CN106222148A - OsCIPK23 and encoding gene application in regulation and control plant ammonium content and regulating plant growth thereof - Google Patents

Abstract

The invention discloses the application of OsCIPK23 and its coding gene in regulating plant ammonium content and regulating plant growth. OsCIPK23 is the protein whose amino acid sequence is sequence 1; the OsCIPK23 coding gene is as follows 1) or 2) or 3): 1) the coding sequence is the cDNA molecule or DNA molecule of sequence 2 in the sequence listing; 2) and 1) limited nucleoside The acid sequence has 75% or more identity, and the cDNA molecule or genomic DNA molecule encoding OsCIPK23; 3) hybridizes with the nucleotide sequence defined in 1) under stringent conditions, and the cDNA molecule or genomic DNA molecule encoding OsCIPK23 . Experiments have proved that inhibiting the expression of the gene encoding OsCIPK23 can inhibit the growth of plant roots and reduce the content of ammonium ions in plants, and OsCIPK23 and its encoding genes can be used to regulate plant ammonium content and plant growth.

Description

OsCIPK23 and its encoding gene in regulation of plant ammonium content and regulation of plant growth Applications

technical field

The invention relates to the application of OsCIPK23 and its coding gene in regulating plant ammonium content and regulating plant growth in the field of biotechnology.

Background technique

Nitrogen is an important nutrient element, accounting for 2% of plant dry weight, and is closely linked to crop production. Nitrate and ammonium ions are the main sources of nitrogen nutrients for higher plants. Rice is an important crop that mainly absorbs ammonium nitrogen. Adjusting ammonium absorption directly affects the growth of rice, which is very critical in agricultural production practice. Therefore, research on ammonium absorption has both theoretical and practical significance.

Calcineurin B-like(CBL)-interacting protein kinase(CIPK) is a key downstream factor of Ca ²⁺ signaling. CBL specifically binds calcium ions and activates its binding kinase protein CIPK to transmit downstream signals. The CBL-CIPK complex has been reported to be involved in various plant stress resistance pathways, such as stresses on plants brought about by salt, drought, low temperature and osmotic pressure.

Contents of the invention

The technical problem to be solved by the present invention is how to regulate the plant ammonium content and/or regulate the growth of plant roots.

In order to solve the above-mentioned technical problems, the present invention firstly provides the application of the substance regulating CIPK protein activity or the substance regulating plant CIPK protein content in any one of D1)-D16):

D1) regulating plant ammonium content;

D2) regulate plant nitrogen uptake;

D3) regulate the absorption of plant ammonium;

D4) regulating plant growth;

D5) cultivating plants with increased ammonium content;

D6) Cultivate nitrogen uptake capacity to increase plants;

D7) cultivating plants with increased ammonium uptake capacity;

D8) cultivating growth-increasing plants;

D9) preparing and regulating plant ammonium content products;

D10) preparing a product for regulating plant nitrogen uptake;

D11) preparing a product that regulates plant ammonium absorption;

D12) preparing a product for regulating plant growth;

D13) preparing and cultivating plant products with increased ammonium content;

D14) preparing and cultivating plant products with increased nitrogen absorption capacity;

D15) preparing and cultivating plant products with increased ammonium absorption capacity;

D16) Preparation of cultivated growth-enhancing plant products.

The regulation of CIPK protein activity can be performed by one or more methods selected from changing the expression of CIPK protein, changing the expression of CIPK protein coding gene, changing the copy number of CIPK protein coding gene, promoter replacement, promoter mutation and gene mutation accomplish.

In the above application, the CIPK protein can be derived from E1), E2) or E3): E1) plants; E2) monocotyledonous plants; E3) rice. In an embodiment of the present invention, the CIPK protein is a rice-derived protein named OsCIPK23.

In the above application, the substance for regulating the protein content of CIPK in plants may be a substance for regulating the expression of a gene encoding a CIPK protein or a substance for knocking out a gene encoding a CIPK protein.

In the above application, the CIPK protein can specifically be the following A1), A2) or A3):

A1) the amino acid sequence is the protein of sequence 1;

A2) A protein having the same function as the amino acid sequence shown in Sequence 1 in the sequence listing through substitution and/or deletion and/or addition of one or several amino acid residues;

A3) A fusion protein obtained by linking a tag at the N-terminal or/and C-terminal of A1) or A2).

