CN118995791A - Application of positive control OsNRT-P2 gene in improving excellent crop yield - Google Patents

Abstract

The invention belongs to the technical field of plant genetic engineering, and particularly relates to application of a positive control OsNRT-P2 gene in improving the excellent yield of crops. The invention can not only improve the yield of crops, but also improve the protein content and nitrogen utilization efficiency of the fruits or seeds of the crops by over-expressing OsNRT-P2 genes in the crops. When the crop is rice, the rice seeds obtained by utilizing the GSP1 and GSP2 nucleotide sequences or genes have the advantages of yield increase, excellent yield and high nitrogen utilization rate, develop a channel for genetic breeding to increase yield and excellent yield and high nitrogen utilization rate of the rice, provide a new production idea for genetic breeding to improve yield, improve nutrition configuration of the rice and improve high nitrogen utilization rate of the rice, and further ensure grain safety.

Description

Application of positive control OsNRT-P2 gene in improving excellent crop yield

Technical Field

The invention relates to the technical field of plant genetic engineering, in particular to application of a positive control OsNRT-P2 gene in improving the excellent yield of crops.

Background

Rice is one of the main grain crops in the world, and the yield increase and the excellent yield are important to the global grain safety. The optimal yield and the yield increase of the rice can effectively meet the grain requirements of the growing population of the world, and the grain supply is ensured to be stable; improving economic benefits and living standard of farmers, promoting development of rural areas, reducing the number of poor population and enhancing social stability. In addition, the optimal yield and the yield increase are also beneficial to reducing the excessive development and damage to land resources and protecting the ecological environment.

In recent years, a series of achievements are obtained by widely researching the variation of the high-yield and high-yield related traits and the genetic basis of the variation, and the genetic improvement leaf of the target gene becomes one of the most feasible solutions of high yield and is important for sustainable agriculture in the future. There have been a great deal of research on obtaining mutants by gene knockout to find many genes related to high and superior rice yield. However, many studies have successively demonstrated that many genes of interest do not necessarily achieve functional enhancement due to various reasons such as the fact that their final function of expression is hindered by other intracellular restriction factors during positive regulation, that the normal regulation mechanisms within the cell are deregulated, that intracellular toxic effects occur, and that cells have been weighted at an adaptive expression level.

Therefore, how to provide an application of positively regulating and controlling the gene related to the optimal yield of crops to actually realize the optimal yield effect of crops is a problem to be solved by those skilled in the art.

Disclosure of Invention

In order to provide a gene capable of improving crop yield, protein content and nitrogen utilization rate and application thereof, the invention provides the following technical scheme:

Application of positive control OsNRT-P2 gene in one or more of improving crop yield, improving crop protein content and improving crop nitrogen utilization efficiency;

The nucleotide sequence of OsNRT-P2 gene is shown as SEQ ID NO. 1.

Preferably, the means of upregulation comprises overexpression.

Preferably, the increasing the crop protein content comprises increasing the crop fruit protein content and/or the seed protein content.

Preferably, in the application as described above the crop is rice.

The invention also provides the application of the biological material for over-expressing OsNRT-P2 genes in one or more of improving crop yield, improving crop protein content and improving crop nitrogen utilization efficiency, wherein the biological material comprises one or more of primer pairs for amplifying OsNRT-P2 genes, a recombinant expression vector containing OsNRT2-P2 genes and a recombinant microorganism containing OsNRT2-P2 genes.

Preferably, the primer pair comprises a forward primer GSP1 and a reverse primer GSP2, wherein the nucleotide sequence of the forward primer GSP1 is shown as SEQ ID NO.2, and the nucleotide sequence of the reverse primer GPS2 is shown as SEQ ID NO. 3.

Preferably, the initial vector of the recombinant expression vector comprises pCAMBIA1300-35S.

Preferably, the OsNRT-P2 gene is inserted between BamHI and HindIII of pCAMBIA1300-35S

The invention also provides a method for cultivating OsNRT-P2 transgenic crops, which comprises the following steps: introducing OsNRT-P2 gene as described above into target crop, to obtain the OsNRT-P2 transgenic crop.

Preferably, the crop is rice.

The invention has the beneficial effects that:

The invention provides application of positive control OsNRT-P2 genes in one or more of improving crop yield, improving crop protein content and improving crop nitrogen utilization efficiency. The rice yield, the rice protein content and the nitrogen utilization efficiency in the positive regulation plant system are obviously increased, the single plant yield is increased by 20%, the rice protein content is increased by 10%, and the nitrogen utilization efficiency of the rice is also increased by 25%; the invention opens up a channel for genetic breeding to increase yield and excellent yield and nitrogen high-efficiency utilization of rice, provides a new production idea for genetic breeding to increase yield, improve nutrition configuration of rice and increase nitrogen high-efficiency rice, and further ensures grain safety.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments will be briefly described below.

