CN114941007A

CN114941007A - Recombinant expression vector and application thereof

Info

Publication number: CN114941007A
Application number: CN202210624604.8A
Authority: CN
Inventors: 李畅; 李广鑫; 刘红恩; 赵鹏; 秦世玉; 睢福庆
Original assignee: Henan Agricultural University
Current assignee: Henan Agricultural University
Priority date: 2022-06-02
Filing date: 2022-06-02
Publication date: 2022-08-26
Also published as: CN117004640A

Abstract

The invention belongs to the technical field of rice cultivation, and particularly relates to a recombinant expression vector and application thereof. The recombinant expression vector comprises gene sequences such as SEQ ID No.1 and OsAMT1.1, and is applied to rice cultivation, so that the cultivated transgenic rice can obviously improve the growth of the rice in low-phosphorus and normal-phosphorus environments, the nitrogen and phosphorus content of each part is obviously increased, and the rice yield can be increased.

Description

Recombinant expression vector and application thereof

Technical Field

The invention belongs to the technical field of rice cultivation, and particularly relates to a recombinant expression vector and application thereof.

Background

The rice is one of three main grain crops in China, and has important significance for agricultural production and guaranteeing national grain safety. In the actual production process, the yield of crops is reduced due to the deficiency of nutrients, moisture and the like. Nitrogen (Nitrogen, N) and Phosphorus (P) are macronutrients essential for plant growth and development, and the effectiveness of Phosphorus in soil is low due to chelation of metal ions present in soil and immobilization of microorganisms. According to investigation, 74% of the soil in China is deficient in phosphorus. Therefore, the rice yield is improved by improving the nitrogen and phosphorus nutrition condition of the rice, and the method has great significance for guaranteeing national grain safety.

NH ₄ ⁺ And NO ₃ ^- Is the main existing form of inorganic nitrogen in soil and can be directly absorbed and utilized by plants. Research shows that the pH value of the rhizosphere can be increased when the plant root system absorbs nitrate, and H can be released when the plant root system absorbs ammonium salt ⁺ Thereby reducing rhizosphere pH and acidifying the soil; phosphorus in soil is one of the main factors restricting agricultural production because of poor mobility. How to improve the nitrogen and phosphorus nutrition of plants is always the key point of research in the field of efficient utilization of plant nutrients.

Disclosure of Invention

In view of the above problems, the present invention aims to provide a recombinant expression vector and use thereof, wherein the recombinant expression vector is used in rice cultivation, can significantly improve rice growth, significantly increase nitrogen and phosphorus contents of each part, and increase rice yield in low-phosphorus and normal-phosphorus environments.

In order to achieve the purpose, the invention can adopt the following technical scheme:

the invention provides a recombinant expression vector, which at least comprises gene sequences shown as SEQ ID No.1 and OsAMT 1.1.

Specifically, one of the ways for improving the phosphorus nutrition status of plants is to release the fixed phosphorus by acidifying the soil by releasing organic acid, etc., so that the phosphorus can be absorbed and utilized by the root system. The present invention therefore hopes to increase NH of rice ₄ ⁺ The rhizosphere soil is acidified, so that the effectiveness of phosphorus in the soil can be improved, and the double high-efficiency absorption of the nitrogen and the phosphorus is finally realized. Existing research tableThe Ming rice high-affinity ammonium transporter gene OsAMT1.1 can transport NH under the low-ammonium and high-ammonium nutritional conditions ₄ ⁺ Thus can be used for increasing NH in soil ₄ ⁺ Potential genes for uptake. Research on Hoque et al (2006) and Ranathunge et al (2014) shows that overexpression of the OsAMT1.1 gene in rice can cause ammonium poisoning under the high-ammonium nutrition condition, so that the rice grows short and small. Therefore, the expression of the OsAMT1.1 gene is driven by finding a proper promoter, and NH can be increased within a certain range ₄ ⁺ Absorbing while avoiding excessive absorption causing NH ₄ ⁺ Symptoms of poisoning by increasing NH ₄ ⁺ Absorb and release more H ⁺ The ions acidify the soil and further increase the absorption of phosphorus.