In order to make the protein in A1) easy to purify, the amino-terminal or carboxy-terminal of the protein consisting of the amino acid sequence shown in Sequence 1 in the Sequence Listing can be linked with the tags shown in Table 1.

Table 1. Sequence of tags

Label Residues sequence Poly-Arg 5-6 (usually 5) RRRRR Poly-His 2-10 (usually 6) HHHHHH FLAG 8 DYKDDDDK Strep-tag II 8 WSHPQFEK c-myc 10 EQKLISEEDL

For the CIPK protein in A2) above, the substitution and/or deletion and/or addition of one or several amino acid residues is a substitution and/or deletion and/or addition of no more than 10 amino acid residues.

The CIPK protein in the above A2) can be synthesized artificially, or its coding gene can be synthesized first, and then obtained by biological expression.

The gene encoding the CIPK protein in the above A2) can be obtained by deleting the codon of one or several amino acid residues in the DNA sequence shown in sequence 2, and/or carrying out missense mutations of one or several base pairs, and /or obtained by linking the coding sequence of the tag shown in Table 1 at its 5' end and/or 3' end.

In the above-mentioned application, the substance for regulating the plant CIPK protein content can be CIPK protein or its related biological material; the biological material is any one of the following B1) to B9):

B1) a nucleic acid molecule encoding a CIPK protein;

B2) an expression cassette containing the nucleic acid molecule of B1);

B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

B4) A recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing a recombinant vector described in B3);

B5) a transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2);

B6) a transgenic plant tissue containing the nucleic acid molecule described in B1), or a transgenic plant tissue containing the expression cassette described in B2);

B7) a transgenic plant organ containing the nucleic acid molecule described in B1), or a transgenic plant organ containing the expression cassette described in B2);

B8) a nucleic acid molecule that inhibits the expression of a CIPK protein-encoding gene;

B9) An expression cassette, a recombinant vector, a recombinant microorganism or a transgenic plant cell line containing the nucleic acid molecule of B8).

In the above application, the nucleic acid molecule encoding the CIPK protein may be as follows 1) or 2) or 3):

1) The coding sequence is the cDNA molecule or DNA molecule of sequence 2 in the sequence listing;

2) A cDNA molecule or a genomic DNA molecule that has 75% or more identity to the nucleotide sequence defined in 1) and encodes a CIPK protein;

3) A cDNA molecule or a genomic DNA molecule that hybridizes to the nucleotide sequence defined in 1) under stringent conditions and encodes a CIPK protein.

Wherein, the nucleic acid molecule can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA.

Wherein, the DNA molecule shown in sequence 2 encodes the CIPK protein shown in sequence 1.

Those skilled in the art can easily use known methods, such as directed evolution and point mutation methods, to mutate the nucleotide sequence encoding the CIPK protein of the present invention. Those artificially modified nucleotides that have 75% or higher identity with the nucleotide sequence of the CIPK protein isolated in the present invention, as long as they encode the CIPK protein and have the function of the CIPK protein, are all derived from the core of the present invention. Nucleotide sequence and is equivalent to the sequence of the present invention.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "Identity" includes 75% or higher, or 85% or higher, or 90% or higher, or 95% or higher, of the nucleotide sequence of the protein composed of the amino acid sequence shown in the coding sequence 1 of the present invention. Nucleotide sequences of higher identity. Identity can be assessed visually or with computer software. Using computer software, identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

In the above-mentioned application, the stringent conditions are in a solution of 2×SSC and 0.1% SDS, hybridize at 68° C. and wash the membrane twice, each time for 5 minutes, and then in a solution of 0.5×SSC and 0.1% SDS, in Hybridize and wash the membrane twice at 68°C, 15 min each time; or, hybridize and wash the membrane at 65°C in a solution of 0.1×SSPE (or 0.1×SSC) and 0.1% SDS.

The identity of 75% or more may be 80%, 85%, 90% or more.