FIG. 1 is a schematic diagram of a rice gene expression backbone vector according to an embodiment of the present invention;

FIG. 2 shows the relative amounts of OsNRT-P2 expressed by different plant materials in accordance with the third embodiment of the present invention;

FIG. 3 is a schematic diagram of agronomic traits of an overground part length of a wild rice plant and a modified rice plant in a fourth embodiment of the invention;

FIG. 4 is a schematic diagram of agronomic traits of biomass after planting of wild rice and modified rice in embodiment IV of the invention;

FIG. 5 is a schematic diagram of agronomic traits of wild rice and modified rice post-planting yields in embodiment IV of the present invention;

FIG. 6 is a graph showing the protein content of the seed grains of the wild rice and the modified rice planted in the fifth embodiment of the invention;

FIG. 7 is a schematic diagram showing nitrogen utilization efficiency of wild rice and modified rice after planting in accordance with the fifth embodiment of the present invention.

Detailed Description

The invention provides application of positive control OsNRT2-P2 genes in one or more of improving crop yield, improving crop protein content and improving nitrogen utilization efficiency of crops; accession number of OsNRT-P2 gene in MSU database is (LOC_Os04 g 40410), CDS sequence of OsNRT-P2 gene is shown as SEQ ID NO.1, concretely as follows:

SEQ ID NO.1：5'-ATGGCTCGGTTTGGGGCGGTAATTCACCGCGTGTTT CTACCGCTGTTGCTGCTCCTTGTAGTTCTCGGTGCTTGCCATGTCACGCCGGCGGCGGCGGCGGCGGGGGCGCGCCTCTCCGCGCTCGCGAAGGCGCTCGTCGTCGAGGCGTCGCCCCGTGCCGGCCAAGTCCTGCACGCCGGCGAGGACGCCATCACCGTGACATGGTCGCTGAACGCGACGGCGGCGGCGGCGGCGGCCGGGGCGGATGCCGGCTACAAGGCGGTGAAGGTGACCCTGTGCTACGCGCCGGCGAGCCAGGTGGGCCGCGGGTGGCGCAAGGCCCACGACGACCTGAGCAAGGACAAGGCGTGTCAGTTCAAGATCGCCCAGCAGCCGTACGACGGCGCCGGCAAGTTCGAGTACACGGTGGCACGCGACGTCCCGACGGCGTCGTACTACGTGCGCGCCTACGCGCTCGACGCGTCGGGGGCGCGGGTGGCCTATGGCGAGACGGCGCCCTCGGCCAGCTTCGCCGTCGCGGGCATCACCGGCGTCACCGCGTCCATCGAGGTCGCCGCCGGCGTGCTCTCCGCGTTCTCCGTCGCCGCGCTCGCCGTCTTCCTCGTCCTCGAGAACAAGAAGAAGAACAAGTGA-3'.

The mode of up-regulation according to the invention is preferably over-expression.

The crop of the present invention is preferably rice. The improvement of the protein content of the crops is preferably the improvement of the protein content of fruits and/or seeds of the crops, and more preferably the improvement of the protein content of rice.

The invention also provides the application of the biological material for over-expressing OsNRT-P2 genes in one or more of improving crop yield, improving crop protein content and improving crop nitrogen utilization efficiency, wherein the biological material comprises one or more of primer pairs for amplifying OsNRT-P2 genes, a recombinant expression vector containing OsNRT2-P2 genes and a recombinant microorganism containing OsNRT2-P2 genes.

The primer pair of the invention preferably comprises a forward primer GSP1 and a reverse primer GSP2, wherein the nucleotide sequence of the forward primer GSP1 is preferably shown as SEQ ID NO.2, and the nucleotide sequence of the reverse primer GPS2 is preferably shown as SEQ ID NO. 3.

SEQ ID NO.2：5'-gagctcggtacccggggatccATGGCTCGGTTTGGGGCG-3'；

SEQ ID NO.3：5'-acgacggccagtgccaagcttTCACTTGTTCTTCTTCTTGTTCT CG-3'。

The initial vector of the recombinant expression vector is preferably pCAMBIA1300-35S, wherein the pCAMBIA1300-35S contains a 35S promoter of cauliflower mosaic virus (CaMV); when the initial vector is pCAMBIA1300-35S, the OsNRT-P2 gene is preferably inserted between BamHI and HindIII of pCAMBIA1300-35S, more preferably the CDS sequence amplified by the primer set according to the above-mentioned technical scheme is inserted between BamHI and HindIII of pCAMBIA1300-35S to form pCAMBIA1300-35S-OsNRT2-P2. The construction method of the recombinant expression vector is not particularly limited, and a method for constructing a conventional vector in the field, such as an enzyme digestion connection method, is adopted.