Through research and exploration, the promoter containing the rice phosphorus deficiency response gene OsPT6, namely the sequence shown as SEQ ID No.1 can drive OsAMT1.1 gene to reasonably express and increase NH in a certain range ₄ ⁺ While avoiding excessive absorption of NH ₄ ⁺ Symptoms of poisoning by increasing NH ₄ ⁺ Absorb and release more H ⁺ The ions acidify the soil and further increase the absorption of phosphorus.

Further, the OsAMT1.1 gene sequence can comprise a sequence shown as SEQ ID No. 2. Specifically, the recombinant plasmid vector at least comprises a sequence shown as SEQ ID No.1 and is used for driving expression of OsAMT1.1 gene, and the OsAMT1.1 gene sequence is preferably a sequence shown as SEQ ID No. 2.

Further, the expression vector in the recombinant expression vector may be any one of a pTCK303 plasmid vector, a Ti plasmid vector, a Ri plasmid vector, and a plant virus vector. Specifically, the expression vector in the recombinant expression vector can effectively express the target gene in a body, and the target gene of the invention is preferably any one of pTCK303 plasmid vector, Ti plasmid vector, Ri plasmid vector and plant virus vector, so that the stable expression of the target gene can be effectively improved.

Further, the recombinant plasmid vector may further comprise an enhancer and/or a reporter gene. Specifically, the recombinant plasmid vector can be connected with an enhancer in order to improve the expression of the OsAMT1.1 gene driven by the sequence shown in SEQ ID No. 1; to simplify the identification of transformed cells, selectable markers including enzymes resistant to antibiotics, enzymes that can also use compounds that are recognized by color change or luminescence, such as recombinant plasmid vectors, may be used to augment the reporter gene.

Further, the enhancer may be a transcriptional enhancer or a translational enhancer.

Further, the reporter gene may be any one of an antibiotic resistance gene, a GUS reporter gene and a luciferase reporter gene.

The invention also provides application of the recombinant expression vector in improving the nitrogen and phosphorus content of rice.

The invention also provides application of the recombinant expression vector in promoting rice growth.

The invention also provides application of the recombinant expression vector in improving rice yield.

Furthermore, in the application, the recombinant expression vector can be used for transforming rice by an agrobacterium-mediated method, a gene gun method, a pollen tube channel method or other methods, so that the nitrogen and phosphorus content of a plant can be increased, the growth of the rice can be promoted, and the yield can be increased in a low-phosphorus or normal-phosphorus environment.

On the other hand, the invention provides a method for cultivating transgenic rice, which transfers the recombinant expression vector to rice for cultivation by an agrobacterium-mediated method, a gene gun method or a pollen tube channel method.

Compared with the prior art, the invention has the following beneficial effects:

(1) the rice transgenic line cultured by the recombinant plasmid vector provided by the invention can improve the biomass of rice seedling stage under the condition of insufficient phosphorus nutrition supply, and compared with wild rice, the root weight and the overground part dry weight of the transgenic line are respectively increased by 4.0-15.2% and 4.3-9.2%.

(2) The rice transgenic line cultured by the recombinant plasmid vector provided by the invention can obviously improve the growth of rice under the conditions of low phosphorus and normal phosphorus-supply soil, and obviously increase the nitrogen and phosphorus contents of each part, compared with wild WT, the dry weight, the nitrogen content and the phosphorus content of the overground part (comprising flag leaves, inverted two leaves, inverted three leaves and stems) of the transgenic line are respectively increased by 9.6-14.6%, 18.9-27.7%, 13.9-24.5%, 12.7-23.0%, 22.1-28.3% and 25.4-30.8%.

(3) Compared with the wild WT, the rice transgenic line cultured by the recombinant plasmid vector provided by the invention has the advantages that the effective tillering, the single plant yield and the grain nitrogen and phosphorus concentration of the transgenic line are obviously increased, the effective tillering number is increased by 13.6-30.9%, the single plant yield is increased by 14.1-25.8%, and the grain nitrogen and phosphorus concentration is respectively increased by 15.7-18.0% and 15.4-24.3%.

