgctggcgaatggaggggggaagccaccggattggcaggactttgttggtatcataactcttcttgttatcaactcgacaatcagtttcattgaggaaaacaatgctggcaatgctgctgctgcactcatggcccgtcttgcaccaaaggccaaggtgcttcgtgatggccgatggactgaggaagaggcggccatccttgtaccaggggatatcgtaagtattaaacttggagacattatacctgcagatgcgcgtctccttgagggagatcctttgaagatcgatcagtctgccctgactggagaatcattgcctgtcaccaaaggtcctggtgatggtgtctattctggttcgactgtcaagcaaggtgagatcgaagccatagtgattgctactggtgttcacactttcttcggaaaagcagcacaccttgttgactcaactaaccaagttggtcatttccagaaggtcctgacggctattgggaatttctgcatttgctcaattgctgtgggaatgtttgttgagatcattgtaatgtaccctatccagcacagggcataccgccccgggattgacaacctccttgtccttctcattggaggcattcccatagccatgccaacagtcttatctgtgactatggcaattgggtcacaccgcttgtctcaacagggagcgatcacaaagagaatgactgcaattgaggagatggcgggcatggatgttctttgcagtgataaaactggaactttgaccttgaataagctcaccgtggacaagaacctcattgatgtctttgaaagaggaatcactcaggaccaagtgattctcatggctgctagagcatcacgaacagaaaaccaggatgctattgatactgcaatagttgggatgctagctgatccaaaagaggcccgtgctggtattcaagaagttcatttcctgccattcaatcctactgacaaaagaacagcattgacatacattgatggtgatggcaaaatgtatcgtgttagtaagggtgcacctgagcagattctccaccttgctcataacaaaccggagatagagcggagggtccatgctgtgattgacaaatttgcagaacgtggacttagatcgcttgctgtagcataccaggaagtaccagaggggacgaaagaaagccctggtggcccatggcattttgttggtctcatgccactttttgatcctccaaggcacgacagtgctgaaacaattcggcgggcacttaatcttggtgtcaatgtcaagatgatcacaggtgatcagctggcaattggaaaagaaacagggcgtcgcctgggaatgggtacaaacatgtacccttcatccgctttgctgggacagaacaaggatgagtccattgccgctttaccagttgatgatctcattgagaaagctgatggctttgctggtgtattcccagagcacaagtatgagattgtgaaacgtctgcaagcaagaaagcacatatgtggaatgactggtgatggtgtcaatgatgctccagccctaaagaaagctgacattggtattgctgttgctgatgcaactgatgcagccaggagtgcttcagatattgtgctcacagaacctggtcttagtgtgatcatcagtgctgtgcttacaagtcgtgcaattttccagcgtatgaagaactacactatctatgctgtctcaatcaccattcgtatagtgcttggatttatgctacttgcgctcatctggaaattcgatttccccccttttatggtcctaatcatagcaattctaaatgatggtaccatcatgactatatcaaaggatcgggtaaaaccatctccactacctgacagctggaagctggctgagatttttacaactggagttgtcctcggtggatacttggcaatgatgacagtaatcttcttctgggctgcatataagacagactttttcccgagaatatttcatgttgaaagccttgagaagacagctcaagatgatttccaaaaacttgcctctgctgtttacctccaagttagcaccatcagccaagctctcatctttgtcacaaggtcccgcagctggtcatttgttgagcgccctggatttctgctggtctttgctttcttggttgcacagctgattgctacactgattgctgtgtatgctgactgggcgttcacctcaatcaaaggcattgggtggggttgggctggtattgtgtggctctacaatctaatcttctacttcccactcgacattatcaagttcctcatcagatatgctttgagtgggaaagcatgggatcttgtcattgagcaaaggatcgctttcacaaggaagaaggactttggtaaggaggagagggagctcaagtgggcacatgctcagaggaccctccatggactgcagccgcctgatgccaagatgttctcggagaaggctggctacaatgagctcaatcagatggctgaagaggcgaagaggagggcggagattgccaggcttagggagctccatacgctcaaggggcatgtagaatcggttgtgaagctcaaaggcctcgacattgagaccatccagcagtcttacaccgtgtga).