The recombinant microorganism of the present invention is preferably a recombinant microorganism obtained by transforming the recombinant expression vector of the above-described technical scheme into agrobacterium, which is preferably EHA105.

The invention also provides a method for cultivating OsNRT-P2 transgenic crops, which comprises the following steps: introducing OsNRT-P2 gene into target crop, and obtaining the OsNRT-P2 transgenic crop, wherein the crop is preferably monocotyledonous plant, more preferably rice. The method of introducing OsNRT-P2 gene into target crop is not particularly limited in the present invention, and conventional methods in the art may be employed.

The technical solutions provided by the present invention are described in detail below with reference to the drawings and examples for further illustrating the present invention, but they should not be construed as limiting the scope of the present invention. .

Example 1

Construction of OsNRT-P2 Gene overexpression Gene expression vector

1) RNA extraction

Taking 20-50mg of rice plant sample of flower 11 in japonica rice, putting into liquid nitrogen for freezing and grinding to be powder, adding 1ml of reagent Invitrogen Trizol of Saimer's organism company for cell lysis, then adding 0.2ml of chloroform, shaking vigorously for 15s, standing at room temperature for 3min, centrifuging (8000 g,15 min), putting the water phase (upper layer) into a new tube, precipitating RNA in the water phase with isopropanol, standing at room temperature for 10min, centrifuging (800 g,15 min), washing the RNA precipitate with 1ml of 75% ethanol, centrifuging for 5min with 7500g, discarding the supernatant, sucking away ethanol, airing, and adding 30 μl of ddH ₂ O for dissolution.

2) RNA inversion

Reverse transcription kit using norpranII 1st Strand cDNA Synthesis Kit reverse transcription was carried out. The following solutions were mixed in a 200 μl centrifuge tube: mu.l Oligo (dT) 23VN (50. Mu.M), 3. Mu. lRNase-free ddH ₂ O, 5. Mu.l of the above RNA solution were heated at 65℃for 5min, rapidly quenched on ice, and allowed to stand on ice for 2min. Mu.l of the mixture obtained in the previous step was added to 10. Mu.l of 2 XRT Mix and 2. Mu. LHISCRIPT II Enzyme Mix, and the mixture was gently mixed by pipetting.

The first strand cDNA synthesis reaction was performed on a PCR instrument at 50℃for 45min and 85℃for 2 min.

3) Acquisition of the Gene of interest

The following solutions were mixed in a 200 μl centrifuge tube: mu.l of the cDNA solution obtained in the above step, 2. Mu.l of GS P1 (see SEQ ID NO. 2: 5'-gagctcggtacccggggatccATGGCTCGGTTTGGGGCG-3'), 2. Mu.l of GSP2 (see SEQ ID NO. 3: ：5'-acgacggccagtgccaagcttTCACTTG TTCTTCTTCTTGTTCTCG-3')、25μl 2×Phanta Max Master Mix、16μl dd H₂O.) were subjected to PCR according to the procedure described in Table 1.

TABLE 1PCR reaction procedure

4) Recombinant vector production

The pCAMBIA1300-35S vector is subjected to enzyme digestion and recovery by using high-fidelity restriction endonucleases HindIII and BamHI to obtain a linearization vector, homologous recombination is carried out on the linearization vector and OsNRT-P2 CDS solution with a homology arm obtained in the previous reaction in 5 XCE II Buffer and Exnase II (Vazyme ClonExpress II One Step Cloning Kit) environments for 1h, and the pCAMBIA1300-35S-OsNRT2-P2 recombinant vector is obtained, wherein the structure of the rice gene expression skeleton vector is shown in figure 1.

Example two

Taking japonica rice middle flower 11 (ZH 11) as an example, osNRT2-P2 genes are transformed into rice seeds to obtain the japonica rice middle flower 11 transgenic rice.

Transferring 0.5 μg of pCAMBIA1300-35S-OsNRT2-P2 gene expression plasmid prepared in example 2 into competent cells of agrobacterium strain EHA105, sequentially ice-bathing for 5min, liquid nitrogen for 5min, water-bathing for 5min at 37 ℃ and ice-bathing for 5min, adding antibiotic-free LB, and activating for 1h in a shaker at 28 ℃ to obtain agrobacterium strain containing pCAMBIA1300-35S-OsNRT2-P2 gene expression plasmid.