Drawings

FIG. 1 is a schematic diagram of a vector of a rice phosphorus deficiency response gene OsPT6 promoter-initiated rice gene OsAMT 1.1.

FIG. 2 is the growth phenotype of the hydroponic transgenic lines at seedling stage under low phosphorus conditions;

FIG. 3 shows the expression identification of the seedling stage hydroponic transgenic line OsAMT1.1 under the low-phosphorus condition.

FIG. 4 shows the root system and overground biomass of the transgenic line cultivated in water in seedling stage under low-phosphorus condition.

FIG. 5 shows the seedling growth phenotype of seedling hydroponic transgenic lines under normal phosphorus conditions.

FIG. 6 shows the expression identification of the seedling stage hydroponic transgenic line OsAMT1.1 under normal phosphorus condition.

FIG. 7 shows the root system and aerial biomass of the transgenic line cultivated in water in seedling stage under normal phosphorus condition.

FIG. 8 shows the growth phenotype of transgenic lines grown in pots under low phosphorus conditions.

FIG. 9 shows the biomass of the above-ground parts of the transgenic lines cultivated in pots under low-phosphorus conditions.

FIG. 10 shows the total nitrogen content of each part of the transgenic lines cultivated in pots under low-phosphorus conditions.

FIG. 11 shows the total phosphorus content of each part of the transgenic lines cultivated in pots under low-phosphorus conditions.

FIG. 12 shows the growth phenotype of transgenic lines grown in pots under normal phosphorus conditions.

FIG. 13 shows the biomass of the above-ground parts of the transgenic lines cultivated in pots under normal phosphorus conditions.

FIG. 14 shows the total nitrogen content of each part of the ground of the potted transgenic lines under normal phosphorus conditions.

FIG. 15 shows the total phosphorus content of each part of the transgenic lines cultivated in pots under normal phosphorus conditions.

FIG. 16 shows the nitrogen and phosphorus concentrations in the wild type and transgenic line brown rice under field conditions.

Detailed Description

The examples are given for the purpose of better illustration of the invention, but the invention is not limited to the examples. Therefore, those skilled in the art can make insubstantial modifications and adaptations to the embodiments described above without departing from the scope of the present invention.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure. Unless the context has a significantly different meaning, the singular forms of expressions include the plural forms of expressions. As used herein, it is understood that terms such as "comprising," "having," "including," and the like are intended to refer to the presence of features, numbers, operations, components, parts, elements, materials, or combinations thereof. The terms of the present invention are disclosed in the specification and are not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials or combinations thereof may be present or may be added. As used herein, "/" can be interpreted as "and" or "depending on the circumstances.

For a better understanding of the present invention, the following further illustrates the contents of the present invention with reference to specific examples, but the contents of the present invention are not limited to the following examples.

Example 1 preparation of expression Rice Gene OsPT6 promoter promoting Rice Gene OsAMT1.1 recombinant vector

(1) Cloning of rice gene OsPT6 promoter sequence

1) Extraction of genomic DNA of Rice

When the rice (Nipponbare) seedlings grow to 3 leaves, about 0.1g of leaves are weighed, ground by liquid nitrogen, fully ground and added into a 2ml centrifuge tube, 1ml of DNA extracting solution (purchased from TIANGEN, China) is rapidly added, and after fully shaking and shaking, the rice genome DNA is extracted.