In the above-mentioned application, the expression cassette (CIPK gene expression cassette) described in B2) that contains the nucleic acid molecule encoding the CIPK protein refers to the DNA that can express the CIPK protein in the host cell, and the DNA can not only include the initiation of CIPK gene transcription A terminator that terminates the transcription of the CIPK gene may also be included. Further, the expression cassette may also include an enhancer sequence. Promoters that can be used in the present invention include, but are not limited to: constitutive promoters, tissue, organ and development specific promoters, and inducible promoters. Examples of promoters include, but are not limited to: Cauliflower Mosaic Virus Constitutive Promoter 35S: Wound-Inducible Promoter from Tomato, Leucine Aminopeptidase ("LAP", Chao et al. (1999) Plant Physiol 120: 979-992); chemically inducible promoter from tobacco, pathogenesis-related 1 (PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-thiohydroxy acid S-methyl ester)); tomato Protease inhibitor II promoter (PIN2) or LAP promoter (both inducible with methyl jasmonate); heat shock promoter (US Patent 5,187,267); tetracycline-inducible promoter (US Patent 5,057,422) ; Seed-specific promoters, such as millet seed-specific promoter pF128 (CN101063139B (Chinese patent 200710099169.7)), seed storage protein-specific promoters (for example, the promoters of phaseolin, napin, oleosin and soybean beta conglycin (Beachy et al. (1985) EMBO J. 4:3047-3053)). They can be used alone or in combination with other plant promoters. All references cited herein are cited in their entirety. Suitable transcription terminators include, but are not limited to: Agrobacterium nopaline synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator and nopaline and octopine Synthase terminators (see, e.g.: Odell et al. (1985) Nature 313:810; Rosenberg et al. (1987) Gene, 56:125; ^Guerineau et al. (1991) Mol. Gen. Genet, 262:141; Proudfoot (1991) Cell, 64:671; Sanfacon et al. Genes Dev., 5:141; Mogen et al. (1990) Plant Cell, 2:1261; Munroe et al. (1990) Gene, 91:151; Ballad et al. (1989) ) Nucleic Acids Res. 17:7891; Joshi et al. (1987) Nucleic Acids Res., 15:9627).

The existing expression vector can be used to construct the recombinant vector containing the expression cassette of the CIPK gene. The plant expression vectors include binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment and the like. Such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA Company), etc. The plant expression vector may also include the 3' untranslated region of the foreign gene, that is, the polyadenylation signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylic acid signal can guide polyadenylic acid to be added to the 3' end of the mRNA precursor, such as Agrobacterium crown gall tumor induction (Ti) plasmid gene (such as nopaline synthase gene Nos), plant gene (such as soybean The untranslated region transcribed at the 3′ end of the storage protein gene) has similar functions. When using the gene of the present invention to construct plant expression vectors, enhancers can also be used, including translation enhancers or transcription enhancers, and these enhancer regions can be ATG initiation codons or adjacent region initiation codons, etc. The reading frames of the sequences are identical to ensure correct translation of the entire sequence. The sources of the translation control signals and initiation codons are extensive and can be natural or synthetic. The translation initiation region can be from a transcription initiation region or a structural gene. In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as adding genes (GUS gene, luciferase gene, etc.) genes, etc.), antibiotic marker genes (such as the nptII gene that confers resistance to kanamycin and related antibiotics, the bar gene that confers resistance to the herbicide phosphinothricin, and the hph gene that confers resistance to the antibiotic hygromycin , and the dhfr gene that confers resistance to methotrexate, the EPSPS gene that confers resistance to glyphosate) or the chemical resistance marker gene (such as the herbicide resistance gene), the mannose-6- that provides the ability to metabolize mannose Phosphate isomerase gene. Considering the safety of the transgenic plants, the transformed plants can be screened directly by adversity without adding any selectable marker gene.

In the above application, the vector can be a plasmid, cosmid, phage or viral vector. Specifically, the plasmid can be a pTCK303 vector.

In the above applications, the microorganisms can be yeast, bacteria, algae or fungi. Wherein, the bacteria can be Agrobacterium, such as Agrobacterium LBA4404.

In the above applications, the transgenic plant cell lines, transgenic plant tissues and transgenic plant organs do not include propagation materials.

In the above application, the regulation of plant growth may be inhibition of plant growth. The regulation of plant ammonium content can be to reduce plant plant ammonium content. The nucleic acid molecule that inhibits the expression of the CIPK protein-coding gene can be a DNA molecule that is reverse complementary to any fragment of the DNA molecule shown in Sequence 2 in the Sequence Listing or its encoded RNA. The nucleic acid molecule that inhibits the expression of the CIPK protein-coding gene can specifically be the DNA molecule shown in the 981st-1327th nucleotide of Sequence 2 in the Sequence Listing or its encoded RNA.