The prepared EHA105 strain containing the gene expression plasmid is used for transforming the calli of flowers 11 in japonica rice, and the specific steps are as follows:

Callus induction and subculture of flower 11 in japonica rice can be achieved by conventional methods known to those skilled in the art. Agrobacterium containing pCAMBIA1300-35S-OsNRT2-P2 gene expression plasmid was shake-cultured in AAM liquid medium containing 50mg/L kanamycin (Kan) and 50mg/L rifampicin (Rif) at 28℃under light shielding 200r/min to D600 nm of 0.4-0.6, and the cells were collected by centrifugation and resuspended to D600 nm=0.1 with liquid NB-As. Selecting callus with proper size and good state, immersing in a 50ml centrifuge tube filled with agrobacterium heavy suspension for 30min, sucking excessive bacterial liquid on sterile filter paper, and blow-drying the bacterial filter paper on the callus surface collapse on an ultra clean bench. After blow-drying, the calli were transferred to solid NBD-As medium with sterile filter paper laid on it and dark-cultured at 25 ℃.

3 Days later, the callus is soaked in sterile water containing 150mg/L TIMENTIN for 10 minutes, redundant moisture of the callus is sucked to be dried, collapse bacteria filter paper on the surface of the callus is dried on an ultra clean bench, and the callus is transferred to NBD-As culture medium for recovery, and is cultivated for 4 days under illumination at 32 ℃. The cells were then transferred to NBD-T medium containing the screening antibiotic hygromycin for continued screening culture 1 time every 2 weeks. When the catalyzed adventitious bud grows to 3-5 cm, cutting the seedling, transferring the seedling into a rooting culture medium RE2 for rooting induction, and obtaining the seedling.

Example III

The leaves of the seedlings obtained in the second example were selected to extract total plant DNA, and the hygromycin resistance gene was identified by PCR using conventional procedures well known to those skilled in the art to obtain three successfully transformed positive seedlings. Then, total RNA of the selected hygromycin positive seedlings is extracted, reverse transcription is carried out to obtain cDNA, and the SYBR qPCR MasterMix of Northenan biotechnology Co., ltd is used for qPCR detection, and OsActin gene is used as an internal reference gene for comparison, and qPCR identification primers are as follows:

OsNRT2-P2 qPCRForwardPrimer 5'-3'

SEQ ID NO.4：TGTTGCTGCTCCTTGTAGTTCTC

OsNRT2-P2 qPCR Reverse Primer 5'-3'

SEQ ID NO.5：CTTCTTCTTGTTCTCGAGGACGA

OsActin qPCRForward Primer 5'-3'

SEQ ID NO.6：GGGTTCACAAGTCTGCCTATTGT

OsActin qPCRReverse Primer 5'-3'

SEQ ID NO.7：ACGGGACACGACCAAGGA

The relative expression levels of OsNRT-P2 in transgenic rice with 3 homozygous lines OsNRT-P2 over-expressed by wild type Zhonghua 11 (ZH 11) and the obtained OsNRT-P2-OE 1, osNRT-P2-OE 4 and OsNRT-P2-OE 10 are shown in FIG. 2. The relative expression levels of OsNRT2-P2-OE1, osNRT2-P2-OE4 and OsNRT2-P2-OE10 at OsNRT-P2 were increased 1-fold, 1.75-fold and 1.5-fold relative to ZH11, respectively.

Example IV

Rice plant height, biomass and rice yield detection

The rice seeds of wild medium flower 11 rice (ZH 11), osNRT2-P2-OE1, osNRT-P2-OE 4 and OsNRT2-P2-OE10 are respectively cultivated into soil according to the conventional cultivation method of farmers, the agronomic management during the growth period of the rice is implemented according to the daily management method of farmers, only the slow-release nitrogen-phosphorus-potassium compound fertilizer is applied in the whole growth period, and the total nitrogen application amount is 10.4g/m ². After the rice is mature, the agronomic characters of the length, biomass and yield of the overground parts of the rice are measured and are respectively shown in figures 3, 4 and 5.