2) Cloning of the full Length of OsPT6 Gene

The promoter of OsPT6(AF536966) gene of rice is searched from a gene database of NCBI website (http:// www.ncbi.nlm.nih.gov /), a primer sequence (as follows) is designed by using software Primer5.0, a PT6 gene promoter sequence with the total length of 2860bp is amplified from rice genome DNA,

P1:5’-TTCTTTGTTCTTCCTCCAGGC-3’(SEQ ID No.3)

P2:5’-GCCAGCTTAATTGCTTGCTTTG-3’(SEQ ID No.4)；

using the rice genomic DNA obtained in step 1) as a template, PCR amplification was carried out using high fidelity enzyme (PrimeStarHSDNApolymerase available from Takara Inc.) according to the following PCR protocol: pre-denaturation at 94 deg.C for 2min, denaturation at 94 deg.C for 30s, renaturation at 53 deg.C for 30s, extension at 72 deg.C for 2min, 30 cycles, 5min at 72 deg.C, and constant temperature at 4 deg.C. The DNA fragment was separated by agarose electrophoresis, cut into gel, recovered and cloned into pEASY-Blunt vector (purchased from Takara), and the promoter sequence (SEQ ID No.1) of OsPT6, a rice gene having a complete coding region, was obtained after the sequencing was completed.

(2) Cloning of rice gene OsAMT1.1 sequence

1) Extraction of Total RNA

Rice (Nipponbare) seedlings grow to 3-leaf stage, after treating with 0.2m ammonium nitrogen for 24 hours, immediately taking roots, rapidly placing the roots in liquid nitrogen for freezing preservation, weighing about 0.1g of the roots, grinding the roots with liquid nitrogen, fully adding the ground roots into a 1.5ml centrifuge tube, rapidly adding 1ml of Trizol reagent (purchased from Invitrogen, USA), fully shaking the roots uniformly, and extracting total RNA after shaking the mixture fully.

2) Cloning of OsAMT1.1 Gene full Length

OsAMT1.1 gene sequence OsAMT1.1(LOC _ Os04g43070) of rice was retrieved from a gene database of rice genome annotation program website (http:// rice. plant biology. msu. edu /). Designing a primer (as follows) to amplify the full-length sequence of OsAMT1.1 from a tissue cDNA library,

P3:5’-ATGGCGACGTGCGCGGCGGAC-3’(SEQ ID No.5)

P4:5’-TTACACTTGGTTGTTGCTGTTGG-3’(SEQ ID No.6)；

using the total RNA obtained in step 1) as a template, synthesizing a first cDNA strand by reverse transcription, and performing PCR amplification by using high fidelity enzyme (PrimeStarHSDPA polymerase from Takara), wherein the PCR program is as follows: pre-denaturation at 94 deg.C for 2min, denaturation at 94 deg.C for 30s, renaturation at 53 deg.C for 30s, extension at 72 deg.C for 1min, 30 cycles, 5min at 72 deg.C, and constant temperature at 4 deg.C. After agarose electrophoresis separation, gel cutting and recovery, the gene is cloned to pEASY-Blunt vector (purchased from Takara company), and after the sequencing is correct, the full-length sequence (SEQ ID No.2) of the rice high-affinity nitrate transport protein gene OsAMT1.1 with a complete coding region is obtained.

(3) Preparation of recombinant vector for expressing rice gene OsPT6 promoter to start rice gene OsAMT1.1

1) Construction of rice gene OsPT6 promoter initiated OsAMT1.1 vector

Designing a primer sequence (as follows) by using software Primer5.0 according to the promoter sequence of the rice gene OsPT6 obtained in the first step and a sequence shown as SEQ ID No. 1:

P5:5’-GCAAGCTTTTCTTTGTTCTTCCTCCAGGC-3’(HindIII)(SEQ ID No.7)

P6:5’-GAGGTACCGCCAGCTTAATTGCTTGCTTTG-3’(KpnI)(SEQ ID No.8)