The invention provides a plant expression vector containing the gene OsAHA3 related to the alkali stress resistance of rice. The plant expression vector is preferably pCsV1300 as a basic vector, and the gene OsAHA3 is inserted into a polyclonal site of the basic vector, which is XbaI and BamHI.

In the present invention, the method for preparing the plant expression vector preferably comprises the steps of: designing primers according to CDS sequences of rice alkali-resistant stress genes OsAHA3 and related information of a multiple cloning site of a binary expression vector pCsV1300, and adding corresponding enzyme cutting sites to amplify OsAHA3 genes; the accurate rice alkali-resistant stress gene OsAHA3 verified by sequencing is cut by restriction enzymes XbaI and BamHI, a DNA fragment containing the complete reading frame of OsAHA3 is recovered, the binary vector pCsV1300 is also cut by restriction enzymes XbaI and BamHI, the linearized vector is purified and recovered, and the recovered DNA fragment is connected to the restriction enzyme cutting site of the genetic transformation vector pCsV 1300. And then transforming the recombinant vector into escherichia coli, extracting plasmids, and performing PCR and enzyme digestion verification.

The invention provides a primer pair for amplifying a rice alkali stress-resistant related gene OsAHA3, which comprises an upstream primer and a downstream primer, wherein the nucleotide sequence of the upstream primer is shown as SEQ ID NO. 3; the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 4.

The invention provides the OsAHA3 and the plasma membrane H⁺-the use of an ATPase protein, said plant expression vector or said primer pair for the resistance of plants to alkali stress. In the embodiment of the invention, a nucleotide sequence of a rice alkali-resistant stress gene OsAHA3 is constructed to obtain a plant expression vector and transform rice, and Na is contained₂CO₃The rice nutrient solution is subjected to alkali stress treatment and then the phenotype, the survival rate and the like of the transgenic rice are observed, and the result shows that the coding plasma membrane H⁺The gene OsAHA3 of ATPase can improve tolerance of rice to alkali stress.

The invention provides the OsAHA3 and the plasma membrane H⁺-the use of an ATPase protein, said plant expression vector or said primer pair in a transgenic plant resistant to alkali stress. The application of the present invention is applicable to any kind of plants, preferably including crops, vegetables and fruit trees. For purposes of illustration, the O isThe biological function of sAHA3 for alkali resistance, in the examples of the present invention, rice is used as a representative of the plant to illustrate the overexpression and alkali resistance of OsAHA3 in plants, but this should not be construed as a limitation to the technical solution of the present invention.

In the invention, during the alkali stress resistance, the pH value of the alkali solution in the environment is preferably 8-12, and more preferably 10.5-11. In the method for constructing the alkali-stress-resistant transgenic plant, the primer pair is preferably adopted to amplify to obtain the OsAHA3, the OsAHA3 or the plant expression vector is introduced into a plant callus cell, and the plasma membrane H is overexpressed⁺-ATPase protein, differentiating and rooting to obtain transgenic positive plants. Obtaining homozygous T2 generation rice transgenic plant through hygromycin screening and molecular identification. The stress resistance analysis is carried out on the obtained homozygous transgenic rice plant, and the result shows that the alkali stress resistance of the over-expression plant is obviously higher than that of the wild Kitaake.

The following examples are combined to provide a rice plasma membrane H related to alkali stress resistance⁺The ATPase proteins and their uses are described in detail, but they are not to be understood as limiting the scope of the invention.

Example 1

Cloning of rice alkali-resistant stress gene OsAHA3

Extraction of RNA

Total RNA from rice leaves was extracted using TRIzol reagent (Invitrogen, USA).

(1) The rice Kitaake leaves growing to the three-leaf one-heart stage were taken, placed in a 2ml tube containing 2 steel balls, and placed in liquid nitrogen.

(2) Grinding the sample by using a tissue disruptor, pre-cooling an adapter required by the sample grinding in liquid nitrogen in advance, putting the tube filled with the sample into the adapter, and grinding the sample into powder by using a machine with the frequency of 1400rpm and the sample grinding time of generally 90 s.

(3) 1ml of TRIzol reagent extract was added to the sample tube, and the sample and extract were rapidly mixed using a vortex apparatus.

(4) And (3) placing the lysate at 15-25 ℃ for 10min to ensure that the sample is fully lysed, and simultaneously sucking out the magnetic beads by using a magnet.