In an embodiment of the present invention, B9) the recombinant vector is that the sequence 2 in the sequence table is inserted between the SpeI and SacI restriction sites of the pTCK303 vector from the 981st to 1327th nucleotide at the 5' end, and the sequence The vector obtained by inserting the reverse complementary fragment of sequence 2 in the list from the 981st to 1327th nucleotide at the 5' end between the SalI and KpnI restriction sites of the pTCK303 vector.

In the above application, the regulation of plant growth may be the regulation of plant root growth; the plant ammonium content may be the ammonium content in plant roots.

In the above application, the plant can be M1) or M2): M1) monocot or dicot; M2) rice.

In order to solve the problems of the technologies described above, the present invention also provides the following M1 or M2 methods:

M1. A method for cultivating transgenic plants with reduced ammonium content and/or reduced root length, including reducing the activity of the CIPK protein in the target plant, reducing the content of the CIPK protein in the target plant, inhibiting the expression of the gene encoding the CIPK protein or knocking out the CIPK protein The coding gene of the transgenic plant is obtained to obtain a transgenic plant; the transgenic plant has the following characteristics of M1a) and/or M1b): M1a) the ammonium content of the transgenic plant is lower than that of the target plant; M1b) the root length of the transgenic plant is lower than the plant of interest;

M2. A method for cultivating transgenic plants with increased ammonium content and/or increased root length, including increasing the activity of the CIPK protein in the target plant, increasing the content of the CIPK protein in the target plant, and promoting the expression of the gene encoding the CIPK protein to obtain a transgenic plant The transgenic plant has the following characteristics of M2a) and/or M2b): M2a) the ammonium content of the transgenic plant is lower than that of the target plant; M2b) the root length of the transgenic plant is lower than that of the target plant.

Wherein, the gene encoding the CIPK protein may be the nucleic acid molecule encoding the CIPK protein.

In the above method, the transgenic plant is prepared by introducing the nucleic acid molecule that inhibits the expression of the gene encoding the CIPK protein into the target plant.

In the above method, the plant ammonium content may be the ammonium content in plant roots.

In the above method, the plant can be M1) or M2): M1) monocot or dicot; M2) rice.

In order to solve the above technical problems, the present invention also provides the biological material.

In the growth of the root in the present invention, the root may be a main root. The root may be an underground part of the plant in the ammonium content of the root. The ammonium content may specifically be the concentration of ammonium ions.

In the present invention, the transgenic plant is understood to include not only the first-generation transgenic plant obtained by transforming the target plant with the TaZnF2 gene, but also its progeny. For transgenic plants, the gene can be propagated in that species, or transferred into other varieties of the same species, particularly including commercial varieties, using conventional breeding techniques. The transgenic plants include seeds, callus, whole plants and cells.

Experiments have proved that the CIPK protein of the present invention and its coding gene can regulate the ammonium content and root growth of plants: the 981st-1327th nucleoside from the 5' end of the CIPK protein coding gene shown in sequence 2 in the sequence listing The expression of the CIPK protein coding gene in the transgenic plants obtained after the reverse complementary fragment of the acid was introduced into the plants was significantly decreased, the tap root length of the transgenic plants was significantly smaller than that of the wild type plants, and the ammonium ion concentration in the roots of the transgenic plants was significantly smaller than that of the wild type plants. It shows that inhibiting the expression of the CIPK protein coding gene can inhibit the growth of plant roots and reduce the ammonium ion content in plants, and can regulate plant ammonium content and plant growth with substances that regulate CIPK protein activity or regulate plant CIPK protein content.

Description of drawings

Figure 1 is a schematic diagram of the partial structure of the recombinant vector used to knock out the OsCIPK23 gene.

Figure 2 shows the differences in the expression of OsCIPK23 gene in the roots of wild-type and transgenic plants (OsCIPK23 RNAi).

Figure 3 is the statistical results of the main root length of wild-type and transgenic plants (OsCIPK23 RNAi).

Figure 4 shows the difference in ammonium content between wild-type and transgenic plants (OsCIPK23 RNAi).

detailed description

The present invention will be further described in detail below in conjunction with specific embodiments, and the given examples are only for clarifying the present invention, not for limiting the scope of the present invention.

The experimental methods in the following examples are conventional methods unless otherwise specified.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

The restriction endonucleases used in the following examples are all Takara products.