PCR amplification was carried out using the pEASY-Blunt vector for cloning the OsPT6 promoter obtained in the first step as a template, using high fidelity enzyme (PrimeStarHSDNApolymerase available from Takara, Inc.) according to the following PCR protocol: pre-denaturation at 94 deg.C for 2min, denaturation at 94 deg.C for 30s, renaturation at 53 deg.C for 30s, extension at 72 deg.C for 2min, 30 cycles, 5min at 72 deg.C, and constant temperature at 4 deg.C. And carrying out agarose electrophoresis separation and cutting the gel back to obtain PCR products of OsPT6 promoters with enzyme cutting sites HindIII and KpnI added to the F end and the R end respectively. The recovered product was double-digested with restriction enzymes HindIII and KpnI, and the overexpression vector pTCK303 plasmid was double-digested with HindIII and KpnI, and then the digested PCR fragment and vector were recovered, respectively. After recovery, the linearized vector and the digested PCR fragment are connected at 4 ℃ overnight by T4 ligase, transformed into Escherichia coli DH5a competent cells, spread on LB solid medium containing 50 ug/mL kanamycin to grow for 12h, then a positive colony is picked, the plasmid is extracted, and after HindIII and KpnI double digestion verification, the fragment size is correct, the bacterial liquid is subjected to DNA sequencing, and the clone containing correct sequencing is named as pOsPT6-pTCK 303.

Designing a primer sequence (as follows) by using software Primer5.0 according to the full-length sequence of the rice gene OsAMT1.1 obtained in the second step and a sequence shown as SEQ ID No. 2:

P7:5’-TTGGTACCATGGCGACGTGCGCGGCGGAC-3’(KpnI)(SEQ ID No.9)

P8:5’-GAGAGCTCTTACACTTGGTTGTTGCTGTTGG-3’(SacI)(SEQ ID No.10)

and (3) performing PCR amplification by using a high fidelity enzyme (PrimeStarHSDPA polymerase purchased from Takara) by taking the pEASY-Blunt vector with the full length of the cloned OsAMT1.1 gene obtained in the second step as a template, wherein the PCR program is as follows: pre-denaturation at 94 deg.C for 2min, denaturation at 94 deg.C for 30s, renaturation at 53 deg.C for 30s, extension at 72 deg.C for 1min, 30 cycles, 5min at 72 deg.C, and keeping constant temperature at 4 deg.C. Agarose electrophoresis separation, gel cutting recovery and obtaining the OsAMT1.1 gene full-length PCR products of which the F end and the R end are respectively added with enzyme cutting sites KpnI and SacI. The recovered product was double-digested with restriction enzymes KpnI and SacI, and the pOsPT6-pTCK303 plasmid was double-digested with KpnI and SacI, and then the digested PCR fragment and vector were recovered, respectively. After recovery, the linearized vector and the digested PCR fragment are connected at 4 ℃ overnight by T4 ligase, transformed into Escherichia coli DH5a competent cells, spread on LB solid medium containing 50 ug/mL kanamycin to grow for 12h, positive colonies are picked, plasmids are extracted, and after the sizes of the fragments are verified to be correct by KpnI and SacI double digestion, the bacterial liquid is subjected to DNA sequencing, and the clone containing the correct sequencing is named as pOsPT6-OsAMT1.1-pTCK 303. Namely an expression vector, the schematic diagram of the vector is shown in figure 1.

Example 2 acquisition and testing of transgenic lines

(1) Cultivation of transgenic rice line

Transfecting agrobacterium with the recombinant vector in the example 1, infecting rice callus, culturing for 60 hours, and obtaining T through selective culture, differentiation, rooting and seedling hardening ₀ Transgenic plants are generated. For all transgenic materialThe propagation is carried out, and T1 generation and T2 generation materials which are stably inherited are obtained.

(2) Transgenic line test of rice

Rice materials were classified into WT (wild control group), PA1 (rice gene OsPT6 promoter-initiated OsAMT1.1 transgenic line), PA2 (rice gene OsPT6 promoter-initiated OsAMT1.1 transgenic line), PA3 (rice gene OsPT6 promoter-initiated OsAMT1.1 transgenic line), and OX (overexpressed OsAMT1.1(35S strong promoter)).

Respectively containing 0.02mM phosphorus source (KH) ₂ PO ₄ ) (Low phosphorus treatment) with 0.2mM phosphorus source (KH) ₂ PO ₄ ) (Normal phosphorus treatment) the hydroponic nutrient solution was cultured for four weeks.