(5) The cells were transferred to a bench-top high-speed centrifuge and centrifuged at 12,000g at 4 ℃ for 10 min.

(6) The supernatant was pipetted into a new 1.5ml tube and 0.2ml of chloroform was added to each sample. And (4) tightly covering the cover, fully shaking each sample by using a vortex instrument for 30s, and standing for 2-15 min at 15-25 ℃.

(7) Centrifuging at 12,000g for 15min at the temperature of 2-8 ℃.

(8) After centrifugation, 3 layers were formed and the top layer of colorless liquid was transferred to a new centrifuge tube.

(9) 500ml of isopropanol was added to each sample, turned upside down, and mixed well. Standing at 15-25 ℃ for 5-10 min to fully precipitate RNA.

(10) Centrifuging at 12,000g for 10min at the temperature of 2-8 ℃, and removing the supernatant.

(11) 1ml of 75% aqueous ethanol solution prepared with DEPC water was added, and the RNA precipitate was rinsed thoroughly by turning upside down to remove the supernatant.

(12) Centrifuging at 7500g for 5min at 2-8 ℃, and removing supernatant.

(13) Centrifuging 7500g for a short time at the temperature of 2-8 ℃, sucking away excessive ethanol by using a liquid-transferring gun, and drying in the air for 5-10 min (not too dry, otherwise not easy to dissolve).

(14) Adding 30 μ l DEPC treated water to dissolve RNA,

(15) the concentration of each sample, A, was determined using a NanoDrop2000 assay₂₆₀/A₂₈₀The value is qualified when the value is 2.0-2.2. Mu.l of RNA was subjected to agarose gel electrophoresis for detection. The samples were stored in an ultra low temperature freezer at-80 ℃ for future use.

Synthesis of cDNA

Mu.g of RNA was taken for reverse transcription, and the RNA was reverse transcribed into cDNA using TransScript One-Step gDNA Removal and cDNAsyntheses SuperMix reverse transcription kit (TransGen Biotech, China).

The reaction system for cDNA synthesis is as follows:

the above liquids were gently mixed and centrifuged briefly. The reaction was stopped at 42 ℃ for 30min and 85 ℃ for 5s, and the cDNA concentration was determined using NanoDrop 2000.

Obtaining the full Length of OsAHA3 Gene cDNA

A pair of specific primers (OsAHA3-F/OsAHA3-R) for amplifying a full-length ORF is designed, and a restriction enzyme cutting site is added at the 5' end of the primer to facilitate the construction of a next vector, so that a pair of primers (OsAHA3-XbaI-F/OsAHA3-BamHI-R) for obtaining a rice alkali-resistant stress gene OsAHA3 by the next PCR amplification is obtained. The cDNA of the expected size is successfully obtained by taking the reverse transcription cDNA as a template. PCR reaction procedure: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 1min, annealing at 58 ℃ for 30s, extension at 72 ℃ for 3min, 32 cycles; stretching for 10min at 72 ℃.

OsAHA3-XbaI-F：5’-gctctagaatggctgagaaggagggca-3’(SEQ ID No.3)

OsAHA3-BamHI-R：5’-cgggatcctcacacggtgtaagactgctgg-3’(SEQ ID No.4)。

(restriction sites and protecting bases are underlined in the primers)

After the reaction is finished, electrophoresis is carried out, products are recovered, the recovered fragments are connected into a vector pMD18-T, escherichia coli is transformed, and a single clone is selected for sequencing to obtain the full length cDNA with a complete reading frame, no mismatching and no frame shifting, wherein the nucleotide sequence is shown as SEQ ID NO.2, and the amino acid sequence is shown as SEQ ID NO. 1.

Example 2

Construction of rice alkali-resistant stress gene OsAHA3 overexpression vector

(1) The binary vector pCsV1300 was digested with XbaI and BamHI, and the large fragment (vector) was recovered by running the gel.

(2) The T vector containing the rice alkali-resistant stress gene OsAHA3 obtained in example 1 was digested with XbaI and BamHI, and the digested vector was run on a rubber bath to recover a DNA fragment (gene) containing the rice alkali-resistant stress gene OsAHA 3.

(3) The recovered vector is routinely ligated to the gene.