Embodiment 1, the construction that is used for knocking out the recombinant vector of OsCIPK23 gene

The nucleotide sequence of the OsCIPK23 (LOC_Os07g05620) gene is nucleotides 1-1353 of sequence 2, and the encoded protein is OsCIPK23, and the amino acid sequence of the protein is sequence 1.

RNA from rice Nipponbare was extracted and reverse transcribed into cDNA. Using this cDNA as a template and using 5'-GTCGACACTAGTCACACTCTTTGAAAAGCAATC-3' and 5'-GGTACCGAGCTCCATCGCCTGCGATTATGCTAC-3' as primers to amplify, a 347bp PCR product was obtained. 1327 nucleotides; the PCR product is a forward fragment.

The PCR product was digested with SpeI and SacI, and the resulting digestion product containing sequence 2 from the 981st to 1327th nucleotide at the 5' end was named restriction product 1; the pTCK303 vector was digested with SpeI and SacI (Wang Zhen, ChenChangbin, Xu Yunyuan, Jiang Rongxi, Han Ye, Xu Zhihong and Chong Kang. 2004. APractical Vector for Efficient Knockdown of Gene Expression in Rice (Oryzasativa L.) Plant Molecular Biology Reporter 22:409-417), to obtain the pTCK303 vector Skeleton: Ligate the digested product 1 with the pTCK303 vector backbone, insert the sequence 2 from the 981st to 1327th nucleotides of the 5' end forwardly between the SpeI and SacI sites of the pTCK303 vector to obtain an intermediate vector;

Then use SalI and KpnI to digest the PCR product, and the resulting digested product containing sequence 2 from the 981st to 1327th nucleotide at the 5' end is named restriction product 2; use SalI and KpnI to digest the intermediate vector, Obtain the backbone of the intermediate vector; connect the restriction product 2 to the backbone of the intermediate vector, and insert the sequence 2 from the 981st to 1327th nucleotide at the 5' end in reverse between the SalI and KpnI sites of the intermediate vector to obtain a recombinant vector, which is RNA interference vector.

After sequencing, the recombinant vector is inserted between the SpeI and SacI restriction sites of the pTCK303 vector from the 981st to 1327th nucleotide (B) of the sequence 2 in the sequence listing, and the sequence 2 in the sequence listing is from The reverse complementary fragment (A) of the 981st-1327th nucleotide at the 5' end was inserted into the vector obtained between the SalI and KpnI restriction sites of the pTCK303 vector, named pTCK-OsCIPK23 RNAi (the schematic diagram of the structure is shown in Figure 1 ), which is an RNA interference carrier. The promoter of Ubiquitin gene (pUbiquitin) is used in the recombinant expression vector with inverted repeats.

Embodiment 2, interfering with OsCIPK23 gene to obtain transgenic rice

1. Obtaining of RNA interference transgenic lines

1) Obtaining of RNA interference transgenic lines

The pTCK-OsCIPK23 RNAi obtained in Example 1 was transformed into Agrobacterium LBA4404 (Takara Biocompany, Cat. 9115) to obtain recombinant bacteria. The plasmid was extracted from the recombinant bacteria and sent for sequencing. The recombinant bacteria containing the pTCK-OsCIPK23 RNAi obtained in Example 1 was named LBA4404/pTCK-OsCIPK23 RNAi.

LBA4404/pTCK-OsCIPK23 RNAi was transformed into rice Nipponbare (hereinafter also referred to as wild-type rice), and hygromycin was screened to obtain 25 _T0 -transformed OsCIPK23 RNAi rice lines, which were RNA interference transgenic lines.

2) Molecular identification

Molecular identification was carried out on the 25 T ₀ generation transgenic OsCIPK23 RNAi rice (OsCIPK23 RNAi) and wild-type rice (WT) obtained above, the total RNA extracted from various rice roots was reverse-transcribed, and the following primers were used for RT-PCR method Identification:

OsCIPK23-F:TGGGCTTTAATGTACAGAAG

OsCIPK23-R:TCACATCTTTCAGGCCATTG

The internal reference gene is Actin, and the internal reference primer is

Actin-F:TCCATCTTGGCATCTCTCAG

Actin-R: GTACCCGCATCAGGCATCTG

The results are shown in Figure 2. The average relative expression of OsCIPK23 in the T ₀ generation of OsCIPK23 RNAi rice (OsCIPK23 RNAi) was lower than the average relative expression of OsCIPK23 in wild-type rice (EV), proving that the OsCIPK23 gene in OsCIPK23RNAi rice was knocked out Or the expression of OsCIPK23 gene is suppressed. In Fig. 2, #3 and #5 are two lines of OsCIPK23 RNAi.