1) Hydroponic nutrient solution culture

Under the condition of low phosphorus, the cultured rice is shown in figure 2, and the rice gene OsPT6 promoter starts OsAMT1.1 transgenic lines (PA1, PA2 and PA3) to grow better than the control Nipponbare wild type rice (WT); the growth condition of an overexpression OsAMT1.1(35S strong promoter) strain (OX) is poorer than that of a control Nipponbare wild type rice (WT); the expression of OsAMT1.1 is driven by the strong 35S promoter, so that ammonium toxicity is caused.

Under the condition of low phosphorus, qRT-PCR identification finds that the gene expression situation is shown in figure 3, and compared with control Nipponbare wild type rice (WT), the expression of OsAMT1.1 at the root and the overground part of OsAMT1.1 transgenic lines (PA1, PA2 and PA3) started by a rice gene OsPT6 promoter is obviously improved; as a positive control, an overexpression OsAMT1.1(35S strong promoter) strain (OX) is obviously higher than that of a wild type OsAMT 1.1.

Under the condition of low phosphorus, the dry weights of the root system and the overground part are shown in figure 4, and compared with the control Nipponbare wild type rice (WT), the dry weights of the root system and the overground part of the OsAMT1.1 transgenic line (PA1, PA2 and PA3) started by the rice gene OsPT6 promoter are respectively increased by 4.0-15.2 percent and 4.3-9.2 percent; and the dry weights of the root system and the overground part of the over-expression OsAMT1.1(35S strong promoter) strain (OX) are smaller than that of the control Nipponbare wild type rice (WT).

Under normal phosphorus conditions, as shown in FIG. 5, the rice gene OsPT6 promoter initiates OsAMT1.1 transgenic lines (PA1, PA2 and PA3) of the rice cultured in nutrient solution, and the growth conditions are almost the same as those of the control Nipponbare wild-type rice (WT); the growth of the over-expressed OsAMT1.1(35S strong promoter) strain (OX) is poor compared with that of the control Nipponbare wild type rice (WT).

Under the condition of normal phosphorus, qRT-PCR identification shows that the gene expression condition is shown in figure 6, and compared with control Nipponbare wild type rice (WT), the rice gene OsPT6 promoter starts the expression of OsAMT1.1 transgenic lines (PA1, PA2 and PA3) roots and overground parts OsAMT1.1 almost; as a positive control, an overexpression OsAMT1.1(35S strong promoter) strain (OX) is adopted, and the expression of OsAMT1.1 is obviously higher than that of a wild type.

Under normal phosphorus conditions, the dry weights of the root system and the overground part are shown in FIG. 7, and compared with the control Nipponbare wild type rice (WT), the promoter of the rice gene OsPT6 starts the OsAMT1.1 transgenic line (PA1, PA2 and PA3) to have almost the same dry weight of the root system and the overground part; and the dry weights of the root system and the overground part of the over-expression OsAMT1.1(35S strong promoter) strain (OX) are smaller than that of the control Nipponbare wild type rice (WT).

2) Soil culture test of potted plants

The low-phosphorus and normal-phosphorus soil samples used in the potted plant soil culture test are taken from Changxing test base of Zhejiang university, wherein the soil phosphorus concentrations are as follows: low phosphorus (Olsen Pi ≈ 4.0mg P/kg), normal phosphorus (Olsen Pi ≈ 7.3mg P/kg soil). Air-drying the soil, sieving and grinding, wherein 15Kg of soil is filled in each barrel, and ammonium chloride and potassium chloride are respectively used as a nitrogen source and a potassium source for normal supply; after seeds germinate for 3 weeks, single plants of five-leaf and one-heart rice seedlings with consistent growth vigor are selected for transplanting, and 5 times of transplanting are set.