(4) The ligation product was transformed into E.coli competent DH 5. alpha. and a single clone was picked for PCR detection.

(5) And (4) for overnight culture of the monoclonal antibody which is detected to be positive, extracting the plasmid for enzyme digestion verification, and using the plasmid with the verification result consistent with the expected fragment for subsequent experiments.

Example 3

Agrobacterium-mediated rice genetic transformation system and identification

(1) Selecting mature and full rice variety Kitaake seeds, and shelling; sterilizing with 75% alcohol for 1-2 min, and pouring out the alcohol; washing with sterilized distilled water for 2 times; adding 0.15% mercuric chloride (containing 0.1% Tween 20) and soaking for 15-18 min, and shaking for several times; mercury mercuric oxide was poured off, and washed with sterilized distilled water 5 times. Inoculating the sterilized seeds into an induction callus culture medium, and culturing for 5-10 days at 32 ℃ under illumination.

(2) The expression vector containing the rice alkali-resistant stress gene OsAHA3 obtained in example 2 is transformed into agrobacterium. In the first 2d of infection, Agrobacterium was streaked onto LB medium containing 50mg/l kanamycin antibiotic and incubated at 28 ℃.

(3) Before infection, scraping the activated agrobacterium into a suspension culture medium, performing shake culture at 28 ℃ and 180rpm for 3-3.5 h, and then adjusting the concentration of a bacterial liquid to OD by using the suspension culture medium₆₀₀0.1 to 0.2. And (5) placing the callus induced for 5-10 days into the agrobacterium tumefaciens suspension, and infecting for 1.5-10 min. The bacterial solution was decanted and the wound surface was blotted dry with sterile filter paper. Covering sterilized filter paper on the surface of the callus, and drying for 30min by an ultra-clean bench. After drying, transferring the callus into a co-culture medium with a layer of sterilized filter paper covered on the surface, firstly culturing in the dark at 20 ℃ overnight, and then transferring into an incubator at 25 ℃ for continuous dark culture for 2 d.

(4) After the co-culture was completed, the callus was transferred to an empty sterilized container with forceps. And (3) repeatedly washing and healing the wound for 7-8 times by using sterilized distilled water, wherein the wound can be quickly washed for the first 3 times, and the wound can be soaked for 3-5 min each time when the wound is washed for the second 3-4 times. Finally, the callus is soaked in sterilized distilled water containing 500mg/l Carbenicillin (Cn) for 30 min. Pouring out the carbenicillin solution, sucking the water on the surface of the callus as much as possible by using a sterilized filter paper, covering a layer of sterilized filter paper on the surface of the callus, and drying for 1h by using an ultra-clean bench.

(5) Placing the cleared callus on a screening culture medium containing hygromycin for 32 ℃, and culturing for 14d by illumination.

(6) After screening for 14 days, the resistant calli were transferred to a differentiation medium and cultured at 28 ℃ with a photoperiod of 14h light/10 h dark.

(7) And when the resistance callus forms a regenerated seedling with the height of 3-4 cm on a differentiation culture medium, transferring the regenerated seedling into a rooting culture medium for culture until a complete transgenic rice plant is formed. The inbred progeny of the transgenic rice can be screened from homozygous transgenic plants by conventional screening with hygromycin.

Example 4

Detection of OsAHA3 expression level in T2 generation homozygous transgenic rice

Taking leaves of wild Kitaake and transgenic rice plants which grow to the three-leaf one-heart stage, and extracting RNA for analyzing the relative expression quantity of genes. RT-qPCR was performed using a Real-time PCR instrument (ABI, USA). RNA extraction was performed as described above for cDNA synthesis. The cDNA was then diluted 10-fold and subjected to RT-qPCR according to the instructions of kit THUNDERBIRD SYBR qPCRMixWithout Rox (TOYOBO, Japan). Primers used for RT-qPCR were designed by Primer Express 3.0, OsACT1 was used as an internal reference gene, and the following primers were used:

qRT-OsAHA3-F：5’-tgaaagccttgagaagacagc-3’(SEQ ID No.5)；

qRT-OsAHA3-R：5’-aagaaagcaaagaccagcaga-3’(SEQ ID No.6)；

OsACT1-F：5’-ctcccccatgctatccttcg-3’(SEQ ID No.7)；

OsACT1-R：5’-tgaatgagtaaccacgctccg-3’(SEQ ID No.8)。

reaction conditions of RT-qPCR: pre-denaturation at 95 ℃ for 10 min; denaturation at 95 ℃ for 15sec, annealing at 60 ℃ for 1min, and this step is repeated for 40 cycles. According to equation 2^-△△CtWhere △ Ct ═ c (Ct)_{Target gene}-Ct_{Internal reference gene})，△△Ct＝(△Ct_Sample(s)-△Ct_Control). Ct is the fluorescence threshold. The results of RT-qPCR showed 3 independent genes compared to the wild typeThe expression level of OsAHA3 in OsAHA3 overexpression plants (OsAHA3OX-1, OsAHA3OX-2 and OsAHA3OX-3) is increased by more than about 20 times (see figure 2).

Example 5

Alkali resistance test of OsAHA3 overexpression plants

Wild type Kitaake and homozygous OsAHA3 overexpressing transgenic plants (OsAHA3OX-1, OsAHA3OX-2 and OsAHA3OX-3) grown to trilobal one-heart stage were transferred to plants containing 25mM Na₂CO₃Treatment for 6 days in nutrient solution (pH 10.95), phenotype was observed and survival was counted.

The results show that compared with wild Kitaake, the OsAHA3 overexpression plant has enhanced tolerance to alkali stress, and the survival rate is obviously higher than that of the wild Kitaake (as shown in figures 3-4).

From the above, it can be seen that the present invention provides plasma membrane H which plays an important role in the response to alkali stress⁺-ATPase protein and its coding sequence. The OsAHA3 overexpression vector is successfully transformed into rice by an agrobacterium-mediated transformation method, and a homozygous T2 generation transgenic rice plant is obtained. Under the alkali stress, the survival rate of OsAHA3 overexpression plants OsAHA3OX-1, OsAHA3OX-2 and OsAHA3OX-3 is obviously higher than that of wild Kitaake. The above results indicate that the plasma membrane H of rice⁺The ATPase gene OsAHA3 can improve tolerance of rice to alkali stress.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> northeast university

<120>Plasma membrane H related to alkali stress resistance of rice⁺-ATPase proteins and uses thereof

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<170>SIPOSequenceListing 1.0

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<213> Artificial Sequence (Artificial Sequence)

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Met Ala Glu Lys Glu Gly Asn Leu Asp Ala Val Leu Lys Glu Ala Val