Using the same method, the empty vector pTCK303 was transformed into wild-type rice to obtain the T ₀ generation of pTCK-transformed rice.

The OsCIPK23 RNAi-transformed rice of the T ₀ generation and the pTCK-transferred rice of the T ₀ generation were sown and subcultured to obtain the OsCIPK23 RNAi-transferred rice of the T ₁ generation and the pTCK-transferred rice of the T ₁ generation, respectively.

2. RNA interference transgenic phenotype observation

The T1 generation of OsCIPK23 RNAi _- transferred rice (OsCIPK23 RNAi) and wild-type rice WT were sowed in MS medium. The T1 generation of _pTCK rice was used as the control.

There were 10 strains for each strain, and the experiment was repeated 3 times, and the results were averaged.

On the 4th day of sowing, the length of the main root of each line was counted, and the results are shown in Figure 3. The length of the main root of the T1 generation of OsCIPK23 RNAi _- transferred rice was significantly smaller than that of the wild-type rice (EV), and the T1 _- generation of pTCK-transferred rice was significantly different from that of the wild-type rice. There was no significant difference in tap root length. The taproot lengths of the two OsCIPK23 RNAi _- transformed rice plants in the T1 generation were 3.2±0.32cm and 2.67±0.25cm respectively, and the taproot lengths of wild-type rice were 4.3±0.45cm. These results showed that the knockout of OsCIPK23 gene or the suppression of the expression of OsCIPK23 gene can reduce the taproot length of rice. In Fig. 3, #3 and #5 are two lines of OsCIPK23 RNAi.

On the 4th day of sowing, the ammonium ion concentration in the roots of each strain was measured.

The results are shown in Figure 4. The concentration of ammonium ions in the roots of T1 _- transformed OsCIPK23 RNAi rice was significantly lower than that of wild-type rice (EV), and there was no significant difference between the roots of T1 _- transformed pTCK rice and wild-type rice. The ammonium ions in the roots of the two T ₁ generation OsCIPK23RNAi-transformed rice were 1.73±0.1mg/g FW and 1.54±0.14mg/g FW, respectively, and the length of the taproot of wild-type rice was 1.96±0.13mg/g FW. These results showed that the knockout of OsCIPK23 gene or the suppression of the expression of OsCIPK23 gene can reduce the concentration of ammonium ions in rice roots. In Fig. 4, #3 and #5 are two lines of OsCIPK23 RNAi.

Claims (10)

1. the material of regulation and control CIPK protein active or the material of regulation and control plant CIPK protein content are at D1)-D16) any one In application:

D1) regulation and control plant ammonium content；

D2) absorption of plant nitrogen is regulated and controled；

D3) absorption of plant ammonium is regulated and controled；

D4) regulating plant growth；

D5) cultivate ammonium content and increase plant；

D6) cultivate nitrogen absorbability and increase plant；

D7) cultivate ammonium absorbability and increase plant；

D8) cultivate growth and increase plant；

D9) preparation regulation and control plant ammonium content product；

D10) product that preparation regulation and control plant nitrogen absorbs；

D11) product that preparation regulation and control plant ammonium absorbs；

D12) regulating plant growth product is prepared；

D13) ammonium content is cultivated in preparation increases plant product；

D14) nitrogen absorbability is cultivated in preparation increases plant product；

D15) ammonium absorbability is cultivated in preparation increases plant product；

D16) growth is cultivated in preparation increases plant product.

Application the most according to claim 1, it is characterised in that: CIPK protein source is in E1), E2) or E3): E1) plant Thing；E2) monocotyledon；E3) Oryza sativa L.；

The material of described regulation and control plant CIPK protein content is the material that regulation and control CIPK protein coding gene is expressed.