The rice plants were potted in low-phosphorus and normal-phosphorus soils, and the results are shown in fig. 8-15, the promoter OsAMT1.1 of the phosphorus deficiency-transferring response gene OsPT6 of Nipponbare rice can obviously improve the growth conditions of rice under the conditions of low-phosphorus and normal-phosphorus soil, which are as follows:

under the condition of low phosphorus, the growth condition of rice is shown in figure 8, the rice gene OsPT6 promoter starts OsAMT1.1 transgenic lines (PA1, PA2 and PA3), and compared with the control Nipponbare wild type rice (WT), the growth condition is slightly better; the growth of the over-expressed OsAMT1.1(35S strong promoter) strain (OX) is poor compared with that of the control Nipponbare wild type rice (WT).

In the case of low phosphorus, the dry matter weight of each part of the overground part of rice (including Flag Leaf (FL), inverted two leaf (2ndL), inverted three leaf (3rdL) and stem (stem)) is as shown in FIG. 9, the rice gene OsPT6 promoter activates OsAMT1.1 transgenic lines (PA1, PA2 and PA3), and the dry matter weight of each part of the overground part (including Flag Leaf (FL), inverted two leaf (2ndL), inverted three leaf (3rdL) and stem (stem)) is increased to different degrees compared with the control Nipponban wild type rice (WT); the root system and the overground part dry weight of the over-expression OsAMT1.1(35S strong promoter) strain (OX) are almost the same as those of the control Nipponbare wild type rice (WT).

Under the condition of low phosphorus, the nitrogen content of the rice is shown in figure 10, and the rice gene OsPT6 promoter starts OsAMT1.1 transgenic lines (PA1, PA2 and PA3), compared with the control Nipponbare wild type rice (WT), the nitrogen content of the overground part (including flag leaves, inverted two leaves, inverted three leaves and stalks) is respectively increased by 18.9% -27.7%, 13.9% -24.5%, 22.1% -28.3% and 25.4% -30.8%. The nitrogen content of the aerial parts (including Flag Leaf (FL), inverted two leaf (2ndL), inverted three leaf (3rdL) and stem (stem)) of the overexpression OsAMT1.1(35S strong promoter) strain (OX) is almost the same as that of the control Nipponbare wild type rice (WT).

Under the condition of low phosphorus, the phosphorus content of the rice is shown in figure 11, the rice gene OsPT6 promoter starts OsAMT1.1 transgenic lines (PA1, PA2 and PA3), and compared with the contrast Nipponbare wild type rice (WT), the nitrogen content of overground parts (including flag leaves, inverted two leaves, inverted three leaves and stalks) is respectively increased by 18.9% -27.7%, 13.9% -24.5% and 22.1% -28.3%, and 25.4% -30.8%; the nitrogen content of the overground part (comprising Flag Leaves (FL), inverted two leaves (2ndL), inverted three leaves (3rdL) and stalks (stem)) of an overexpression OsAMT1.1(35S strong promoter) strain (OX) is almost the same as that of a control Nipponbare wild type rice (WT).

Under normal conditions, the growth conditions of rice are shown in figure 12, the rice gene OsPT6 promoter starts OsAMT1.1 transgenic lines (PA1, PA2 and PA3), and compared with the control Nipponbare wild type rice (WT), the growth conditions are better; the growth condition of an overexpression OsAMT1.1(35S strong promoter) strain (OX) is poorer than that of a control Nipponbare wild type rice (WT).

Under normal conditions, the dry matter weight of each part of the overground part of the rice is shown in figure 13, the promoter of the rice gene OsPT6 starts OsAMT1.1 transgenic lines (PA1, PA2 and PA3), and the dry matter weight of each part of the overground part (comprising Flag Leaf (FL), inverted two leaves (2ndL), inverted three leaves (3rdL) and stem (stem)) is increased to different degrees compared with that of the control Japanese sunny wild type rice (WT); and the dry weights of the root system and the overground part of the over-expression OsAMT1.1(35S strong promoter) strain (OX) are smaller than that of the control Nipponbare wild type rice (WT).