1 5 10 15

Asp Leu Glu Asn Ile Pro Leu Glu Glu Val Phe Glu Asn Leu Arg Cys

20 25 30

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

35 40 45

Phe Gly Pro Asn Lys Leu Glu Glu Lys Glu Glu Ser Lys Phe Leu Lys

50 55 60

Phe Leu Gly Phe Met Trp Asn Pro Leu Ser Trp Val Met Glu Ala Ala

65 70 75 80

Ala Ile Met Ala Ile Ala Leu Ala Asn Gly Gly Gly Lys Pro Pro Asp

85 90 95

Trp Gln Asp Phe Val Gly Ile Ile Thr Leu Leu Val Ile Asn Ser Thr

100 105 110

Ile Ser Phe Ile Glu Glu Asn Asn Ala Gly Asn Ala Ala Ala Ala Leu

115 120 125

Met Ala Arg Leu Ala Pro Lys Ala Lys Val Leu Arg Asp Gly Arg Trp

130135 140

Thr Glu Glu Glu Ala Ala Ile Leu Val Pro Gly Asp Ile Val Ser Ile

145 150 155 160

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

165 170 175

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

180 185 190

Thr Lys Gly Pro Gly Asp Gly Val Tyr Ser Gly Ser Thr Val Lys Gln

195 200 205

Gly Glu Ile Glu Ala Ile Val Ile Ala Thr Gly Val His Thr Phe Phe

210 215 220

Gly Lys Ala Ala His Leu Val Asp Ser Thr Asn Gln Val Gly His Phe

225 230 235 240

Gln Lys Val Leu Thr Ala Ile Gly Asn Phe Cys Ile Cys Ser Ile Ala

245 250 255

Val Gly Met Phe Val Glu Ile Ile Val Met Tyr Pro Ile Gln His Arg

260 265 270

Ala Tyr Arg Pro Gly Ile Asp Asn Leu Leu Val Leu Leu Ile Gly Gly

275 280 285

Ile Pro Ile Ala Met Pro Thr Val Leu Ser Val Thr Met Ala Ile Gly

290 295 300

Ser His Arg Leu Ser Gln Gln Gly Ala Ile Thr Lys Arg Met Thr Ala

305 310 315 320

Ile Glu Glu Met Ala Gly Met Asp Val Leu Cys Ser Asp Lys Thr Gly

325 330 335

Thr Leu Thr Leu Asn Lys Leu Thr Val Asp Lys Asn Leu Ile Asp Val

340 345 350

Phe Glu Arg Gly Ile Thr Gln Asp Gln Val Ile Leu Met Ala Ala Arg

355 360 365

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

370 375 380

Met Leu Ala Asp Pro Lys Glu Ala Arg Ala Gly Ile Gln Glu Val His

385 390 395 400

Phe Leu Pro Phe Asn Pro Thr Asp Lys Arg Thr Ala Leu Thr Tyr Ile

405 410 415

Asp Gly Asp Gly Lys Met Tyr Arg Val Ser Lys Gly Ala Pro Glu Gln

420 425 430

Ile Leu His Leu Ala His Asn Lys Pro Glu Ile Glu Arg Arg Val His

435 440 445

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

450 455460

Ala Tyr Gln Glu Val Pro Glu Gly Thr Lys Glu Ser Pro Gly Gly Pro

465 470 475 480

Trp His Phe Val Gly Leu Met Pro Leu Phe Asp Pro Pro Arg His Asp

485 490 495

Ser Ala Glu Thr Ile Arg Arg Ala Leu Asn Leu Gly Val Asn Val Lys

500 505 510

Met Ile Thr Gly Asp Gln Leu Ala Ile Gly Lys Glu Thr Gly Arg Arg

515 520 525

Leu Gly Met Gly Thr Asn Met Tyr Pro Ser Ser Ala Leu Leu Gly Gln

530 535 540

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

545 550 555 560

Lys Ala Asp Gly Phe Ala Gly Val Phe Pro Glu His Lys Tyr Glu Ile

565 570 575

Val Lys Arg Leu Gln Ala Arg Lys His Ile Cys Gly Met Thr Gly Asp

580 585 590

Gly Val Asn Asp Ala Pro Ala Leu Lys Lys Ala Asp Ile Gly Ile Ala

595 600 605

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

610 615620

Thr Glu Pro Gly Leu Ser Val Ile Ile Ser Ala Val Leu Thr Ser Arg

625 630 635 640

Ala Ile Phe Gln Arg Met Lys Asn Tyr Thr Ile Tyr Ala Val Ser Ile

645 650 655

Thr Ile Arg Ile Val Leu Gly Phe Met Leu Leu Ala Leu Ile Trp Lys

660 665 670

Phe Asp Phe Pro Pro Phe Met Val Leu Ile Ile Ala Ile Leu Asn Asp

675 680 685

Gly Thr Ile Met Thr Ile Ser Lys Asp Arg Val Lys Pro Ser Pro Leu

690 695 700

Pro Asp Ser Trp Lys Leu Ala Glu Ile Phe Thr Thr Gly Val Val Leu

705 710 715 720

Gly Gly Tyr Leu Ala Met Met Thr Val Ile Phe Phe Trp Ala Ala Tyr