Application the most according to claim 1 and 2, it is characterised in that: CIPK protein is following A1), A2) or A3):

A1) aminoacid sequence is the protein of sequence 1；

A2) aminoacid sequence shown in sequence in sequence table 1 is passed through replacement and/or the disappearance of one or several amino acid residue And/or add and have the protein of identical function；

A3) at A1) or N end A2) or/and C end connects the fused protein that label obtains；

The material of described regulation and control plant CIPK protein content is CIPK protein or its relevant biological material；Described biomaterial For following B1) to B9) in any one:

B1) nucleic acid molecules of CIPK protein is encoded；

B2) containing B1) expression cassette of described nucleic acid molecules；

B3) containing B1) recombinant vector of described nucleic acid molecules or containing B2) recombinant vector of described expression cassette；

B4) containing B1) recombinant microorganism of described nucleic acid molecules or containing B2) recombinant microorganism of described expression cassette or contain B3) recombinant microorganism of described recombinant vector；

B5) containing B1) the transgenic plant cells system of described nucleic acid molecules or containing B2) transgenic plant of described expression cassette Cell line；

B6) containing B1) Transgenic plant tissue of described nucleic acid molecules or containing B2) the transgenic plant group of described expression cassette Knit；

B7) containing B1) the transgenic plant organ of described nucleic acid molecules or containing B2) the transgenic plant device of described expression cassette Official；

B8) nucleic acid molecules that suppression CIPK protein coding gene is expressed；

B9) containing B8) expression cassette of described nucleic acid molecules, recombinant vector, recombinant microorganism or transgenic plant cells system.

4. according to described application arbitrary in claim 1-3, it is characterised in that: the nucleic acid molecules of described coding CIPK protein For following 1) or 2) or 3):

1) the cDNA molecule of sequence 2 or DNA molecular during coded sequence is sequence table；

2) with 1) nucleotide sequence that limits has 75% or more than 75% homogeneity, and the cDNA molecule of coding CIPK protein Or genomic DNA molecule；

3) under strict conditions with 1) nucleotide sequence hybridization that limits, and the cDNA molecule of coding CIPK protein or genome DNA molecular；

Described regulating plant growth is suppression plant growing；Described regulation and control plant ammonium content is for reducing plant ammonium content；Institute State suppression CIPK protein coding gene express nucleic acid molecules be with the DNA molecular shown in sequence in sequence table 2 in arbitrary The DNA molecular of section reverse complemental or the RNA of its coding.

5. according to the application described in claim 3 or 4, it is characterised in that: described suppression CIPK protein coding gene is expressed Nucleic acid molecules is the 981st DNA molecular shown in-1327 nucleotide of sequence 2 or the RNA of its coding in sequence table.

6. according to described application arbitrary in claim 1-5, it is characterised in that: described regulating plant growth is regulation and control plant roots Growth；Described plant ammonium content is the ammonium content in plant roots；

And/or, described plant is M1) or M2): M1) monocotyledon or dicotyledon；M2) Oryza sativa L..

The method of the most following M1 or M2:

M1, the method cultivating the transgenic plant that ammonium content reduces and/or root length reduces, including reducing CIPK egg in purpose plant The activity of white matter, reduce the expression of the encoding gene of the content of CIPK protein in purpose plant, suppression CIPK protein or strike Except the encoding gene of CIPK protein, obtain transgenic plant；Described transgenic plant has following M1a) and/or M1b) Feature: M1a) described transgenic plant ammonium content is less than described purpose plant；M1b) described transgenic plant root length is less than institute State purpose plant；

M2, the method cultivating the transgenic plant that ammonium content increases and/or root length increases, including increasing CIPK egg in purpose plant In the activity of white matter, increase purpose plant, the expression of the encoding gene of the content of CIPK protein, promotion CIPK protein, obtains Obtain transgenic plant；Described transgenic plant has following M2a) and/or feature M2b): M2a) described transgenic plant ammonium Content is less than described purpose plant；M2b) described transgenic plant root length is less than described purpose plant；

CIPK protein is arbitrary described CIPK protein in claim 1-3；

The encoding gene of described CIPK protein is the nucleic acid molecules encoding CIPK protein described in claim 3 or 4.

Method the most according to claim 7, it is characterised in that: described transgenic plant is by will be with claim 3-5 In arbitrary described suppression CIPK protein coding gene nucleic acid molecules described purpose plant of importing of expressing prepare.

9. according to the method described in claim 7 or 8, it is characterised in that: described plant ammonium content is the ammonium content in plant roots；

And/or, described plant is M1) or M2): M1) monocotyledon or dicotyledon；M2) Oryza sativa L..

10. arbitrary described biomaterial in claim 3-5.