As can be seen from FIG. 3, the height of the aerial parts of wild-type medium-flower 11 rice (ZH 11) is about 104cm, and the heights of three different OsNRT2-P2 over-expression homozygous lines such as OsNRT2-P2-OE1, osNRT2-P2-OE4 and OsNRT-P2-OE 10 are 109 cm-118 cm;

as can be seen from FIG. 4, the aerial part dry weight of wild type medium flower 11 rice (ZH 11) is about 13.5g, while the biomass of the homozygous strain overexpressed by three different OsNRT-P2 is improved by 18.5% -48.1% compared with the wild type;

As can be seen from FIG. 5, the yield per plant of wild type medium flower 11 rice (ZH 11) was about 6.5g, whereas the yield per plant of three different OsNRT-P2 overexpressing homozygous lines was improved by about 15% over that of wild type medium flower 11 (ZH 11).

In conclusion, over-expression OsNRT of the gene OsNRT-P2 can obviously increase the overground plant height and biomass of rice, the overall growth vigor is obviously better than that of wild type, and the rice yield is also obviously improved.

Example five

Rice protein content detection

Adopting the same rice material as in the fourth embodiment, after measuring the agronomic characters in the fourth embodiment, harvesting rice, drying the overground part and weighing the rice, performing digestion with 98% concentrated sulfuric acid, titrating with 15% H ₂O₂ to clear solution after the rice is boiled to black homogenate, and then continuing digestion until H ₂O₂ is completely driven. The obtained completely cracked sample solution was filtered through a nylon filter membrane and collected in a clean tube, and the ammonium content in the solution was measured by indophenol blue colorimetry in an enzyme-labeled instrument, and the results are shown in fig. 6.

As can be seen from FIG. 6, the grain protein content of wild-type medium flower 11 (ZH 11) is about 6.3-7 mg/g, while the grain protein content of three different OsNRT-P2 over-expressed homozygous lines is improved by about 8% -15% compared with wild-type medium flower 11.

The nitrogen utilization rate of the rice material is calculated by adopting a nitrogen utilization rate calculation formula, and the result is shown in figure 7.

The nitrogen utilization efficiency is calculated according to the following formula:

nitrogen utilization efficiency = amount of nitrogen fertilizer absorbed/amount of nitrogen fertilizer applied in rice plants.

As shown in FIG. 7, the nitrogen utilization efficiency of wild-type medium flower 11 (ZH 11) is about 47%, but the nitrogen utilization efficiency of three different OsNRT-P2 over-expressed homozygous lines can reach 58% -70%, and the nitrogen utilization efficiency of three different OsNRT-P2 over-expressed homozygous lines is improved by 11% -33% compared with that of wild-type medium flower 11 (ZH 11).

In conclusion, over-expression of the rice OsNRT-P2 gene obviously improves the nitrogen content and yield in rice, improves the nitrogen utilization efficiency of the rice, and has promotion effect on agronomic traits such as plant height, biomass and the like.

The foregoing is merely a preferred embodiment of the invention, and it should be noted that modifications could be made by those skilled in the art without departing from the principles of the invention, which modifications would also be considered to be within the scope of the invention.

Claims (10)

1. Application of positive regulation of OsNRT2-P2 gene in increasing one or more of crop yield, crop protein content and crop nitrogen utilization efficiency; The nucleotide sequence of the OsNRT2-P2 gene is shown in SEQ ID NO.1. 2. The use according to claim 1, characterized in that the positive regulation method includes overexpression. 3. The use according to claim 1, characterized in that increasing the protein content of crops comprises increasing the protein content of crop fruits and/or seeds. 4. The use according to any one of claims 1 to 3, characterized in that the crop is rice. 5. Use of biological materials overexpressing the OsNRT2-P2 gene in one or more of increasing crop yield, increasing crop protein content and improving nitrogen utilization efficiency of crops, characterized in that the biological materials include one or more of a primer pair for amplifying the OsNRT2-P2 gene, a recombinant expression vector containing the OsNRT2-P2 gene, and a recombinant microorganism containing the OsNRT2-P2 gene. 6. The biomaterial according to claim 5, characterized in that the primer pair comprises a forward primer GSP1 and a reverse primer GSP2, the nucleotide sequence of the forward primer GSP1 is shown as SEQ ID NO.2, and the nucleotide sequence of the reverse primer GPS2 is shown as SEQ ID NO.3. 7. The biomaterial according to claim 5, characterized in that the initial vector of the recombinant expression vector comprises pCAMBIA1300-35S. 8. The biomaterial according to claim 7, characterized in that the OsNRT2-P2 gene is inserted between BamHI and HindIII of pCAMBIA1300-35S. 9. A method for cultivating an OsNRT2-P2 transgenic crop, comprising: introducing the OsNRT2-P2 gene according to claim 1 into a target crop to obtain the OsNRT2-P2 transgenic crop. 10. The method according to claim 9, wherein the crop is rice.