725 730 735

Lys Thr Asp Phe Phe Pro Arg Ile Phe His Val Glu Ser Leu Glu Lys

740 745 750

Thr Ala Gln Asp Asp Phe Gln Lys Leu Ala Ser Ala Val Tyr Leu Gln

755 760 765

Val Ser Thr Ile Ser Gln Ala Leu Ile Phe Val Thr Arg Ser Arg Ser

770 775 780

Trp Ser Phe Val Glu Arg Pro Gly Phe Leu Leu Val Phe Ala Phe Leu

785 790 795 800

Val Ala Gln Leu Ile Ala Thr Leu Ile Ala Val Tyr Ala Asp Trp Ala

805 810 815

Phe Thr Ser Ile Lys Gly Ile Gly Trp Gly Trp Ala Gly Ile Val Trp

820 825 830

Leu Tyr Asn Leu Ile Phe Tyr Phe Pro Leu Asp Ile Ile Lys Phe Leu

835 840 845

Ile Arg Tyr Ala Leu Ser Gly Lys Ala Trp Asp Leu Val Ile Glu Gln

850 855 860

Arg Ile Ala Phe Thr Arg Lys Lys Asp Phe Gly Lys Glu Glu Arg Glu

865 870 875 880

Leu Lys Trp Ala His Ala Gln Arg Thr Leu His Gly Leu Gln Pro Pro

885 890 895

Asp Ala Lys Met Phe Ser Glu Lys Ala Gly Tyr Asn Glu Leu Asn Gln

900 905 910

Met Ala Glu Glu Ala Lys Arg Arg Ala Glu Ile Ala Arg Leu Arg Glu

915 920 925

Leu His Thr Leu Lys Gly His Val Glu Ser Val Val Lys Leu Lys Gly

930 935 940

Leu Asp Ile Glu Thr Ile Gln Gln Ser Tyr Thr Val

945 950 955

<210>2

<211>2871

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>2

atggctgaga aggagggcaa cctcgacgcc gtcctcaagg aggccgtcga cttggagaat 60

attcccctcg aggaagtgtt tgagaacctg agatgcagcc gcgagggtct cacaactcag 120

caggcgcagc agcgcctcga aatctttggc cccaacaagc ttgaggagaa ggaggagagc 180

aagttcctca agtttttggg gttcatgtgg aatccactct catgggtcat ggaagctgca 240

gctatcatgg ccattgcgct ggcgaatgga ggggggaagc caccggattg gcaggacttt 300

gttggtatca taactcttct tgttatcaac tcgacaatca gtttcattga ggaaaacaat 360

gctggcaatg ctgctgctgc actcatggcc cgtcttgcac caaaggccaa ggtgcttcgt 420

gatggccgat ggactgagga agaggcggcc atccttgtac caggggatat cgtaagtatt 480

aaacttggag acattatacc tgcagatgcg cgtctccttg agggagatcc tttgaagatc 540

gatcagtctg ccctgactgg agaatcattg cctgtcacca aaggtcctgg tgatggtgtc 600

tattctggtt cgactgtcaa gcaaggtgag atcgaagcca tagtgattgc tactggtgtt 660

cacactttct tcggaaaagc agcacacctt gttgactcaa ctaaccaagt tggtcatttc 720

cagaaggtcc tgacggctat tgggaatttc tgcatttgct caattgctgt gggaatgttt 780

gttgagatca ttgtaatgta ccctatccag cacagggcat accgccccgg gattgacaac840

ctccttgtcc ttctcattgg aggcattccc atagccatgc caacagtctt atctgtgact 900

atggcaattg ggtcacaccg cttgtctcaa cagggagcga tcacaaagag aatgactgca 960

attgaggaga tggcgggcat ggatgttctt tgcagtgata aaactggaac tttgaccttg 1020

aataagctca ccgtggacaa gaacctcatt gatgtctttg aaagaggaat cactcaggac 1080

caagtgattc tcatggctgc tagagcatca cgaacagaaa accaggatgc tattgatact 1140

gcaatagttg ggatgctagc tgatccaaaa gaggcccgtg ctggtattca agaagttcat 1200

ttcctgccat tcaatcctac tgacaaaaga acagcattga catacattga tggtgatggc 1260

aaaatgtatc gtgttagtaa gggtgcacct gagcagattc tccaccttgc tcataacaaa 1320

ccggagatag agcggagggt ccatgctgtg attgacaaat ttgcagaacg tggacttaga 1380

tcgcttgctg tagcatacca ggaagtacca gaggggacga aagaaagccc tggtggccca 1440

tggcattttg ttggtctcat gccacttttt gatcctccaa ggcacgacag tgctgaaaca 1500

attcggcggg cacttaatct tggtgtcaat gtcaagatga tcacaggtga tcagctggca 1560

attggaaaag aaacagggcg tcgcctggga atgggtacaa acatgtaccc ttcatccgct 1620

ttgctgggac agaacaagga tgagtccatt gccgctttac cagttgatga tctcattgag 1680

aaagctgatg gctttgctgg tgtattccca gagcacaagt atgagattgt gaaacgtctg 1740

caagcaagaa agcacatatg tggaatgact ggtgatggtg tcaatgatgc tccagcccta 1800

aagaaagctg acattggtat tgctgttgct gatgcaactg atgcagccag gagtgcttca 1860