Under normal conditions, the nitrogen content of rice is shown in fig. 14, the rice gene OsPT6 promoter starts OsAMT1.1 transgenic lines (PA1, PA2 and PA3), and compared with the contrast Nipponbare wild type rice (WT), the nitrogen content of overground parts (including flag leaves, inverted two leaves, inverted three leaves and stems) is respectively increased by 18.9% -27.7%, 13.9% -24.5% and 22.1% -28.3%, 25.4% -30.8%; the nitrogen content of the overground part (comprising Flag Leaves (FL), inverted two leaves (2ndL), inverted three leaves (3rdL) and stalks (stem)) of an overexpression OsAMT1.1(35S strong promoter) strain (OX) is reduced compared with that of a control Nipponbare wild type rice (WT).

Under normal conditions, the phosphorus content of rice is shown in fig. 15, the rice gene OsPT6 promoter starts OsAMT1.1 transgenic lines (PA1, PA2 and PA3), and compared with the control Nipponbare wild type rice (WT), the phosphorus content of overground parts (including flag leaves, inverted two leaves, inverted three leaves and stems) is respectively increased by 18.9% -27.7%, 13.9% -24.5% and 22.1% -28.3% and 25.4% -30.8%; the phosphorus content of overground parts (comprising Flag Leaves (FL), inverted two leaves (2ndL), inverted three leaves (3rdL) and stalks (stem)) of an overexpression OsAMT1.1(35S strong promoter) strain (OX) is reduced compared with that of a control Nipponbare wild type rice (WT).

3) Field test

Sowing the transgenic rice plant line T2 for about 4 weeks, transplanting, and performing field test, wherein during the test, field fertilization management is normally performed, and the field test shows that the effective tillering number of the transgenic rice line with the phosphorus deficiency response gene OsPT6 promoter for starting OsAMT1.1 is obviously increased by 13.6-30.9%; further improving the yield of the single plant, and increasing 14.1-25.8% compared with the wild type (as shown in Table 1); meanwhile, the absorption and distribution of nitrogen and phosphorus nutrition of the rice are improved, and the nitrogen and phosphorus concentrations in the brown rice are respectively increased by 15.7-18.0% and 15.4-24.3% compared with the wild type WT (as shown in figure 12).

TABLE 1 agronomic trait statistics of transgenic lines T2 generations

In conclusion, the invention discovers that the promoter for expressing the phosphorus deficiency response gene OsPT6 of the rice can obviously improve the nitrogen and phosphorus content of each part of the rice under the condition of different soil phosphorus concentrations by starting the rice gene OsAMT1.1, promotes the growth of the rice and finally improves the grain yield of the rice.

Finally, the above embodiments are only intended to illustrate the technical solution of the present invention and not to limit the same, and although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention, which shall be covered by the claims of the present invention.

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Claims

1. The recombinant expression vector is characterized by comprising gene sequences shown as SEQ ID No.1 and OsAMT 1.1.

2. The recombinant expression vector according to claim 1, wherein the OsAMT1.1 gene sequence comprises a sequence shown as SEQ ID No. 2.

3. The recombinant expression vector of claim 1 or 2, wherein the expression vector in the recombinant expression vector is selected from any one of a pTCK303 plasmid vector, a Ti plasmid vector, a Ri plasmid vector and a plant virus vector.

4. The recombinant expression vector according to claim 1 or 2, further comprising an enhancer and/or a reporter gene.

5. The recombinant expression vector of claim 4, wherein the enhancer is a transcriptional enhancer or a translational enhancer.

6. The recombinant expression vector according to claim 4, wherein the reporter gene is any one of an antibiotic resistance gene, a GUS reporter gene and a luciferase reporter gene.

7. The use of the recombinant expression vector of any one of claims 1 to 6 for increasing nitrogen and phosphorus content in rice.

8. Use of the recombinant expression vector of any one of claims 1 to 6 for promoting rice growth.

9. Use of the recombinant expression vector of any one of claims 1 to 6 for increasing rice yield.

10. A method for breeding transgenic rice, comprising transferring the recombinant expression vector of any one of claims 1 to 6 to rice by Agrobacterium-mediated transformation, biolistic transformation or pollen tube pathway transformation.