gatattgtgc tcacagaacc tggtcttagt gtgatcatca gtgctgtgct tacaagtcgt 1920

gcaattttcc agcgtatgaa gaactacact atctatgctg tctcaatcac cattcgtata 1980

gtgcttggat ttatgctact tgcgctcatc tggaaattcg atttcccccc ttttatggtc 2040

ctaatcatag caattctaaa tgatggtacc atcatgacta tatcaaagga tcgggtaaaa 2100

ccatctccac tacctgacag ctggaagctg gctgagattt ttacaactgg agttgtcctc 2160

ggtggatact tggcaatgat gacagtaatc ttcttctggg ctgcatataa gacagacttt 2220

ttcccgagaa tatttcatgt tgaaagcctt gagaagacag ctcaagatga tttccaaaaa 2280

cttgcctctg ctgtttacct ccaagttagc accatcagcc aagctctcat ctttgtcaca 2340

aggtcccgca gctggtcatt tgttgagcgc cctggatttc tgctggtctt tgctttcttg 2400

gttgcacagc tgattgctac actgattgct gtgtatgctg actgggcgtt cacctcaatc 2460

aaaggcattg ggtggggttg ggctggtatt gtgtggctct acaatctaat cttctacttc 2520

ccactcgaca ttatcaagtt cctcatcaga tatgctttga gtgggaaagc atgggatctt 2580

gtcattgagc aaaggatcgc tttcacaagg aagaaggact ttggtaagga ggagagggag 2640

ctcaagtggg cacatgctca gaggaccctc catggactgc agccgcctga tgccaagatg 2700

ttctcggaga aggctggcta caatgagctc aatcagatgg ctgaagaggc gaagaggagg 2760

gcggagattg ccaggcttag ggagctccat acgctcaagg ggcatgtaga atcggttgtg 2820

aagctcaaag gcctcgacat tgagaccatc cagcagtctt acaccgtgtg a 2871

<210>3

<211>27

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>3

gctctagaat ggctgagaag gagggca 27

<210>4

<211>30

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>4

cgggatcctc acacggtgta agactgctgg 30

<210>5

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>5

tgaaagcctt gagaagacag c 21

<210>6

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>6

aagaaagcaa agaccagcag a 21

<210>7

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>7

ctcccccatg ctatccttcg 20

<210>8

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>8

tgaatgagta accacgctcc g 21

Claims (9)

1. Plasma membrane H related to alkali stress resistance of rice⁺-an ATPase protein characterized in that its amino acid sequence is represented by SEQ id No. 1.

2. A gene OsAHA3 related to the alkali stress resistance of rice is characterized in that the nucleotide sequence is shown as SEQ ID NO. 2.

3. A plant expression vector comprising the rice alkali stress-resistant related gene OsAHA3 as set forth in claim 2.

4. The plant expression vector of claim 3, wherein the plant expression vector is pCsV1300 as a base vector, and the gene OsAHA3 is inserted into the base vector at a multiple cloning site for XbaI and BamHI.

5. A primer pair for amplifying the rice alkali stress resistant related gene OsAHA3 as claimed in claim 2, comprising an upstream primer and a downstream primer, wherein the nucleotide sequence of the upstream primer is shown as SEQ ID NO. 3; the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 4.

6. The OsAHA3 of claim 2, the plasma membrane H of claim 1⁺-use of an ATPase protein, the plant expression vector of claim 3 or 4 or the primer pair of claim 5 for the resistance to alkali stress in plants.

7. The OsAHA3 of claim 2, the plasma membrane H of claim 1⁺-use of an ATPase protein, the plant expression vector of claim 3 or 4 or the primer pair of claim 5 in a transgenic plant resistant to alkali stress.

8. The use according to claim 6 or 7, wherein the plant comprises a crop, a vegetable and a fruit tree.

9. The use of claim 6 or 7, wherein the pH value of the alkaline solution in the environment is 8-12 during the alkali stress resistance.

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